Role of Tartaric and Malic Acids in Wine Oxidation - American

May 8, 2014 - KEYWORDS: wine oxidation, oxygen, iron, tartaric acid, malic acid, reduction potential. □ INTRODUCTION. Wine is repeatedly exposed to ...
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Role of Tartaric and Malic Acids in Wine Oxidation John C. Danilewicz* 44 Sandwich Road, Ash, Canterbury, Kent CT3 2AF, United Kingdom S Supporting Information *

ABSTRACT: Tartaric acid determines the reduction potential of the Fe(III)/Fe(II) redox couple. Therefore, it is proposed that it determines the ability of Fe to catalyze wine oxidation. The importance of tartaric acid was demonstrated by comparing the aerial oxidation of 4-methylcatechol (4-MeC) in model wine made up with tartaric and acetic acids at pH 3.6. Acetic acid, as a weaker Fe(III) ligand, should raise the reduction potential of the Fe couple. 4-MeC was oxidized in both systems, but the mechanisms were found to differ. Fe(II) readily reduced oxygen in tartrate model wine, but Fe(III) alone failed to oxidize the catechol, requiring sulfite assistance. In acetate model wine the reverse was found to operate. These observations should have broad application to model systems designed to study the oxidative process in foods and other beverages. Consideration should be given to the reduction potential of metal couples by the inclusion of appropriate ligands. KEYWORDS: wine oxidation, oxygen, iron, tartaric acid, malic acid, reduction potential

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INTRODUCTION Wine is repeatedly exposed to oxygen during its production, initially as it is racked for clarification and stabilization and then during filtration and at bottling. Oxygen may be introduced deliberately with copper (Cu) to remove reductive odors.1 In red wine oxygen is also slowly introduced during maturation in barrels or during micro-oxygenation,2 and finally oxygen may gain access to wine from and through closures.3 The principal initial reactants are polyphenols and ethanol.4−6 Polyphenols are oxidized to quinones, which can react to produce pigments7 and also add nucleophiles such as odorant thiols.8,9 Ethanol is oxidized to acetaldehyde, which cross-links flavanols and flavanols with anthocyanins, again to produce polymers and pigments.10−12 These reactions are initially sought to a limited degree in red wine but not in white wine as they lead to browning and loss of aroma.13,14 A better understanding of the mechanism of these oxidative processes and how they may be modified by sulfite and other possible additives is therefore highly desirable for the winemaker. Oxygen in its normal triplet state does not react directly with polyphenols, because it cannot accept electron pairs, its outer electrons being unpaired with parallel spins. It accepts electrons singly, such as from reduced metals and from free radicals.4,5 Studies in model wines have shown that iron is required to catalyze catechol oxidation, and this is markedly enhanced by small amounts of copper.15 Addition of these metals to real wines has also been shown to accelerate oxidation, whereas it is slowed and can be eventually stopped in white wine by their removal.16 Iron, in its reduced ferrous state (Fe(II)), is first oxidized to the ferric state (Fe(III)) by oxygen.17 It was thought that oxygen was initially reduced to hydroperoxyl radicals (HO2•) and it was proposed that these could oxidize polyphenols to semiquinones, which were further oxidized to quinones, possibly by Fe(III) (Scheme 1).5 However, this mechanism does not appear to operate as oxygen is reduced independently of polyphenol oxidation in model wine. It is proposed that Fe(II) reduces an intermediate Fe−oxygen complex to produce hydrogen peroxide without the formation © XXXX American Chemical Society

Scheme 1. Proposed Mechanism for the Oxidation of Catechols Involving Hydroperoxyl Radicals

