Possible Role of High-Molecular-Weight Salivary Proteins in Astringency Development.

Since the initial findings that food tannin/salivary protein interaction and subsequent precipitation is the main cause of the astringency development, numerous studies have concentrated on the supramolecular characterization of these bindings. Most of these works have focused on the low-molecular-weight salivary proteins, in particular proline-rich proteins, hardly considering the involvement of the high-molecular-weight salivary proteins (HMWSPs). Herein, different techniques such as fluorescence quenching, Isothermal Titration Calorimetry and HPLC-MS-DAD were employed to determine the occurrence of molecular interactions between three HMWSPs, namely, mucin, α-amylase and albumin, and a complex extract of tannins composed mainly of flavan-3-ols. The obtained results prove the capability of the three HMWSPs to effectively interact with the flavan-3-ol extract, involving different forces and action mechanisms. Flavan-3-ols are capable of interacting with mucins by a mechanism that includes the formation of stable ground-state complexes that led to approximately 90% flavan-3-ol precipitation, while for albumin and α-amylase, the interaction model of a "sphere of action" was established, which represented only 20% flavan-3-ol precipitation. These data highlight the relevance of including HMWSPs in astringency analyses, paying special heed to the role of mucins in the interaction and subsequent precipitation of dietary tannins.

Potential Use of Torulaspora delbrueckii As a New Source of Mannoproteins of Oenological Interest.

In this work, three MP extracts obtained from Torulaspora delbrueckii were added to red wine, and the changes in phenolic composition, color, and astringency were evaluated by HPLC-DAD-ESI-MS, tristimulus colorimetry, and sensory analysis, respectively. The MP extracts modified wine phenolic composition differently depending on the type of MP. Moreover, two MP extracts were able to reduce wine astringency. The fact that the MP-treated wines showed an increased flavanol content suggests the formation of MP-flavanol aggregates that remain in solution. Furthermore, the formation of these aggregates may hinder the interaction of flavanols with salivary proteins in the mouth. The effect of these MPs might be associated with their larger size, which could influence their ability to bind flavanols and salivary proteins. However, one of the astringent-modulating MPs also produced a loss of color, highlighting the importance of assessing the overall impact of MPs on the organoleptic properties of wine.

Supramolecular Study of the Interactions between Malvidin-3-O-Glucoside and Wine Phenolic Compounds: Influence on Color.

Supramolecular study of the interactions between the major wine anthocyanin, malvidin-3-O-glucoside (Mv3G) and different wine phenolic compounds (quercetin 3-O-ß-glucopyranoside (QG), caffeic acid, (-)-epicatechin, (+)-catechin, and gallic acid) has been performed at two different molar ratios (1:1 and 1:2) in acidic medium where flavylium cation predominates (pH ≤ 2). Color variations have been evaluated by differential colorimetry using CIELAB color space. These studies have been complemented with isothermal titration calorimetry assays and molecular dynamics simulations. The color of Mv3G flavylium cation is modified by the interaction with QG toward more bluish and intense colors. Interaction constants between the anthocyanin and the different phenolic compounds were obtained, ranging from 9.72 × 108 M-1 for QG to 1.50 × 102 M-1 for catechin. Hydrophobic interactions and H-bonds are the main driving forces in the pigment/copigment aggregation, except for the interactions where caffeic acid is involved, in which hydrophobic interactions acquire greater preponderance.

Role of Anthocyanins in the Interaction between Salivary Mucins and Wine Astringent Compounds.

Wine astringency is a very complex sensation whose complete mechanism has not been entirely described. Not only salivary proline-rich proteins (PRPs) are involved in its development; salivary mucins can also play an important role. On the other hand, it has been described that anthocyanins can interact with PRPs, but there is no information about their potential role on the interactions with mucins. In this work, the molecular interactions between salivary mucins (M) and different wine phenolic compounds, such as catechin (C), epicatechin (E) and quercetin 3-ß-glucopyranoside (QG), as well as the effect of the anthocyanin malvidin 3-O-glucoside (Mv) on the interactions with mucins, were assessed by isothermal titration calorimetry (ITC). Results showed that the interaction between anthocyanin and mucins is stronger than that of both flavanols analyzed, since the affinity constant values were 10 times higher for anthocyanin than for catechin, the only flavanol showing interaction in binary assay. Moreover, at the concentration at which polyphenols are usually found in wine, flavonols seem not to be involved in the interactions with mucins. These results showed, for the first time, the importance of wine anthocyanins in the mechanisms of astringency involving high-molecular-weight salivary proteins like mucins.

Effect of the addition of soluble polysaccharides from red and white grape skins on the polyphenolic composition and sensory properties of Tempranillo red wines.

Soluble polysaccharides from white (PSW) and red (PSR) grape skins were obtained to be evaluated as potential modulators of the unbalanced astringency of a Tempranillo red wine. The modulation of astringency was evaluated by a sensory panel and it seemed to be related to the changes in the polyphenolic profile. Isothermal Titration Calorimetry (ITC) studies, employed to characterize flavan-3-ol-polysaccharide interactions, showed that PSR decreased noticeably wine astringency causing a great flavan-3-ol loss (ca. 40 %), since they interacted more spontaneously with the flavan-3-ols (ca. ΔGtotal = -2.14 × 104 cal/mol) than PSW (ca. ΔGtotal = -1.32 × 104 cal/mol). The strength of these interactions seems to be related to the polysaccharide molecular size and to the presence of arabinogalactans in the structure. On the contrary, PSW showed no relevant effects on wine astringency. Furthermore, potential variations of color were also assessed and no deleterious effect was observed after the addition of any polysaccharide.

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.

The biological activity of the wine anthocyanins delphinidin and petunidin is mediated through Msn2 and Msn4 in Saccharomyces cerevisiae.

