Polymeric Pigments in Red Wines

C H A P T E R

14 Polymeric Pigments in Red Wines Joana Oliveira, Victor de Freitas and Nuno Mateus REQUIMTE - LAQV - Department of Chemistry and Biochemistry, Faculty of Science, Oporto University, Oporto, Portugal

ABBREVIATIONS HPLC DAD MS/MS LC MS NMR LC/DAD MS MALDI-TOF A F A-F F-A HRMS

High performance liquid chromatography diode array detection mass spectrometry/mass spectrometry Liquid chromatography mass spectrometry Nuclear magnetic resonance Liquid chromatography diode array detection mass spectrometry Matrix-assisted laser desorption/ionization-time of flight Anthocyanins Flavanols Anthocyanin-flavanol adducts Flavanol-anthocyanin adducts High resolution mass spectrometry

14.1 INTRODUCTION Anthocyanins are polyphenolic compounds derived from plant secondary metabolism that are present in vegetables, flowers, fruits, and some beverages such as red wines, being responsible for their red, violet, and blue colors. These pigments in aqueous solutions occur in different forms in equilibria that are dependent on the pH (Brouillard and Lang, 1990; Santos et al., 1993; Brouillard and Delaporte, 1977) (Fig. 14.1). At low pH values, anthocyanins are present in their red flavylium cation form. When the pH increases for values between 3 and 6, the flavylium cation form is hydrated yielding to the colorless hemiketal form that is in equilibrium with the pale yellow cis-chalcone form through tautomerization. Simultaneously, the flavylium cation is deprotonated to the respective violet neutral quinoidal base that at higher pH values can be deprotonated yielding the blue anionic quinoidal base (Brouillard and Delaporte, 1977). Considering all this, at wine pH (3 4) these pigments would be expected to be present mainly in their noncolored hemiketal form. However, flavylium cation is the main form present in red wines. This is the result of its stabilization by different copigmentation mechanisms such as self-association and interaction with other wine components (Liao et al., 1992; Trouillas et al., 2016; Brouillard and Dangles, 1994; Gonzalez-Manzano et al., 2009). In addition, copigmentation has been described as the first mechanism involved in the formation of polymeric anthocyanin-derived pigments in red wines during aging (Brouillard and Dangles, 1994). Furthermore, the reactivity of anthocyanins is strongly affected by different parameters, namely their concentration, temperature, presence of oxygen, and pH, with this latter being the most relevant one. This is correlated with the fact that each equilibrium form of anthocyanins presents different activated positions, thereby affecting their reactivity. In fact, the flavylium cation form presents electrophilic characteristics at carbons C-2 and C-4 from the ring C that can undergo nucleophilic attack by water (Brouillard and Delaporte, 1977; Santos et al., 1993) or sulfur dioxide (Berke´ et al., 2000) (Fig. 14.2). At the same time, the hydroxyl group at carbon C-5 presents

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14. POLYMERIC PIGMENTS IN RED WINES R1

R1 OH

O

Quinoidal base (violet) O

O

O

H+

R2

O R2

pKa ~ 7 O-glucose

O-glucose

OH

Anionic quinoidal base (blue)

OH

pKa~ 4

H+

R1

Flavylium cation (red) HO

OH

O R2

O-glucose OH

3 < pH < 6 H2O/ H+ R1

R1 OH

OH

OH HO

OH

O

HO

O

R2

R2

Tautomerization O-glucose

O-glucose

Hemiketal (noncolored)

OH

OH

Cis-chalcone (yellow)

FIGURE 14.1 Equilibrium forms of anthocyanins in aqueous solutions at different pH values (R1 and R2 5 H, OH or OMe). Adapted from Brouillard, R., Lang, J., 1990. The hemiacetal-cis-chalcone equilibrium of malvin, a natural anthocyanin. Can. J. Chem. 68, 755 761.

