The Pennsylvania State University. The Graduate School. College of Agricultural Sciences STUDIES ON THE REACTION OF WINE FLAVONOIDS

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1 The Pennsylvania State University The Graduate School College of Agricultural Sciences STUDIES N THE REACTIN F WINE FLAVNIDS WITH EXGENUS ACETALDEHYDE A Dissertation in Food Science by Marlena K. Sheridan 2016 Marlena K. Sheridan Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2016

2 The dissertation of Marlena K. Sheridan was reviewed and approved* by the following: Ryan J. Elias Associate Professor of Food Science Dissertation Advisor Chair of Committee Joshua D. Lambert Associate Professor of Food Science John Coupland Professor of Food Science Michela Centinari Assistant Professor of Horticulture Robert F. Roberts Professor of Food Science Head of the Department of Food Science *Signatures are on file in the Graduate School

3 ABSTRACT Red wine quality is known to improve with oxidation, typically as a result of exposure to oxygen. These benefits are based on the reaction of acetaldehyde with flavonoids in red wine. Acetaldehyde forms ethylidene bridges between flavonoids resulting in polymeric pigments and modified tannins, contributing to color stability and improved mouthfeel of red wine. Winemakers often use oxygenation techniques in order to take advantage of the benefits of acetaldehyde. These methods are based on the reduction of oxygen in several metal-catalyzed steps resulting in acetaldehyde; undesirable, reactive intermediates are also formed as side products of this mechanism. As these reactive species can result in deleterious effects, oxygen exposure is a relatively risky technique in order to gain the benefits of acetaldehyde. As a replacement for oxygenation techniques, I investigated the use of exogenous acetaldehyde additions to improve the color stability and mouthfeel of red wine. I first determined the viability of exogenous acetaldehyde treatment of a red wine by examining the effects of the treatment during alcoholic fermentation. Two levels of acetaldehyde (100 mg/l and 1000 mg/l) were added to a red wine over eight days of fermentation. After the completion of fermentation, wines were analyzed for their color stability and protein precipitation. High acetaldehyde treatment significantly increased the concentration of polymeric pigments and decreased the amount of protein precipitated by tannin. These results demonstrate the ability of exogenous acetaldehyde treatment to improve color stability and mouthfeel of a red wine. In order to understand the role of wine components in the reaction of acetaldehyde with wine flavonoids, I assessed the effect of ph, dissolved oxygen, and sulfur dioxide (S 2 ) in model wine solutions. The rate of reaction of acetaldehyde with catechin was significantly increased with lower ph and was not affected by dissolved oxygen. Interestingly, the reaction of iii

4 acetaldehyde with flavonoids was slowed but not prevented by the addition of S 2 as determined by monitoring the rate of reaction and the formation of polymeric pigments. These results demonstrate that acetaldehyde is reactive, not inert as previously assumed, in its sulfonate form. Based on the efficacy of acetaldehyde in a system with an equimolar concentration of bisulfite in the previous study, I then explored the reactivity of aldehydes from bisulfite adducts. I synthesized α-hydroxyalkylsulfonates from bisulfite and several aldehydes found in wine: formaldehyde, acetaldehyde, propionaldehyde, isobutyraldehyde, and benzaldehyde. The reactivity of aldehydes from their free and bound (sulfonate) forms with catechin was determined. The results demonstrate a clear relationship between reactivity of an aldehyde from its sulfonate and the dissociation constant (K d ) of that sulfonate. The bridged catechin oligomers from these aldehydes were also characterized by MALDI-TF MS, several for the first time. This work was the first evidence of the reactivity of aldehydes, including acetaldehyde, from their sulfonates. In order to confirm the application of exogenous acetaldehyde in red wine production, I investigated the treatment of red wine with exogenous acetaldehyde, the acetaldehyde-bisulfite adduct, and oxygenation after alcoholic fermentation. A reasonably low concentration of exogenous acetaldehyde (500 µm, 22 mg/l) significantly improved all measures of color stability evaluated, including color density and polymeric pigments. The sulfonate did not have the same effect as acetaldehyde, but did increase several parameters of color stability. The comparison between exogenous acetaldehyde and oxygenation demonstrated the inefficiency of the formation of acetaldehyde from oxygen. While both treatments consumed monomeric anthocyanins, only exogenous acetaldehyde significantly increased the concentration of polymeric pigments. iv

5 verall, I demonstrated that exogenous acetaldehyde treatment is a viable alternative to oxygenation for improving the color stability of red wines. At low concentrations (22 mg/l), winemakers could use acetaldehyde to significantly improve color stability and wine quality. Acetaldehyde is also likely to contribute to beneficial reactions of flavonoids in wine when found as a sulfonate, as I showed its reactivity upon the addition of bisulfite and when added as a sulfonate. While further work is needed to optimize its application during red wine production, this work suggests that exogenous acetaldehyde treatment could be an effective method of improving color stability for winemakers. v

6 TABLE F CNTENTS List of Figures...x List of Tables... xiii Acknowledgements...xv Chapter 1 Literature Review Introduction Description of Acetaldehyde Wine xidation xygen in Wine Micro-xygenation of Wine Formation of Acetaldehyde xidation Side Reactions Sulfur Dioxide in Wine Reaction of Bisulfite with Acetaldehyde Reactions of Acetaldehyde with Wine Flavonoids Anthocyanins Description of Wine Anthocyanins Role of Anthocyanins in Wine Reactions of Anthocyanins with Acetaldehyde Tannins Description of Wine Tannins Role of Tannins in Wine Reactions of Tannins with Acetaldehyde Impact of Reactions of Acetaldehyde with Flavonoids on Wine Quality Significance and Hypotheses Significance Hypotheses and Aims...30 Chapter 2 Exogenous Acetaldehyde as a Tool for Modulating Wine Color and Astringency during Fermentation...31 vi