Polyphenols are considered to be responsible for some of the health benefits derived from the consumption of red wine. These protective effects might probably be explained in the context of the xenohormesis theory that considers plant metabolites as interspecific chemical signals. However, the complexity of the polyphenolic constituents of different wines makes it difficult to clarify the specific contribution of polyphenols to such effects. In the present work, we fractionated the polyphenols of a red wine and evaluated the effect of each polyphenolic fraction on the growth pattern of the yeast Saccharomyces cerevisiae. We observed a different contribution of the phenolic fractions to the xenohormetic response of S. cerevisiae, the fractions that were enriched with red pigments being the most protective against oxidative insults. Moreover, we found that red wine phenolic fractions exert their biological activity through the activation of the Yap1 and Msn2 stress-responsive regulators. Above all, the anthocyanins delphinidin 3-glucoside and petunidin 3-glucoside were found to improve significantly the growth rate of S. cerevisiae in an Msn2-, Msn4-dependent manner, indicating that the stress regulators Msn2 and Msn4 participate in the xenohormetic activity of the wine polyphenols delphinidin and petunidin.

Blending strategies for wine color modification â : Color improvement by blending wines of different phenolic profiles testified under extreme oxygen exposures.

Limited oxygenation and over-oxidation experiments were designed to compare the phenolic and chromatic characters of base wines Cabernet Franc (CF), Cabernet Sauvignon (CS), and their counterparts that blended with modifier wines Marselan (MA) and Petit Verdot (PV). In both limited oxygenation and over-oxidation conditions, all blend wines generally contained higher C*ab, a* and Red%, and lower hab, b* and Yellow% than their base wine counterparts, because MA contributed flavonols (copigments) and anthocyanins, and PV contributed flavanols (anthocyanin derived pigments precursors). Chromatic changes that can be perceived by human eye (ΔE*ab) in CF based blend wines were more obvious than that of CS based blend wines, which indicate that base wine with lower phenolic concentrations and weak phenolic profiles (CF) might be more prone to be chromatically modified than base wine with higher phenolic concentrations and distinct phenolic profiles (CS). Chemical influences of different blending strategies on anthocyanin derivatives' formations were depending on phenolic profiles of the modifier wines and base wines, and also on the oxygen exposure. The results suggest that the chromatic improvement of base wines could be realized by blending modifier wines under different oxygen exposures.

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.

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.

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.

Interactions between wine phenolic compounds and human saliva in astringency perception.

Astringency is a complex perceptual phenomenon involving several sensations that are perceived simultaneously. The mechanism leading to these sensations has been thoroughly and controversially discussed in the literature and it is still not well understood since there are many contributing factors. Although we are still far from elucidating the mechanisms whereby astringency develops, the interaction between phenolic compounds and proteins (from saliva, oral mucosa or cells) seems to be most important. This review summarizes the recent trends in the protein-phenol interaction, focusing on the effect of the structure of the phenolic compound on the interaction with salivary proteins and on methodologies based on these interactions to determine astringency.

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.

Interaction between Wine Phenolic Acids and Salivary Proteins by Saturation-Transfer Difference Nuclear Magnetic Resonance Spectroscopy (STD-NMR) and Molecular Dynamics Simulations.

The interaction between phenolic compounds and salivary proteins is highly related to the astringency perception. Recently, it has been proven the existence of synergisms on the perceived astringency when phenolic acids were tested as mixtures in comparison to individual compounds, maintaining constant the total amount of the stimulus. The interactions between wine phenolic acids and the peptide fragment IB712 have been studied by saturation-transfer difference (STD) NMR spectroscopy. This technique provided the dissociation constants and the percentage of interaction between both individual and mixtures of hydroxybenzoic and hydroxycinnamic acids and the model peptide. It is noteworthy that hydroxybenzoic acids showed higher affinity for the peptide than hydroxycinnamic acids. To obtain further insights into the mechanisms of interaction, molecular dynamics simulations have been performed. Results obtained not only showed the ability of these compounds to interact with salivary proteins but also may justify the synergistic effect observed in previous sensory studies.

Molecular Approach to the Synergistic Effect on Astringency Elicited by Mixtures of Flavanols.

The interactions between salivary proteins and wine flavanols (catechin, epicatechin, and mixtures thereof) have been studied by HPLC-DAD, isothermal titration microcalorimetry, and molecular dynamics simulations. Chromatographic results suggest that the presence of these flavanol mixtures could facilitate the formation of precipitates to the detriment of soluble aggregates. Comparison between the thermodynamic parameters obtained showed remarkably higher negative values of ΔG in the system containing the mixture of both flavanols in comparison to the systems containing individual flavanols, indicating a more favorable scenario in the mixing system. Also, the apparent binding constants were higher in this system. Furthermore, molecular dynamics simulations suggested a faster and greater cooperative binding of catechin and epicatechin to IB714 peptides when both types of flavanols are present simultaneously in solution.

Effect of the addition of flavan-3-ols on the HPLC-DAD salivary-protein profile.

The interaction between monomeric flavan-3-ols and salivary proteins has been studied using HPLC-DAD. A chromatographic method has been described and seven protein fractions were collected. The peptides and proteins present in each fraction have been identified using nLC-MS-MS analysis. The interaction between saliva and catechin, epicatechin and gallocatechin has been studied. These compounds interact in a discriminated way with salivary proteins: catechin causes a decrease of some fractions, epicatechin causes the decrease or increase of fractions while gallocatechin seems to cause an increase of two fractions. This variable behavior is explained, for the decrease in the chromatographic area, by the precipitation of salivary proteins and, for the increase of the area, by the formation of soluble complexes and/or for the formation of new peaks.

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.