R1

R1

El

OH

OH

B

B HO

HO

O

A

C

R2

Nu O-Glc

OH

El

O

Nu Flavylium cation - red-

A

H2O

C

OH

R2

O-Glc

El

OH

Hemiketal form - noncolored-

FIGURE 14.2 General scheme of the main reactive positions in anthocyanins according to the equilibrium form present (flavylium cation or hemiketal form) (R1 and R2 5 H, OH or OMe).

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nucleophilic features in the presence of electrophilic species (Oliveira et al., 2009a; He et al., 2006; Fulcrand et al., 1998). On the other hand, carbons C-6 and C-8 from the ring A can act as nucleophiles in the presence of electrophilic species when anthocyanins are present in their hemiketal form (Sousa et al., 2010). For instance, during red wine aging the color changes from an intense red color to a more brick-red hue due to the reactivity of anthocyanins with other phenolic compounds present in wines giving rise to the formation of more stable polymeric pigments displaying colors ranging from yellow to turquoise blue (He et al., 2010, 2006; Bakker and Timberlake, 1997; Fulcrand et al., 1996; Gomez-Alonso et al., 2012; Schwarz et al., 2003a, 2003b; Mateus et al., 2003a, 2003b; Oliveira et al., 2007, 2010, 2014b; Timberlake and Bridle, 1976; Dallas et al., 1996; Rivas-Gonzalo et al., 1995; Salas et al., 2003; Atanasova et al., 2002). The formation of these pigments during wine aging will be discussed in this chapter alongside their stability in solution and their influence in wine color.

14.2 POLYMERIC PIGMENTS IN RED WINES The intense red color displayed by young red wines is due to the presence of high concentrations of anthocyanins extracted from grapes during vinification. During wine aging and maturation, the concentration of anthocyanins starts to decrease leading to the formation of polymeric anthocyanin derivatives. Furthermore, polymeric pigments are described to play an important role in the long-term color stability of aged red wines (Boulton, 2001), although their full identity and origin in red wines is not completely determined.

14.2.1 Anthocyanin-Derived Pigments Found in Red Grapes and Wines The presence of dimeric and trimeric anthocyanins in grape skins was first evidenced by Vidal and coworkers using mass spectrometry (MS) techniques (Vidal et al., 2004). Then, Pati et al. (2009) detected 22 anthocyanin dimers in grape skins from Cabernet Sauvignon, Montepulciano, and Malvazia varieties using high performance liquid chromatography diode array detection (HPLC DAD) MS/MS. Dimeric structures were also reported by Salas et al. (2005) and Alcalde-Eon et al. (2007) in red wines using the same technique. A few years later, Oliveira et al. (2013a) established the presence of an A-type oenin trimer (Fig. 14.3) in a young Port wine using liquid chromatography (LC) MS and nuclear magnetic resonance (NMR) spectroscopy. Moreover, the origin of these oligomeric pigments in red wine was postulated to result from their extraction from grape skins during the winemaking process, as they were detected in grape skins (Oliveira et al., 2013a; Vidal et al., 2004; Pati et al., 2009) and grape pomace (Oliveira et al., 2015) using LC/DAD MS and/or Matrix-assisted laser desorption/ionizationtime of flight spectrometry.