7 2.1 Abstract Introduction Materials and Methods Materials Wine Production Acetaldehyde Measurement Pigment Analysis Total Phenolics Measurement Protein Precipitation Analysis Salivary Protein Precipitation Analysis Statistical Analysis Results and Discussion Acetaldehyde Concentrations in Must and Wine Effect of Exogenous Acetaldehyde on Wine Color Effect of Exogenous Acetaldehyde on Tannin-Protein Interactions Conclusions Acknowledgements...45 Chapter 3 Reaction of Acetaldehyde with Wine Flavonoids in the Presence of Sulfur Dioxide Abstract Introduction Materials & Methods Materials Reaction Mixture Preparation Flavonoid Analysis Pigment Analysis Reaction Product Characterization Statistical Analysis Results and Discussion Reaction of Acetaldehyde with Flavanols Reaction of Acetaldehyde with M3G and Flavanols Characterization of Products with MALDI-TF MS Acknowledgements...72 vii

8 Chapter 4 Reactions of Free and S 2 -Bound Aldehydes with (+)-Catechin Abstract Introduction Materials and Methods Materials Bisulfite Adduct Synthesis Reaction Mixture Preparation Catechin Analysis Reaction Product Characterization Statistical Analysis Results and Discussion Consumption of Catechin by Aldehydes Consumption of Catechin by Aldehyde-Bisulfite Adducts Characterization of Catechin-Aldehyde Products Acknowledgements...95 Chapter 5 Improving Red Wine Color Stability with Exogenous Acetaldehyde Abtract Introduction Materials and Methods Materials Bisulfite Adduct Synthesis Model Reaction Mixture Preparation Catechin Analysis Treatment of Red Wine with Free and Bound Acetaldehyde Treatment of Red Wine with Endogenous and Exogenous Acetaldehyde Pigment Characterization Statistical Analysis Results and Discussion Activity of Acetaldehyde-Bisulfite Adduct in Model Solutions Effect of Acetaldehyde and Acetaldehyde-Bisulfite Adduct on Red Wine Color Effect of Exogenous Acetaldehyde and xygenation on Red Wine Color Acknowledgements viii

9 Chapter 6 Conclusions and Recommendations for Future Work Reaction of Flavonoids with Acetaldehyde Reactivity of Aldehydes from α-hydroxyalkylsulfonates Exogenous Acetaldehyde as Wine Treatment Concluding Remarks References ix

10 List of Figures Figure 1.1. Formation of acetaldehyde from oxygen (Adapted from Danilewicz) Figure 1.2. Formation of reactive intermediates during the reduction of H 2 2 (Adapted from Danilewicz) Figure 1.3. Reaction of quinone with wine components (Adapted from Kreitman) Figure 1.4. Reactions of the hydroxyl radical ( ) and 1-hydroxyethyl radical (1- HER) Figure 1.5. Equilibrium and pk a values for sulfur dioxide Figure 1.6. Reactions of bisulfite in wine Figure 1.7. Reaction of acetaldehyde with bisulfite to form 1-hydroxyethanesulfonate Figure 1.8. Flavonoid Structures Figure 1.9. Reaction of acetaldehyde with a representative flavonoid Figure Equilibrium of anthocyanin forms Figure Reaction of malvidin-3-glucoside with bisulfite Figure Formation of polymeric pigments from malvidin-3-glucoside Figure Flavan-3-ol monomers found in grapes Figure Flavan-3-ol monomer and oligomer (i.e., a condensed tannin) Figure 2.1. Mechanism of astringency by tannin-protein complex formation, aggregation, and precipitation Figure 2.2. Direct and indirect condensation products of flavanols and anthocyanins occurring in wine Figure 2.3. Tannin content of BSA-tannin precipitates quantified by ferric chloride reaction in (+)-catechin equivalents (CE). Error bars represent one standard deviation of the mean, and results in with different letters (a, b) are significantly different (p < 0.05) Figure 2.4. Tannin content of saliva-tannin precipitates quantified by ferric chloride reaction in (+)-catechin equivalents (CE). Error bars represent one standard x

11 deviation of the mean, and results in with different letters (a, b) are significantly different (p < 0.05) Figure 3.1. Reaction of acetaldehyde with bisulfite and with representative flavonoids to form an ethylidene-bridged adduct Figure 3.2. Catechin concentrations in model wine after treatment with 20 mg/l acetaldehyde at ph 2.0, 2.5, 3.0, and 3.5 as determined by HPLC-DAD Figure 3.3. Catechin concentrations in model wine after treatment with 20 mg/l acetaldehyde with and without oxygen present at ph Figure 3.4. Catechin concentrations in model wine (ph 2.5) after treatment with acetaldehyde and S 2 additions under aerobic (A) and anaerobic (B) conditions. Low S 2 samples (A+Low S 2 ) contained 40 mg/l TS 2 and High S 2 samples (A+High S 2 ) contained 80 mg/l TS Figure 3.5. Catechin concentrations in all samples containing catechin over 12 days: catechin only (C), catechin with acetaldehyde (C+A), catechin with acetaldehyde and 40 mg/l S 2 (C+A+S 2 ), M3G and catechin with acetaldehyde (M3G+C+A), and M3G and catechin with acetaldehyde and 40 mg/l S 2 (M3G+C+A+S 2 ) Figure 3.6. Malvidin 3-glucoside (M3G) concentrations in all samples containing M3G over 12 days: M3G only (M3G), M3G with acetaldehyde (M3G+A), M3G with acetaldehyde and 40 mg/l S 2 (M3G+A+S 2 ), M3G and catechin with acetaldehyde (M3G+C+A), M3G and catechin with acetaldehyde and 40 mg/l S 2 (M3G+C+A+S 2 ), M3G and GSE with acetaldehyde (M3G+G+A), and M3G and GSE with acetaldehyde and 40 mg/l S 2 (M3G+G+A+S 2 ) Figure 3.7. Select color parameters from the modified Somers assay for control and treatment groups (acetaldehyde and acetaldehyde+s 2 ) at 12 days: A) Total anthocyanins, B) Degree of ionization of anthocyanins, C) Color density. Values represent the average of three experimental replicates ± standard deviation. Columns in the same group with different letters indicate significant differences (p<0.05) Figure 3.8. MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with acetaldehyde Figure 3.9. Reaction of catechin with acetaldehyde to form ethylidene-bridged oligomers followed by cleavage to form vinyl catechin moieties Figure Reaction of catechin with M3G and acetaldehyde to form an ethylidenebridged polymeric pigment and subsequent rearrangement to a pyranoanthocyanins Figure 4.1. Mechanism of reaction of aldehydes with catechin Figure 4.2. Reactions of aldehydes with bisulfite and catechin xi

