3.3.1. Occurrence, general chemistry, and sensory aspects.

Tannins, also known as proanthocyanidins, and/or condensed tannins,5 encompass oligomeric (degree of polymerization ≤ 2 and < 5) and polymeric flavonoids (degree of polymerization ≥ 5) made up by the five monomeric flavan-3-ols shown in Figure 7 [12, 119]. As defined by Bate-Smith and Swain (1962), tannins are "water soluble compounds having molecular weights between 500 and 3000 and, besides giving the usual phenolic reactions, they have special properties such as the ability to precipitate alkaloids, gelatin and other proteins" [120]. Haslam [12] further noted that tannins could also form complexes with polysaccharides and that molecular weights as high as 20,000 can be found in nature. These annotations certainly pertain to grapes and wines. Wine tannins readily interact with native or yeast-derived polysaccharides [121, 122], and this interaction can modulate sensory properties such as astringency [121, 123] (Figure 9). Furthermore, based on the degree of polymerization, molecular weights as high as 5000, 5200, and 22,000 have been reported in seed, wine, and skin extracts, respectively, from V. vinifera [124], highlighting the polymeric nature of grape and wine tannins.

Oligomeric and polymeric tannins are defined by the connectivity and nature of the interflavanic bond. This refers to a covalent connection between two flavan-3-ols, also referred as "subunits"; the average number of constitutive subunits in the oligomer or polymer (assuming a normal distribution of tannin sizes as a function of molecular weight) is referred to as degree of polymerization. A technique based on HPLC analysis, known as phloroglucinolysis, allows for the calculation of the so-called mean degree of polymerization (mDP) [125].

Two types of interflavanic bonds have been observed. Interflavanic bonds connecting either the carbon at position 4 in the extension subunit and the carbon at position 8 in the terminal subunit (C4 ! C8), or the carbon at position 4 in the extension subunit and the carbon at position 6 in the terminal subunit (C4 ! C6), are collectively known as B-type linkages [12]. The flavan-3-ol subunits can also be connected by two single bonds, one between C4 and C8, and another between the hydroxyl groups at C7 and C2, forming an ether bond known as Atype linkage [126, 127]. The occurrence of these three types of bonds is not uniformly distributed within grape and wine tannins. The C4 ! C8 bonds are found in skins, seeds, and wines [68]; however, C4 ! C6 and A-type linkages are formed as a result of rearrangements and intra and/or intermolecular oxidation reactions during winemaking [128].

<sup>5</sup> The relevant literature reviewed here uses several terms to refer to red wine tannins, including "tannin," "condensed tannin," and "proanthocyanidin." The term "tannin" will be used throughout this review.

Figure 9. Interaction between proline-rich proteins, a tannin trimer, and a polysaccharide fragment. Narrow black sawlike lines are suggested sites of hydrogen bonding. Wide gray lines are suggested regions of hydrophobic interactions. Redrawn and modified from Ref. [123].

The three connectivities described (C4 ! C8, C4 ! C6, and C7 ! C2) can, in turn, display the usual two stereochemistries of monomeric flavan-3-ols, with the R stereochemistry referred to as α and the S stereochemistry as β [129]. As a result of the enantiomeric and conformational properties of the flavan-3-ols subunits, the number of possible structures increases in a factorial fashion as the number of subunits in the oligomer or polymer increases. For example, considering only the C4 ! C8 linkages, about 105 unique tannin structures have been estimated to exist.

Oligomeric tannins include tannin dimers, trimers and tetramers of different connectivities. B-type dimers isolated from grapes and wines are composed of (+)-catechin and (-)-epicatechin and include the dimers B1 (epicatechin-(4β!8)-catechin), B2 (epicatechin-(4β!8)-epicatechin), B3 (catechin-(4α!8)-catechin), and B4 (catechin-(4α!8)-epicatechin) (Figure 10). Moreover, dimers having a C4 ! C6 connectivity (B5 to B8 series) and epigallocatechin have also been observed [130]. Likewise, the α and β dimers B1, B2, B3, and B4 of epicatechin-3-O-gallate and either catechin or epicatechin have also been identified in seeds, skins, and wines [131–133]. Dimers, trimers, tetramers, and pentamers containing A-type bonds were recently found in grape seeds of both white and red V. vinifera varieties [127]. Tannin trimers found in grapes and wines consist of three flavan-3-ol units linked by two C4 ! C8 interflavan bonds (C-type) or one C4 ! C6 interflavan bond in the terminal or extension subunits, and one C4 ! C6 interflavan bond in the remaining subunit (T-type) [130, 134, 135]. A nonexhaustive account of the most common connectivities in dimers and trimers observed in V. vinifera grapes and wines is shown in Figure 10.

The three connectivities described (C4 ! C8, C4 ! C6, and C7 ! C2) can, in turn, display the usual two stereochemistries of monomeric flavan-3-ols, with the R stereochemistry referred to as α and the S stereochemistry as β [129]. As a result of the enantiomeric and conformational properties of the flavan-3-ols subunits, the number of possible structures increases in a factorial fashion as the number of subunits in the oligomer or polymer increases. For example, considering only the C4 ! C8 linkages, about 105 unique tannin structures have been estimated to exist.

Figure 9. Interaction between proline-rich proteins, a tannin trimer, and a polysaccharide fragment. Narrow black sawlike lines are suggested sites of hydrogen bonding. Wide gray lines are suggested regions of hydrophobic interactions.

