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A Technical Guide for Wine Producers
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RECENT ARTICLES | WYNBOER HOME
Chemical and sensory properties
of grape and wine phenolics Part I
Introduction
Phenolic compounds are major wine constituents that are responsible for some wine organoleptic properties including, in particular, colour and astringency. In addition, their health contribution is attracting considerable interest in the international scientific community. The phenolic composition of grapes is greatly influenced by grape-growing conditions, whereas the phenolic composition of the resulting wine depends not only on the composition of the grapes used, but also on the wine-making conditions, which influence the extraction of the various constituents into the wine and their subsequent reactions.
For red grapes roughly 30-40% of the total phenolic material is located in the skins and 60-70% in the seeds. It is important to note that, even with prolonged skin contact and maceration, phenolic extraction from skins is less than 50% of the amount available, while close to 60% of the available seed phenolics are extracted during fermentation. The anthocyanin content can vary in young wines from 100mg/L in Pinot Noir to 1500 mg/L in varieties such as Shiraz and Cabernet Sauvignon, to a minimum of 50 mg/L or less during aging. The total amount of phenols range from approximately 1 to 5 g/L in a light to full bodied red wine.
Structural diversity of the main phenolic compounds present in grapes and wine
Phenolic compounds exhibit a wide diversity of structures, but they can be divided into flavonoids and non-flavonoids. The flavonoid compounds in wine are mostly extracted from the skins and seeds of grapes during fermentation.
Flavonoids are characterized as molecules possessing two phenolic groups joined by a pyran (oxygen-containing) ring structure (Figure 1). All of these phenolic classes have a large number of structures that differ in the number and position of hydroxy (-OH) and methoxy (-OCH3) groups on the basic skeleton. Each structure can also be variously substituted (e.g. glycosylated, acylated, esterified). The main flavonoid species important to the chemical reactions and sensory properties of red wine are the anthocyanins (e.g. malvidin-3-O-glucoside) and flavanols (e.g. catechin and epicatechin). The next most abundant compounds are flavonols, their yellow pigments in the skins of both red and white grapes.
The non-flavonoids are represented by mostly phenolic acids (benzoic acids, hydroxycinnamic acids) and their esters and are present in the pulp of grapes at low levels (Figure 2). The various acids are differentiated by substitution of their benzene ring.
The important hydroxycinnamates in grapes are the tartaric esters of caffeic, p-coumaric and ferulic acid, namely caftaric, coutaric, and fertaric acid (position R2 = H, replaced by tartaric acid). Post harvest hydrolysis, especially by pectin esterase, frees at least part of the hydroxycinnamates of grapes from their tartrate portion. The hydroxybenzoic acids are primarily degradation products; the most important is gallic acid.
(+)-Catechin (R1 = H; R2 = OH) and (-)-epicatechin (R1 = OH; R2 = H) form dimers, trimers and various higher oligomers (procyanidins) through interflavan (C4-C6/C4-C8) linkages (Figure 3). Furthermore, some units are substituted by gallic acid on the 3-hydroxyl group. An infinite number of isomers are possible, which explains why it is so difficult to separate these molecules.
Skin tannins [30 units] differ from seed tannins [2 to 16 units] by their lower amounts of galloylated derivatives and higher average molecular weights. It is possible to isolate and separate the following components in grapes and wine: (+)-catechin, (-)-epicatechin, dimeric, trimeric, oligomeric and condensed procyanidins. Tannins in the stalks are relatively similar to those in the seeds.
In young red wines, the procyanidins are primarily in the dimeric or trimeric form, whereas in older wines, polymers of eight to ten or more units are found (Ribéreau-Gayon, 1972).
The role of anthocyanins in the colour of red wine
Anthocyanins are the red pigments in grape skins, which play an important role in the colour of wine. There are several forms of anthocyanins that exist in equilibrium at wine pH, with only 25% or less in the red, flavylium form (Figure 4) (Singleton, 1988). Sulphur dioxide (SO2) also forms a colourless bisulphite addition compound with the flavylium ion. The reaction is reversible in that depletion of sulphur dioxide in the wine will lead to the formation of free anthocyanin. A process called co-pigmentation can also influence this equilibrium.
