3.2. Flavan-3-ols

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

Isolated anthocyanins are tasteless or indistinctly flavored [40]; however, upon reaction with oligomeric or polymeric tannins during winemaking, oligomeric and polymeric pigments are

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 prefer-

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)

formed (Figure 14) and these can in turn modulate astringency (Section 3.4).

swings [39].

3.1.2. Extraction during winemaking

160 Phenolic Compounds - Natural Sources, Importance and Applications

entially adsorbed by wine lees [51].

and 30 days (extended maceration). Adapted from Refs. [43, 55].

### 3.2.1. Occurrence, general chemistry, and sensory aspects

In V. vinifera grapes and wines, flavan-3-ols occur both in seeds and skins as five monomers: (+)-catechin, (-)-epicatechin, (+)-gallocatechin, (+)-epigallocatechin, and (-)-epicatechin-3-Ogallate [64–66] (Figure 7). The glycosyl derivatives of four of these monomers have also been

<sup>4</sup> Cold soak, also known as prefermentative cold soak, is a winemaking technique in which the onset of alcoholic fermentation is delayed by keeping the must (and thus the fermentation solids) at temperatures ranging from 5 to 15C by means of a cooling system in jacketed fermenters or by the use of solid CO2 (dry ice). During cold soak, the extraction of water-soluble compounds is sought, as opposed to the extraction that takes place in the presence of ethanol during alcoholic fermentation. The duration of cold soak is defined by the winemaker and it can be as short as 2 days and as long as 14 days.

Figure 7. Monomeric flavan-3-ols found in V. vinifera grapes and wines.

recently reported in Merlot grapes and wines [67]. Flavan-3-ols occur in several isomeric forms. The carbons at the C2 and C3 positions of the flavan-3-ol backbone are two asymmetric centers, such as the five monomeric flavan-3-ols are grouped into two diastereomers pairs, with configurations 2R:3S for (+)-catechin and (+)-gallocatechin and 2R:3R for (-)-epicatechin, (-)-epigallocatechin, and ()-epicatechin-3-O-gallate [68]. These different isomeric configurations, in turn, have an impact on bioavailability [69], antioxidant and radical scavenging properties [70], and, ultimately, on sensory properties [71, 72], as further discussed later.

In seeds, flavan-3-ols are located in thin-walled cells between the external cuticle and the inner lignified layers. The (sometime observed) seed browning during berry ripening is thought to be the result of both monomeric flavan-3-ols and tannins undergoing oxidation [73]. Seeds contain only (+)-catechin, ()-epicatechin, and (-)-epicatechin-3-O-gallate [64, 74]. In the skins, flavan-3-ols occur in the subepidermal cell as shapeless or spherical inclusions free in the vacuoles but also associated with the tonoplast [75]. Skins contains (+)-catechin, (-)-epicatechin, (-)-epicatechin-3-O-gallate, and, additionally, (-)-epigallocatechin [76], as well as trace amounts of (+)-gallocatechin and (-)-epigallocatechin gallate [66, 77].

Quantitative differences also occur within the berry tissues. Seeds concentrate the vast majority of flavan-3-ols of the berry [66, 75–79]. For example, flavan-3-ol concentrations of about 179 mg/kg fresh weight (FW) have been found in Cabernet Sauvignon seeds whereas skins of the same variety only have 4.8 mg/kg FW [80]; similar results are reported for other varieties [81, 82]. In wines, the content of monomeric flavan-3-ols varies from 29 to 41 mg/L in Tempranillo and Graciano wines [78], 107 to 176 mg/L in Pinot Noir wines [83], 182 mg/L in Tannat wines [84], and up to 288 mg/L in Cabernet Sauvignon wines [43].

Due to the reduced state of the C ring of the flavan-3-ol structure, and thus favorable oneelectron donation properties, flavan-3-ols can react with several wine electrophiles. The condensation of monomeric flavan-3-ols with anthocyanins either by a direct covalent reaction between them [85, 86] or mediated by acetaldehyde [87, 88] is one of the main reactions with impact on color during wine aging and is further addressed in Section 3.4.

Flavan-3-ols are colorless and do not absorb light in the visible spectrum, but have a peak of UV absorbance between 270 and 280 nm [89, 90]. Catechin and epicatechin are both susceptible to enzymatic [91] and nonenzymatic oxidation [92, 93], with both mechanisms resulting in a change in spectral properties from colorless to yellow hues [89, 94]. The end products of these reactions are quinones. Quinones are powerful electrophiles that can readily react with wine nucleophiles such as sulfur compounds (e.g., volatile thiols, common in Sauvignon Blanc wines) and hydrogen sulfide, thus decreasing their volatility and their influence on wine aroma [95, 96].

Flavan-3-ols have defined taste attributes. Flavan-3-ols have a marked bitter taste, and their influence on the development of the bitterness sensation was recognized as early as 1966, when Rossi and Singleton isolated an ether-soluble fraction from grape seeds containing (+)-catechin, (-)-epicatechin, and (-)-epicatechin gallate [97]. Addition of this fraction at 200 mg/L to a white wine showed no contribution to astringency but significantly increased bitterness. Later, it was found that the chiral difference between the two main wine flavan-3-ols produces a significant difference in temporal perception of bitterness: (-)-epicatechin is significantly more bitter and had a significantly longer duration of bitterness than (+)-catechin [71, 98, 99]. The more planar conformation of the C ring of (-)-epicatechin compared with the less planar (+)-catechin may facilitate the diffusion of the molecule to the gustative receptor; in addition, the higher lipophilicity of epicatechin (relative to catechin) could also explain its higher comparative bitterness [71, 100].

Although it is assumed that both (+)-catechin and (-)-epicatechin cannot precipitate proteins, protein-induced precipitation of these flavan-3-ols has been confirmed by peptide models of salivary proline-rich-proteins (PRPs) whereby PRPs did indeed interact with flavan-3-ols having masses below 500 Da [101, 102]. Further confirmation was reported in model wine studies whereby a time-intensity sensory procedure found both (+)-catechin and epicatechin to elicit astringency [98, 103]. Also, (-)-epicatechin-3-O-gallate and (+)-catechin were found to precipitate PRPs when the molar ratio of flavan-3-ols to protein exceeded 27 [104]. In Cabernet Sauvignon wines, the perception of astringency was at least partially explained by the simultaneous occurrence of a comparatively higher concentration of flavan-3-ols and tannins [105]. Astringency of monomeric flavan-3-ols may be the result of cooperative precipitation of, or cooperative binding with, proteins due to the presence of one 1,2-dihydroxy or 1,2,3-trihydroxy groups [103, 106], and this may be enhanced by the presence of tannins [105]. These studies highlight the potential role of flavan-3-ol monomers on astringency perception in red wines.