Oligomeric tannins include tannin dimers, trimers and tetramers of different connectivities. B-type dimers isolated from grapes and wines are composed of (+)-catechin and (-)-epicatechin and include the dimers B1 (epicatechin-(4β!8)-catechin), B2 (epicatechin-(4β!8)-epicatechin), B3 (catechin-(4α!8)-catechin), and B4 (catechin-(4α!8)-epicatechin) (Figure 10). Moreover, dimers having a C4 ! C6 connectivity (B5 to B8 series) and epigallocatechin have also been observed [130]. Likewise, the α and β dimers B1, B2, B3, and B4 of epicatechin-3-O-gallate and either catechin or epicatechin have also been identified in seeds, skins, and wines [131–133]. Dimers, trimers, tetramers, and pentamers containing A-type bonds were recently found in grape seeds of both white and red V. vinifera varieties [127]. Tannin trimers found in grapes and wines consist of three flavan-3-ol units linked by two C4 ! C8 interflavan bonds (C-type) or one C4 ! C6 interflavan bond in the terminal or extension subunits, and one C4 ! C6 interflavan bond in the remaining subunit (T-type) [130, 134, 135]. A nonexhaustive account of the most common connectivities in dimers and trimers observed in V. vinifera grapes and

wines is shown in Figure 10.

Redrawn and modified from Ref. [123].

166 Phenolic Compounds - Natural Sources, Importance and Applications

Figure 10. Overview of some of the most abundant tannin dimers and trimers observed in V. vinifera grapes and wines. Only tannins based on (+)-catechin and (-)-epicatechin are shown; however, dimers and trimers containing (-)-epigallocatechin and (-)-epicatechin-3-O-gallate also occur in V. vinifera.

Polymeric tannins have an mDP ≥ 5 subunits. Early studies by Czochanska and colleagues on tannins from 22 plant sources with molecular weights ranging from 1500 to 5000 elucidated the structure and stereochemistry of the interflavanic bond in these polymers by 13C NMR and confirmed the presence of C4 ! C8, C4 ! C6, and A-type (C7 ! C2) linkages [136]. Therefore, the observations described previously for oligomers also apply to polymeric tannins.

The term "proanthocyanidin," commonly used in the literature to refer to tannins, stems from the ability of these compounds to release anthocyanins upon cleavage of the interflavanic bond under heat and acidic conditions. Based on the last reaction, tannins are classified as procyanidins, when they release cyanidin (characteristic of tannins containing (+)-catechin, (-)-epicatechin, and (-)-epicatechin-3-O-gallate), and prodelphinidins, when they release delphinidin (as is the case of tannins containing (-)-epigallocatechin) [137]. The spectral features mentioned for monomeric flavan-3-ols also apply for oligomeric and polymeric tannins.

Oligomeric and polymeric tannins occur both in skins and seeds, although their quantitative and qualitative composition, as well as their molecular weight and distribution, differ within these tissues and also in the resulting wines. On a whole berry basis, up to 80% of the total extractable tannin pool of the berry is located in the seeds [12]. In seeds at physiological maturity, seed tannins measured by protein precipitation vary from 2.15 to 4 mg/g FW for varieties such as Cabernet Sauvignon, Merlot, and Syrah [43, 47, 138–140]. Conversely, skin tannins vary between 0.35 and 1 mg/g FW [43, 47, 139, 140]. In wines, total tannins (by protein precipitation) measured in 1325 commercial red wines ranged from 30 to 1895 mg/L, with a mean concentration of 544 mg/L [141].

Seed-derived polymeric tannins are co-located with the flavan-3-ols in the cells below the external cuticle [142]. Seed tannins associated with cell walls reportedly have a higher mDP than their cellular counterparts [143]. The mDP of seed tannins of different grape varieties vary between 2 and 22 [73, 78, 134, 143, 144–146], which represents a 11-fold variation. Seed tannins are composed of (+)-catechin, (-)-epicatechin, and epicatechin-3-O-gallate, with monomeric flavan-3-ols, dimers (a portion of them bearing A-type linkages), and trimers being the predominant species [74, 130, 134]. The percentage of galloylation of seed tannins has been estimated to be around 30% [26]. In the skins, tannins occur as vacuolar components in subepidermal, thickwalled cells, but they are also found in the tonoplast and cell walls [75, 147]. Skin tannins associated with cell walls have a higher mDP than those found as free cytoplasmic components; however, tannins found in the cytoplasm are more abundant [147]. Skin tannins typically have the highest molecular weight within the berry, varying from 6 up to 85 subunits in varieties such as Cabernet Franc, Cabernet Sauvignon, Graciano, Merlot, Pinot Noir, Syrah, Carmenère, and Tempranillo [78, 124, 134, 144–148]. This represents a 14-fold variation. Skin tannins are composed primarily of (-)-epicatechin, (-)-epigallocatechin, and (+)-catechin. However, trace amounts of epicatechin-3-O-gallate have also been found [147], giving a percentage of galloylation estimated to typically be <5% [26].

Wine tannins are composed of all the five monomeric flavan-3-ol subunits, with (-)-epicatechin, followed by (+)-catechin and (-)-epigallocatehin being the major constituents [78]. Moreover, the proportion of each subunit is affected by both grape variety and winemaking technique, and, like with flavan-3-ols, a relationship between an increased percentage of galloylated subunits and maceration length has been observed [43, 113, 135]. For wines, the reported mDP values vary between 2 and 17 subunits [47, 124, 148–151]. However, oligomeric tannins with an mDP < 5 are the major constituents of wine tannins [43, 109, 124, 152] (Figure 8a and Figure 8b). Taking into consideration only the reported mDP values and subunit composition of seeds, skins, and wines, it seems as though tannin composition and distribution in wine resembles more of that of the seeds than that of the skins and that this feature is amplified as maceration time increases (Figure 8b).