Co-pigmentation is defined as the enhancement of colour due to the formation of a complex between pigments (anthocyanins) and co-pigments. It is now apparent that co-pigmentation can account for between 30 and 50% of the colour in young wines and that it is primarily influenced by the levels of several specific, non-coloured phenolic components or co-factors (Boulton, 1996). The flavonols have been found to be the most effective copigments.
The significance of this phenomenon on pigment extraction and colour retention during fermentations, the rate of subsequent pigment polymerisation, and the possible protection of anthocyanins from oxidation are only now being suggested.
Chemical reactions of anthocyanins and tannins during winemaking and aging
The changes from the purple red of young wines to the tawny hue observed in older red wines are attributed to the conversion of grape anthocyanins to new, more stable polymeric pigments. The average wine monomeric anthocyanin concentration decreased about 30% (range 0 to 61%) between two and nine months while polymeric anthocyanins tripled. The percent of total 520 nm absorbance contributed by polymeric compounds ranged from 50% to 85% of wine colour, respectively after one year and ten years of aging.
Polymerisation reactions
There are several possible methods of polymerisation by which the pigments of young wines may be altered and stabilised during aging. The complex pigments formed are not very sensitive to variations in pH and SO2 (Somers and Evans, 1977). Furthermore, non-coloured phenolics may also proceed to pigmented species. Small quantities of monomeric pigments are formed in wine during fermentation and maturation by cycloaddition mechanisms between anthocyanins and various yeast-derived metabolites. Compared with other pigments in wine, these new molecules are present in very small quantities. However, they are very stable and concentrations, unlike those of free anthocyanins that condense with tannins or break down under certain conditions, hardly vary during aging.
Condensation reactions involved in wine aging are classically described as anthocyanin-tannin additions, that can either be direct, generating orange xanthylium salts, or involve acetaldehyde, leading to purple pigments. Direct addition reaction takes place even in the absence of air, as it requires no or only small amounts of oxygen and has been shown to take place very slowly (Dallas et.al., 1996). Indirect condensation, involving acetaldehyde (Bakker et.al., 1993) could involve both anthocyanin-flavanol and flavanol-flavanol condensation (Saucier et.al., 1997a; Saucier et.al., 1997b). Acetaldehyde in wine is produced either biochemically by yeast metabolism during fermentation, or by the oxidation of ethanol by phenolic compounds in the presence of oxygen. The rate of formation and accumulation of acetaldehyde mediated polymerisation increase with a decrease in pH and an increase in temperature. Polymerisation is also much slower in the presence of sulphur dioxide. Other tannin reactions include, on one hand, acid-catalysed bond making and bond-breaking processes characteristic of proanthocyanidin chemistry (Haslam, 1980) and, on the other hand, oxidation reactions leading to browning (Cheynier et.al., 1997). Pigments become more resistant to oxidation with extended polymerisation with the monomeric anthocyanins the most easily oxidized (Yokotsuka and Singlelton, 2001).
Proteins and polysaccharides found in musts and wines originate from the grape berry or yeast. The latter can be released into the wine during alcoholic fermentation or post fermentation while aging wine on yeast lees. Phenolic compounds are associated or loosely bound with proteins (Yokotsuka et.al., 1991) and polysaccharides (Waters et.al., 1991). These interactions may lead to the formation of bottle deposits (Peng et.al., 1996).
