Caracterización de los procesos químicos asociados a la oxidación del vino

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SUMMARY Characterization of chemical processes associated with wine oxidation Introduction Nowadays, small amounts of oxygen are used in winemaking all over the world to obtain high quality wines1. A mild oxidation is known to produce important improvements in red wines, such as color stabilization2, 3, softening of astringency and bitterness4, aroma modulation and decrease of vegetative aromas4, 5. On the other hand, too low oxygen exposure levels are likely to be detrimental due to increased occurrence of reduced aroma off-flavors6 and harsher mouthfeel7-9; while too high oxygen exposure levels can degrade some oxygen-sensitive key aroma compounds in wine and can also produce new oxygen-related off-flavors, such as methional and phenylacetaldehyde (with aromas reminding of baked potato, and honey)10, 11. Oxygen is a very necessary element for the maturation of young wine but it can be detrimental to already aged red wines or for most white and rosé wines. As the interaction of oxygen with wine is largely dependent on wine composition, exposing a wine to a given amount of oxygen can lead to unpredictable results. Sulfur dioxide (SO2) is the most important additive used to prevent wine oxidation, not only for its antioxidant properties, but also because of its antimicrobial and antioxidasic characteristics. As this molecule can produce allergic reactions, there are restrictions about the maximum levels at which it can be used and its replacement is a major overall aim of the industry12. Wine oxidation chemistry has been broadly studied 6, 13-17. The general mechanism establishes that oxygen accepts electrons from iron and copper ions, which act as catalysts, forming a superoxide ion O2˙− (hydroperoxyl radical HO-O˙ at wine pH). Phenolic compounds with a catechol group are oxidized by this radical to a quinone, and the radical is reduced to hydrogen peroxide. If sulfur dioxide is present, it reacts with hydrogen peroxide, giving sulfate and water, and with the quinone, reducing it back to the catechol or forming a sulfonated product. If there is no sulfur dioxide in the wine, hydrogen peroxide can take part in more oxidation steps, such as the Fenton reaction18. In this case, iron and copper ions interact with hydrogen peroxide to form the hydroxyl radical HO˙, a strong oxidant that reacts with many organic compounds in wine and it mainly oxidizes ethanol to acetaldehyde. Bearing all this in mind, the main objective of this PhD Thesis is to characterize the chemical processes that concur during wine oxidation, paying particular attention to the kinetics of oxygen consumption, to their relationship to sulfur dioxide levels and to the major chemical changes induced or caused by oxidation. The ultimate goals are to understand the factors driving the major transformations associated with the oxidation of real wines, both reds, whites and rosés, and to produce qualitative and quantitative criteria to help winemakers in the decision making about oxygen exposure and antioxidant addition in the winery. Chapter 1: Free and Total Sulfur Dioxide Determination in Wine by Headspace Gas Chromatography with Mass Spectrometry Detection (HS-GCMS) The crucial role played by SO2 in wine oxidation13, makes its accurate quantitative determination at low levels (mg/L) essential to understand the chemistry of wine oxidation. The reference method for SO2 determination is the aspiration-oxidation method, which is usually referred as Rankine method19, 20 which is known to be not very sensitive and which becomes inaccurate at levels below 10 mg/L. In this procedure, free sulfur dioxide is distilled from the acidified sample and it is oxidized in a hydrogen peroxide solution. The sulfuric acid formed is subsequently titrated with sodium hydroxide. The remaining sample is heated and refluxed to release bound sulfur dioxide and the previous oxidative absorption-titration procedure is repeated for the determination of bound sulfur dioxide. This method is not very complex, the material is inexpensive, and it is not interfered by pigments or by acetic acid21. However it is laborious and time-consuming and becomes inaccurate at low levels of free sulfur dioxide19, 21. Besides, some undefined losses of sulfur dioxide are possible and a high bias may occur in the free sulfur dioxide determination due to the possibility that the reversibly bound SO2 dissociates as the free form is removed19. A new direct Headspace-GCMS method, using a standard CTC-autosampler, has been optimized and validated. The method quantifies in two independent runs free and total SO2 and free