12 Figure 4.3. Consumption of catechin by aldehydes. Values represent the average of three experimental replicates ± standard deviation Figure 4.4. Consumption of catechin by aldehyde-bisulfite adducts. Values represent the average of three experimental replicates ± standard deviation Figure 4.5. Catechin consumed after 28 days by aldehydes and aldehyde-bisulfite adducts. Values represent the average of three experimental replicates ± standard deviation. Values with different letters indicate significant differences (p<0.05) Figure 4.6. Correlation between log K d of aldehydes-bisulfite adducts and the percent of catechin consumed by the bisulfite adduct compared to the free aldehyde Figure 4.7. MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with formaldehyde Figure 4.8. MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with acetaldehyde Figure 4.9. MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with propionaldehyde Figure MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with isobutyraldehyde Figure MALDI-TF mass spectrum recorded in positive reflectron mode of catechin treated with benzaldehyde Figure 5.1. Reaction of malvidin-3-glucoside with catechin and acetaldehyde Figure 5.2. Formation of acetaldehyde from oxygen (Adapted from Danilewicz) Figure 5.3. xidation reactions leading to acetaldehyde formation (Adapted from Danilewicz) Figure 5.4. Catechin consumption at ph 2 4 by acetaldehyde (A) and 1- hydroxyethanesulfonate (B) xii

13 List of Tables Table 1.1. Concentrations of acetaldehyde in alcoholic beverages. 6, Table 1.2. Sources for introduction of atmospheric oxygen in wine. 1,11, Table 1.3. Anthocyanins found in V. vinifera grapes Table 2.1. Color parameters from the HTP modified Somers assay of treatment groups post-primary fermentation. a Table 3.1. Color parameters from the modified Somers assay for control and treatment groups (Acetaldehyde or Acetaldehyde and S 2 ) at 12 days. a Table 3.2. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with acetaldehyde Table 3.3. Predicted and observed m/z values recorded in positive reflectron mode MALDI-TF MS of catechin and M3G treated with acetaldehyde Table 3.4. Predicted and observed m/z values recorded in positive reflectron mode MALDI-TF MS of GSE treated with acetaldehyde Table 3.5. Predicted and observed m/z values recorded in positive reflectron mode MALDI-TF MS of GSE and M3G treated with acetaldehyde Table 4.1. dor thresholds and characteristics of aldehydes Table 4.2. Dissociation constants (K d ) of aldehyde-bisulfite adducts Table 4.3. Aldehyde-bisulfite adducts synthesized and their synthetic yields Table 4.4. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with formaldehyde Table 4.5. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with acetaldehyde Table 4.6. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with propionaldehyde Table 4.7. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with isobutyraldehyde Table 4.8. Predicted and observed m/z values as recorded in positive reflectron mode MALDI-TF MS of catechin treated with benzaldehyde xiii

14 Table 5.1. Color parameters from the modified Somers assay for control, bisulfite treated, acetaldehyde treated, and sulfonate treated wines after 8 weeks Table 5.2. Color parameters from the Harbertson-Adams assay for control, bisulfite treated, acetaldehyde treated, and sulfonate treated wines after 8 weeks. a Table 5.3. Color parameters from the modified Somers assay for control, acetaldehyde treated, and oxygenated (periodic aeration) wines after 8 weeks. a Table 5.4. Color parameters from the Harbertson-Adams assay for control, acetaldehyde treated, and oxygenated (periodic aeration) wines after 8 weeks. a xiv

15 Acknowledgements I would like to express my sincere gratitude to my advisor, Dr. Ryan Elias, for giving me the opportunity to join his lab and explore what was a brand new field to me. I appreciated his guidance throughout this process and his trust in allowing me the freedom to pursue my own interests. I would also like to thank my committee members, Dr. Josh Lambert, Dr. John Coupland, and Dr. Michela Centinari for their guidance. Their diverse knowledge and insights helped me to envision a larger context for my project and challenged me to expand my own thinking. I would like to acknowledge Ms. Denise Gardner for her unending support and help during wine production. Her impeccable organization, work ethic, and optimism made the wine production for this project possible, and even enjoyable. I would also like to acknowledge Dr. Tatiana Laremore for her assistance with MALDI-TF analyses and for her constant supply of chocolates. Finally, I would like to thank my lab mates and classmates for teaching me, supporting me, and commiserating with me during my time at Penn State. I have the utmost appreciation for my family and friends who have encouraged me from near and far. Thank you for believing in me and making these years in State College great ones. xv

16 Chapter 1 Literature Review 1.1 Introduction In recent years, wine research has progressed as analytical techniques and knowledge of wine have improved. A large portion of this research has focused on underlying mechanisms for observed benefits of wine processing in order to better their outcomes. xygen management in particular has been a focus due to its significant impact on wine quality. xygenation is known to improve red wine characteristics associated with color and mouthfeel based on the oxidation and modification of flavonoids. However, oxygen also leads to deleterious effects associated with chemical and microbial instability. As a risky yet rewarding treatment, oxygenation has been investigated in an effort to enhance the ability of winemakers to take advantage of this process. Developments in oxygenation include micro-oxygenation as a means to achieve precise and predictable oxidation. This technique allows winemakers greater control of oxygen concentrations but does not eliminate many of the risks associated with its presence. Accelerated aging techniques have also been attempted including heating, ultrasound, irradiation, high pressure, and electrolysis. 1,2 These methods all include undesirable side effects. Acetaldehyde is in an intermediate oxidation product that results in the modification of red wine polyphenols to improve mouthfeel and color stability and has not been thoroughly investigated. Addition of exogenous acetaldehyde could be used as a tool for winemakers that would replace risky oxygen exposure as a less expensive alternative. 1

