Incorporation of Malvidin-3-Glucoside into High Molecular Weight Polyphenols during Fermentation and Wine Aging

Transcription

1 Incorporation of Malvidin-3-Glucoside 139 Incorporation of Malvidin-3-Glucoside into High Molecular Weight Polyphenols during Fermentation and Wine Aging Alejandro Zimman 1,2 and Andrew L. Waterhouse 1* Abstract: A decrease in anthocyanin concentration throughout the late stages of fermentation and aging has been well documented and is associated with the formation of polymeric pigments or pigmented tannins. In order to understand the formation of polymeric pigments, a 3 H-labeled anthocyanin (malvidin-3-glucoside) was added to a fermenting juice and to a young wine. During fermentation, there was a rapid disappearance of tritium from solution 24 hr after the addition, suggesting that half of the anthocyanins were associated with grape solids. During the early stages of aging, there were negligible losses of radioactivity, with a steep decline in malvidin-3-glucoside. In the aging experiment, most of the anthocyanins appeared to incorporate into polymeric structures with negligible losses elsewhere. Furthermore, these polymers changed over time, altering their visible absorbance. Aging variables such as wine ph, additional tannin levels, and copigmentation had little or no effect. However, temperature had pronounced effects during polymer pigment formation. Key words: polymeric pigment, anthocyanin, tannin, fermentation Red wines made from Vitis vinifera grapes contain anthocyanin monoglycosides. Among all anthocyanins, malvidin-3-glucoside is found with the highest concentration (Ribereau-Gayon 1982). A decrease in anthocyanin concentration is noticed even before the end of fermentation. This decline continues throughout maturation and aging in the bottle (Nagel and Wulf 1979). Concomitantly, wine color also changes, but does not disappear, and the common visual description is a shift from red-purple to red-brown color. Anthocyanin chemistry is quite diverse. At low ph, the flavylium (red) form is predominant and increasing the ph will favor three other forms: chalcone, pseudobase (or carbinol), and quinonoidal (Brouillard 1982). Noncovalent interactions of anthocyanins with other compounds result in changes in maximum wavelength absorption as well as in absorptivity (Boulton 2001). Anthocyanins participate in many reactions in wine that yield different products. Covalent reaction with SO 2 results in a colorless anthocyanin and its structure has been elucidated by nuclear magnetic resonance (Berke et al. 1998). Other small molecules such as pyruvic acid also react with anthocyanins (Bakker and Timberlake 1997, Benabdeljalil et al. 2000, Sarni-Manchado et al. 1997), but most interest has focused on condensation with high molecular weight compounds referred to as 1 Department of Viticulture and Enology, University of California, Davis, CA ; 2 Current address: Pathology and Laboratory Medicine, University of California, Los Angeles, CA *Corresponding author [Tel: (530) ; fax: (530) ; alwaterhouse@ucdavis.edu] Acknowledgments: The authors wish to thank the American Vineyard Foundation for financial support. The authors also thank Juan Marcó at Kendall-Jackson, Oakville for winemaking advice. Manuscript submitted February 2003; revised May, July 2003 Copyright 2004 by the American Society for Enology and Viticulture. All rights reserved. tannins or proanthocyanidins (Haslam 1980, Remy et al. 2000, Ribereau-Gayon 1983, Singleton and Trousdale 1992, Somers 1966). The proposed mechanisms of condensation are based on the ability of an anthocyanin to react as an electrophile or nucleophile. As an electrophile, the flavylium species can react with C-6 or C-8 of the flavan-3-ol units of tannins, followed by oxidation to regenerate the flavylium structure (Santos-Buelga et al. 1995, Somers 1971). As a nucleophile, the nucleophilic positions C-6 or C-8 of an anthocyanin in the carbinol form will react with the carbocations generated after acid-catalyzed cleavage of tannins (Haslam 1980). Two alternative mechanisms are based on condensation through an acetaldehyde bridge reacting (1) at the nucleophilic positions of the anthocyanin and the flavan-3-ol (Es-Safi et al. 1999, Rivas-Gonzalo et al. 1995) or (2) at the nucleophilic position of a flavan-3-ol and the electrophilic position of an anthocyanin, which results in a new heteroaromatic ring (Asenstorfer et al. 2001, Francia-Aricha et al. 1997). The elucidation of the actual mechanism or mechanisms that produce most of the pigmented tannin is not known because the isolation and chemical characterization of pigmented tannins is difficult. One of many problems is that the products do not have the same absorbance as the original anthocyanins, while another is the high number of products formed. The chemical fate of anthocyanins in a complex mixture of products may be also examined by radioactive tracing. This approach is commonly used in pharmacology to study distribution and metabolic fate of compounds after ingestion, including flavonoids (Kohri et al. 2001). In addition, tracing experiments can be applied to monitor changes during storage of food, and, to our knowledge, there is only one report where a radioactive anthocyanin was used for 139