acetaldehyde. Free forms are directly determined after sample acidulation. Total forms require the previous incubation at 100°C. This new method has lower limits of detection (1 mg L-1) and higher accuracy (s = ± 0.6 mg L-1) and requires lower volume of wine and reagents than the reference method. Besides, less sample preparation is required, the analysis is simpler and takes a short time. For validation, 30 wines were analyzed and compared to the reference method of aspiration-oxidation, finding a good correlation for free and total SO2 (P<0.0001). However, if acetaldehyde is present in a molar ratio above 1:1.5 with SO2, the accurate determination of total SO2 it is not possible. Thus, only the new method for determining free SO2 is used in the following chapters. Chapter 2: Oxygen Consumption by Red Wines. Part I: Consumption Rates, Relationship with Chemical Composition, and Role of SO2 The central role of oxygen in the process of wine maturation has been long known22 and it is nowadays generally accepted that moderate exposure of wine to oxygen can be beneficial for wine quality6-9, 23-25, while too low or too high exposure levels are likely to be detrimental6-9. In the present study, the oxygen consumption rates of different red wines and the loss of SO2 have been measured under carefully controlled oxidation conditions. Different PLS models made it possible to obtain an initial assessment of the influence of key chemicals and specific chemical profiles on the ability of wine to consume oxygen. Fifteen Spanish red wines were extensively characterized in terms of SO2, color, antioxidant indexes, metals, and polyphenols and were subjected to five consecutive sensor-controlled cycles of air saturation at 25 °C. Simple parameters (color parameters, SO2, antioxidant indexes and acetaldehyde) were determined at the end of each saturation, while the complete series of analysis was repeated on the oxidized samples. The oxidation process in cycles of wines is highly reproducible. Plots of O2 consumption vs time revealed that within each cycle, O2 consumption rates cannot be interpreted by simple kinetic models. Plots of cumulated consumed O2 vs time made it possible to define a fast and highly wine-dependent initial O2 consumption rate and a second and less variable average O2 consumption rate which remains constant in saturations 2 to 5. Both rates have been satisfactorily modelled, and in both cases they were independent of Fe and SO2 levels and highly dependent on Cu levels and polyphenolic profile. Besides, initial rates were related to absorbance at 620 nm and tannins rich in epigallocatechin; and average rates were also related to Mn, pH, Folin and protein precipitable proanthocyanidins (PPAs). The study of SO2 consumption reveals important clues about the potential action of different antioxidants present in wine. In particular, it has been found that SO2 is used less efficiently during the first saturation in some wines most of which also showed a high initial O2 consumption rate. Besides, wines containing high levels of acetaldehyde made also a less efficient use of their SO2, particularly when the levels of free SO2 come down. The first observation suggests either that some other antioxidants more active than SO2 are present in some wines or that SO2 is less available in the first saturation. Correlation data and PLS modeling strongly suggest that tannins rich in epigallocatechin structural units and/or blue pigments are involved in that unexpected behavior. Chapter 3: Oxygen Consumption by Red Wines. Part II: Differential Effects on Color and Chemical Composition Caused by Oxygen Taken in Different Sulfur Dioxide-Related Oxidation Contexts Recent studies about the chemical processes involved in wine oxidation have made it possible to begin to understand the pivotal role played by sulfur dioxide (SO2) in wine oxidation 14, 15, 26-30. Its reaction with quinones, in competition with other wine nucleophiles31, is apparently essential for the oxidation to progress, since the redox equilibrium between o-diphenol and o-quinone is for most simple o-diphenols strongly displaced to the phenol form 14, 15. The outcome of the SO2-quinone reaction depends on several factors, such as the SO2 concentration, the type and concentration of competitive nucleophiles 31 and the own stability of the quinone itself 15, 29. These three factors will essentially dictate the change in the polyphenolic composition caused by the uptake of a certain amount of oxygen as well as the proportion of SO2 consumed per molecule of O2. Chemical changes caused by oxidation of 15 red wines during 5 consecutive air-saturation cycles have been assessed. Phenolic