17 1.1.1 Description of Acetaldehyde Acetaldehyde is a volatile compound that is ubiquitous in nature. It is present in the atmosphere as a product of plant respiration and combustion. 3 Acetaldehyde is also formed in the body during the metabolism of alcohol. 3 Acetaldehyde is commonly used as a flavor additive and is generally recognized as safe (GRAS). 4 It has an odor threshold of 0.5 mg/l and this odor is typically fruity and pleasant at low concentrations but pungent at higher concentrations. 3,5 A common source of acetaldehyde is alcoholic beverages as acetaldehyde is formed during fermentation and chemical oxidation of ethanol. The flavor threshold of acetaldehyde in wine is mg/l. 6 A summary of acetaldehyde levels in these beverages is found in Table ,7 Table 1.1. Concentrations of acetaldehyde in alcoholic beverages. 6,7 Beverage Total Acetaldehyde (mg/l) Red Wine White Wine Sweet Wine Sherry Port Brandy Cognac Beer 5 63 Apple Wine/Cider Whisky Rum 0 68 Vodka 0 13 Tequila Sake

18 1.2 Wine xidation Acetaldehyde is formed as an ethanol oxidation product in wine typically after oxygen exposure. Direct oxidation of phenolics does not occur at wine ph (3-4) since the phenolate anion form is not present (pk a 9-10) Instead, oxidation of wine results in products like acetaldehyde that can react with phenolics. The reduction of oxygen to form acetaldehyde is the goal of deliberate oxygen exposure, though this mechanism involves many other reactions and intermediates that should not be discounted xygen in Wine xygen is easily incorporated into wine throughout production during the course of several processing steps and deliberate oxygenation techniques. Upon exposure to air, oxygen will be dissolved quickly; wine stirred with air will become saturated in ca. 30 seconds. 11,12 The dissolved oxygen (D) concentration in wine saturated with air is 8.4 mg/l at 20 C. Wine production steps can introduce D levels that vary from very low concentrations (e.g., pumping) to saturation (crushing or pressing) (Table 1.2). 11,13,14 Table 1.2. Sources for introduction of atmospheric oxygen in wine. 1,11,13 15 peration Increase in Dissolved xygen (mg/l) Crushing, Pressing Saturation Racking Pumping Centrifugation 1.0 Filtration Bottling 0.8 Cold Stabilization 1.3 3

19 xygen is also quickly consumed in red wine; after saturation, D of a red wine will be reduced to below 1 mg/ml in about 6 days at 30 C. 8,16 The D of wine in storage tanks or barrels is typically between 0.02 and 0.05 mg/l. 11 Red wine has an oxygen consumption capacity of up to 800 mg/l. 11 While techniques like barrel aging are used to incorporate a relatively controlled amount of oxygen, the exposure of red wine to oxygenation is still highly variable. Instead, winemakers can use micro-oxygenation (MX) under highly controlled conditions in order to introduce small amounts of D over time Micro-xygenation of Wine MX involves the deliberate, gradual addition of D before malolactic fermentation (MLF) (10-30 ml/l/month) or after MLF (1-5 ml/l/month). 17,18 MX is used to improve wine color, aroma, and mouthfeel. 18 MX is based on the premise that the rate of oxygen dosing is less than the rate of oxygen consumption in the wine; therefore, in theory, no increase in D should be measureable during an appropriately administered MX application. 19 However, it has been shown that even low doses of oxygen (1 ml/l/month) result in increased concentrations of D in red wine. 20 When applied before MLF, MX is more effective due to an abundance of monomeric anthocyanins. This allows better color stabilization and also limits tannin lengthening as anthocyanins form terminal subunits. 21 Additionally, MLF has been shown to decrease the concentration of residual acetaldehyde from MX as malolactic bacteria can metabolize acetaldehyde. 22 This may be detrimental to wines if there is insufficient time for acetaldehyde to react before it is depleted by MLF. 23,24 Studies on the use of MX with red wine have been reviewed recently. 18,19,25,26 Generally, MX is considered to be beneficial for wines that have high phenolic contents and 4

20 especially for wines that have a high proportion of free anthocyanins. 21,27 Wines with relatively low phenolic contents are more prone to the risk of over-treatment, which leads to the loss of desirable aromas, undesirable changes in wine color, and growth of aerobic organisms like acetic acid bacteria. 18,27 These deleterious effects are also a risk if the rate of oxygen addition is not chosen correctly. MX is also expensive, especially for the initial investment, with prices around $1,000 per tank and total costs up to a couple hundred thousand dollars for large wineries. 28,29 Even when MX is implemented correctly, it is difficult to get reproducible results and to monitor the process without the use of specialized, sensitive equipment. 20 Though MX is a useful tool for controlling oxidation, there are still many risks and costs of its use in winemaking. Periodic aeration has recently been explored as an alternative to MX. 30 In this treatment, oxygen is added weekly by aerating a small volume of wine to D saturation and then reintroducing it to the bulk wine. In this way, the same dose of total oxygen can be added without the use of MX equipment but with the overall benefits of oxidation. Periodic aeration may be a useful and less expensive alternative to MX for winemakers Formation of Acetaldehyde Acetaldehyde in wine can result from either biological (enzymatic) or strictly chemical (non-enzymatic) means. Under the latter scenario, acetaldehyde is formed by several metalcatalyzed steps that lead to the eventual oxidation of ethanol (Figure 1.1). Metal ions first take part in the reaction by forming an Fe(II)- 2 adduct, wherein an electron transfer occurs to form an Fe(III)-superoxo complex, and subsequently hydrogen peroxide (H 2 2 ). 31 Hydrogen peroxide is further reduced by the Fenton reaction to form a hydroxyl radical ( ). This radical is sufficiently reactive to interact with organic material in a concentration-based manner. The 5

