2. Classification, occurrence, and general reactivity

discussed both in the popular press and in academia [1–4]. So this begs the question: why all the hype surrounding phenolics in wine? Perhaps the first reason stems from the fact the most phenolics bear color. Color in food and beverages have always captivated human beings. Louis Pasteur, the prominent French chemist and microbiologist, used the term "wine color" a whopping 119 times in its seminal treatise "Etudes sur le vin" [5]. Pigments were among the first organic compounds studied in wine, perhaps as an unconscious acknowledgement to the fact that it is color, through the sense of vision, the first attribute human beings appraise when approaching a glass of wine. The term "oenin" to characterize the grape anthocyanin malvidin-3-glucoside, one of the main pigments found in grapes and wines, was first used in 1915 [6]. The amphoteric nature of anthocyanins and their pH-dependent colored forms was recognized as early as 1806: "The colouring matter of the Alicant raisin is the same as that of the red fruits and common red wines; it has the singular property of becoming red by the acids; although blue by nature, it becomes green with the alkalis…" [7]. Tannins, which contribute indirectly to wine color, were also discussed by Pasteur [5] and later in 1895, by E. Manceau, then a scientists at Möet et Chandon (France) who published a method to study tannins in, expectedly,

Another possible explanation for the early interest in the study of phenolics in wine is the fact that specific phenolic compounds were early recognized as determinants of the flavor and mouthfeel properties of red wines. The tactile sensation of astringency and the taste sensation of bitterness in red wines were recognized, again, by Pasteur [5], but no link to phenolics was made at the time. Later in 1958, E.C. Bate-Smith, a prominent British phytochemist, stated that tannins in wines were responsible for the "liquoring properties and body" of wines and were "intimately" concerned with the perception of quality [9]. Interest on the sensory aspects of phenolic compounds in wines quickly sparkled a series of studies on how to maximize the

Indeed, Eugene W. Hilgard, a German-born UC-Berkeley professor, conducted perhaps the earliest studies on the effect of different processing techniques during the fermentation of red grapes in California (USA). His findings, though made between 1885 and 1890, were accurate and were confirmed decades later using much more advanced analytical tools. Hilgard noted that, for example, during red winemaking "maceration1 of the wine on the pomace after fermentation increases tannins but adds nothing to color" [10]. Hilgard also noted that "it is quite certain that, according to the method of fermentation used, the extraction of the pomace and the consequent tint of the wine may seriously differ" [11]. This chapter expands on these later thoughts, i.e., the factors that underpin the extraction and retention of phenolic compounds into wine, along with the chemical and sensory implications they brought about to the finished wines. Before discussing how phenolics are extracted during red winemaking, it is

Maceration is a crucial step during red winemaking whereby the fermentation solids (skins, seeds, lees, and eventually stems) are kept in contact with the fermenting must/wine. It is during maceration that phenolic compounds, free aroma, and aroma precursors are extracted into wine. Winemakers also refer to maceration as "skin contact time" or "maceration

Champagne wines [8].

1

time."

extraction of phenolics into wine during winemaking.

154 Phenolic Compounds - Natural Sources, Importance and Applications

The term "polyphenol," sometimes also referred to as phenol or flavonoid, encompasses about 4000 bioactive compounds of plant and fungal origin which have more than one aromatic phenol ring within the structure as opposed to nonflavonoids or simple phenols [12, 13]. Phenolic compounds are very reactive in wines due to the high electron density of the aromatic ring (s), which is enhanced by the presence of one or more electron-donating hydroxyl groups [12, 14]. As a result, the electron density at the -ortho and -para positions activates the carbons on those sites with partial negative charges, thus enabling the structure to undergo electrophilic substitutions at these positions (Figure 1). The reactivity of the phenol ring is further increased by the weak acidic character of the hydroxyl group that can donate a proton (H<sup>+</sup> ) due to the partial migration of electrons from the oxygen in the hydroxyl to the aromatic ring [14]. In addition, the pK<sup>a</sup> of the most acidic phenolic hydrogen is above 7, which explains why polyphenols are easily deprotonated forming phenolate ions at physiological pH. Phenolate ions can donate electrons directly to oxygen, thereby forming reactive semiquinones [15]. However, at pH values between 3 and 4, such as the ones normally encountered in red wines, phenols are largely protonated and as such cannot directly donate electrons to oxygen [15–17].