acids, flavanols, flavonols, anthocyanins, condensed tannins, antioxidant and pigments indexes, chromatic parameters and aroma compounds were analyzed. According to evidences found in chapter 2 (there are two different OCRs, some wines initially do not consume SO2 efficiently and a low SO2 efficiency is found in the last saturation), the hypothesis of the existence of at least 3 different mechanisms is proposed: preSO2, centered in SO2 and radical forming. In order to investigate the existing relationship between the effects caused by O2 and the levels and consumption rates of wine SO2, the total oxygen consumed by the wines (16−25 mg/L) was subdivided into different nonmutually exclusive categories. The first one is the oxygen consumed with no equivalent SO2 consumption (O2notSO2). The second is the O2 consumed in the first saturation without equivalent SO2 consumption (O2preSO2), and finally the O2 consumed at low levels of free SO2, named radical forming oxygen, subdivided into two subgroups: oxygen consumed at free SO2 levels below 5 and below 2 mg/L (O2atSO2<5 and O2atSO2<2). Regarding the changes in specific polyphenols, in general there was a neat increment of many phenolic acids and polymeric pigments, while flavanols, flavonols and anthocyanins decrease. The categories found most influential on chemical changes were O2preSO2 and O2atSO2<5 (or radical forming O2). Chromatic changes were strongly related to both O2 categories, even though anthocyanidin degradation was not related to any O2 category. Radical forming O2 prevented both formation of red pigments and reduction of epigallocatechin and other proanthocyanidins, induced accumulation of phenolic acids probably formed from oxidative degradation of anthocyanins and other phenolics, and caused losses of β-damascenone and whiskylactone without evidence of acetaldehyde formation. O2preSO2 seemed to play a key role in the formation of blue pigments and in the decrease of Folin index and of many important aroma compounds. Chapter 4: Oxygen Consumption Rates and Changes in Red Wines After a Single Air-Saturation In previous experiments (chapters 2 and 3), red wines turned out to behave differently in the first air-saturation than in the following ones, which made us to address the first step of oxidation in an independent experiment. Eight commercial wines from different vintages were extensively studied in terms of kinetics, sulfur dioxide efficiency and changes in the phenolic matter during this first saturation. Wines were selected in a previous experiment looking for differential behavior regarding SO2 consumption during the first saturation. Results confirmed that red wines can consume oxygen at quite different initial OCRs (Oxygen Consumption Rates). Taking into account simple and multivariate relationships between initial OCRs and initial composition, and between initial OCRs and major changes during the oxidation, it can be concluded that initial OCR of a red wine depends critically on: 1. Its content in copper, which acts as a catalyst with a positive effect on OCR 2. Its absorbance at 620 nm and its content in epigallocatechin, both with a positive effect on OCR. As changes in these components were not critically related to OCR, it should be thought that part of these compounds should be regenerated more efficiently at higher rates. These compounds would behave, therefore, not only as oxidation substrates but as catalysts in the oxidation of SO2 3. Its contents in gallic acid and in tannins with a catechin terminal unit, both with a negative effect on OCR. As these compounds decrease at significantly higher levels in wines with low OCRs, it may be thought that they are substrates consumed in the oxidation, likely by reaction to SO2, and that such consumption takes place at low rates 4. Its contents in total acetaldehyde, with a negative effect on OCR. Levels of this compound also increase significantly at a larger extent in wines consuming oxygen slowly. Such dependence, taking into account the “apparent” null effects of free and total SO2 on OCRs, may suggest that it is the consequence of the inverse relationship between the wine content in total acetaldehyde and the wine content in Acetaldehyde Reactive Polyphenols (ARPs). These ARPs would have a major positive effect on oxygen consumption This set of results shows that the ratio (gallic acid + tannins with catechin terminal units)/(epigallocatechin + A620nm) has a strong influence on initial OCRs and that different OCRs imply different changes in the profile of tannins. While slow O2 consumptions are linked to strong decreases in tannins with catechin terminal units, fast O2 consumptions are linked to decreases in tannins with epicatechin-3-O-gallate terminal