21 hydroxyl radical therefore reacts with ethanol to yield acetaldehyde, as ethanol is the most abundant organic compound present in wine (~2 M). 8 10,31,32 Acetaldehyde is also formed as a byproduct of yeast metabolism. 6 Sugar is the primary substrate for the formation of acetaldehyde, which takes place mainly during the growth period of yeast. There are large differences in the amount of acetaldehyde produced between species and strains of Saccharomyces cerevisiae. 6,33 Fe(II) Fe(II) 2 Fe(II) Fe(II) Fe(III) 2Fe(III) + H 2 2 Fe(II) CH 3 CH 0.5 H CH 3 CH H 2 H Et Fe(III) Figure 1.1. Formation of acetaldehyde from oxygen (Adapted from Danilewicz). 31 In studies on MX or other sources of D, there is no consensus on resulting acetaldehyde concentrations. Due to the complexity of the reactions that lead to acetaldehyde generation and its quick consumption, the concentration of acetaldehyde in a wine sample is not representative of the acetaldehyde formed up to that point. Previous studies have shown that the acetaldehyde concentration remains low during oxygenation until it reaches a point where it begins to increase significantly (1 mg/l/day). 34 This lag phase may represent the consumption of free sulfur dioxide or available polyphenols, but this has not been investigated. It is, however, clear that large amounts of acetaldehyde are formed with the excessive application of MX. 35 The benefits of MX have been seen even when acetaldehyde concentrations do not appear to 6

22 increase with treatment. 36 nce oxygen is no longer being introduced, the acetaldehyde in a wine will be consumed xidation Side Reactions xygen is reduced to water in a series of steps that involve several reactive intermediates (Figure 1.2). While acetaldehyde is formed as a product of these reactions, quinones, hydroxyl radicals, and hydroxyethyl radicals are also generated, which can be detrimental to wine quality. 10,38,39 R Fe(II) H 2 2 H 2 R Fe(III) H Et CH 3 CH CH 3 CH Figure 1.2. Formation of reactive intermediates during the reduction of H 2 2 (Adapted from Danilewicz). 10 Quinones are highly electrophilic reactive oxygen species formed during the oxidation of some phenolic compounds. These species react quickly with nucleophiles found in wine (e.g., amino acids, bisulfite (HS - 3 ), thiols (RSH), ascorbic acid, and flavanols) (Figure 1.3). Reaction of quinones with flavanols can lead to browning as they form polymers. 40,41 In the presence of bisulfite or ascorbic acid, quinones will be reduced back to hydroquinones. 42,43 The reaction of quinones with amino acids is followed by Strecker degradation to form aldehydes including methional and phenylacetaldehyde. 44 The reaction of quinones with thiols significantly impacts 7

23 wine aroma; the consumption of thiols as catechol adducts removes these desirable aroma compounds Reaction with bisulfite, glutathione (GSH), and ascorbic acid has been shown to occur at a very fast rate followed by other thiols and amino acids. 50 Strecker Degradation Aldehydes H 3 S Amino Acids HS 3 - R R R RSH RS R Flavanols Ascorbic Acid R R H R Figure 1.3. Reaction of quinone with wine components (Adapted from Kreitman). 51 Hydroxyl radicals are formed by the breakdown of hydrogen peroxide (Figure 1.2). These radicals are highly reactive and therefore react in a non-selective manner with wine components based on concentration. 10,52 Ethanol is therefore the most likely target followed by glycerol and tartaric acid. The reaction products of ethanol, glycerol, and tartaric acid are acetaldehyde, glyceraldehyde, and glyoxylic acid, respectively (Figure 1.4). 53,54 1-Hydroxyethyl 8

24 radical (1-HER), an intermediate in the oxidative pathway from ethanol to acetaldehyde, is also a reactive oxygen species. 10,55 1-HER also reacts with the α,β-unsaturated side chains of cinnamic acids, including caffeic acid and ferulic acid, forming an allylic alcohol. 56 Furthermore, recent studies have demonstrated that 1-HER can also contribute to the oxidative loss of thiols that are important to wine aroma. 57 The reactions of these two radicals, as well as quinones, contribute to side reactions and possible deleterious effects of oxidation in wine. Glyceraldehyde H Glycerol Ethanol Cinnamic Acid C 2 Glyoxylic Acid H Tartaric Acid 1-HER R-SH Thiol Acetaldehyde R-S Figure 1.4. Reactions of the hydroxyl radical ( ) and 1-hydroxyethyl radical (1-HER) Sulfur Dioxide in Wine Sulfur dioxide (S 2 ) is added to wine to prevent chemical and microbial spoilage. S 2 is found in equilibrium of three forms: molecular S 2, bisulfite, and sulfite (Figure 1.5). The molecular form is responsible for microbial preservation of wine. The majority of S 2, however, is found as the bisulfite ion at wine ph; 96% will be the bisulfite form at ph Bisulfite provides chemical stability, as it is able to quench oxidation reactions as well as to react with oxidation products. Bisulfite reacts readily with hydrogen peroxide and quinones (Figure 1.6). 9

25 Reaction with hydrogen peroxide prevents the oxidation of ethanol to acetaldehyde and reaction with quinones regenerates their catechol. 42,58,59 S 2 therefore protects other wine components from oxidation by quenching reactive oxygen species. Bisulfite also reacts with anthocyanins, bleaching their color. The role of this reaction will be discussed in greater detail in later sections. pk a = 1.86 pk a = 7.2 S 2 + H 2 HS H + S H + Molecular S 2 Bisulfite Sulfite Figure 1.5. Equilibrium and pk a values for sulfur dioxide. Anthocyanin-S 3 H Anthocyanin H H 2 S 4 H 3 C H S HS 3 - R H 3 S R R Figure 1.6. Reactions of bisulfite in wine Reaction of Bisulfite with Acetaldehyde Bisulfite is known to react with compounds to prevent possible aromatic faults associated with oxidation by forming non-volatile adducts. Bisulfite readily reacts with carbonyl compounds, including acetaldehyde, to form α-hydroxyalkylsulfonates (Figure 1.7). Excessive acetaldehyde can lead to a characteristically oxidized aroma and is perceived as a fault in wine, though the sulfonate is reported to have no detectable aroma. 60 In this way, bisulfite can both 10

