3.1.1. Occurrence, general chemistry, and sensory aspects

Anthocyanins occur as vacuolar components in the berry skin tissue (and in the mesocarp of the so-called teinturier varieties) and are present as monomers of six glycosylated forms, namely malvidin, cyanidin, petunidin, peonidin, delphinidin, and pelargonidin [26, 27]. Glycosylation occurs at the C3 position and renders the molecule water-soluble [28]. Acylation in turn occurs in the C6 position of the glucose moiety by esterification with an aromatic (p-coumaric, caffeic, ferulic, and sinapic acids) or an aliphatic acid (acetic, malic, malonic, oxalic, and succinic acids) (Figure 3). The acylation of the sugar might promote the chemical stability of the anthocyanin molecule [29], possibility through stacking of the acyl groups with the pyrilium ring of the flavylium cation, thereby reducing the susceptibility to the nucleophilic attack of water [30].

Anthocyanins are red pigments responsible for the color of red wines and owe their spectral properties to the resonant structure given by a 10-electron system, partially delocalized between the pyran C and A rings, as well as to the extended conjugated system of unsaturated bonds in the structure [13, 31]. As a result, the maximum absorbance (λmax) of these compounds varies between 475 and 540 nm depending upon the aglycone (known as anthocyanidin) and substitution patterns of the C ring [26]. The λmax between 475 and 540 nm confers anthocyanins blue to red color hues. O-methylation in aglycones such as malvidin, petunidin, and peonidin causes a slight reddening effect (Figure 4) and reduces the reactivity of the nearby phenolic hydroxyl groups, thereby increasing the stability of the molecule [32]. On the other hand, the increase in the number of free hydroxyl groups in the B ring increases blueness (i.e., bathochromic shift), which in turn renders the structure more polar but less stable. Acylation has also been suggested to produce a bathochromic shift, giving more purple tones, possibly as a result of intramolecular copigmentation reactions [30]. These and other features can be observed in Figure 4 for purified anthocyanins isolated from Cabernet Sauvignon grapes after preparative HPLC fractionation of anthocyanins and confirmation by mass spectroscopy.

Figure 3. Basic anthocyanin structure, acyl groups, and the main six anthocyanindins found in V. vinifera grapes and wines.

Figure 4. Preparative HPLC chromatogram showing separation of anthocyanin monoglucosides and acyl-glucosides of Cabernet Sauvignon grapes from Washington State (USA). Anthocyanin fractions are shown approximately above of each major peak and were collected directly from the elutant at pH ~ 1.8. Peak assignment: (1) delphinidin-3-O-glucoside, (2) cyanidin-3-O-glucoside, (3) petunidin-3-O-glucoside, (4) peonidin-3-O-glucoside, (5) malvidin-3-O-glucoside, (6) delphinidin-3-O-acetyl-glucoside, (7) petunidin-3-O-aceyl-glucoside, (8) peonidin-3-O-acetyl-glucoside, (9) malvidin-3- O-acetyl-glucoside, (10) malvidin-3-(6-O-caffeoyl)-glucoside, (11) petunidin-3-(6-O-coumaroyl)-glucoside, (12) peonidin-3-(6-O-coumaroyl)-glucoside (shoulder), and malvidin-3-(6-O-coumaroyl)-glucoside.

The flavylium cation of the anthocyanins is responsible for the chromatic properties of young red wines, with a molar extinction coefficient (ε) of 29,500 M-1 cm-1 for malvidin-3-glucoside in 0.1% HCl methanolic solution [33]. As a result of this, some of the color observed in aged red wines can still be attributable to the flavylium cation. However, upon crushing and during winemaking, anthocyanins undergo a variety of electrophilic and nucleophilic substitutions giving rise to cycloaddition and condensation products, and oligomeric and polymeric pigments (Section 3.4 and Figure 13). These transformations invariably have an impact on wine color, and it is usually during maceration and postmaceration when the most noticeable changes in wine color take place (Figure 5). For example, at the beginning of maceration, the intense purplered color and hyperchromic shift (i.e., increase in absorbance) observed in red wines is the result of intra- and intermolecular and self-association copigmentation reactions [34]. Subsequently, with the disruption of copigmentation mediated by increasing ethanol levels, perceived color decreases and shifts toward more red tones [35]. As maceration winds up and aging ensues, the incorporation of anthocyanins into vitisins A, B, vinyl-catechol derivatives, and xanthylium salts, to name some possible reactions that lead to the formation of the socalled pyranoanthocyanins (Figure 13), causes a shift in color from deep-red to orange or brick-orange hues [36]. It is also expected that the incorporation of anthocyanins into

Figure 5. Evolution of the full-length visible spectrum of a Malbec wine produced with different maceration times, ranging from 4 hours to 21 days. The vertical line indicates the 520-nm wavelength, which is approximately the wavelength of maximum absorbance for most anthocyanins. Notice the drop in the absorbance values of the full spectrum in the wine produced with 21 days maceration relative to the wine produced with 8 days of maceration.