of an intermediate capable of oxidizing polyphenols, such as the hydroperoxyl radical (Scheme 2).18 Hydrogen peroxide is rapidly reduced by Fe(II) in the Fenton reaction to produce highly reactive hydroxyl radicals (HO•). These radicals react at diffusion-controlled rates, which means that they react with the first potential substrate they encounter, and due to its highest concentration, ethanol is the principal target. Abstraction of a hydrogen atom yields the 1-hydroxyethyl radical.19 Oxygen is simultaneously taken up, and it is proposed that it adds to this carbon-centered radical to yield the hydroxyethylperoxyl radical.18 A possibility is that this radical could be reduced by Fe(II), as two Fe(II) react for each oxygen that is consumed. Elimination of hydrogen peroxide would then produce acetaldehyde. This speculative mechanism would result in the regeneration of hydrogen peroxide once initially produced by polyphenol oxidation and so provide an additional entry point for oxygen in the overall oxidative process. Fe(III) will oxidize pyrogallol, but catechols, such as (+)-catechin, which are less powerful reductants, do not react in model wine, or at least the reaction is extremely slow.17 However, it is found that sulfite promotes the oxidation of (+)-catechin, and it is proposed that it draws the reaction forward by reacting with the quinone, Received: February 13, 2014 Revised: May 7, 2014 Accepted: May 8, 2014

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Scheme 2. Revised Mechanism of Oxygen Reduction Coupled to Catechol Oxidation Mediated by Fe Redox Cycling; Participation of Sulfite in Regenerating the Catechol and Removing Hydrogen Peroxide

which is presumably formed in very small amounts.16,20 Importantly, sulfite also reacts rapidly with hydrogen peroxide and so prevents ethanol oxidation.15 For some time it was accepted that sulfite simply reacted with oxygen, but it is now apparent that its action is far more complex. As with polyphenols, oxygen cannot react directly with sulfite. It requires the catalytic intervention of Fe. Sulfite is first oxidized by Fe(III) to the sulfite radical (SO3•−), which then reacts rapidly with oxygen to produce the peroxomonosulfate radical (SO5•−). This radical is a stronger oxidant capable of oxidizing sulfite directly to generate further sulfite radicals and so continue the chain reaction.21−23 Sulfate radicals (SO4•−) are also produced. These radicals are very powerful oxidants of similar oxidizing capability to hydroxyl radicals and so will also oxidize ethanol to acetaldehyde.15 Far from being a reductant, sulfite alone will generate a highly oxidizing system in the presence of oxygen. Similarly, polyphenols alone produce a powerful oxidant by generating hydrogen peroxide. It has been proposed that polyphenols and sulfite complement each other, in that polyphenols would prevent sulfite oxidation by intercepting peroxomonosulfate radicals, whereas sulfite reduces quinones back to polyphenols and removes any hydrogen peroxide that is produced.16 However, more recent results have confirmed that sulfite autoxidation is so slow that it is unlikely to occur in wine conditions.18 In any event, under ideal conditions when a catechol is oxidized in model wine, the SO2:O2 molar reaction ratio is 2:1, one mole of SO2 reacting with the quinone and the other with the hydrogen peroxide. However, in real wine the ratio may be less than 2:1, and this has been ascribed to substances that compete with sulfite for quinones.20 In addition to the above reactions, sulfite forms nonvolatile adducts with carbonyl compounds,24 such as acetaldehyde, phenylacetaldehyde, and methional, produced during the winemaking process, which otherwise would produce unpleasant (oxidized) aromas.25 Importantly, SO2 also prevents microbial growth both directly as molecular SO2 and indirectly as sulfite,24,26 by preventing the growth of aerobic bacteria by facilitating polyphenol oxidation, so removing oxygen.18 A detailed examination of wine oxidation, therefore, reveals the key involvement of iron throughout the process. It is proposed that the ability of Fe to redox cycle is made possible by its co-ordination to tartaric and malic acids, which determine the reduction potential of the Fe(III)/Fe(II) couple in relation to that of the oxygen and catechol couples.18,27 In this paper the importance of tartaric and malic acids is demonstrated by

comparing the mechanism of catechol oxidation in tartrate, malate, and acetate model wines at pH 3.6. Acetic acid coordinates less strongly with Fe(III) than the diacids and so should raise the reduction potential of the Fe couple.