26 prevent and mask wine oxidation. The portion of S 2 that has reacted with acetaldehyde, other carbonyls, or other wine components (e.g., anthocyanins) is referred to as bound S 2. All other forms constitute the free S 2 portion; together, bound and free S 2 comprise total S 2. During production, wine will have free S 2 present in order to protect the wine from chemical and microbial instability. H 3 C H H S H 3 C H S Figure 1.7. Reaction of acetaldehyde with bisulfite to form 1-hydroxyethanesulfonate. The reaction of acetaldehyde with bisulfite is fast and results in the strongly bound adduct, 1-hydroxyethanesulfonate (K d = 2.4 x 10-6 M). 1,61 Most research has therefore assumed that 1-hydroxyethanesulfonate is inert with respect to acetaldehyde reactivity. However, this acetaldehyde-bisulfite adduct has been shown to have antioxidant activity in beer; the formation of radicals from ethanol oxidation was slowed by the sulfonate indicating similar activity to S 2 itself. 62 In most wines, there is excess S 2 to acetaldehyde so that there will be free S 2 present. While further oxidation consumes S 2 and may free some acetaldehyde, reaction products from acetaldehyde are seen in wines over time with free S 2 present throughout. This suggests that acetaldehyde is able to react with wine components in the presence of free S 2. The reactivity of acetaldehyde from its bound form has not been explored. 11

27 1.3 Reactions of Acetaldehyde with Wine Flavonoids Reactions of acetaldehyde, specifically those with phenolics, are complicated but are critical to understanding the complexities of wine chemistry. Acetaldehyde mediates many oxidation reactions as the direct oxidation of phenolics is unlikely at wine ph Direct condensation reactions can occur via quinone intermediates (Figure 1.3). The reactions of acetaldehyde with specific polyphenols will be reviewed here. The total polyphenol content of red grapes is in the range of mg/100 g (fresh weight basis). 63 Red wine contains a large number of polyphenols, between 1000 and 3000 mg/l gallic acid equivalents total polyphenol content. 64 These polyphenols include flavonoids, which are important for red wine chemistry and quality. Some of the most important reactions of acetaldehyde in wine are those with flavonoids. Flavonoids are compounds with a characteristic three-ring structure: two aromatic rings (A and B) connected by a pyran ring (C) (Figure 1.8). The saturation of the C ring determines the class of flavonoids. Anthocyanins contain a fully unsaturated C ring, also known as a pyrilium cation. Flavanols contain a fully saturated C ring. Anthocyanins and flavanols are the major classes of flavonoids found in grapes and wine. 65,66 These flavonoids and their reaction products will be discussed in greater detail in later sections ' 2' 4' 8 B 1 1' 5' A C 2 3 6' 5 4 R Flavonoid Anthocyanin Flavanol Figure 1.8. Flavonoid Structures 12

28 Flavonoids can react with acetaldehyde by the mechanism shown in Figure 1.9. The first step is the acid-catalyzed protonation of acetaldehyde to form an electrophilic carbocation. The nucleophilic A ring of a flavonoid, represented here as a substituted resorcinol, then adds to the protonated acetaldehyde to form a carbocation after the loss of water. The addition of another nucleophilic flavonoid forms an ethyl-bridged flavonoid dimer. This ethylidene bridge between flavonoids is characteristic of the reaction of acetaldehyde with flavonoids. However, acidcatalyzed cleavage of that bridge results in the formation of a vinyl moiety on one of the bridged flavonoids. By this mechanism, bridged anthocyanins and flavanols can form as well as those with vinyl moieties. R 1 R 2 H 3 C H H H H H + -H + H R 2 H 3 C H R 1 H + H R 1 R 2 H R 1 R 2 + R 3 R 4 H + H R 3 H R 4 R 1 R 2 H R 3 -H + R 4 Figure 1.9. Reaction of acetaldehyde with a representative flavonoid. 13

29 1.3.1 Anthocyanins Description of Wine Anthocyanins Anthocyanins are a class of pigmented flavonoids that contribute colors of red, purple, or blue. Their environment (e.g., ph, complexation with phenolics) and intrinsic structural characteristics determine their observed color. 67,68 The amount of anthocyanins found in grapes varies significantly from 30 to 750 mg/100 g depending on the variety and growing conditions. 69 Anthocyanins have been studied for their health benefits including their ability to act as anticancer, antimicrobial, and antiviral agents. 70 Anthocyanins are found in the skin of red grapes and are derived from six aglycones, or anthocyanidins pelargonidin, cyanidin, delphinidin, peonidin, petunidin, and malvidin (Table 1.3). 67,71,72 These grape anthocyanins are found as 3-glucosides in Vitis vinifera grapes and their 3,5-diglucosides are also found in American species (V. riparia, V. rupestris, etc.) and French- American hybrids. 68 The aglycone anthocyanidins differ in their substitution on the B-ring with variations in the number and position of hydroxyl and methoxyl groups present. The anthocyanins with more methoxyl groups appear to be more red, thus malvidin is the reddest pigment present. 68 Malvidin-3-glucoside is also the most abundant anthocyanin in young, red, V. vinifera wines accounting for 50-90% of the anthocyanins present depending on the variety, growing conditions, and winemaking practices. 71 Acylated monoglucoside anthocyanins are also present in wine, which are acylated by p-coumaric, caffeic and acetic acids. The reactions of acylated monoglucosides will not be reviewed here but have been discussed elsewhere. 1,71,73,74 14