Anthocyanin and tannin assays
As wine ages the absorbance maximum l ~ 520 nm declines in intensity corresponding to an increase in the yellow colour at l ~ 420 nm. These two values are suitable for studying wines with some age, but do not always cover the relatively deep colours of young wines. The blue component (620 nm), attributed to the quinodal forms of free and combined anthocyanins must be taken into account in assessing these colours (Glories, 1984). The wine hue or tint, measured as the ratio of absorbance's A420/ A520 typically increases from 0.4-0.5 in young wines to around 0.8-0.9 in mature red wine (Somers and Evans, 1977). This indicates the increased contribution of polymeric pigments to the wine colour. Several methods for the assessment of colour in red wines have generally recognized the ionisable and bleachable nature of the anthocyanins and have distinguished these from the polymeric forms that are generally less sensitive to pH and less bleached by bisulphite. It is now clear that the contribution of colour due to co-pigmentation is also bleached with SO2. The SO2 bleaching methods will attribute all the colour loss to pigments when as much as half of it could be from copigmented forms in young wines, and these forms have extinction values several times those of the free anthocyanins. At high dilutions, typically 24:1, virtually all of the copigmented anthocyanins have returned to the ionisation equilibrium involving the flavylium cation, the pseudobase, and the chalcone forms. This loss in colour with dilution can be used in calculation of the fraction of colour that is due to co-pigmentation. An extensive review about co-pigmentation and its role in the colour of red wine by (Boulton, 2001) was recently published.
These spectrophotometric measures can be applied to wine fractions to determine the total amount of pigments sensitive to bisulphite bleaching. Somers used a dilution of wine in 1M HCl to push the anthocyanin equilibrium to the flavylium form and break up any possible colour enhancement by co-pigmentation to measure the total amount of potential colour.
Various spectrophotometric and spectroscopic methods have also been used to determine the total polyphenol content of wine. The Folin-Ciocalteu method has been the one of general preference (Singleton and Rossi, 1965), usually with an arbitrary standard such as gallic acid. The Folin-Ciocalteu determines total phenols (and other easily oxidised substances) by producing a blue colour from reducing yellow heteropolyphosphomolybdate-tungstate anions. Somers used the expression OD280 - 4 as an index of total polyphenols in red wine (Somers and Evans, 1977), while Glories developed a set of polyphenolic measurements as discussed below. The PVPP index is used to quantify phenolic polymers (pigmented and non-pigmented) based on the fact that free anthocyanins and other monomers can be washed of a PVPP (polyvinylpyrrolidone) column with dilute alcohol, while the rest will stick to the surface. Bakker compared the methods used by Glories and Somers to calculate total polyphenols and found a correlation coefficient of 0.956 (Bakker et.al., 1986). The following indexes indicate different tannin attributes. The HCl index is based on the instability of procyanidins in a concentrated HCl medium, where the precipitation speed depends on the degree of polymerisation and therefore reflects the state of polymerisation of tannins in the wine. The dialysis index relates to the structure of the tannin with bulky or highly charged molecules passing through the pores of a dialysis membrane more slowly than small molecules with lower charges. A high dialysis index indicates that there are bulky, generally polymerised pigments, the HCl index will then also be high. In wines with a particularly high anthocyanin content, the HCl index may be low, although the dialysis index is high. The gelatin index is based on the capacity of tannins to react with proteins, forming stable combinations. This reactivity is responsible for the sensation of astringency when tasting red wine.
It was originally believed that the ability of polyphenols to bind to proteins would be useful for the quantitation of the polyphenols, but no correlation could be found between the amount of proteins precipitated and phenolics present. The affinity of procyanidins towards polyprolines, casein and gelatins appeared essentially determined by the number of o-substituents and also with the degree of polymerisation of the polyphenols and galloylation (Haslam et.al., 1992).
The relationship between the maximum quantity of proteins precipitated and the quantity of tannins may be considered characteristic of the tannic strength. The maximum reactivity occurs with procyanidins that have a molecular weight around 2500 (eight flavanol units).
Isolation and separation of phenolic compounds in grapes and wine
Analysing the structures of the various phenolic compounds are currently limited to dimeric, trimeric or, possibly, tetrameric procyanidins. The methods currently used to determine phenolic composition consist of rough fractionations into four or five classes of compounds with similar characteristics.