14.2.2 Anthocyanin-Derived Pigments Formed in Red Wines During Aging Acetaldehyde is the main aldehyde (90%) present in wines as a result of yeast metabolism during the first stages of alcoholic fermentation, being also produced throughout the wine aging process from ethanol oxidation (Liu and Pilone, 2000). In fortified wines like Port wines, this compound and other aldehydes (propionaldehyde, 2-methylbutyraldehyde, isovaleraldehyde, methylglyoxal, benzaldehyde) are present in higher amounts due to the addition of wine spirit (40 260 mg/L of acetaldehyde) to stop the fermentation (Pissarra et al., 2005). Sherry wines also present high levels of acetaldehyde (90 500 mg/L) due to the fact that this wine style is produced under oxidative conditions (Zea et al., 2015). Moreover, acetaldehyde is extremely reactive and can react with different phenolic compounds present in grapes and wines (e.g., anthocyanins and flavanols) increasing the number of different chemical pathways that can occur in red wines starting from anthocyanins and/or flavanols to yield polymeric pigments (Pissarra et al., 2003; Jurd, 1969; Atanasova et al., 2002; Timberlake and Bridle, 1976; GarciaViguera et al., 1994; Rivas-Gonzalo et al., 1995). The acetaldehyde-mediated polymerization between either only flavanols or with anthocyanins is the most well documented reaction in the literature (Rivas-Gonzalo et al., 1995; Francia-Aricha et al., 1997; Es-Safi et al., 1999a, 1999b; Santos-Buelga et al., 1999; Escribano-Bailon et al., 2001; Remy-Tanneau et al., 2003; Salas et al., 2003; Pissarra et al., 2003, 2004b; Saucier et al., 1997). The condensation of anthocyanins (A) with flavanols (F) occurs directly (Remy-Tanneau et al., 2003) or is mediated by aldehydes (Pissarra et al., 2003, 2004b) (Fig. 14.4).

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14. POLYMERIC PIGMENTS IN RED WINES OMe OH

HO

O OMe H O OH

H

HO

OH OH

O

OH OMe OH

O

O OMe

O HO

OH OH

O

OH

OH OMe

OH

O

O OMe

O

HO

OH

OH O

OH OH

FIGURE 14.3 Structure of the malvidin-3-glucoside trimer isomer detected in a young red wine and in red grape skin. Adapted from Oliveira, J., da Silva, M.A., Jorge Parola, A., Mateus, N., Bra´s, N.F., Ramos, M.J., et al. 2013a. Structural characterization of a A-type linked trimeric anthocyanin derived pigment occurring in a young Port wine. Food Chem. 141, 1987 1996.

Two mechanisms were described in red wines for the direct condensation between anthocyanins and flavanols yielding flavanol-(4,8)-anthocyanin (F-A1) and anthocyanin-(4,8)-flavanol (A1-F) adducts. The first consists of the nucleophilic attack of the carbon C-6/C-8 of the anthocyanin (hemiketal form) to the electrophilic carbocation present at carbon C-4 of a flavanol unit produced during the cleavage of a proanthocyanidin in the slightly acidic conditions (Somers, 1971; Jurd, 1969; Salas et al., 2003). The occurrence of F-A1 pigments in red wines was reported in the literature by Salas et al. (2005) using LC/DAD/electrospray ionization-mass spectrometry (ESIMS) spectrometry (Salas et al., 2004a). F-A1 acetaldehyde-derived pigments, namely F-methylmethine-F-A1 and methylmethine-(F-A1) were also found to occur in a 2-year-old Port wine using HPLC-ESI-MS (Nave et al., 2010b). In addition, the formation of A-F dimers in red wines is described to result from the nucleophilic attack of the carbon C-6/C-8 of a flavanol unit to the electrophilic carbon C-4 of an anthocyanin (flavylium cation form) giving rise to a flavene structure product (colorless) (Remy et al., 2000) that can evolve to the colorless bicyclic form [A-type, A-(O)-F] (Jurd, 1969; Remy et al., 2000; Remy-Tanneau et al., 2003) or undergo oxidation to give the red pigment A1-F (Duen˜as et al., 2005; Liao et al., 1992), which could dehydrate to the orange-yellow xanthylium salt. This latter was only detected by UV vis spectroscopy in wine-like model solutions (Santos-Buelga et al., 1995, 1999). On the other hand, the formation of aldehyde-mediated polymeric pigments starts with the addition of the protonated aldehyde (acidic conditions) to the nucleophilic carbon C-6/C-8 of a flavanol unit. Then, the formed adduct undergoes the nucleophilic attack of the carbon C-6/C-8 of an anthocyanin (hemiketal form). The last step includes a dehydration reaction to yield the anthocyanin-alkyl/aryl-flavanol dimer (Pissarra et al., 2003; Timberlake and Bridle, 1976; Sousa et al., 2007). Besides, these pigments were also reported to be at the origin of the pyranoanthocyanin-flavanol compounds found in red wines during aging (Francia-Aricha et al., 1997; Mateus et al., 2002, 2003a). Moreover, a dimeric oenin acetaldehyde-mediated condensation product [malvidin-3-glucoside-(8,8)-malvidin3-glucoside] was also found to occur in wine-like model solutions and in red wines (Atanasova et al., 2002).