As noted previously, upon crushing and during winemaking, tannins not only undergo modifications in their original molecular weight, but they can also undergo further reactions and re-arrangements with anthocyanin, to form the so-called polymeric pigments (Section 3.4), as well as with other flavan-3-ols and/or tannins dimers and trimers [153].

under heat and acidic conditions. Based on the last reaction, tannins are classified as procyanidins, when they release cyanidin (characteristic of tannins containing (+)-catechin, (-)-epicatechin, and (-)-epicatechin-3-O-gallate), and prodelphinidins, when they release delphinidin (as is the case of tannins containing (-)-epigallocatechin) [137]. The spectral features mentioned for monomeric

Oligomeric and polymeric tannins occur both in skins and seeds, although their quantitative and qualitative composition, as well as their molecular weight and distribution, differ within these tissues and also in the resulting wines. On a whole berry basis, up to 80% of the total extractable tannin pool of the berry is located in the seeds [12]. In seeds at physiological maturity, seed tannins measured by protein precipitation vary from 2.15 to 4 mg/g FW for varieties such as Cabernet Sauvignon, Merlot, and Syrah [43, 47, 138–140]. Conversely, skin tannins vary between 0.35 and 1 mg/g FW [43, 47, 139, 140]. In wines, total tannins (by protein precipitation) measured in 1325 commercial red wines ranged from 30 to 1895 mg/L, with a mean concentration of 544

Seed-derived polymeric tannins are co-located with the flavan-3-ols in the cells below the external cuticle [142]. Seed tannins associated with cell walls reportedly have a higher mDP than their cellular counterparts [143]. The mDP of seed tannins of different grape varieties vary between 2 and 22 [73, 78, 134, 143, 144–146], which represents a 11-fold variation. Seed tannins are composed of (+)-catechin, (-)-epicatechin, and epicatechin-3-O-gallate, with monomeric flavan-3-ols, dimers (a portion of them bearing A-type linkages), and trimers being the predominant species [74, 130, 134]. The percentage of galloylation of seed tannins has been estimated to be around 30% [26]. In the skins, tannins occur as vacuolar components in subepidermal, thickwalled cells, but they are also found in the tonoplast and cell walls [75, 147]. Skin tannins associated with cell walls have a higher mDP than those found as free cytoplasmic components; however, tannins found in the cytoplasm are more abundant [147]. Skin tannins typically have the highest molecular weight within the berry, varying from 6 up to 85 subunits in varieties such as Cabernet Franc, Cabernet Sauvignon, Graciano, Merlot, Pinot Noir, Syrah, Carmenère, and Tempranillo [78, 124, 134, 144–148]. This represents a 14-fold variation. Skin tannins are composed primarily of (-)-epicatechin, (-)-epigallocatechin, and (+)-catechin. However, trace amounts of epicatechin-3-O-gallate have also been found [147], giving a percentage of galloylation estimated

Wine tannins are composed of all the five monomeric flavan-3-ol subunits, with (-)-epicatechin, followed by (+)-catechin and (-)-epigallocatehin being the major constituents [78]. Moreover, the proportion of each subunit is affected by both grape variety and winemaking technique, and, like with flavan-3-ols, a relationship between an increased percentage of galloylated subunits and maceration length has been observed [43, 113, 135]. For wines, the reported mDP values vary between 2 and 17 subunits [47, 124, 148–151]. However, oligomeric tannins with an mDP < 5 are the major constituents of wine tannins [43, 109, 124, 152] (Figure 8a and Figure 8b). Taking into consideration only the reported mDP values and subunit composition of seeds, skins, and wines, it seems as though tannin composition and distribution in wine resembles more of that of the seeds than that of the skins and that this feature is amplified as maceration time

flavan-3-ols also apply for oligomeric and polymeric tannins.

168 Phenolic Compounds - Natural Sources, Importance and Applications

mg/L [141].

to typically be <5% [26].

increases (Figure 8b).

The ability of wine tannins to interact with proteins provides the physiological basis of the sensation of astringency, of paramount importance in red wines. Astringency is a tactile (not a taste) sensation with a marked temporal aspect that appears as a pucker feeling (typically in the upper lip) and/or feeling of dryness in the palate, which arises from a sudden loss of lubrication of the oral epithelium [154–157]. Astringency is not confined to a particular region of the mouth but is a diffuse surface phenomenon, which typically develops between 15 and 20 s [158, 159].

The development of astringency necessitates the presence of both tannins and proteins capable of interact with each other. The wine provides the tannins. The proteins are provided by the subject's saliva. The saliva of humans and other mammals contains proline-rich proteins (PRPs) [160]. Salivary PRPs constitute 70% of the proteins in saliva and are made up of three types: the acidic, glycosylated, and basic proteins, which comprise roughly 30, 23, and 17% of unstimulated saliva, respectively [161]. While acidic- and glycosylated proline-rich proteins have specific biological roles, basic PRPs display high affinity for tannins [162, 163]. Basic PRPs have a relatively preserved sequence across a wide range of species of mammals, composed of a 19 aminoacid sequence dominated by proline, glutamine, and glycine [164]. This sequence is repeated with variations between 5 and 15 times to result in a protein of ~150 amino acids in length that is extended and random-coiled in confirmation [164, 165]. The flexibility of the protein is further improved by the existence of several low-energy conformations [166, 167]. The biological role of astringency in mammals has been intensively debated but there is now evidence that astringency may be an evolutionary defense mechanism against dietary anti-feeding factors [168]. Tannins can have a variety of harmful effects on animals, including sequestration of iron and inhibition of digestive enzymes [160]. The role of PRPs may be to bind to the tannins and precipitate them, thereby preventing harmful effects in the gastrointestinal tract [169].

The interaction between PRPs and tannins results from hydrogen bonding between the tertiary amide or carbonyl groups of the proline subunits and the hydroxyl groups of the tannin [167, 170]. However, hydrophobic interactions whereby the hydrophobic face of the aromatic ring of the phenol interacts with the pyrrolidine ring of the protein may also cooperatively aid in the complexation process [163, 170, 171] (Figure 9). Likewise, the galloyl ring in epicatechin-3-O-gallate and tannins containing this flavan-3-ol provides supplementary aromatic surfaces that may engage in hydrophobic complexation with the proline ring as well [163, 172]. From this perspective, increase in the percentage of galloylation in a given tannin should result in enhanced perceived astringency.

