**3. Assuring wine color in the long term**

## **3.1 Copigmentation**

Considering the anthocyanin equilibria in aqueous solution and the wine pH (pH 3–4), anthocyanin should be mainly present in a colorless form. Nevertheless, there are some mechanisms, known under the generic name of copigmentation, that contribute to stabilize the colored forms.

The phenomenon of copigmentation is due to non-covalent hydrophobic interactions between the aromatic nuclei of anthocyanins, with other molecules called copigments, normally colorless. It is a spontaneous and exothermic process consisting of the stacking of the copigment on the planar flavylium ion or quinoidal forms of anthocyanins [26]. Structurally, it manifests itself in a vertical anthocyanin-copigment stacking giving rise to a sandwich-like structure, establishing weak Van der Waals-type bonds and hydrophobic interactions between the compounds (**Figure 3**).

These sandwich-like groups generate a hydrophobic environment, due to the glucose molecules of the stacked anthocyanins, and in this way the nucleophilic attack of water molecules is prevented, and therefore the hydration of the anthocyanins is slowed, shifting the balance towards the colored form and therefore increasing the percentage of colored anthocyanins [27, 28].

Copigmentation also changes the absorbance spectrum of the anthocyanin chromophore, leading to an increase in the absorbance in the visible range (hyperchromic effect) and a displacement of the visible wavelength of maximum absorption (bathochromic effect).

*Anthocyanins and Wine Color: A Complex Story DOI: http://dx.doi.org/10.5772/intechopen.105162*

#### **Figure 3.**

*Schematic representation of the anthocyanin copigmentation.*

There are different types of compounds that can act as anthocyanin copigments, such as amino acids, nucleotides, carbohydrates or phenolic compounds. Flavonoids, in particular flavanols and flavonols, and hydroxycinnamic derivatives are the compounds that could most act as co-pigments due to their larger presence in wines.

Some authors show that the contribution of copigmentation in the color of young red wines can reach 30–50%, since at the pH of the wine, about 80% of the anthocyanins are in a colorless hydrated form [29].

The stability of the co-pigmentation complex in wines is limited and can be affected by several parameters:

Storage temperature: the increase in temperature produces a decrease in the copigmentation process and therefore affects the color [29–31].

pH: by increasing the pH there is a lower contribution of the anthocyanins in the form of the flavillium cation, therefore the co-pigmentation complex is also lowered [29, 32].

Ethanol content: the increase in ethanol favors the rupture of the copigmentation complex, although in the range of usual ethanol concentrations in wine, this influence is limited [29, 33].

Nature and structure of the cofactor: In wine, several compounds can act as cofactors such as hydroxycinnamic acids (caffeic, p-coumaric and ferulic acids), flavonols and flavanols. Flavonols are very good cofactors, although they are found in small quantities in wines [34, 35]. Flavan-3-ols, both monomers and oligomers intervene in co-pigmentation, although polymeric flavan-3-ols are less effective

as co-pigments. [30, 36, 37]. Among monomers, epicatechin is considered a better co-pigment than catechin [27, 38], dimer procyanidins with C4–C6 bonds appear to behave as better co-pigments than their respective dimers with C4–C8 bonds, possibly due to their more open and flexible conformation and the esterification of flavanols with gallic acid, as in the case of (–) - epicatechin-3-O-gallate, improves their capacity as copigments [27, 36].

Molar cofactor / anthocyanin ratio: increasing this ratio produces an increase in the effects produced by copigmentation [29, 30, 34, 39]. For this reason, copigmentation can be improved in wines by the prefermentative addition of substances that could act as cofactors. Some authors reported that the prefermentative addition of (+)-catechin produces increases in wine color from 9 to 13% and from 36 to 60% with caffeic acid [34, 35]. However this addition is not a regulated practice in enology. As an example of authorised practices, the addition of enological tannins also led to an improvement of wine chromatic characteristics and its stability [40]. Also, a covinification of some grape varieties could be an interesting practice for obtaining a richer phenolic composition. In this regard, covinification of Tempranillo and Graciano varieties results in higher copigmentation values than in the corresponding monovarietal wines [41]. Also, the addition of wood derivatives during the maceration process has been evaluated as an alternative technological application to modulate the phenolic composition and color characteristics of red wines, especially in warm climate regions [42].