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(A)

R1

OH

OH

OH HO

HO

O

O R2

R R1 OH

OH

O HO

OH HO

OH HO

OH O

OH OH

OH

O R2

O

OH

R O HO

OH OH

O

HO

OH

OH

OH

A+-F dimer

F-A+ dimer

(B) OH HO R O

R1

OH

OH HC

HO OH

HO

R3 O R2 O HO

OH

OH O

OH OH

Anthocyanin-alkyl/aryl-flavanol adduct

FIGURE 14.4 (A) General structure of the anthocyanin-flavanol (A1-F) and flavanol anthocyanin (F-A1) adducts formed by the direct condensation of anthocyanins with flavanols (R1 and R2 5 H, OH or OMe). (B) Anthocyanin-alkyl/aryl-flavanols adducts R1 and R2 5 H, OH or OMe; R3 5 H; CH3; CH(CH3)2; CHCH3CH2CH3; CH2CH(CH3)2; CH2CH3; Ph. Adapted from Pissarra, J., Mateus, N., Rivas-Gonzalo, J., Buelga, C.S., de Freitas, V., 2003. Reaction between malvidin 3-glucoside and (1)-catechin in model solutions containing different aldehydes. J. Food Sci. 68, 476 481.

14.2.3 A-Type Vitisin-Derived Pigments Formed in Red Wines During Aging Over the years, different anthocyanin-derived compounds (pyranoanthocyanins) displaying orange colors have been described in the literature from the reaction of anthocyanins with small molecules produced by yeasts during fermentation, namely acetaldehyde (Bakker and Timberlake, 1997; Oliveira et al., 2009a), pyruvic acid (Fulcrand et al., 1996), oxaloacetic acid (Araujo et al., 2017), acetoacetic acid (He et al., 2006), diacetyl (GomezAlonso et al., 2012), vinyl-phenols (Fulcrand et al., 1996), and hydroxycinnamic acids (Schwarz et al., 2003b; Schwarz and Winterhalter, 2003). These compounds are likely to contribute to the red/orange hues observed in red wines during aging. Carboxypyranoanthocyanins (A-type vitisins) are described in the literature as the main pyranoanthocyanins formed in red wines during aging (Mateus and de Freitas, 2001) from the reaction between anthocyanins and pyruvic (Fulcrand et al., 1996) and/or oxaloacetic (Araujo et al., 2017) acids. In red Port wines, the levels of Atype vitisins increase after wine fortification with wine spirit and start to decrease after around 100 days. The formation of these anthocyanin-pyruvic acid adducts occurs concomitantly with the decrease of anthocyanins (Mateus and de Freitas, 2001). Although these orange pigments are not polymeric by themselves, they may contribute to the polymeric fraction of red wine pigments, as they were reported in the literature to react with other wine components (Mateus et al., 2003b, 2004; Oliveira et al., 2007, 2010). Indeed, some years ago two anthocyanin-derived pigments, namely a vinylpyranomalvidin-3-glucoside-procyanidin dimer and the respective coumaroylated derivative (A-type portisins), presenting an unusual bluish color in acidic pH conditions were identified in a young Port red wine (Mateus et al., 2003b) (Fig. 14.5). Studies performed in wine model solutions suggested that these bluish pigments can be formed in red wines from the reaction between A-type vitisins and flavanols (in the presence of acetaldehyde) or vinyl-flavanols (Mateus et al., 2003b, 2004). The structure of A-type