The development of astringency follows a three-stage process, consistent with the in-mouth temporal development of this sensation [173, 174]. In the first stage, the binding of multidentate tannins to several sites on the protein causes the randomly coiled protein to coil around the phenol, compacting the protein. In the second stage, the tannin fractions of the protein-tannin complexes cross-link forming polyphenol bridges and creating protein dimers. During the third stage, the dimers aggregate to form larger complexes that eventually precipitate out of solution. In wines, the spectrum of subtle differences in astringency sensations was compiled in the "red wine mouthfeel wheel" in which astringency is categorized in 7 subqualities and 30 subattributes [175], which highlights the complex nature of this sensation in red wines.

Perhaps the most determining factor on astringency development in red wines is, simply, tannin concentration [105, 176–178]. However, additional effects include wine pH and ethanol content [179, 180], viscosity [181, 182], and the presence of sugars [183, 184] and polysaccharides [121, 185]. Also, the tannin stereochemistry, composition, and connectivity affect the perception of astringency. For example, the dimer (+)-catechin-(+)-catechin linked via a C4 ! C8 interflavan bond has lower astringency than its C4 ! C6 counterpart or than the C4 ! C8 (+)-catechin-(-)-epicatechin dimer [103]. Second to tannin concentration, tannin size (molecular weight) also modulates the development of astringency [186–189]. Precipitation of salivary PRPs is enhanced by the presence of high molecular weight tannins that have some structural flexibility, as in the case of tannins containing freely rotating interflavan bonds and gallolyl groups, due to a larger number of available binding sites for interaction with the proline residues [102]. Larger tannins can also engage in self-association, thereby promoting complex aggregation. However, the relationship between polymer size and perceived astringency does not progress linearly. For example, at equimolecular concentrations, Vidal et al. found that a wine-like solution containing tannins with an mDP of 70 is only two times more astringent than one with an mDP of 3 [188]. Empirical evidence that astringency elicited by grape tannins is mostly concentration-driven can be found by comparatively assessing astringency in seeds (overall high tannin concentration, with tannins of low molecular weight) with that of skins (relative lower tannin concentration than seeds but with tannins of high molecular weight). Seeds typically elicit a much higher astringency sensation than skins in spite of being composed of low molecular weight tannins and this may be due to the fact that seed tannins are typically at much higher concentrations than skin tannins on a berry fresh weight basis.

Lastly, astringency perception in red wines is also influenced by factors extrinsic to the nature of the tannin and PRP's interactions, including salivary flow rate [159], sensitivity to the bitter agent 6-n-propylthiouracil (PROP), known as PROP status [190], and frequency of exposure [183, 191]. Sensitivity to PROP seems to have equivocal effects on astringency development; however, individuals with low salivary flow rate (1.92 g/min) perceive astringency later and with higher intensity than individuals with high salivary flow rate (3.73 g/min) [159].

Some oligomeric and polymeric tannins can also interact with gustatory receptors, and they can thus elicit a mild bitter response [26]. Time-intensity sensory studies have shown that bitterness perception decreases from flavan-3-ols monomers to trimers [103]. More generally, bitterness decreases as the polymer size increases, probably because of the difficulty of large polymers to diffuse inside the taste bud pores [26].

### 3.3.2. Extraction during winemaking

Tannin dimers and trimers follow extraction kinetics similar to those reported for flavan-3-ols, and as such these oligomers can be extracted in the absence of ethanol, for example during prefermentative cold soak [111]. In Tempranillo wines after a 2-day cold soak, the concentration of tannin dimers, trimers, and tetramers increased from 22, 23, and 0 mg/L, respectively, to 27, 30, and 6 mg/L, respectively, after postfermentative maceration for 1 week [109]. In another study, the levels of the B2 dimer and the C trimer increased from about 6 mg/L at day 2 to about 22 mg/L (B2 dimer) and to 11 mg/L (C trimer) after 20 days of maceration in the white variety Viura [108]. These results suggests that as the oligomers increases in size, their extraction and retention into wine progresses more slowly.

During the third stage, the dimers aggregate to form larger complexes that eventually precipitate out of solution. In wines, the spectrum of subtle differences in astringency sensations was compiled in the "red wine mouthfeel wheel" in which astringency is categorized in 7 subqualities and 30 subattributes [175], which highlights the complex nature of this sensation

Perhaps the most determining factor on astringency development in red wines is, simply, tannin concentration [105, 176–178]. However, additional effects include wine pH and ethanol content [179, 180], viscosity [181, 182], and the presence of sugars [183, 184] and polysaccharides [121, 185]. Also, the tannin stereochemistry, composition, and connectivity affect the perception of astringency. For example, the dimer (+)-catechin-(+)-catechin linked via a C4 ! C8 interflavan bond has lower astringency than its C4 ! C6 counterpart or than the C4 ! C8 (+)-catechin-(-)-epicatechin dimer [103]. Second to tannin concentration, tannin size (molecular weight) also modulates the development of astringency [186–189]. Precipitation of salivary PRPs is enhanced by the presence of high molecular weight tannins that have some structural flexibility, as in the case of tannins containing freely rotating interflavan bonds and gallolyl groups, due to a larger number of available binding sites for interaction with the proline residues [102]. Larger tannins can also engage in self-association, thereby promoting complex aggregation. However, the relationship between polymer size and perceived astringency does not progress linearly. For example, at equimolecular concentrations, Vidal et al. found that a wine-like solution containing tannins with an mDP of 70 is only two times more astringent than one with an mDP of 3 [188]. Empirical evidence that astringency elicited by grape tannins is mostly concentration-driven can be found by comparatively assessing astringency in seeds (overall high tannin concentration, with tannins of low molecular weight) with that of skins (relative lower tannin concentration than seeds but with tannins of high molecular weight). Seeds typically elicit a much higher astringency sensation than skins in spite of being composed of low molecular weight tannins and this may be due to the fact that seed tannins are typically

at much higher concentrations than skin tannins on a berry fresh weight basis.

with higher intensity than individuals with high salivary flow rate (3.73 g/min) [159].

polymers to diffuse inside the taste bud pores [26].