Preparative methods based on the separation of procyanidins on a molecular weight basis have been developed. These methods include liquid chromatography on hydrophobic stasionary phases such as Fractogel TSK HW-40 (Ricardo da Silva et.al., 1991) and Sephadex LH-20 (Kantz and Singleton, 1991). Ion exchange has also been effective in the separation of monomeric anthocyanins and smaller pigments (Spagna and Pifferi, 1992). Strong absorption of polymeric phenols to most types of resins, results in the loss of an unknown amount of material and low resolution limits separation to a combination of dimers and trimers.
Separation of procyanidins has been achieved with HPLC (high performance liquid chromatography) columns used coupled with UV-visible detectors (Ricardo da Silva, 1990) for identification. However, analysis of tannins becomes increasingly difficult as their molecular weight increase and separation only up to the tetramer have been achieved. HPLC separation of wine phenolics usually requires prior purification, which makes quantification difficult, as some material is always lost. In most direct HPLC methods the polymeric phenols elute as an increase in the baseline. A method based on Price (Price et.al., 1995) has been optimised for the analysis of grape seed and red wines tannins (Peng et.al., 1999; Peng et.al., 2001), which enables the non-pigmented polymeric phenols and the polymeric pigments to elute as a single peak, at the end of the run.
Characterization and identification of grape and wine polyphenols
The major methods for the identification of grape procyanidins are electrospray ionisation mass spectrometry (ESI-MS) and 1H NMR and 13C NMR spectrometry.
Nevertheless, degradation by thiolysis is essential to confirm the structure of polyphenolic compounds (Thompson et.al., 1972). This consists of heating the procyanidins in the presence of an acid and a nucleophilic agent such as phloroglucinol or toluene-ĉ-thiol (Kennedy et.al., 2000). The degradation allows the distinction between terminal units (released as flavan-3-ols) and extension units (released as their benzylthioethers), and, thus, gives a measure of the mean degree of polymerisation (mDP) and the respective amounts of each constitutive unit. The ester linkage of gallic acid are retained during thiolysis and enzymatic hydrolysis with tannase are used to release gallic acid and thus determine the degree of galloylation (Prieur et.al., 1994).
The molecular weight (MW) profiles of proanthocyanidin polymers (condensed tannins) can also be determined by gel permeation chromatography (GPC). The flavanol polymers are mostly derivatized as they are too polar to be separated with a large range of gel permeation chromatography substrates (Williams et.al., 1983). Complete acid hydrolysis of procyanidins with butanol and hydrochloric acid yield the resulting anthocyanidins (Porter et.al., 1986) and can also be used as and indication of chain length, but is not as specific or sensitive as thiolysis (Matthews et.al., 1997).
Electrospray ionisation (ESI) is the best-suited mass spectrometry method for the analysis of phenolic compounds and is used either directly or on line after HPLC separation. Application of ESI-MS made it possible to detect and partly characterize high molecular weight procyanidins in the extracts, which are eluted as unresolved peaks under current HPLC (LC) techniques (Peng et.al., 2001). However, mass spectrometry does not allow to distinguish between catechin and epicatechin-based structures or to determine the position of interflavan C-C linkages. Finally, although for a given compound, the signal intensity is proportional to the concentration, it depends on the tendency of the molecule to become ionised, which is greatly influenced by its structure. Therefore, ESI-MS cannot be used for quantification. MALDI-TOF (matrix-assisted laser desorption/ionisation time-of-flight) is a new soft ionisation technique with a wider applicable mass range and has been successful in detecting apple procyanidins up to the decamer level as the single charged species (Ohnishi-Kameyama et.al., 1997). The use of other techniques complementary to LC-MS is essential for total characterization and identification of these complex flavanol polymers.
Given the above, nuclear magnetic resonance (NMR) is undoubtedly the method of choice to identify phenolic compounds (Laouenan et.al., 1997), but so far, it can only be applied to pure compounds, which are very difficult to obtain in sufficient amount.
Contribution of phenolic compounds and their reactants to wine bitterness and astringency
Phenols play an important role in red wine colour, bitterness, astringency, as well as other tactile or 'mouth-feel' characteristics. The colour and taste changes occurring during the course of wine aging are believed to result from the conversion of grape anthocyanins and proanthocyanidins to other polymeric species.