units. Molar ratios between consumed SO2 and consumed O2 (SO2:O2) found in the first saturation were well below the theoretical 2:1 figure. Simple and multivariate correlations suggest that the proportion of SO2 consumed in the oxidation or SO2 efficiency is related to: 1. The wine content in total SO2, which suggests that free SO2 levels are not critical 2. The wine content in epigallocatechin, free or in tannins, with a positive effect on SO2 consumption. As these compounds decrease also at higher levels in wines consuming more SO2, it should be concluded that these compounds are main substrates of oxidation and that its consumption takes place strictly following the SO2-centered oxidation cycle 3. The wine anthocyanin/tannin ratio, with a strong negative effect on SO2 consumption. This suggests that at similar levels of tannins, the presence of anthocyanins is linked to a consumption of O2 concurrent with a poor SO2 consumption, which could suggest that part of the free SO2 determined is actually bound to anthocyanins and therefore is less accessible to react with quinones or H2O2 4. The wine content in methionine, with a negative effect on SO2 consumption. As this amino acid decreases during oxidation at higher levels when SO2 is just slightly consumed, while its sulfone accumulates, it can be concluded that methionine competes with SO2 as antioxidant. This would demonstrate the existence of oxidation pathways not related at all to SO2 consumption. Data suggest that other amino acids, such as phenylalanine or aroma compounds, such as whiskey lactone, could be also degraded when O2 is consumed with a poor SO2 consumption Finally, from a multivariate point of view, it can be said that changes in the wine chemical composition due to oxidation are clearly different from changes due to aging, and those changes during the first saturation are much stronger in young wines than in aged wines. In general terms, tannins are oxidized differently according to the variety: Garnacha wines had a larger decrease in total tannins when oxidized, whereas Tempranillos had an important drop in mDP (mean degree of polymerization). Chapter 5: Oxygen Consumption Rates in White and Rosé Wines: Measurement, Dependence of Wine Chemical Composition, Role of SO2 and Chemical Changes. Oxygen management is crucial in winemaking, since it can cause significant improvements or irreversible defects. White and rosé wines are most often not exposed to oxygen on purpose, except for some specific styles of wines. Therefore, if normal whites and rosés are accidentally exposed to air, their quality will be damaged32. By nature, white and rosé wines have much smaller antioxidant capacities than reds, and have also a different polyphenolic profile, since they contain relatively high levels of benzoic and hydroxycinnamic acids but they have few flavonoid phenolics, tannins or other polymeric material. Consequently, a greater amount of sulfur dioxide (SO2) is usually required to prevent oxidation spoilage. This chapter focuses on the study of the oxygen consumption rates of white and rosé wines and in the chemical changes produced related to oxidation. In a similar experiment to that described in the second chapter, wines underwent five consecutive cycles of air saturation at 25 °C, and oxygen was monitored periodically. Changes in SO2, color, antioxidant indexes, metals, polyphenols and aroma compounds were determined. In spite of the fact that oxygen consumption rates were not constant and did not follow first order kinetics, averaged rates at different times could be modelled. Models related copper, flavonols and hydroxycinnamic acids with higher oxygen consumption rates. Surprisingly, sulfur dioxide consumption is lower than in reds, in spite of the higher levels of this antioxidant and of the smaller OCRs. In contrast to reds, SO2 consumption is highly related to the initial amount of free SO2. A possible explanation for this lack of efficiency in the use of this antioxidant is the easiness with which acetaldehyde accumulates in whites and rosés, which hampers the release of free SO2 from its adducts. Also relevant to determine SO2 consumption were pH, Mn, phenolic indexes (all positively) and gallic acid (negatively). The antioxidant capacity of whites and rosés, measured in this experiment by Folin-Ciocalteau and TEAC assays, decreased following a linear trend with oxygen consumption. Browning, measured as the increase of absorbance at 420 nm, is highly correlated to the decrease in several flavanols such as catechin and procyanidins B1 and B2. Many significant changes in aroma compounds are related to specific SO2 consumption patterns. 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