Introduction

Colour is an important feature of red wine, being the first attribute to be perceived by wine consumers, and is directly associated with its quality. Red wines have a very complex matrix due to the wide variety of compounds extracted from grapes and to the metabolites released by yeast during the fermentation process. In specific cases, such as fortified wines like Port wines, the complexity is even higher owing to the addition of wine spirit to stop the fermentation. Wine spirits possess a great variety of compounds such as higher alcohols, esters, and mainly aldehydes (acetaldehyde, 2-methylpropanal, 2-methylbutanal and benzaldehyde, among others) (Pissarra et al., 2005). Moreover, wine ageing in oak barrels, addition of oak chips, or must fermentation in contact with oak staves can also have an impact on the chemical transformation of wine components. During these processes, different compounds can be extracted from oak, such as ellagitannins, phenolic acids, and furanic and phenolic aldehydes (Gonzalez-Centeno et al., 2016; Garcia et al., 2012), that can participate in wine colour stabilization. Among these molecules, aldehydes should have a higher impact on wines due to their increased reactivity towards flavonoids like flavan-3-ols and anthocyanins (Pissarra et al., 2003; Sousa et al., 2012)The latter are the main pigments in many flowers and fruits (e.g. grapes) being responsible for the great diversity of colours (i.e. red, violet and blue) found in nature and the red/violet colour observed in young red wines. In aqueous solution, the colour of anthocyanins is dependent on their structure and is strongly affected by the pH (Brouillard and Lang, 1990). At very acidic pH (pH~1), anthocyanins are present in their flavylium cation form which has a red colour. When the pH is raised to 3-4, the flavylium cation is involved in two parallel reactions in equilibrium: deprotonation to form the violet quinonoidal base and hydration at the C-2 position yielding the non-coloured hemiketal form. These forms are also in equilibrium with the cis- and trans-chalcone forms which present a yellow colour (Pina, 1998). With a pH rise to values up to 6, the quinonoidal base can be ionized to form the respective blue anionic quinoidal form [(Brouillard and Lang, 1990; Santos et al., 1993) (Figure 1). According to these equilibria, it would be expected that at wine pH (3.2-3.8), anthocyanins would be present in a great extent in their non-coloured hemiketal form. Nevertheless, there are some mechanisms to stabilize the coloured forms of anthocyanins. These include self-association (Gonzalez-Manzano et al., 2008; Mistry et al., 1991), intra and intermolecular copigmentation (Mistry et al., 1991; Yoshida et al., 2009) and metal complexation mechanisms. Copigmentation helps to stabilize the flavylium form and protects it from the nucleophilic attack by water, thereby preventing the formation of colourless forms (Goto and Kondo, 1991; Yoshida et al., 2009, Dangles et al., 1994). However, some years ago, Asenstorfer and his co-workers using paper electrophoresis postulated that at wine pH, malvidin-3-glucoside is present as the uncharged quinonoidal base as major coloured component of the equilibria (Asenstorfer et al., 2003).

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During wine ageing, anthocyanins participate in several reactions (i.e. reduction, oxidation, and polymerization) involving other wine molecules, leading to the formation of more stable anthocyanin-derived compounds (Oliveira et al., 2009; Oliveira et al., 2014).

Bearing all this, this paper will focus on reviewing the current knowledge of red wine colour, especially concerning colour stabilization mechanisms and colour evolution during wine ageing and maturation.

Colour stabilization

In red wines made from Vitis vinifera L. grapes, the monomeric anthocyanins present are 3-O-glucosides of delphinidin, cyanidin (in very small amounts), petunidin, peonidin and malvidin. They differ from each other on the hydroxylation and/or methoxylation pattern of ring B, which affects directly the hue and colour stability (Mateus et al., 2001). In red wines, the glucose moiety can also be esterified at the hydroxyl group present at the C-6 position with different acids, namely acetic, p-coumaric and caffeic acids 30. Malvidin-3-O-glucoside and its derivatives are usually the most abundant anthocyanins in young red wines (Mateus et al., 2001).

1. Interaction with cell-wall components

Due to the strong influence of anthocyanins on red wine quality, many studies have been conducted to determine how anthocyanins are extracted during winemaking. In red grapes, these pigments are mainly located in skins . Inside the cells, anthocyanins are found in the vacuoles enclosed by tonoplast and cytoplasmic lipid membranes, which are in turn encapsulated by the skin cell-wall (CW) (Figure 2) (Rodríguez et al., 2004). During winemaking, the rate and the extent of anthocyanin extraction depend not only on their concentration but also on the grape skin CW polysaccharide composition (Busse-Valverde et al., 2012). In fact, during the maceration process, anthocyanins and soluble and insoluble CW polysaccharide fragments resulting from pectolytic enzyme activity come into contact and a substantial proportion of anthocyanins may be adsorbed by the CW in suspension in the must. Such interactions affect the extractability of anthocyanins during fermentative maceration, their solubilization and the final colour of the wines (Padayachee et al., 2012; Bautista-Ortín et al., 2016) (Figure 2).