Monomeric and polymeric flavan-3-ols
Monomeric and polymeric flavan-3-ols are the primary contributors to the astringency and bitterness of red wine and have been reviewed extensively (Gawel, 1998). Generally the so-called condensed tannins are more astringent than the smaller oligomers, while the monomers, dimers and trimers evoke more bitterness than the condensed tannins. This concept has been used to explain the "hardness" of young wines and the "softness" of older wines, as a result of their oligomeric to polymeric procyanidin ratios. A further complication is the observation that astringency is not necessarily experienced in isolation. There is also a concern that many people confuse bitterness and astringency or have difficulty in distinguishing between them.
Non-flavonoid phenols
In red wines the concentration of cinnamic acid derivatives is considerably lower than that of the flavonoid phenols and they probably only contribute to bitter nuances in wine. The estimated threshold values for the hydroxycinnamic acids and their tartaric acid esters differ greatly between studies (Gawel, 1998), most indicate that they are present in wine at concentrations near or below their detection threshold in water. This would then indicate that individually they have little or no sensory impact on wine, but even if individual non-flavonoids do not have a sensory impact, their combined effect may have one. The flavonol, quercetin is also found in sensorially significant concentrations in red wine. However, quercetin, like the hydroxycinnamates, appears to primarily elicit a bitter taste with weak astringency.
Pigments
White grapes fermented in the presence of skins and seeds under typical red winemaking conditions result in wines with vastly different sensory profiles to those of red wines; they tend to be coarser and lower in astringency. Pure anthocyanins have been reported informally to have only a very mild indistinct taste. Singleton and Trousdale (Singleton and Trousdale, 1992) suggested that the incorporation of anthocyanins in polymeric procyanidins is primarily responsible for the distinctive astringency of red wines. Kantz (Kantz and Singleton, 1991) suggested that the sugar of the anthocyanin and perhaps the polarity of the flavylium cation both increase the solubility and decrease the precipitability of the resultant anthocyanin-bearing tannin molecule. Thus anthocyanins seem to increase the amount of tannin retained in wine. Ribéreau-Gayon (Ribéreau-Gayon and Glories, 1980) also reported that these pigmented tannins are less reactive towards proteins and accordingly less astringent. Others have found anthocyanins to contribute to the mouth-feel of red wine, enhancing the mouth-coating sensation of roundness and fullness as well as raising perceived astringency. Brossaud (Brossaud et.al., 2001) found that the addition of an anthocyanin mixture to seed and skin tannin extracts increased the astringency of the solution over the astringency of either fraction alone, but had no effect on bitterness. In contrast some have found anthocyanin-tannin combinations not very astringent but with a marked bitterness, especially in young wines. Clearly no defined attributes can yet be attributed to anthocyanins and their reactants. Astringency is a complex system of tactile impulses and the role of anthocyanins during the aging process of wine needs to be investigated.
Sensory perception of mouth-feel (e.g. astringency) and bitterness attributes
Some wine components have astringent sub-qualities themselves or have separate taste qualities, which indirectly affect astringency perception by altering the salivary flow rate and composition of the individual's saliva.
Both organic and inorganic acids have been shown to be astringents in their own right. Other wine components affect the sensory perception of astringency and/or bitterness. Ethanol increase bitterness, but suppresses astringency of phenols (Noble, 1990), while raising the acidity (and perceived sourness) increase the intensity (Kallithraka et.al., 1997) and duration of astringency. Astringency was also reduced by an increase in viscosity, while bitterness was not affected, (Ishikawa and Noble, 1995). Red wine contains a number of viscosity-enhancing components; they comprise of ethanol, glycerol, polysaccharides, monosaccharides and non-reducing sugars.
Astringency is not confined to a particular region of the mouth or tongue but is perceived as a diffuse stimulus, which requires appreciable time for development. It has generally been accepted that astringency arises when lubricating salivary proteins are precipitated as a protein-tannin complex and the lubricating effect is lost. It is possible that the presence of this precipitate on the tongue and soft palate also contributes to the overall mouth-feel sensation. Another possibility is that the fraction of the astringent material remaining in solution contributes to the sensation and, at least in some cases, this might involve interaction with a bitter receptor. Protein complexation and precipitation of tannins lower the astringency of wines, in addition to possible effects on the texture and mouth-feel properties together with the polysaccharides present.