3.3.2. Extraction during winemaking

Lastly, astringency perception in red wines is also influenced by factors extrinsic to the nature of the tannin and PRP's interactions, including salivary flow rate [159], sensitivity to the bitter agent 6-n-propylthiouracil (PROP), known as PROP status [190], and frequency of exposure [183, 191]. Sensitivity to PROP seems to have equivocal effects on astringency development; however, individuals with low salivary flow rate (1.92 g/min) perceive astringency later and

Some oligomeric and polymeric tannins can also interact with gustatory receptors, and they can thus elicit a mild bitter response [26]. Time-intensity sensory studies have shown that bitterness perception decreases from flavan-3-ols monomers to trimers [103]. More generally, bitterness decreases as the polymer size increases, probably because of the difficulty of large

Tannin dimers and trimers follow extraction kinetics similar to those reported for flavan-3-ols, and as such these oligomers can be extracted in the absence of ethanol, for example during prefermentative cold soak [111]. In Tempranillo wines after a 2-day cold soak, the concentration

in red wines.

170 Phenolic Compounds - Natural Sources, Importance and Applications

Extraction of tannins (mDP ≥ 5) into wine during winemaking has been followed using different analytical approaches, ranging from protein precipitation [47, 58] to acid-catalyzed depolymerization followed by HPLC analysis [111, 146]. Expectedly, the comparison of extraction patterns established by both methods is tenuous at best, as protein precipitation and HPLC greatly differ in the amount, composition, and molecular weight of the tannins that each method quantify. Moreover, extraction patterns of skin and seed tannins are not necessarily similar and they certainly diverge from each other as maceration progresses; as such it is often difficult to unequivocally ascertain the source of tannin extraction during the time course of maceration. Using acid-catalyzed depolymerization and HPLC, the extraction of skin tannins into wine was found to follow a Boltzmann sigmoid model [146]. In this model, a lag phase of initially slow extraction is observed due to the period of time required for the tannins to diffuse out of the berry cells and into the fermentor. The extent of this lag phase may be modulated by the degree of berry crushing or the ethanol concentration. This lag phase is followed by a plateau concentration, which is reached when tannin concentration is at its apparent maximum. Likewise, the extraction of tannins from seeds has also been modeled using a Boltzmann sigmoid extraction pattern, but this model has only proved valid for the first 6 days of contact with the fermentation solids [111]. In model wine experiments containing only seeds with varying ethanol levels, these same authors observed an initial slow extraction of tannins, which was attributed to the period of time required for these tannins to diffuse out of the seed cells and into the solution. As with skins, this lag period was followed by a plateau, the value of which increased with ethanol content, suggesting an initial effect of the ethanol in the degradation of the outer protective layers of the seeds. However, from day 6 to day 10, tannin extraction into wine increased linearly. The authors attributed this observation to the hydration of seed cells and subsequent cellular leakiness, leading to a somewhat abrupt release of seed tannins.

In Merlot wines, tannins measured by protein precipitation increased almost linearly during the first 7 days of maceration. Moreover, extended maceration for 20 days increased tannin extraction from a mean of 469 mg/L in control wines to 985 mg/L in the extended maceration wines [58]. Also, in Merlot wines, protein precipitable tannins were followed at 2-day intervals during a 30-day maceration period. Tannin extraction occurred quickly and almost linearly from day 2 to day 10, then reached a plateau between day 10 and 22, and was followed by an almost linear increase up to the peak of extraction [47] (Figure 11a). While the observed plateau between day 10 and 20 is consistent with seed hydration, the ensuing linear increase is consistent with the extraction of seed tannins [111]. Indeed, after 30 days of maceration, the proportion of seedderived tannins in finished wines was estimated to be about 73% in Cabernet Sauvignon [43] and between 73 and 80% in Merlot wines [47, 116]. It has also been suggested that postfermentation extraction of tannins could be the result of a desorption mechanism whereby ethanol may disrupt the noncovalent interactions of the previously extracted tannins that were bound to cell-wall material [192]. It is unclear if this linear increase is the result of one or a

Figure 11. Extraction patterns of protein precipitable tannins during a 30-day maceration period in (a) Merlot and (b) Cabernet Sauvignon wines. CE: catechin equivalents. Adapted from Refs. [43, 47].

combination of both mechanisms, but in the range of 11–14.4% ethanol this supposed "desorptive" effect seems to be inexistent [47, 116]. It is also unclear if the extraction patterns shown in Figure 11 will apply to other varieties with vastly different seed and skin phenolic composition, such as Pinot Noir, Sangiovese, Tempranillo, or Nebbiolo.

The extraction into wine and the ensuing fate of oligomeric and polymeric tannins during postmaceration and aging is also governed by the matrix composition, including the presence (or lack thereof) of anthocyanins and the presence of other compounds known to bind with wine tannins, primarily mannoproteins from yeast origin, polysaccharides, and other cell-wall components. Indeed, wine tannins can also react via noncovalent interactions with cell-wall material present during fermentation. These noncovalent interactions include hydrogen bonding and hydrophobic interactions. For example, the hydrogen-bonding-mediated interaction between tannins and polysaccharides is illustrated in Figure 12. As tannins increase in molecular weight, each additional monomer increases the number of sites that can form hydrogen bonds between the tannin and cell-wall components [192–195]. An increase in polymerization also increases the hydrophobic character of the tannin. The presence of galloylated units, more common on seed than in skins tannins, enhances the tannin-polysaccharide interaction as well, as highly galloylated tannins may be encapsulated by hydrophobic pockets and pores in the polysaccharide network [195, 196]. These findings had led to the hypothesis that the failure to recover high molecular weight tannins in wine is the result of tannin-cell-wall interactions occurring during winemaking and that the binding capacity of the cell walls is influenced by both tannins and polysaccharide structure and composition [192]. A series of studies conducted by Bindon and colleagues had confirmed this hypothesis. Bindon et al. found a significant relationship between the tannin molecular mass and the proportion of tannins adsorbed by skin cell-wall polysaccharides, with the end result being that high molecular weight tannins (>15,000 g/mol) are not extractable and/or removed from the wine by interaction with cell-wall components [194, 197–199]. In practical terms, the proportion of bound (and thus "missing") tannins during Cabernet Sauvignon winemaking have been estimated to range from 17 to 29% of those originally measured in the fruit [200, 201].