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MATERIALS AND METHODS

Materials. Water (Emsure, Fe ≤ 1 μg/L, Cu ≤ 0.4 μg/L; E. Merck, Darmstadt, Germany), Cu(II) sulfate pentahydrate, Fe(III) chloride hexahydrate, sodium hydroxide, L-(+)-tartaric acid (BDH AnalaR grade), L-(−)-malic acid, acetic acid (BDH, GPR), and ethanol (96% GPR grade) were obtained from VWR International (Lutherworth, UK). 4-Methylcatechol (4-MeC, 95+%) and Fe(II) sulfate heptahydrate (99+% ACS reagent) were obtained from Sigma-Aldrich (Poole, Dorset, UK), and potassium metabisulfite (Kadifit) was from Erbslöh Geisenheim AG (Geisenheim, Germany). UV−vis spectra were taken with a Jenway 7315 spectrometer (Keison Products, Chelmsford, UK) Preparation of Model Wine Solutions. L-(+)-Tartaric acid (5.0 g, 3.33 × 10−2 mol/L), L-(−)-malic acid (4.46 g, 3.33 × 10−2 mol/L), and acetic acid (3.81 mL, 6.66 × 10−2 mol/L) were each dissolved in water (∼800 mL) in a 1 L volumetric flask. Ethanol was added to give a 12% (v/v) final concentration, and the pH was increased to 3.60 with 2.5 N sodium hydroxide; also, water was added progressively to the mark as the required pH was approached. Solutions of FeSO4, FeCl3, 4-MeC, and K2S2O5 were freshly made up in water and (+)-catechin in 1:1 H2O/EtOH by volume before use and added in small volumes (25−200 μL) to the model wines as required. Reaction of Fe(II) with Oxygen in Tartrate and Acetate Model Wine. Fe(II) (20 mg/L) was added to the model wine (3 × 20 mL) containing Cu(II) (0.3 mg/L). The resulting solutions were immediately transferred to three 100 mL bottles fitted with screw caps, stored in the dark, and shaken periodically to maintain air saturation. Fe(III) concentration was determined from the absorbance at 334 nm in triplicate. Oxidation of 4-MeC in Model Wines. 4-MeC (1.0 g/L) was added to model wines (500 mL) containing Cu(II) (0.15 mg/L), followed by SO2 (∼60 mg/L) and Fe(II) (5.0 mg/L). Initial free SO2 concentration was determined in duplicate, and the resulting solution was immediately divided between three 500 mL stoppered flasks, stored in the dark with periodic shaking to maintain air saturation. Oxidation was followed by measuring SO2 concentration in triplicate on the basis of a 2:1 SO2/O2 molar reaction ratio. Spectroscopic Measurements. Spectra were taken using 10 mm quartz cuvettes against a model wine blank. The small notch that was observed in spectra at ∼248 nm coincided with a sharp increase in absorbance of the model wine. It was, therefore, assumed that it was due to a large decrease in light transmission through the instrument, and spectra at lower wavelengths were not taken to be reliable. Fe(III) calibration curves were obtained by dissolving FeCl3 hexahydrate (48.4 mg) in 10 mL of model wine; 50−400 μL of this solution was then B

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Figure 1. Proposed structures Fe complexes: (a) Fe(III)−catecholate complex;29,30 (b) Fe(III)−tartrate monomer; (c) Fe(III)−tartrate dimer at wine pH.31,32

the O2/H2O2 couple (E3.6 = 565 mV).18,27 As a result, Fe(II) is rapidly oxidized to Fe(III) in air-saturated model wine containing a small amount of Cu(II) (Figure 2). In contrast,

diluted to 20 mL with model wine to give the calibration standards. Such solutions were stable for at least 9 h when stored in the dark. Measurement of SO2. Free SO2 concentration was measured with the modified Ripper procedure, using potassium iodate and starch− KI.28 When measurements were taken in triplicate, mean values (±SD) were calculated and figures drawn using Excel software (Microsoft, Redmond, WA, USA). Where error bars denoting ± SD are not shown, they were smaller than the data point symbol dimensions. Experiments were conducted at ambient temperature (18.5−20.5 °C).