### **3.2 Formation of anthocyanin-derived and polymeric compounds**

During aging, the color due to copigmentation decreases [34, 43]. This is also accompanied by a decrease in monomeric anthocyanin concentration, since they can be involved in degradation reactions, such as oxidation, but color not always decrease, even when copigmentation diminish. Studies have shown that the reaction of anthocyanins with other wine components leads to the formation of red anthocyaninderived compounds and polymerized compounds.

Pyranoanthocyanins: They are anthocyanin-derived compounds, formed by nucleophilic cycloaddition of a polarizable double bond of a compound with an anthocyanin. The formation of the pyran ring in the anthocyanin structure makes it less sensitive to pH variations than the anthocyanins from which they originate, due to the fact that the C4 position is blocked. Moreover, these compounds have shown to express more color than the corresponding anthocyanin [44].

**Figure 4** shows the different types of pyranoanthocyanins that appear in wines, whose structure will depend on the compound that incorporates to the anthocyanin:


Their concentration in wines will depend on the initial wine anthocyanin composition, the concentration of precursors, as well as the presence of small amounts of oxygen, since an oxidation stage intervenes in their formation [44].

The pyranoanthocyanins generated by the cycloaddition of acetaldehyde or pyruvic acid to the monomeric anthocyanin make up the so-called vitisins or carboxypyranoanthocyanins.

The carboxypyranoanthocyan obtained by the cycloaddition of pyruvic acid and malvidin-3-glucoside is called vitisin A. Furthermore, pyruvic derivatives of cyanidin, delphinidin, petunidin and peonidin have been identified in wine, both from monoglycosides and their acylated forms [44–47]. Vitisin A has a λmax of 511 nm.

The structure of vitisin B is the decarboxylated structure of vitisin A, generated by the cycloaddition of acetaldehyde to malvidin-3-glucoside. Later, coumarylvitisin B was also detected in wine [44, 46–50]. Vitisin B has a maximum wavelength ~ 20 nm shorter than vitisin A.

The formation of vitisin A and B depends on the anthocyanin composition, pH, concentration of pyruvic acid and acetaldehyde. Pyruvic acid and to a lesser extent acetaldehyde appear during alcoholic fermentation by the action of yeasts. Vitisin A is the most rapidly formed carboxypyranoanthocyanin and the first to be detected in young wines [51], On the other hand, vitisin B begins to be synthesized at the end of alcoholic fermentation, since acetaldehyde production is proportional to the amount of fermented sugar, being higher at the end of alcoholic fermentation.

Vitisins are more stable than monomeric anthocyanins and present a slower degradation rate than monomeric anthocyanins. After one year of aging, the amount of monoglycoside anthocyanins decreases between 80 and 90%, while vitisin A decreases between 15 and 25% and after 30 months, the monomeric anthocyanins in the wine have practically disappeared, while there are still between 20 and 35% of the initial amount of vitisins [52], even increases have been found in oxidative aging increases significantly [47, 53].

The phenylpyranoanthocyanins are formed by the addition of hydroxycinnamic acids (p-coumaric, caffeic, ferulic and synapic acid) or their decarboxylation products (4-vinylphenol, 4-vinylcatechol, 4-vinylguayacol and 4-vinyl syringol) to a monomeric anthocyanin.

The flavanylpyranoanthocyanins were initially synthesized in a model solution from malvidin-3-glucoside, acetaldehyde and flavanols (both monomeric such as (+) - catechin and (–) - epicatechin, as dimeric such as procyanidin B2 and B3), obtaining small amounts of malvidin- 3-glucoside-vinyl (epi) catechin and malvidin-3-glucoside-vinyldicatechin. Later they were identified in Port wines and red wines [44–47, 50, 54]. Like the previous pyranoanthocyanins (carboxypyranoanthocyanins and phenylpyranoanthocyanins), these pigments are more resistant to changes in pH or discoloration by hydration than free anthocyanins and do not suffer discoloration by SO2.