Figure 12. Hydrogen-bonding-mediated interaction between the hydroxyl groups of the tannin trimer C1 and the oxygen atoms and glycosidic linkages of the polysaccharide homogalacturonan. Redrawn and modified from Ref. [192].

### 3.4. Polymeric pigments and other anthocyanin-derived pigments

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

combination of both mechanisms, but in the range of 11–14.4% ethanol this supposed "desorptive" effect seems to be inexistent [47, 116]. It is also unclear if the extraction patterns shown in Figure 11 will apply to other varieties with vastly different seed and skin phenolic

Figure 11. Extraction patterns of protein precipitable tannins during a 30-day maceration period in (a) Merlot and (b)

The extraction into wine and the ensuing fate of oligomeric and polymeric tannins during postmaceration and aging is also governed by the matrix composition, including the presence (or lack thereof) of anthocyanins and the presence of other compounds known to bind with wine tannins, primarily mannoproteins from yeast origin, polysaccharides, and other cell-wall components. Indeed, wine tannins can also react via noncovalent interactions with cell-wall material present during fermentation. These noncovalent interactions include hydrogen bonding and hydrophobic interactions. For example, the hydrogen-bonding-mediated interaction between tannins and polysaccharides is illustrated in Figure 12. As tannins increase in molecular weight, each additional monomer increases the number of sites that can form hydrogen bonds between the tannin and cell-wall components [192–195]. An increase in polymerization also increases the hydrophobic character of the tannin. The presence of galloylated units, more common on seed than in skins tannins, enhances the tannin-polysaccharide interaction as well, as highly galloylated tannins may be encapsulated by hydrophobic pockets and pores in the polysaccharide network [195, 196]. These findings had led to the hypothesis that the failure to recover high molecular weight tannins in wine is the result of tannin-cell-wall interactions occurring during winemaking and that the binding capacity of the cell walls is influenced by both tannins and polysaccharide structure and composition [192]. A series of studies conducted by Bindon and colleagues had confirmed this hypothesis. Bindon et al. found a significant relationship between the tannin molecular mass and the proportion of tannins adsorbed by skin cell-wall polysaccharides, with the end result being that high molecular weight tannins (>15,000 g/mol) are not extractable and/or removed from the wine by interaction with cell-wall components [194, 197–199]. In practical terms, the proportion of bound (and thus "missing") tannins during Cabernet Sauvignon winemaking have been estimated to

composition, such as Pinot Noir, Sangiovese, Tempranillo, or Nebbiolo.

Cabernet Sauvignon wines. CE: catechin equivalents. Adapted from Refs. [43, 47].

172 Phenolic Compounds - Natural Sources, Importance and Applications

range from 17 to 29% of those originally measured in the fruit [200, 201].

An overview of the variety of reactions anthocyanins can undergo during maceration and aging is presented in Figure 13. Upon crushing (and releasing from vacuoles) and during aging, anthocyanins can readily react with a variety of electrophiles and nucleophiles, including other anthocyanins [202], flavan-3-ols via direct condensation [203–206], or acetaldehydemediated [204, 205, 207, 208], dimeric or trimeric tannins [86, 209], lactic acid [204], glyceraldehyde [210], acetaldehyde [211, 212], pyruvic acid [212, 213], glyoxylic acid [214, 215], and a number of other aldehydes, including furfural, 5 hydroxymethylfurfural, isovaleraldehyde, benzaldehyde, propionaldehyde, isobutyraldehyde, formaldehyde, and 2-methylbutyraledehyde [215, 216]. Aldehydes are formed either from the metabolism of Saccharomyces cerevisiae during alcoholic fermentation [212, 217] or from the metal-catalyzed oxidation of

Figure 13. Main chemical pathways involving anthocyanins during winemaking and aging of red wines. Malvidin-3 glucoside is shown as the starting anthocyanin, but other anthocyanins may also participate in these reactions. Redrawn and adapted from Ref. [219] and complemented with data from Refs. [26, 204, 220, 221].

several wine substrates, especially ethanol (to acetaldehyde), most particularly thorough the Fenton Reaction [17, 218]. All these anthocyanin-derived products have spectral properties different from that of the native anthocyanins (Figure 13). Ultimately, these reactions together with the formation of oligomeric and polymeric pigments account for the evolution of wine color, from deep purple, due to monomeric anthocyanins and self-association copigmentation reactions in young wines, to orange, brick-red tones as the wine ages.

other anthocyanins [202], flavan-3-ols via direct condensation [203–206], or acetaldehydemediated [204, 205, 207, 208], dimeric or trimeric tannins [86, 209], lactic acid [204], glyceraldehyde [210], acetaldehyde [211, 212], pyruvic acid [212, 213], glyoxylic acid [214, 215], and a number of other aldehydes, including furfural, 5 hydroxymethylfurfural, isovaleraldehyde, benzaldehyde, propionaldehyde, isobutyraldehyde, formaldehyde, and 2-methylbutyraledehyde [215, 216]. Aldehydes are formed either from the metabolism of Saccharomyces cerevisiae during alcoholic fermentation [212, 217] or from the metal-catalyzed oxidation of

174 Phenolic Compounds - Natural Sources, Importance and Applications

Figure 13. Main chemical pathways involving anthocyanins during winemaking and aging of red wines. Malvidin-3 glucoside is shown as the starting anthocyanin, but other anthocyanins may also participate in these reactions. Redrawn

and adapted from Ref. [219] and complemented with data from Refs. [26, 204, 220, 221].