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RESULTS AND DISCUSSION In the absence of stronger competing ligands such as tartaric acid, catechol forms a 1:1 complex, [Fe(L2−)(H2O)4]+, with Fe(III) at pH 2−3, which exhibits two broad absorption bands at 429 and 700 nm, εmax = 880 and 1000, respectively.29,30 Bound water molecules in the complex are rendered more acidic, and one is likely to deprotonate to give [Fe(L2−)(OH−)(H2O)3] at wine pH, where LH2 stands for the catechol (Figure 1a).30 However, the complex is unstable and decomposes over ∼24 h to a mixture of Fe(II) and quinone,29 which in turn decomposes further.27 Fe(II) does not form a catecholate complex below pH ∼7.29 Fe(III) coordinates strongly with tartaric acid, forming a monomeric complex Fe(L2−)+ at low pH (Figure 1b), with an absorption maximum at 346 nm, where LH2 is tartaric acid and L2− is where both carboxyl groups are ionized. Above pH 2 the dimeric complex, [Fe2(L2−)2(−3H+)]−, is formed, becoming the dominant species at pH ∼3.5 (Figure 1c), which has a less pronounced absorption maximum that is shifted to 335 nm. At pH 5 the analogous trimer, [Fe3(L2−)3(−6H+)]3− is produced, which displays no clear maximum but a flat shoulder at ∼300 nm.31,32 The exact structure of these complexes has not been established. However, the water molecules attached to two Fe(III) are the most acidic, and the dioxygen olate bridge system is drawn here to explain the loss of the three protons on dimerization (Figure 1c).33 Fe(II) binds much more weakly with tartaric acid to form a monomeric complex.31 The greater affinity of tartaric acid for Fe(III) displaces the redox equilibrium toward Fe(III), making it a weaker oxidant and hence Fe(II) a stronger reductant.18 Consequently, the reduction potential of the Fe(III)/Fe(II) couple was found to be lowered to 385 mV by cyclic voltammetry in model wine pH 3.3.18 This value was the same as found in aqueous tartaric acid.34 Because the reduction potential was found to decrease by 130 mV per pH unit increase in the wine pH range,34 it should be 345 mV at pH 3.6, which places it well below that of

Figure 2. Oxidation of Fe(II) (20 mg/L, 3.58 × 10−4 mol/L) at pH 3.6 in model wines saturated with aerial oxygen in the presence of Cu(II) (0.3 mg/L): (a) tartrate model wine; (b) acetate model wine.

under the same conditions, Fe(II) failed to reduce oxygen in the acetate model wine (Figure 2), which is consistent with an increase in the reduction potential of the Fe(III)/Fe(II) couple. The reduction potential of the Fe(III)/Fe(II) couple in aqueous NaClO4/H2SO4, where Fe(III) and Fe(II) exist as the hexaaquo complexes, [Fe(H2O)6 ]3+ and [Fe(H2O)6 ]2+, was found to be 687 mV at pH 3.3 by cyclic voltammetry, which also places it above that of the oxygen couple. Consequently, Fe(II) was also found not to reduce oxygen in this system.18 Raising the reduction potential even further by adding 2,2′-bipyridyl to tartrate model wine has the same effect.18 Inhibition of oxidation has also been demonstrated by electron paramagnetic resonance spectroscopy, where addition of 2,2′-bipyridyl as well as FerroZine to the tartrate model wine and real wine prevented the production of 1-hydroxyethyl radicals.35 It is also apparent that the reduction potential of the Fe couple is lowered to well below that of the quinone/4-MeC couple (E3.6 = 577 mV) and, as observed previously at higher tartaric acid concentration,18 Fe(III) is no longer able to oxidize 4-MeC at wine pH. When Fe(III) is added as FeCl3 to tartrate model wine, a weak absorption band (λmax = 335 nm, εmax = 2050) (Figure 3, curve a) is observed, which can be ascribed to the dimer (Figure 1c). When 4-MeC is added, the resulting C

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not reduce Fe(III) in tartrate model wine, or at least the reaction is extremely slow.18 Fe(III) is reported to form a trimeric complex in acetate buffer at pH 1.9−3.31,31 and addition of Fe(III) as FeCl3 to acetate model wine gave a similar absorbance curve as in the tartrate model wine (Figure 4, curve a; λmax = 334−338 nm,