Figure 4. Preparative HPLC chromatogram showing separation of anthocyanin monoglucosides and acyl-glucosides of Cabernet Sauvignon grapes from Washington State (USA). Anthocyanin fractions are shown approximately above of each major peak and were collected directly from the elutant at pH ~ 1.8. Peak assignment: (1) delphinidin-3-O-glucoside, (2) cyanidin-3-O-glucoside, (3) petunidin-3-O-glucoside, (4) peonidin-3-O-glucoside, (5) malvidin-3-O-glucoside, (6) delphinidin-3-O-acetyl-glucoside, (7) petunidin-3-O-aceyl-glucoside, (8) peonidin-3-O-acetyl-glucoside, (9) malvidin-3- O-acetyl-glucoside, (10) malvidin-3-(6-O-caffeoyl)-glucoside, (11) petunidin-3-(6-O-coumaroyl)-glucoside, (12) peonidin-

Figure 3. Basic anthocyanin structure, acyl groups, and the main six anthocyanindins found in V. vinifera grapes and wines.

3-(6-O-coumaroyl)-glucoside (shoulder), and malvidin-3-(6-O-coumaroyl)-glucoside.

158 Phenolic Compounds - Natural Sources, Importance and Applications

oligomeric and polymeric tannins (to form the so-called polymeric pigments) should cause a reduction in the molar extinction coefficient of the flavylium form, thus leading to a decrease in color saturation as winemaking progressed [37]. In the case of pyranoanthocyanins, these do not necessarily appear to have lower molar extinction coefficients relative to the native anthocyanins [38], and, in fact, their molar extinction coefficient is much more stable toward pH swings [39].

Isolated anthocyanins are tasteless or indistinctly flavored [40]; however, upon reaction with oligomeric or polymeric tannins during winemaking, oligomeric and polymeric pigments are formed (Figure 14) and these can in turn modulate astringency (Section 3.4).

### 3.1.2. Extraction during winemaking

The diffusion of anthocyanins into the must requires the breakdown of two biological barriers, namely the cell wall, including the degradation of the pectic substances in the middle lamella, and the tonoplast of the vacuoles of the skin subepidermal cells [41]. Normal operations during crushing ensure the breakdown of cellular walls, and native enzymatic reactions allow for the degradation of pectic substances and polysaccharides in the middle lamella. The diffusion process is favored by the water-soluble nature of anthocyanins, resulting in maximum extraction rates and a peak of extraction within the first 3 to 7 days of maceration [35, 42–48] as observed in Figure 6. The rate of extraction of the different anthocyanins seems to be similar [45, 47] and proportional to their original concentration in grapes. However, some studies have found that wines have a relative higher amount of malvidin-3-glucoside than that originally present in the grapes [49, 50]. Establishment of unequivocal extraction and retention patterns of different anthocyanin forms may be complicated by the fact that acylated anthocyanins may undergo acid hydrolysis upon extraction into the fermenting must/wine [50], thereby releasing the monoglucoside forms and/or by the fact that, for example, acylated derivatives are preferentially adsorbed by wine lees [51].

Figure 6. Overview of the extraction of and evolution of anthocyanins during (a) prefermentative cold soak (CS), maceration, end of malolactic fermentation (end MLF), and up to 24 months of bottle ageing (B) of Malbec wines processed with a maceration length of 15 days (control) and with cold soak for 7 days + 15 days of maceration (cold soak), and (b) maceration and bottle aging of Cabernet Sauvignon wines processed with a maceration length of 10 days (control) and 30 days (extended maceration). Adapted from Refs. [43, 55].

The early extraction of anthocyanins may also influence the solubility and retention of oligomeric and polymeric tannins through the formation of polymeric pigments [52–54]. Following the peak of anthocyanin extraction, a variable drop in concentration, which can be as high as 60% from peak concentration, is typically observed [42, 43, 55] (Figure 6a). Loss of anthocyanins during maceration in fermenting must and wines has been attributed to a variety of factors, including ionic adsorption by the negatively charged yeast cell walls and yeast lees during postmaceration [56, 57], adsorption onto bitartrate crystals and particulate matter [35], incorporation into small and large polymeric pigments [43, 58], formation of pyranoanthocyanins [56, 59], and oxidative cleavage of the heterocyclic C-ring leading to direct anthocyanin degradation [60]. A decrease in copigmentation as a result of an increasing concentration of ethanol in the fermenting must (which increases the hydrophobic character of the medium thereby disrupting the copigmentation complex) also contributes to both the loss of anthocyanins and a decrease in wine color [34, 35, 61]. At the end of maceration, the levels of anthocyanins recovered in the wine, relative to the grape initial content, have been reported to be around 40% [26].

Counter to what intuition may suggest, there appears to be a negative relationship between maceration length and anthocyanins retained in the resulting wine [43, 47, 58, 62, 63] (Figure 6b). Moreover, analysis of anthocyanins recovered in the pomace hardly increased the recovery yield, suggesting that a major proportion of anthocyanins were converted to other species or were irreversibly adsorbed on the solid material during maceration [26, 47]. This discrepancy was originally attributed to the enhanced formation of polymeric pigments in wines undergoing extended maceration. One study found that Merlot wines produced with extended maceration were lower in anthocyanins but higher in polymeric pigments relative to control wines [58]. However, the formation of polymeric pigments during extended maceration cannot fully explain the observed anthocyanin loss [47]. This suggests that, in addition to anthocyanin losses by adsorption, degradation reactions may be at play during extended maceration. Ultimately, the extraction patterns during winemaking and aging and thus the final concentration of anthocyanins are both modulated by the variety and the maceration technique the winemaker decides to put in place, which can be as simple as delaying the onset of alcoholic fermentation (e.g., the technique known as cold soak<sup>4</sup> ) or increasing maceration time (e.g., extended maceration) (Figure 6a and b, respectively).