Previous works have shown that the anthocyanin content of a given cultivar is not always positively correlated with the anthocyanin concentration in the resulting wine (Romero-Cascales et al., 2005). This lack of correlation was attributed to the partial retention of these anthocyanins in the skin cells due to the barrier effect of the CW polymers (Ortega-Regules et al., 2006; Rolle et al., 2009; Rolle et al., 2012). The grape CW have a very complex structure, composed essentially of a network of cellulose microfibrils inter-knotted with a hemicellulosic polysaccharide matrix consisting mainly of xyloglucan polymers. Pectic polysaccharides and other components such as enzymes, structural proteins, ions and low molecular weight polyphenols are also embedded within this matrix (Pinelo et al., 2006). Pectic polysaccharides are a group of polysaccharides rich in galacturonic acid and classified in three types of polymers: homogalacturonan polymer and two branched side chain polymers containing a rhamnosyl residue, the so-called rhamnogalacturonans I and II (Vicens et al., 2009 ; Nunan et al., 1997). The grape skin CW are made up of 30% neutral polysaccharides (cellulose, xyloglucan, arabinan, galactan, xylan and mannan), 20% acidic pectic polysaccharides (of which 62% are methyl esterified) and approximately 15% insoluble proanthocyanidins . During fruit ripening, the degradation of CW polysaccharides by pectolytic enzymes and other enzymes able to catalyze the hydrolysis of bonds in plant CW polysaccharides increases the extractability of anthocyanins from grape skins.

Figure – Schematic representation of the grape cell structure evidencing the presence of the vacuoles where anthocyanins are located and schematic representation of the primary cell wall evidencing the most important polysaccharides.

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Different studies using purified grape CW material or pure cellulose and cellulose-pectin composites as CW models have also demonstrated the high affinity of these CW materials for anthocyanins and highlighted that these interactions may affect anthocyanin extraction (Padayachee et al., 2012; Bindon et al., 2014). Likewise, it was evidenced that some anthocyanin structural features may influence their extractability from grape skins. In particular, a lower extraction yield was reported for coumaroylated anthocyanins compared to the non-acylated ones (Fournand et al., 2006). Furthermore, the results reported by Saura-Calixto et al. suggest that wine polymeric polysaccharides interact with two major groups of polyphenolic compounds: free wine polyphenols and wine polyphenols associated to polysaccharides. About 35 to 60% of total wine polyphenols in red wine and about 10% in white wine are associated to polysaccharides (polyphenol-polysaccharide complexes). Moreover, anthocyanins were the major constituents of red wine polysaccharide-polyphenol complexes, while phenolic acids were the major constituents in white wine complexes

The retention of polyphenols in CW polysaccharides depends on the conformational flexibility and on the molecular weight of the phenolic moiety. Also, physical features of CW polysaccharides like porosity and chemical composition can also influence the eventual association between CW polysaccharides and anthocyanins. Different polarities and the hydrophobic nature of these polysaccharides influence the degree of retention or adsorption of anthocyanins (Renard et al., 2001; Le Bourvellec, 2004).. Hydrogen bonds and hydrophobic interactions are the two main driving forces proposed to explain the nature of complex formation between anthocyanins and plant CW polysaccharides . It seems likely that the adsorption of anthocyanins involves the establishment of a number of low energy non-covalent interactions derived from a combination of hydrogen bonding between the hydroxyl groups of phenols and the oxygen atoms of the cross-linking ether bonds of sugars present in the CW polysaccharides and hydrophobic interactions between sugar rings and phenols (Fernandes et al., 2014; Phan et al., 2015) (Figure 3). By Saturation Transfer Difference – Nuclear Molecular Resonance (STD-NMR) and Molecular Dynamics (MD) studies, Fernandes et al. described that anthocyanins interact with polysaccharides over a two-stage process – a rapid but limited phase followed by a slower but more extensive binding, which may be due to deposition of anthocyanin molecules onto the occasional sites where primary binding had already occurred . Likewise, some polysaccharides have the ability to form gel-like structures or to develop secondary structures in solution forming hydrophobic pockets able to encapsulate and complex polyphenols (Gonçalves et al., 2012; Le Bourvellec et al., 2005). Differences in polysaccharides based on galactose and arabinose, together with changes in the cellulose content and degree of methylation of pectins, may also affect the extractability rate of anthocyanins from grapes into the must (Ortega-Regules et al., 2006). According to Hernández-Hierro et al. (2012), the ripeness degree and, to a lesser extent, the soluble solids content also influence the amount of anthocyanins released and generally, non-acylated anthocyanins were better extracted than the acylated ones. On the other hand, total insoluble CW material has shown to exhibit the biggest opposition to anthocyanin extraction, slightly higher for non-acylated anthocyanins, while the highest amounts of cellulose, rhamnogalacturonans II and polyphenols were positively correlated with anthocyanin extraction. Xyloglucans, homogalacturonans and rhamnogalacturonans I evidenced opposite behaviour to anthocyanin extraction (Hernández-Hierro et al., 2014).