Figure 3. UV−vis spectra in tartrate model wine: (a) Fe(III) (10 mg/ L, 1.79 × 10−4 mol/L); (b) 4-MeC (44.5 mg/L, 3.58 × 10−4 mol/L); (c) spectrum taken immediately after mixing the two; (d) spectrum 21 h after mixing the two with the addition of SO2 (32 mg/L, 5.0 × 10−4 mol/L). Figure 4. UV−vis spectra in acetate model wine: (a) Fe(III) (20 mg/ L, 3.58 × 10−4 mol/L); (b) 4-MeC (44.5 mg/L, 3.58 × 10−4 mol/L); (c) spectrum taken 2 min after mixing the two; (d) spectrum of the same mixture taken after 12 h.

spectrum is the sum of the two individual spectra (curve a plus b) and remains unchanged for at least 4 days (curve c). Not only is the catechol unable to reduce Fe(III), but it is also too weak a ligand to compete with tartaric acid for Fe(III) at the relative concentration used in this experiment, as the absorbance due to the Fe(III)−tartrate dimer at 335 nm remains unchanged over many days. (+)-Catechin and gallic acid behaved similarly and were not oxidized by Fe(III) in the same system (Supporting Information Figures SI 1 and SI 2). However, with pyrogallol, which is a stronger reductant, addition of Fe(III) caused a steady increase in absorbance in the 300−350 nm range, presumably due to oxidation products as the reaction mixture progressively yellowed. This spectral change overlapped with the Fe(III) absorption band, and it was not possible to follow the extent of reduction (Supporting Information Figure SI 3). Previously, it had been shown by monitoring oxygen uptake that, in contrast to (+)-catechin, pyrogallol is oxidized in model wine without sulfite assistance.17 The importance of reduction potentials in polyphenol oxidation is illustrated by the addition of FerroZine to model wine. This Fe(II) selective ligand raises the reduction potential of the Fe couple to such an extent that Fe(III) is then able very rapidly to oxidize catechols.18,19 It has been shown that when (+)-catechin is exposed to oxygen in tartrate model wine, enough oxygen is taken up to oxidize the initial Fe(II) that is present but oxidation proceeds no further. However, oxygen uptake continues when sulfite is added.17 The rate of oxidation in model systems containing sulfite increases with increasing Fe and catechol concentration, and it is, therefore, concluded that sulfite functions by promoting catechol oxidation, drawing forward the thermodynamically unfavorable reaction by reacting with the quinone.16,20 It has also been shown that, although 4-MeC does not reduce Fe(III) in tartrate model wine, it does so in the presence of sulfite.18 This is a key reaction as it allows the metal to redox-cycle, and the effect is also seen in the above experiment. Addition of sulfite to a nonreacting mixture of 4MeC and Fe(III) results in the reduction of Fe(III); the concentration of Fe(III) stabilizes from 10 to 3.4 mg/L after 21 h (Figure 3, curve d). It has been found that sulfite alone does

εmax = 1273) but with a slight shoulder at ∼400 nm. However, catechols interacted very differently with Fe(III) in the acetate system. Addition of 4-MeC (Figure 4, curve b) resulted in an immediate loss of absorption at ∼335 nm and formation of a new broad absorption band at ∼400 nm, εmax = 690 (curve c). It is proposed that this absorption is due to the formation of the catecholate complex (Figure 1a) discussed above, as the catechol is now capable of competing with the weaker monodentate acetate ligand. As previously reported, this absorption band faded over 12 h, and it is concluded that electron transfer within the complex results in the reduction of Fe(III) by the catechol.29,30 (+)-Catechin was found to behave similarly in this system. Addition of (+)-catechin (3.58 × 10−4 mol/L) to Fe(III) (1.79 × 10−4 mol/L) in acetate model wine instantly abolished the absorption band at 334 nm due to the Fe(III)−acetate complex with the formation of a blue-green Fe(III)−(+)-catechin complex, with broad absorption maxima at ∼426 and ∼700 nm. The color progressively changed to green over 1.3 h, with intensification of the broad absorption band at ∼426 nm, which shifted to ∼392 nm over 31 h, as the catechin is oxidized (Supporting Information Figure SI 4). Sulfite was also found to affect oxidation in the acetate model wine but in a different manner. Although Fe(II) does not react with oxygen in this system (Figure 2), it does so on addition of sulfite. Addition of an equimolar amount of sulfite to a solution of Fe(II) (20 mg/L, 3.58 × 10−4 mol/L) in air-saturated acetate model wine resulted in a steady increase in absorbance at 336 nm as Fe(III) was produced, 8.6 mg/L after 106 min (Figure 5). Under the same conditions sulfite alone did not reduce Fe(III) over 17.5 h. These results were unexpected as sulfite is generally viewed as a reductant rather than facilitating Fe(II) oxidation. Perhaps sulfite is able to coordinate with Fe(III)/ Fe(II) in the presence of acetate and lower the reduction potential of the couple so that it may reduce oxygen. However, as in tartrate model wine, Fe(II) is very rapidly oxidized by H2O2. From the absorbance at 338 nm it was shown that all of the Fe(II) (3.58 × 10−4 mol/L) was oxidized within 1 min in D