The age of the wines influences the concentration of flavanylpyranoanthocyanin with a flavanol unit, which increased with the years of aging of the wine, being the most important pigments in 6-year-old wines, possibly due to the degradation of catechindimer flavanylpyrantocyanins whose concentrations they were lower in older wines [54].

#### **3.3 Polymeric compounds**

Other reactions that may occur with anthocyanins are the formation of polymeric pigments, that are, in general, more stable to pH variations, and temperatures than monomeric anthocyanins, providing stability to the color of wine. These polymerization reactions mainly involve anthocyanins and polymeric flavan-3-ols, commonly known as wine tannins [55].

Direct condensations between phenolic compounds: The condensations between anthocyanins and tannins are nucleophilic addition reactions, since the phenolic compound (anthocyanin or tannin) can act as an electrophile (compound with a poor electron density) and be attacked by nucleophilic agents (compounds with an excess in electron density). Anthocyanins behave as electrophiles when they are in the form of the flavillium cation, on the other hand, their hydrated forms act as nucleophiles. Also, tannins can intervene as nucleophiles since their monomers have a negative charge density in their C6 and C8 positions, while the breaking of the interflavanol bond (CC) between the monomers that constitute tannin, an intermediate adduct is generated which operates as an electrophile [56]. Therefore, the condensation between anthocyanins and tannins presents two types of products; tannin-anthocyanin (T-A) and anthocyanin-tannin (A-T). They present a color similar to free anthocyanins and their existence has been confirmed in wine [56, 57].

Tannin-anthocyanin condensation (T-A): Procyanidin or tannin in an acid medium such as wine can undergo hydrolysis, a phenomenon that occurs spontaneously, generating an intermediate adduct, a carbocation or active catechin. This is an electrophilic structure that reacts with anthocyanin in the hemiacetal form (nucleophile), giving rise to a colorless compound, which after dehydration presents an orange color. T-A condensation occurs in the absence of oxygen, thus explaining its appearance in wines subjected to reducing environments, such as a bottle or tank [57].

Anthocyanin-tannin condensation (A-T): The mechanism consists of the union of the C4 position of the anthocyanin in the form of a flavillium cation (electrophile) with the negative positions C6 or C8 of the tannins (nucleophile), forming a colorless AT dimer which after its oxidation, facilitated by the presence of oxygen, it takes on a red color (A+-T) [56]. It has been shown in wine that these compounds have greater stability than monomeric anthocyanins, and their percentage increases during the maturation time [57].

*Anthocyanins and Wine Color: A Complex Story DOI: http://dx.doi.org/10.5772/intechopen.105162*

Acetaldehyde-mediated condensation between phenolic compounds: The formation mechanism of this polymerization consists of the activation of the acetaldehyde in the form of carbocation, which reacts with tannin in positions C6 or C8, giving a cationic aldehyde-flavanol adduct. Subsequently, an attack occurs on an anthocyanin in hydrated or hemiacetalic form at its C8 position [56]. Upon deprotonation, the violet colored quinoidal base is formed. Yeasts produce acetaldehyde under anaerobic conditions, at low pH, high sugar concentration, presence of SO2 and at low temperatures. Acetaldehyde is also a by-product of the metabolism of acetic and lactic bacteria [58] and it can also be generated in wine by the oxidation of ethanol. The presence of this condensation compounds in wines produces an increase in color [59–61] and they are more resistant to SO2 discoloration or hydration than free anthocyanins, not because the C2 and C4 positions of the anthocyanin are occupied, but because of steric hindrances produced by the monomers surrounding the anthocyanin [59]. Its formation is faster than direct condensation between anthocyanin-tannins, but these compounds are less stable. The rupture of the ethyl bridge leads again to the acetaldehyde-flavanol cation adduct (ethylflavanol) that can subsequently intervene in new condensations or generate, after dehydration, pyranoanthocyanins (flavanylpyranoanthocyanins) [59]. For this reason, the presence of this compounds can play an important role as more stable pyranoanthocyanic precursors.