Polymeric pigments encompass a variety of winemaking artifacts formed by the covalent association, either direct or mediated by an aldehyde (e.g., acetaldehyde, glyceraldehyde, and glyoxilic acids) between anthocyanins and tannins. Relative to intact anthocyanins, polymeric pigments share two fundamental features: they are (1) partially resistant to bisulfite bleaching and (2) more resilient to pH changes [222, 223]. This is because polymerization protects by steric hindrance of the chromophore of the anthocyanin from the attack of water and other nucleophiles, oxidation, or the bleaching effect of SO2 [222, 224]. The presence of anthocyanins during maceration increases the solubility and retention of tannins [52–54], an observation classically attributed to the formation of polymeric pigments. Singleton hypothesized that the glucose moiety in the anthocyanin and the polarity of the flavylium cation may decrease the precipitability of the resulting polymeric pigment [225]. In a subsequent experiment in which white wines were produced with different portions of added tannins and anthocyanins, the retention of tannins and formation of polymeric pigments increased in the presence of added anthocyanins [53]. However, this later study also found that the stoichiometric addition of anthocyanins relative to tannins approached an ideal proportion, where excessive anthocyanins did not increase pigmented polymer formation. This finding suggests that the proportion of anthocyanins and tannins during maceration can condition anthocyanin and tannin stability and the subsequent formation of polymeric pigments.

Polymeric pigments modulate wine color, long-term color stability, and possibly astringency changes during winemaking and aging. From the perspective of color, the UV-visible spectra of anthocyanin-flavan-3-ol adducts (i.e., dimers) resulting from condensation with aldehydes is bathochromically shifted (i.e., bluish color) compared to those of their precursors, with a shift in absorbance of 10 nm for linear substituents and of 20 nm for branched substituents [26, 207, 216] (Figure 13). However, the chromatic properties of polymeric pigments are less clear. Indeed, a numerical value for the molar extinction coefficient of polymeric pigments at wine pH is not yet available, but (indirect) experimental evidence suggests that it should be comparatively lower than that of the intact anthocyanins [37, 43, 226]. This, together with the disappearance and/or transformation of anthocyanins, may in turn explain why the color of red wine not only changes in hue but also decreases in saturation during aging.

Evidence for the existence of polymeric pigments in wine is abundant [37, 43, 143, 227–229], and some possible structures are shown in Figure 14. In Pinot Noir wines, pigmented polymers were isolated and characterized as mixtures of dimers to octamers in which the anthocyanin moiety was linked to the flavan-3-ol by B-type and A-type linkages [227]. In a separate study, ultrafiltration and gel adsorption chromatography combined with <sup>1</sup> H, 13C, and 2D-NMR were used to characterize a high molecular weight tannin polymer (>5 kDa) isolated from a Bordeaux red wine [229]. The structural backbone of this polymer consisted of a tannin chain with (-)-epicatechin, (+)-catechin, and (-)-epicatechin-3-O-gallate as extension and terminal subunits. The presence of acetaldehyde bridges was also observed in the A ring of some subunits as well as that of pyranoanthocyanins linked to the backbone via C4 ! C6 or C4 ! C8 linkages. Interestingly, polysaccharides were also found to be present within the structure, although these were not covalently linked to the tannin backbone. Organic and phenolic acids as well as aminoacids were also found to be part of the polymeric fraction structure. This appears to be the first report to completely elucidate the heterogeneous structure of these compounds as they occur in red wines. Lastly, in Cabernet Sauvignon wines, pigmented material was isolated by preparative HPLC [43]. The spectral features of the pigmented material featured a comparatively higher 280 to 520 nm absorbance ratio compared to that of intact anthocyanins (thus indicating the presence of flavan-3-ols) and an mDP between 5 and 10 units. The tannin component of this polymeric material was also put in evidence by subjecting the polymer to protein precipitation with bovine serum albumin (BSA), further indicating the potential astringent properties of this material.

Figure 14. Potential structures of polymeric pigments resulting from the covalent reaction between anthocyanins (A) and tannins (T). Only C4 ! C8 and A-type interflavanic bonds are depicted but C4 ! C6 interflavanic bonds can also occur.

From a sensory standpoint, polymeric pigments affect two key aspects in red wines: color development and mouthfeel modification. Direct sensory evidence of the role of polymeric pigments in astringency changes during aging is, however, relatively recent. Indeed, the observed "lessening" of astringency during aging was thought to be the result of the reaction of anthocyanins with tannins of various sizes to give rise to polymeric pigments [222, 230]. However, the structural complexity and heterogeneity of these pigments prevented their isolation and subsequent chemical and sensory characterization. With the advent of new analytical, semipreparative, and preparative HPLC techniques, the characterization of these compounds has become possible. Work by Vidal and colleagues in 2004 found that polymeric pigments with mDP of ~3 and 9 and bearing an anthocyanin moiety were less astringent than apple tannins with the same mDP but deprived of anthocyanins [40]. Moreover, these authors showed that modifying the molecular structure by introducing an acetaldehyde bridge decreased astringency but also increased bitterness. An explanation for the comparatively lower astringency of polymeric pigments relative to that of intact tannins is that the incorporation of an anthocyanin moiety with its glycoside portion increases the polarity of the polymer [225]. As the development of astringency is partially governed by hydrophobic interactions between salivary PRPs and phenols, the higher hydrophilic character of the pigmented polymer would decrease the interaction of this polymeric material with salivary PRPs and thus reduce perceived astringency.