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was the catechol able to coordinate with Fe(III) to a detectable extent. Furthermore, when Fe(II) was added to air-saturated malate model wine containing a small amount of Cu(II), Fe(II) was rapidly oxidized. From the absorbance measured at 350 nm it was estimated that 94% had been oxidized to Fe(III) over 15 h (Figure 7).

Figure 5. UV−vis spectra in air-saturated acetate model wine: oxidation of Fe(II) (20 mg/L, 3.58 × 10−4 mol/L) at times indicated after addition of SO2 (22.9 mg/L, 3.58 × 10−4 mol/L).

acetate model wine upon the addition of H2O2 (5.1 × 10 −4 mol/L). It is concluded, therefore, that the Fenton reaction would proceed in this as in the tartrate model system. Malic acid has a similar chelating ability with Fe(III) as tartaric acid, forming similar polynuclear complexes at wine pH.36 Below pH 2.6 the mononuclear complex, [(Fe (L2−)]+, is formed, which exhibits a weak absorption maximum at 241 nm, similar to that of the corresponding tartrate complex. However, in the wine pH range the dinuclear, [Fe2 (L2−)3(−H+)2]2−, and trinuclear, [Fe3(L2−)5(−H+)4]5−, complexes are both formed, which differ from the tartrate complexes in that the Fe(III)/ ligand ratios are no longer 1:1.36 No absorption maximum is observed in the 330−350 nm range, absorbance increasing steadily without inflection (Figure 6, curve a). Like tartaric acid,

Figure 7. UV−vis spectra in air-saturated malate model wine: spectra at times indicated after adding Fe(II) (20 mg/L, 3.58 × 10−4 mol/L) to model wine containing Cu(II) (0.3 mg/L).

As discussed above, the oxidation of catechols is dependent on the ability of Fe to redox cycle, that is, the reaction of Fe(II) with oxygen to generate Fe(III) and the ability of Fe(III) in turn to oxidize catechols to regenerate Fe(II). Having compared both halves of the cycle in tartrate and acetate model wines, the overall rates of 4-MeC oxidation were compared in the two systems in the presence of sulfite (Figure 8). In the tartrate model system sulfite reacts with the hydrogen

Figure 6. UV−vis spectra in malate model wine: (a) Fe(III) (20 mg/ L, 3.58 × 10−4 mol/L); (b) 4-MeC (44.5 mg/L, 3.58 × 10−4 mol/L); (c) spectrum taken 24 h after mixing the two.