30 Table 1.3. Anthocyanins found in V. vinifera grapes. 71 R 1 H R 2 R 3 Name R 1 R 2 R 3 Pelargonidin H H H Pelargonidin-3-glucoside H H Glu Cyanidin H H Cyanidin-3-glucoside H Glu Delphinidin H Delphinidin-3-glucoside Glu Peonidin CH 3 H H Peonidin-3-glucoside CH 3 H Glu Petunidin CH 3 H Petunidin-3-glucoside CH 3 Glu Malvidin CH 3 CH 3 H Malvidin-3-glucoside CH 3 CH 3 Glu Role of Anthocyanins in Wine Anthocyanins are readily extracted from grape skins during red wine production since they are water-soluble. The breakdown of grape skin and cell membranes begins during crushing, while maceration, heat of fermentation, ethanol content, and other fermentation conditions encourage further extraction of anthocyanins. 75,76 The total concentration of anthocyanins in young, red wines is about 500 mg/l, though this concentration can be as high as 2000 mg/l. 1,71 15

31 However, as will be discussed in greater detail in this review, monomeric anthocyanins react quickly with other components of wine. nly a few days after crushing, the rate of reaction of anthocyanins exceeds the rate of extraction, and the concentration of solution phase monomeric anthocyanins begins to decrease. 77 Approximately 50 to 70% of anthocyanins will be incorporated into polymeric pigments after one year. 75,78 80 Anthocyanins are also lost to degradation and precipitation reactions. The degradation of anthocyanins generates colorless, low molecular weight compounds. 81 The most important role of anthocyanins in wine is by contributing color, though they may also contribute to taste and mouthfeel under certain conditions. 82 Anthocyanins exist in four forms in solution whose distribution is dependent on ph (Figure 1.10). nly one of these forms is the desired red form, the flavylium cation, which is favored at low ph. 68,83,84 At ph 3.4, only 15% of anthocyanins are in their colored, flavylium state. 1 This proportion decreases at higher ph. The hydration constant (pk h ) of malvidin-3- glucoside is Therefore, at wine ph, the colorless, hydrated form is favored

32 H CH 3 -Glu CH 3 +H 2 -H + H CH 3 -Glu CH 3 Flavylium cation (red) Carbinol pseudobase (colorless) -H + CH 3 CH 3 H CH 3 CH 3 -Glu Quinoidal base (blue) -Glu Chalcone (pale yellow) Figure Equilibrium of anthocyanin forms Anthocyanins also react quickly with the bisulfite form of S 2 forming a colorless adduct in a reaction known as S 2 bleaching (Figure 1.11). 86 At wine ph, bisulfite is by far the most common form (96%) of S 2 so the concentration of total S 2 is closely related to the amount of anthocyanins that remain colored. Consequently, at a constant S 2 concentration, higher concentrations of anthocyanins will lead to greater color intensity. 71 The anthocyanin-bisulfite adduct is stable at wine ph as its dissociation constant (pk s ) is 5. 66,87 However, resistance to S 2 bleaching is seen in anthocyanin-derived pigments formed by oxidation reactions. 17

33 CH 3 CH 3 H CH 3 +HS 3 H CH 3 -Glu -Glu S 3 H Flavylium cation (red) Bisulfite adduct (colorless) Figure Reaction of malvidin-3-glucoside with bisulfite. The color of anthocyanins in their environment is mainly affected by ph and S 2 concentration as described above. Their color can also be stabilized by copigmentation, an inter- or intramolecular association of anthocyanins with other moieties that increases their color density. Copigmentation has been extensively reviewed elsewhere. 1,71,88 90 Perhaps the more important process for stabilization, however, is the conversion of anthocyanins to polymeric pigments. This can occur by several mechanisms associated with oxidation 74, but this review will focus on the role of acetaldehyde in improving color stability of red wine pigments Reactions of Anthocyanins with Acetaldehyde Experiments by Timberlake and Bridle first showed the important role that acetaldehyde plays in increasing the degree of ionization of anthocyanins and lowering the amount of monomeric anthocyanins in red wine. 91 Anthocyanins readily take part in condensation reactions involving acetaldehyde. The hydrated form of the monomeric anthocyanin can act as a nucleophile from the A ring, similar to flavanols. As shown in Figure 1.9, an anthocyanin adds to protonated acetaldehyde to form a carbocation. The addition of another nucleophilic flavonoid 18

34 leads to the formation of an ethylidene bridge between the anthocyanin and flavonoid. This bridging by acetaldehyde was proposed by Timberlake and Bridle in 1976 and confirmed by Fulcrand et al. in ,93 Anthocyanins can react by this acetaldehyde-mediated mechanism with other anthocyanins to form dimers or with other flavonoids, including flavanols. This mixed group of products formed is referred to as polymeric pigments (Figure 1.12). Ethyl-bridged polymeric pigments have been observed in model systems and in red wine Gl CH 3 H CH 3 H Gl CH 3 H CH 3 CH 3 CH 3 H Gl CH 3 CH 3 H H 3 C H H H Gl Gl CH 3 CH 3 H H CH 3 CH 3 Gl Figure Formation of polymeric pigments from malvidin-3-glucoside. These ethylidene bridges formed by reaction with acetaldehyde are unstable and susceptible to acid-catalyzed cleavage (Figure 1.9). 86,99,100 Further reaction of the products from that cleavage form another class of polymeric pigments referred to as pyranoanthocyanins (Figure 1.12). 66,100 These pyranoanthocyanins include vitisins, derivatives of monomeric anthocyanins with an additional pyran ring formed between C4 and the hydroxyl group of C5. 15,66,101 Pyranoanthocyanin-flavanol oligomers were first proposed based on model studies by Francia-Aricha et al. and were observed to be more resistant to discoloration by ph changes and 19