Recently, Weber et al., using size-exclusion chromatography on Sephadex resin, isolated 14 tannin fractions from the 2005 Cabernet Sauvignon wine [37]. Anthocyanins, mainly malvidin-3-glucoside, were found in the first 10 fractions, indicating the pigmented nature of the polymeric fraction. Fractions 1 to 3 were composed of large polymeric pigments as measured by protein precipitation, with low anthocyanin and tannin content; fractions 4 to 7 consisted of anthocyanin-rich pigmented polymers with medium tannin content; and fractions 8 to 14 consisted of small-sized, tannin-like oligomers with very low anthocyanin content but very high tannin content. Upon sensory evaluation of each fraction dissolved in model wine at iso-concentrations of 500 mg/L, fractions with a low amount of anthocyanins elicited higher astringency, suggesting that further incorporation of anthocyanins into polymers should result in a decrease in astringency. In another report, a pigmented polymer isolated from a Bordeaux red wine was fractionated into eight fractions of different molecular weights by gel permeation chromatography [229]. Upon dissolution of these fractions in 1% ethanol at iso-concentrations, astringency was found no to vary in seven of these fractions in spite of differences in mDP and degree of galloylation. However, one fraction consisting of 50% polysaccharides was found to be less astringent. Overall, current evidence suggests that the lessening of astringency along with red wine aging may not be related to a drastic change in the total amount of tannin present. Rather, the structural modification of wine tannins, primarily resulting from the incorporation of anthocyanins, and, secondarily, from the addition of other metabolites such as carbohydrates, proteins, and polysaccharides, may drive changes in perceived astringency during aging.

### 3.4.2. Formation during winemaking

(+)-catechin, and (-)-epicatechin-3-O-gallate as extension and terminal subunits. The presence of acetaldehyde bridges was also observed in the A ring of some subunits as well as that of pyranoanthocyanins linked to the backbone via C4 ! C6 or C4 ! C8 linkages. Interestingly, polysaccharides were also found to be present within the structure, although these were not covalently linked to the tannin backbone. Organic and phenolic acids as well as aminoacids were also found to be part of the polymeric fraction structure. This appears to be the first report to completely elucidate the heterogeneous structure of these compounds as they occur in red wines. Lastly, in Cabernet Sauvignon wines, pigmented material was isolated by preparative HPLC [43]. The spectral features of the pigmented material featured a comparatively higher 280 to 520 nm absorbance ratio compared to that of intact anthocyanins (thus indicating the presence of flavan-3-ols) and an mDP between 5 and 10 units. The tannin component of this polymeric material was also put in evidence by subjecting the polymer to protein precipitation with bovine serum

176 Phenolic Compounds - Natural Sources, Importance and Applications

albumin (BSA), further indicating the potential astringent properties of this material.

Figure 14. Potential structures of polymeric pigments resulting from the covalent reaction between anthocyanins (A) and tannins (T). Only C4 ! C8 and A-type interflavanic bonds are depicted but C4 ! C6 interflavanic bonds can also occur.

Polymeric pigment formation increases progressively during maceration and aging (Figure 15) ultimately leading to color changes, modification of mouthfeel properties, and,

Figure 15. Overview of the formation of polymeric pigments during maceration and bottle aging of Cabernet Sauvignon wines processed with a maceration length of 10 days (control) (a) and 30 days (extended maceration) (b). SPP: small polymeric pigments; LPP: large polymeric pigments. Adapted from Ref. [43].

eventually, precipitation. Singleton and Trousdale reported that white wines produced with added tannins and anthocyanins showed a linear increase in polymeric pigment content after addition of seed tannins in the range of 0 to 1000 mg/L (gallic acid equivalents) and anthocyanins in the range of 0 to 500 mg/L [53]. Using protein precipitation, Harbertson et al. found that large polymeric pigments (LPP), which precipitate BSA, increased by 70% between pressing and 185-day postpressing in Merlot wines [58]. Small polymeric pigments (SPP), which do not precipitate BSA, and are assumed to be composed of tannin-anthocyanin dimers, either of direct condensation or mediated by acetaldehyde [231], comparatively increased 30% from pressing to 185-day postpressing. In this same experiment, wines produced with extended maceration and saignée<sup>6</sup> and containing a higher concentration of tannins gave rise to an enhanced formation of LPP; however, this occurred with a decline in the anthocyanin content of 43% relative to its peak concentration [58]. A similar trend was observed in Merlot wines obtained with extended maceration (30 days), in which a two-fold increase of the total polymeric pigments was observed from day 4 to day 30, along with significant losses of malvidin, delphinidin, petunidin, and peonidin anthocyanin derivatives [47]. Furthermore, this later work demonstrated that the formation of polymeric pigments alone during extended maceration was only partially responsible for the observed anthocyanin loss because an increase in the polymeric pigment content of 13 mg/L from day 4 to day 30 occurred along with a drop in wine anthocyanins of 231 mg/L in this same time frame. In summary, these results suggest a complex relationship between tannin content, anthocyanin extraction (or loss), and polymeric pigment formation during maceration. As shown in Figure 15, a common feature of extended maceration seems to be the formation of polymeric pigments with the ability to precipitate BSA (and by a similar mechanism to elicit astringency), but this occurs at the expense of anthocyanin loss (and, consequently, of wine color saturation) (although this anthocyanin loss is generally not fully explained by the formation

<sup>6</sup> The practice of saignée consists of taking a portion of the must from the bottom of the tank before the onset of alcoholic fermentation with the aim of increasing the solid to volume ratio of the remaining must and then furthering the extraction of phenolics and aroma compounds from seeds and skins.

of polymeric pigments). Altogether, these findings suggest that the presence of anthocyanins invariably leads to the formation of polymeric pigments; yet, the proportion of anthocyanins and tannins during maceration, which is expected to differ widely depending on variety, clone, and viticultural and climatic conditions, as well as winemaking technique, will condition the amount of pigmented tannins that are effectively formed during winemaking and aging.