Figure 8. Free SO2 concentration during the oxidation of 4-MeC (1.0 g/L) in acetate and tartrate model wine maintained at air saturation containing Fe(II) (5.0 mg/L) and Cu(II) (0.15 mg/L).

malic acid coordinates only weakly with Fe(II), both acids being hard ligands that bind preferentially to Fe(III). Consequently, they should modify the catalytic activity of the Fe(III)/Fe(II) couple in a similar manner. When FeCl3 was added to 4-MeC in malate model wine, the same result is obtained as in the tartrate system. The resultant spectrum (Figure 6, curve c) was the sum of that for Fe(III) and 4-MeC, and no change was discernible for 24 h. As in the tartrate system, Fe(III) did not oxidize the model catechol nor

peroxide that is generated as oxygen is reduced and the quinone as the catechol is oxidized, resulting in a 2:1 molar reaction ratio as discussed in the Introduction.15,16,20 The rate of reaction was found to be independent of SO2 concentration, as it remained constant over a wide SO2 concentration range. It is concluded that its rate of reaction with the above oxidation products is fast relative to that of oxidation, which is dependent on oxygen and catechol concentrations. Oxygen concentration E

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autoxidation and, by binding directly to Fe(III), could facilitate polyphenol oxidation. It is possible, therefore, that the mechanisms of oxidation of white and red wines may differ slightly. As concluded in the present study, the ligand would determine the catalytic activity of the metal.

was held constant as the model wines were maintained at air saturation, and catechol concentration changes little as it is in substantial molar excess and is in part regenerated as oxidation proceeds. In the above studies it was shown that Fe(II) is oxidized in acetate model wine, which presumably generates hydrogen peroxide. It was also demonstrated that the catechol/ Fe(III) complex is unstable, spectral changes being consistent with formation of the quinone.29 It is, therefore, assumed that SO2 will react with the hydrogen peroxide and quinone as in the tartrate system, also resulting in a 2:1 molar reaction ratio in acetate model wine. As can be seen, the rates of reaction are both linear, with rate in the tartrate model system a little slower. However, on the basis of the above results, it would have been a mistake to assume that the underlying mechanisms are the same. In the tartrate case, Fe(II) reacts directly with oxygen, but the oxidation of the catechol requires sulfite assistance. In the acetate system the reverse operates. Fe(III) reacts unaided with the catechol; it is Fe(II) oxidation that requires sulfite assistance. Previous studies have provided strong evidence that polyphenol oxidation in wine is dependent on Fe catalysis, in which electrons are transferred from the polyphenol to oxygen by the intermediate redox cycling of the Fe(III)/Fe(II) couple.15,16 The overall rate of reaction will be dependent on either the rate of reaction of Fe(II) with oxygen or the rate of reduction of Fe(III) by the polyphenol, whichever is slower. As discussed above, the rates of these reactions should depend on the reduction potential of the Fe(III)/Fe(II) couple relative to that of the oxygen and polyphenol couples.18 Therefore, tartaric and malic acids do not just provide an acidic environment but also determine the catalytic activity of the Fe redox couple. It would not be valid to base mechanistic arguments on standard reduction potentials, which are determined at pH 0. The reduction potential of the oxygen and polyphenol couples is pH dependent, decreasing linearly by 59 mV per unit increase in pH, one proton adding to neutralize the negative charge as each electron is introduced in the reduction step.27 Both Fe(III) and Fe(II) form octahedral complexes, and the reduction potential of the couple will depend on the relative affinity of ligands for the two oxidation states. The structure of the complexes may also be pH dependent, such as in the case of Fe, by determining the degree of “polymerization”. The pH will also determine the extent to which ligands forming the complex are protonated, so altering the charge surrounding metal ions. Therefore, the reduction potential of the metal couple may not be simply deduced. These findings should apply to other model systems designed to study oxidation in foods and other beverages. Consequently, not only should pH be considered, but metal ligands mimicking those present in the system being studied should be incorporated. Studies in model wine have proved very helpful for exploring the catalytic function of Fe and Cu and the mechanism of action of sulfite in real wine. These present model wine studies should be no less relevant. In white wine tartaric and malic acids are present in very large molar excess compared to any polyphenol that might coordinate with Fe and so will be the dominant Fe ligands. In red wines, with higher polyphenol and lower acid concentrations, it is possible that polyphenols could compete effectively with tartaric acid for Fe(III). However, as hard ligands, which also favor co-ordination to Fe(III) relative to Fe(II), polyphenols would lower the reduction potential of the Fe(III)/Fe(II) couple in a similar manner to the acid.18 Such co-ordination would, therefore, also facilitate Fe(II)

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 44 1304 812 530. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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