A mouth-feel wheel was developed to describe the mouth-feel sensations elicited by red wines (Gawel et.al., 2000). This enables us to better define the contribution of specific phenolic compounds as well as relate phenolic composition to their sensory mouth-feel properties. Gawel (Gawel et.al., 2001) showed that astringency in red wine can manifest itself in many subtle yet complex forms, and that tasters can be trained to reproducibly discriminate and rate the intensities of astringent sub-qualities elicited by red wines.
Tannins extracted from the skins react less with the proteins in gelatin than those from seeds and stalks. The latter, consist exclusively of procyanidins, are polymerised to varying degrees, depending on the maturity of the grapes. They do not contain any of the free anthocyanins or tannin complexes with polysaccharides or proteins that soften the tannins in the skins. The tannic balance of a young red wine comes from a good harmonization of tannins from both origins. However, there is a high risk of excessive astringency if seed tannins dominate, while bitterness is typical of too much extract from the skins, especially if the grapes are insufficiently ripe. It is useful to measure the reactivity of tannins in wine, but this is not the only factor involved in assessing astringency. Other components, such as proteins, polysaccharides, ethanol, glycerol and tartaric acid, either inhibit the reaction and temper its aggressiveness or exacerbate it (Yokotsuka and Singleton, 1987).
The perception of astringency is a highly dynamic process, changing continuously during ingestion and especially following expectoration or swallowing (Noble, 1995). Astringency also increases upon repeated ingestion, with the rate of increase being greater when the time between ingestions is shortened. The rate of increase of astringency with repeated ingestion is unchanged when astringency is allowed to decay to zero. This clearly demonstrates the need for careful experimental design of trials involving astringency, due to the strength of carry-over effects. Experienced wine tasters also use specific descriptive terms to relate the perceived nuances of the astringent sensation to other tasters. It is reasonable to expect that, after swallowing, the reduction in intensity of astringency over time is dependent on the flushing of phenolics from the mouth and the replenishment of saliva, which then acts as lubricant. The rate of clearance of various substances introduced uniformly into the oral cavity has been observed to vary significantly within the various parts of the mouth. Like all other sensory attributes, astringency perception varies greatly between individuals (Fischer et.al., 1994). Tasters also differ in their ability to observe bitterness, with those who experience bitterness intensely having a larger number of bitterness observation seats on the tongue than those who do not as intensely. The tasters who have high saliva flow rates, experience the bitter sensation and astringency later and not as intensely than the tasters who have lower saliva flow rates.
Analytical measure of astringency
The gelatin index seems mainly to be linked to astringency and tannic strength (a PVPP index) to both astringency and bitterness. The extent to which particular proteins were precipitated (as judged by reversed phase HPLC) did not correlate with the reported sensory potency of the flavanols tested (Kallithraka et.al., 2001). The quantity of flavanols precipitated not only did not agree with the relative astringency recorded; the quantity of flavanols remaining in the supernatant might be more closely related to their previously determined astringency than to the amount precipitated. More extensive studies, both sensorial and analytical with a greater number of purified flavanols and a superior method of protein determination are required in order to develop a method to quantitate astringency.
Conclusion
Considerable advances have been made in the elucidation of the structures and distribution of polyphenols in grapes. The relationship that exist between the structure and taste (e.g. astringency, bitterness) of various procyanidins and anthocyanin-procyanidin-derived molecules co-existing in wines, remain to be established. Correlation of the changes in mouth-feel attributes with the changing phenolic composition of wine resulting from different viticultural and winemaking practices (discussed in Part II) applied, will be possible with the utilization of the mouth-feel wheel.
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Anita Oberholster, Department of Enology and Viticulture,
University of Stellenbosch, Stellenbosch
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