The positive relationship between polyphenol content in CW and anthocyanin extraction probably suggests the existence of copigmentation processes referenced before between these components while the extraction is taking place, so they could be able to assist the extraction process (Boulton, 2001).

Figure . Schematic representation of some interactions established between anthocyanins and a low methoxylated pectin model involving hydrophobic interaction and H bonding. Pectin molecule is depicted with sticks and coloured in dark gray (Fernandez et al., 2014)..

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Gonçalves et al. reported a higher retention capacity of acylated anthocyanins by CW polymeric material, compared to the non-acylated anthocyanins. The acylation of the glucose residue was shown to enhance anthocyanin hydrophobicity, suggesting that the association of polymeric material with anthocyanins could be mainly due to hydrophobic interactions. In addition, CH- interaction can occur between the aromatic rings on anthocyanins and carbohydrates (Gonçalves at al., 2012). Aggregation of anthocyanin pigments to yeast CW during fermentation has also been explained to occur as a result of hydrophobic interactions between sugars and phenols, although this adsorption has been found to depend also on the degree of methoxylation and acylation of anthocyanins (Morata et al., 2003). Previous reports also showed that the physical properties of grape skins, like skin hardness, thickness, number of cell layers and CW thickness, and the grape variety are also linked to anthocyanin extractability (Río Segade et al., 2011 ; Río Segade et al., 2008).

Colour evolution during wine ageing and maturation

During ageing and maturation, the colour of wines changes from red/violet to red/orange due to several chemical reactions (oxidation, reduction and polymerization) where anthocyanins participate, thus being the precursors of new compounds. In the first stages of wine evolution, anthocyanins can react with flavan-3-ols (catechin monomers and condensed tannins) directly or indirectly via aldehydes, yielding new purple pigments (anthocyanin-alkyl-catechin) that further lead to the formation of pyranoanthocyanin-catechin compounds displaying an orange colour (Pissarra et al., 2003; Liao et al., 1992; Rivas-Gonzalo et al., 1995; Francia-Aricha et al. 1998) (Figure 5). Acetaldehyde is particularly important for anthocyanin chemical transformation reactions. This aldehyde is present in high amount in wine spirits (40-260 mg/L) and is also formed during fermentation and wine ageing from ethanol oxidation (Pissarra et al., 2005).

Figure 5 – Anthocyanin-derived pigments identified in red Port wines.

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Several pyranoanthocyanin pigments resulting from the reaction of anthocyanins with small molecules arising from grapes or from yeast metabolism have been identified in red wines over the last years, like acetaldehyde (Bakker and Timberlake, 1997), pyruvic acid (Fulcrand et al., 1996), acetoacetic acid (He et al., 2006), diacetyl (Gomez-Alonso et al., 2012), hydroxycinnamic acids (Schwarz et al., 2003; Rentzsch et al., 2007) and p-vinyl-phenols (Hayasaka and Asenstorfer, 2002; Schwarz et al., 2003) (Figure 5).

Pyruvic acid derivatives of anthocyanins (A-type vitisins) are the pyranoanthocyanins detected in higher concentration in Port wines (Mateus and de Freitas, 2001). The characteristic chemical and physical properties of Port wine could be at the origin of higher levels of yeast metabolites likely to yield anthocyanin derivatives. Indeed, when wine spirit is added to stop fermentation, pyruvic acid concentration is expected to be higher than when the fermentation is allowed to go to dryness. In fact, pyruvic acid released by yeast at the beginning of the fermentation is further used in yeast metabolism. Its levels increase right after wine fortification, and the initial formation of anthocyanin-pyruvic acid adducts is concurrent with the degradation of anthocyanin monoglucosides. A-type vitisins were found to be very resistant to wine ageing when compared to the original grape anthocyanins (Oliveira et al., 2013, Mateus and de Freitas, 2001; Asenstorfer and Jones, 2007). Yellowish α-pyranone-anthocyanins were also described to be present in aged red wines derived from the direct oxidation of A-type vitisins (He et al., 2010).