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14. POLYMERIC PIGMENTS IN RED WINES OMe OH O

HO

OMe O HO

OH O

OH O-R1

O

OH OH

HO

O OH

OH

OH

OH O

HO

OH OH

A-type portisins

FIGURE 14.5 General structure of the A-type portisins found to occur in a 2-year-old Port wine (R1 5 H or, coumaroyl group). Adapted from Mateus, N., Silva, A.M.S., Rivas-Gonzalo, J.C., Santos-Buelga, C., de Freitas, V., 2003b. A new class of blue anthocyanin-derived pigments isolated from red wines. J. Agric. Food Chem. 51, 1919 1923.

portisins comprises a pyranoanthocyanin moiety linked to a flavanol (monomer or dimer) by a vinyl linkage (Mateus et al., 2003b, 2004). A few years later, structurally similar compounds (B-type portisins) were identified in Port red wines with a phenolic moiety replacing the flavanol one. B-type portisins were described to result from the reaction of A-type vitisins with vinyl-phenolics (Mateus et al., 2006) or hydroxycinnamic acids, such as p-coumaric, caffeic, ferulic, and sinapic acids (Oliveira et al., 2007). The occurrence of A and B-type portisins in aged red wines points to a second-generation of anthocyanin derivatives in which the main precursors are no longer genuine anthocyanins but rather carboxypyranoanthocyanins (A-type vitisins) involved in the formation of polymeric pigments in the later stages of red wine aging. Furthermore, a few years ago, Oliveira et al. (2010) identified in 9-year-old Port wine and in the respective lees, a family of pyranoanthocyanin dimers presenting an unusual turquoise blue color at acidic pH (Fig. 14.6). The origin of these pigments in red wines was demonstrated in wine-like model solutions to involve the reaction between an A-type vitisin and a methylpyranoanthocyanin (Oliveira et al., 2010). Even though the mechanism of formation is still undetermined, it has been proposed that the origin of pyranoanthocyanin dimers should start with the formation of a charge-transfer complex between the A-type vitisin and the methylpyranoanthocyanin similarly to what was reported by Chassaing et al. (2008) for related compounds. All these anthocyanin-derived pigments may have an important contribution to the color hues and stability in red wine and the knowledge of their chemical pathways are crucial to better understand the color evolution of red wines during aging.

14.3 ANALYSIS OF POLYMERIC PIGMENTS Red wine is unquestionably a very complex matrix which makes the identification of all its molecules and their chemical pathways more difficult to achieve. The main difficulties are associated with the limitation of the techniques used, their detection limits, and the increased complexity of the polymeric structures aimed to be identified. The analytical methods commonly used to study grape and wine polyphenols involve reverse-phase LC coupled with spectrophotometry or MS (Di Stefano and Flamini, 2008; Flamini and de Rosso, 2008; Arapitsas et al., 2014, 2016; Ehrhardt et al., 2014). However, this technique combined with multiple mass spectrometry

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14.4 STABILITY IN SOLUTION AND INFLUENCE IN RED WINE COLOR

R1 OH HO

OR 6

HO

O R2

HO R4 HO

O

O HO

OH

O

OH O

OH OR 5

O R3 O

O

OH

FIGURE 14.6 General structure of the pyranoanthocyanin dimers found to occur in aged Port wines and in respective lees (R1, R2, R3 and R4 5 H, OH or OMe; R5 and R6 5 H, acetyl, coumaroyl, independently of each other). Adapted from Oliveira, J., Azevedo, J., Silva, A.M.S., Teixeira, N., Cruz, L., Mateus, N., et al., 2010. Pyranoanthocyanin dimers: a new family of turquoise blue anthocyanin-derived pigments found in Port wine. J. Agric. Food Chem. 58, 5154 5159.