Phenols can also readily participate in noncovalent interactions with various components of the wine matrix. The hydroxyl groups can act both as H-bond donors or acceptors. The phenol group(s) are also amphiphilic agents, as the hydroxyl groups are hydrophilic but the aromatic ring is hydrophobic, allowing the structure to become engaged in hydrophobic interactions as well [12]. Another distinctive property of phenolics is that, upon variation in the matrix

Figure 1. Activated sites of the phenol ring. Redrawn from Ref. [14].

conditions such as pH [14] or oxidation-reduction potential [18], phenolics can act both as electrophiles (i.e., electron-loving molecules) and nucleophiles, thus readily reacting with electron-rich or electron-deficient compounds, respectively (Figure 2).

Figure 2. (a) Basic flavonoid ring structure and numbering, (b and c) sites for electrophilic substitutions, and (d) sites for nucleophilic substitutions. Redrawn from Refs. [13, 25].

For the purpose of this chapter, occurrence, reactivity, chemical structure, and sensory properties of phenolic compounds are discussed exclusively in the context of Vitis vinifera L. grapes and wines. Also, only the most important polyphenols of the flavonoid group, namely anthocyanins, flavan-3-ols and tannins, as well as their reaction products, will be discussed. Phenolics belonging to the nonflavonoid class include benzoic (e.g., gallic, hydroxibenzoic, protocatechuic, vanillic, and syringic acids) and cinnamic acid derivatives (p-coumaric, cafeic, ferulic, and sinapic acids). Also, the hydroxylated stilbenes are included in the nonflavonoid class, of which trans- and cis-resveratrol (3,5,4-trihydroxystilbene), as well as their glucose derivatives (trans- and cis-piceids), have all been identified in grapes and wines [19, 20]. Although quantitatively much less important than the flavonoid class, nonflavonoids phenolics play a direct role in both chemical and coupled-enzymatic oxidation reactions in white and red wines [21, 22]. Cinnamic acids can act as copigments,2 inducing changes in color through the phenomenon of copigmentation3 [23] and can also impact wine aroma when metabolized by yeast of the genera Brettanomyces/Dekkera to generate volatile ethyl-phenols [24].

Phenolic flavonoids possess a three-ring system, composed of 15 carbon atoms in the form C6-C3-C6 [12, 13]. The central C ring contains oxygen forming a pyran ring that can adopt various oxidation states, and it is fused to two aromatics rings, termed A and B (Figure 2a). In the flavonoid family, the B-ring is fused to the pyran ring at position 2. The A ring is derived from the phloroglucinol structure and is the most conserved portion of the C6-C3-C6 backbone. Furthermore, different substitutions in the B ring define different compounds within a family. The members of the flavonoid class found in grapes and wines all have the same substitution pattern of hydroxyl groups at carbons 5 and 7 of the A ring. On the other hand, differences in the oxidation state and substitution on the C ring define the different flavonoid classes [13]. Thus,

<sup>2</sup> Copigments are typically noncolored phenolic and/or nonphenolic compounds able to engage in copigmentation reactions with anthocyanins.

<sup>3</sup> As defined by Boulton [34], the phenomenon of copigmentation is due to molecular associations between pigments and other (usually non-colored) organic molecules in solution. These associations cause the pigments to exhibit far greater color than would be expected from their concentration. When anthocyanins engage in copigmentation, both hyperchromic (increase of absorbance) and bathochromic (shift of absorbance toward blue hues) are normally observed.

anthocyanins represent the highest oxidation state of the C ring, and on the other extreme, flavan-3-ols represent the most reduced state of the said ring.

From a chemical and sensory standpoint, the three most relevant phenolic classes within the flavonoid family are flavan-3-ols (the "building blocks" of tannins), anthocyanins, and tannins. A heterogeneous family of reaction products resulting from the reaction between anthocyanins and tannins, the so-called polymeric pigments, are not originally present on grapes but formed during winemaking operations and wine aging (Section 3.4). This chapter will focus on the chemical and sensory aspects of flavan-3-ols, anthocyanins, tannins, and polymeric pigments in red wines, providing along the way an overview of their extraction patterns during red winemaking.