A few years ago, a new class of pyranoanthocyanin compounds (vinylpyranoanthocyanins) named portisins, presenting an unusual bluish colour at acidic pH values, was identified in a young Port wine (Mateus et al., 2003) (Figure 5). The studies performed in model solutions have demonstrated that these compounds can be formed in wines from the reaction between A-type vitisins and flavanols in the presence of acetaldehyde (Mateus et al., 2004). Similar compounds were identified in Port wines resulting from the reaction of A-type vitisins with hydroxycinnamic acids such as p-coumaric, caffeic, ferulic and sinapic acids (Oliveira et al., 2007). More recently, a turquoise blue pyranoanthocyanin dimer pigment was found to occur in a 9-year-old Port wine and in the respective lees derived from the reaction of the A-type vitisin with a methylpyranoanthocyanin (Oliveira et al., 2010).

The occurrence of these novel pigments in aged wines points to a second-generation pathway where the main precursors are no longer the native anthocyanins but secondary anthocyanin products such as A-type vitisins involved in the formation of new pigments in subsequent stages of wine ageing that may contribute directly or indirectly to its colour evolution.

Moreover, studies performed in aqueous solutions using UV-Visible spectroscopy and NMR techniques revealed that pyranoanthocyanins present a higher colour stability when compared to their anthocyanin precursors due to the absence of hydration reactions (Oliveira et al., 2009; Oliveira et al. 2013; Oliveira et al., 2014).

On the other hand, polymeric pigments have been described to play an important role in the long-term colour stability of aged red wines (Boulton, 2001). However, there is still a lot to know about their identity and chemical pathways in wines. The first evidence of the presence of oligomeric anthocyanins (dimeric and trimeric) was described by Vidal and co-workers in grape skins using mass spectrometry techniques (Vidal et al., 2004). Later, the dimeric compounds were also reported to occur in red wines by Salas et al. (2005) and Alcalde-Eon et al. (2007) using the same technique. A few years later, Oliveira et al. demonstrated the presence of an A-type malvidin-3-glucoside trimer in a young Port wine using LC-MS and NMR methodologies (Oliveira et al., 2013) (Figure 6). The origin of these compounds in red wine could result from their extraction from grape skin during the winemaking process, as the presence of these trimeric pigments was detected in a grape skin extract by LC/DAD-MS spectrometry (Oliveira et al., 2013). Moreover, this pigment revealed to be much less prone to hydration reactions than the anthocyanin monomers, with hydration accounting for less than 10% of the overall reactivity (Oliveira et al., 2014). A dimeric acetaldehyde malvidin-3-glucoside condensation pigment was also found to occur in red wine (Atanasova, 2002).

All these new families of anthocyanin-derived pigments make an important contribution to colour hue and stability in wine and, in some cases, they could constitute a quality factor.

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Figure 6 – Structure of A-type malvidin-3-glucoside trimer detected in a young wine and in grape skins.

Conclusions

Red wine is undoubtedly a complex matrix, and our understanding of all the molecules and events responsible for wine colour is far from being complete. The main limitations are related to the capability of the techniques used, the detection limits, and the increasing complexity of the structures, among others. Many physical and chemical phenomena have been described herein where anthocyanins were reported to interact/react with each other and many other simpler or more complex phenolic and non-phenolic compounds. All of these phenomena interfere or have influence on red wine colour.

The main characteristic of all copigmentation processes is the capacity to hinder the hydration reaction for all the mechanisms involved in vertical π-stacking of the chromophore, preventing nucleophilic attack by water and colour loss. Of all the copigmentation phenomena, intermolecular copigmentation is the most important in red wines due to the variety and concentration of possible copigments that can be found in solution. Moreover, there are evidences of the occurrence of copigmentation between anthocyanins and grape CW components while the extraction is taking place. The only requirement is the ability to adopt a planar configuration that allows the approach and association with the anthocyanins.

Acknowledgements : This work received financial support from FEDER funds through COMPETE, POPH/FSE, QREN and FCT (Fundação para a Ciência e Tecnologia) post-doctoral scholarships (SFRH/BPD/112465/2015 and FOOD-RL1-PHD-QUINOA-01-02), investigator contract (IF/00225/2015) and grants (PTDC/AGR-TEC/2789/2014, REDE/1517/RMN/2005). This work also received financial support (UID/QUI/50006/2013 - POCI/01/0145/FEDER/007265) from FCT/MEC through national funds and FEDER, under the Partnership Agreement PT2020.

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