35 S These products can be formed either from the reaction of an anthocyanin with a vinyl flavanol, or the reaction of a flavanol with a pyranoanthocyanin already formed by reaction with acetaldehyde. Anthocyanins are believed to act primarily as terminal subunits in reactions with flavanol oligomers as they react mainly from C Reactions between tannins, anthocyanins, and acetaldehyde in model solutions have shown that polymerization of anthocyanins happens readily with tannins present and slowly without them, with color shifts towards violet. 91,93,96, Products of these condensation reactions with anthocyanins also undergo shifts in color from the starting monomeric anthocyanin. Ethyl-linked anthocyanin species are typically more purple (absorbance between 528 and 540 nm) and the rearranged, stable derivatives, pyranoanthocyanins, are orange (absorbance between 480 and 510 nm). 91,95,111,112 ther adducts range from red (absorbance between 515 and 526 nm) for tannin-anthocyanin adducts to blue (absorbance at 575 nm) for flavanyl-vinylpyranoanthocyanins (portisins) Some anthocyanin-tannin adducts, however, may be colorless. 117 Polymeric pigments formed contribute to increased color stability of red wine. These polymeric pigments are resistant to bleaching as they are weak binders of S 2. 15,101 In fact, flavanol-ethyl-anthocyanin adducts are not bleached by S 2 to any degree, although their color is known to be affected by ph with a shift towards blue as ph increases. 95 Pyranoanthocyanins are relatively more stable to S 2 and ph changes due to the addition of a pyran moiety on the C ring which prevents the addition of water or S 2. 84,101,112,118 Polymeric pigments may also have increased color intensity due to intramolecular copigmentation. 86 verall, reactions of acetaldehyde lead to a shift in color from red/purple to tawny/brick red, enhancement of color intensity, and resistance to bleaching by ph changes and S 2 as pyranoanthocyanins and flavanyl-pyranoanthocyanins are formed from monomeric anthocyanins. 1,15,66,73,119 20

36 1.3.2 Tannins Description of Wine Tannins Condensed tannins, or proanthocyanidins, are oligomeric polyphenols composed of flavan-3-ol subunits (catechins). While tannins are common to many foods including tea, chocolate, and many fruits, they are especially important to grape and wine quality. 120 They are also known to confer many health benefits upon consumption Grapes contain between 1 and 4 g/l condensed tannin which are found in the skin and seeds of grapes. 1 These tannins vary in size (degree of polymerization or DP) and subunit composition (e.g., stereochemistry, substitution). 123 The most common catechin subunits found in grape tannins are (+)-catechin, (-)- epicatechin, (-)-epicatechin gallate (ECG), and (-)-epigallocatechin (EGC) (Figure 1.13). The composition of tannins depends on their location in the grape; seed tannins contain no EGC while skin tannins do, skin tannins have 5% ECG while seed tannins have 30%

37 H H (+)-Catechin (-)-Epicatechin H H (-)-Epigallocatechin (-)-Epicatechin gallate Figure Flavan-3-ol monomers found in grapes. Tannins are present in grapes in a wide range of DP, from the monomer (DP 1) to large oligomers up to DP 80 found in grape skin (Figure 1.14). 98,124,125 Tannins in grapes are B-type proanthocyanidins, indicating that they have a single bond (C4-C6 or C4-C8) between subunits. However, A-type proanthocyanidins have been observed in wine. 126 These A-type compounds contain an additional ether bond (C5 or C7 and C2) between subunits and can be formed from B- type via a radical process. 1 22

38 H H H 4 8 n 3 H 4 8 Figure Flavan-3-ol monomer and oligomer (i.e., a condensed tannin) Role of Tannins in Wine Tannins are extracted into red wine during the primary alcoholic fermentation as an increase in ethanol concentration enables dissolution of these large, relatively nonpolar molecules. Tannin extraction requires higher alcohol concentration and temperature than other polyphenols (e.g., anthocyanins) due to their lower aqueous solubility. 77 Practices such as extended maceration can be used to increase tannin extraction from grapes during wine production. 77 Higher alcohol content, S 2, temperature, and skin contact time are known to increase tannin extraction. 75 Tannin concentration in wine has been reported to be between 300 and 700 mg/l catechin equivalents, although quantification methods for tannins are too inconsistent to give reliable concentrations in wine. 64,127,128 23

39 Tannins, and other polyphenols, are important in red wine for their ability to eliminate free radicals and to chelate metal ions, thus contributing to the chemical stability of wine. 1,123 Tannins are also capable of interaction with many proteins. Their interaction with proteins is involved in many of the benefits of tannins for plants and humans Tannin-protein interactions can also lead to haze formation in white wine. 120 The ability of tannins to bind with proteins is arguably most important for their role in the astringency of red wine based on their interaction with salivary proteins. 129 Tannins contribute to the taste and mouthfeel of wine. In particular, larger tannins are perceived as astringent. Astringency is the sensation of drying and/or puckering in the mouth, a sensation that is desirable to a certain extent. Astringent components in wine contribute to a velvety mouthfeel but, when present at exceedingly high concentrations, can lead to wines that are perceived as out of balance and having a grainy or powdery mouthfeel. 130,131 Astringency has been an area of intense interest due to the complexities in its characterization and molecular mechanism. 132 The sensation of astringency is likely due to a loss of lubrication upon the precipitation of protein-tannin complexes. 133,134 The proteins that take part in this interaction are primarily proline-rich proteins (PRPs) 77,135 that are common in saliva with over 20 previously identified. Proline, glycine, and glutamine make up 70 to 80% of PRPs. 136 PRPs are generally unstructured and more likely to be randomly coiled. 137 Astringency is dependent on the tannins in a wine as well as the matrix of the wine. 138 Tannin size is an important determining factor for the perceived astringency. Small tannins are typically more bitter than astringent while large tannins are more astringent and only slightly bitter. 82,139,140 The interaction between tannins and proteins has been shown to be a function of DP ,143,144 While astringency has been shown to be a function of tannin length, other characteristics of tannin structure can influence the interaction with proteins. 82 Astringency also 24