(MS/MS and MSn) analysis is only effective for the structural characterization of low molecular weight compounds (Flamini et al., 2015; Arapitsas et al., 2012a). In the last few years, the number of studies in the field of untargeted grape and wine polyphenolic LC MS analysis has increased in the literature providing interesting information about various enological practices such as grape variety, geographical origin of wines, and their chemical age (Arapitsas et al., 2012a, 2014; Fulcrand et al., 2008; Cuadros-Inostroza et al., 2010). Untargeted methods have been found to have good resolution, high sensitivity, and high-throughput capacity being able to detect/identify a great number of possible compounds in a single run (Arapitsas et al., 2012b). Conversely, targeted analysis is used for the quantitative determination of specific molecules but with limited information on the overall sample metabolome (Cuadros-Inostroza et al., 2010; Vaclavik et al., 2011). On the other hand, high-resolution mass spectrometry (HRMS) has been described as a powerful technique for the analysis of complex samples in many fields. With HRMS techniques it is possible to obtain the exact mass of molecules and to determine their elemental composition, which is crucial for the proper identification of the compounds (Vallverdu-Queralt et al., 2017a,b). Delcambre and Saucier (2012) demonstrated the application of Q-TOF-HRMS analysis of grapes and wines to be used as a footprint for the determination of the grape variety and the geographical origin of the wine. The combination of untargeted analysis with Orbitrap-HRMS or Q-TOF-HRMS could be promising techniques for the identification of polymeric pigments in complex samples such as red wines. In fact, very recently Vallverdu´-Queralt et al. (2017a,b), have demonstrated the ability of these kinds of methodologies to identify numerous polymeric pigments in wine-like model solutions containing oenin and (2)-epicatechin and acetaldehyde.

14.4 STABILITY IN SOLUTION AND INFLUENCE IN RED WINE COLOR Color is one of the most important quality indicators of a red wine. During maturation and aging, the color of red wines changes from an intense red color to a more red-orange hue due to the chemical transformation of genuine anthocyanins extracted from the grape skins during fermentation forming polymeric pigments by the mechanisms discussed previously. Sulfur dioxide (SO2) is commonly used during the winemaking process as an antioxidant and antiseptic inhibiting the growth of undesirable microbial. However, in the case of red wines, SO2 can increase the extractability

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14. POLYMERIC PIGMENTS IN RED WINES

of anthocyanins if added prior to fermentation and improve their stability when added at bottling (Burroughs, 1974). This is correlated to the reversible bleaching of anthocyanins that occurs in red wine due to the formation of colorless anthocyanin-2-bisulphite or anthocyanin-4-bisulphite adducts (Berke´ et al., 2000; Jurd, 1964) (Fig. 14.7). In general, anthocyanin-derived pigments are much more stable than the anthocyanin counterpart towards bleaching by SO2. This is mainly due to the fact that the positions in the pigment structure at which SO2 is likely to react are blocked. Moreover, conversely to anthocyanins, studies of polymeric pigments equilibria in aqueous solutions and their contribution to the overall wine color are limited in the literature (Oliveira et al., 2006b, 2014a, 2014b; Cruz et al., 2010; Salas et al., 2004b; Asenstorfer et al., 2006; Pissarra et al., 2004a; Nave et al., 2010a; Sousa et al., 2007). The study of their chromatic characteristic at different pH values and the determination of the corresponding ionization constants is valuable information about their expected occurrence at red wine pH. In addition, the physicochemical features studied over the years for some polymeric pigments using UV visible spectroscopy and NMR revealed a higher stability of these compounds towards the hydration reactions when compared to their anthocyanin precursors, which can contribute to the color stability of red wines during the aging process. However, some exceptions are observed like the case of F-A1 adducts. The study of these pigments performed in model solutions at different pH values using UV visible spectroscopy showed that the flavanolic unit (catechin) shifts the absorption maximum of the flavylium cation from 518 to 535 nm but has no significant modification on the kinetic (hydration rate) and thermodynamic (hydration constant) properties compared to the oenin (Nave et al., 2010a). Conversely, anthocyanin-alkyl/aryl-flavanol pigments that are described to contribute to the red/violet hues observed in young red wines during the first stages of wine maturation display a purple color with characteristic UV visible spectra that present a λmax in the visible region at 540 nm and a shoulder at 450 nm (Pissarra et al., 2004a, Sousa et al., 2007). Moreover, studies performed in aqueous solutions using UV visible spectroscopy showed that when the pH increases from 2.2 to 5.5, oenin-methylmethine catechin pigment solutions become gradually more violet, while similar solutions of the anthocyanin are almost colorless at pH 4.0 (Escribano-Bailon et al., 2001). This indicates a higher protection against water attack of the oenin moiety of the pigment when compared to the oenin alone (Escribano-Bailon et al., 2001). Similar results were obtained for other anthocyaninalkyl/aryl-flavanol pigments (Sousa et al., 2007). However, these polymeric pigments are more prone to degradation in aqueous solution comparatively to anthocyanins with the cleavage of the methylmethine bridge yielding oenin as a major product (Escribano-Bailon et al., 2001). R1

R1 OH

HO

OH

NaHSO 3

O

HO

O

R2

2

2

R2 SO3Na

4 O-glucose

O-glucose OH

NaHSO3

OH

R1 OH

HO

O R2

4 O-glucose OH

SO3Na

FIGURE 14.7 Bisulfite addition to anthocyanins (R1 and R2 5 H, OH or OMe). Adapted from Berke´, B., Che`ze, C., Deffieux, G., Vercauteren, J., Sulfur Dioxide Decolorization or Resistance of Anthocyanins: NMR Structural Elucidation of Bisulfite-Adducts. In: G.G. Gross, R.W. Hemingway, T. Yoshida, S.J. Branham (Eds.), Plant Polyphenols 2: Chemistry, Biology, Pharmacology, Ecology. Springer US: Boston, MA, 1999; pp 779 790.

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In addition, the titration of the oenin trimer showed that the multistate equilibria of this compound is strongly dominated by acid-base chemistry, with the reaction sequence hydration tautomerization isomerization accounting for less than 10% of the overall reactivity (Oliveira et al., 2014a). So, the lack of reactivity and the higher chromatic stability presented by these oligomeric compounds when compared to anthocyanin monomers can have a direct impact on the overall red wine color during aging. Over the years, the equilibrium forms of pyranoanthocyanin pigments have been studied using NMR and UV visible spectroscopy (Asenstorfer and Jones, 2007; Cruz et al., 2010; Oliveira et al., 2009b, 2011). In aqueous solutions these polymeric pigments have been shown to coexist under different equilibrium forms that are pHdependent. Using NMR spectroscopy it was possible to postulate the absence of hydration reactions and that only proton transfer reactions occur when the pH change in pyranoanthocyanins. In fact, a number of studies based on UV visible spectroscopy have already established that pyranoanthocyanin pigments are protected from the attack by water when compared with their anthocyanin precursors (Oliveira et al., 2006a, 2006b, 2009b, 2011, 2013b, 2014b; Asenstorfer and Jones, 2007; Cruz et al., 2010; Vallverdu-Queralt et al., 2016). An exception was described by Gomez-Alonso et al. (2012) for acetyl-pyranoanthocyanins that at wine pH are present in their noncolored hemiketal form (at carbon C-10). All these polymeric pigments may have a direct or an indirect impact on the color of red wines during aging.

14.5 CONCLUSION Red wine is a complex matrix, which makes the identification of all its molecules and their chemical formation pathways a rather difficult task. Over the years, different families of polymeric pigments have been described from the reaction of anthocyanins present in grapes with other wine components. After the first reactions involving genuine anthocyanins yielding newly-formed anthocyanin derivatives, another stage of pigment formation arises from the reaction of carboxypyranoanthocyanins (A-type vitisins) with other wine components. This has led to the identification of several anthocyanin polymeric pigments. However, this is only the tip of the iceberg on this matter and a lot remains to be done to fully determine the fraction of polymeric pigments present in red wines.

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