2 140 Zimman and Waterhouse that purpose in strawberry juice (Livingston and Markakis 1956). A rapid synthetic method (Zimman and Waterhouse 2002) produced enough tritiated malvidin-3-glucoside to trace the fate of anthocyanins in wine fermentation and aging. This approach can provide information regarding the amount of anthocyanin incorporated, the absorptivity properties of the new structures, and the effect of variables such as temperature, ph, or tannin concentration on the formation of these pigmented tannins. Materials and Methods Chemicals. Malvidin-3-glucoside was purchased from Polyphenols SA (Sandnes, Norway). [3'-O-Methyl- 3 H] malvidin-3-glucoside was synthesized and purified following a method published previously (Zimman and Waterhouse 2002). While tritium labeling can be at risk due to exchange in some situations, hydrocarbons are poor exchangers. An estimate of the half-life of this hydrogen (as the methyl on anisole) would be in excess of 2 x years (Zatsepina et al. 1974). Tritiated water and scintillation cocktail Ultima Gold were purchased from Packard (Meriden, CT). Scintillation cocktail Scintiverse E was from Fisher (Pittsburgh, PA). Chlorogenic acid, epicatechin, and trifluoroacetic acid were from Sigma (St. Louis, MO). High-performance liquid chromatography (HPLC) grade methanol, methylene chloride, and 1-heptanesulfonic acid (sodium salt) were purchased from VWR (West Chester, PA). Fermentation experiment. Cabernet Sauvignon grapes were used in six small-scale fermentations. Each fermentation contained 340 g of berries (22 Brix; 0.94 g and 0.85 ml per berry) that were manually crushed, without cracking the seeds, before inoculation with yeast (Premier Cuvée, Red Star, Milwaukee, WI) (0.525 g yeast added to 140 ml water at 35 C, mixed well, and 20 ml added to each experiment). Fermentations were placed in a water bath set at 30 C. The solids were well mixed with the liquid 3 times per day using a plastic stick. 3 H-malvidin-3-glucoside was added to three of the six fermentations when the absorbance at 520 nm reached a maximum. The addition was done by decanting the liquid into a container, adding µci of tritiated compound, and pouring liquid back into the solids. This process was also repeated for the other three experiments that had no 3 H-malvidin-3-glucoside addition. The end of the fermentation (below 0.5% reducing sugars) was determined using Clinitest reagent tablets (Bayer, Elkhart, IN). After the fermentation was finished, the wine was separated from solids by decanting. Each wine was stored in 50-mL plastic vials at 5 C in the dark for 10 months. Air exposure was reduced to a minimum with a stream of nitrogen before closing the cap and the vial was not opened until analysis. All samples were frozen until analysis except for the aged wine. The skins of 100 berries, from the same batch of grapes used in the fermentation experiments, were separated from the pulp and seeds and then washed three times with icecold water. Anthocyanins were extracted from the skins with acetone:water (70:30) for 24 hr under nitrogen atmosphere. After filtering the solids, acetone was evaporated under reduced pressure at 35 C. The concentration of malvidin-3- glucoside in the water extract was calculated by HPLC as described below. The maximum concentration possible of malvidin-3-glucoside in each fermentation was calculated based on mg of malvidin-3-glucoside extracted per kg of grapes and volume yield (340 g of grapes used in each fermentation gave 140 ml of wine). The extraction procedure was performed three times and the concentration averaged. It was necessary to decolorize the samples to quantify radioactivity as wine pigments quench the fluorescence produced by the scintillation fluid. The bleaching procedure is based on a published method by Smith and Lang (1987). Two ml of bleach (2% NaOCl) was added to 1 ml of wine followed by 0.2 ml of 4M NH 4 OH and left in the hood for 1 hr. Then 0.2 ml of acetic acid (glacial) was added. After 30 min, 10 ml of Scintiverse E was added and samples were stored in the dark for 24 hr before counting them using the quench curve described in the normal-phase HPLC section. To account for the residual luminescence contribution, a blank value was calculated with a wine sample that had no 3 H-malvidin-3-glucoside added. Aging experiment. Cabernet Sauvignon wine from Oakville, CA, was made at commercial scale during fall Fermentation temperature was set up to 30 C with two daily pump-overs in a 24,000-L tank. After fermentation was completed, skins and seeds were in contact for two extra days. Once the wine was pressed, a small volume (20 L) was taken to the laboratory and inoculated with malolactic bacteria (Viniflora-Oenos, CHR Hanse, Denmark). After no malic acid was observed by paper chromatography, the wine was filtered through 0.45 µm poly(tetrafluoroethylene) and used for experimentation. Each experiment was performed in duplicate. The initial volume of wine was 3 ml and stored in glass vials. Several variables that could alter the incorporation of malvidin-3-glucoside were studied. Control: Wine ph was modified to 3.6 with 9 N H 2 SO 4. 3 H-malvidin-3-glucoside was added (38 µci/l), flushing with N 2 before closing the vial. Wine was stored in the dark at 20 C. Temperature: Same as control but stored at two different temperatures, 5 C and 35 C. ph: Same as control but the initial ph was adjusted to two different values before the addition of 3 H-malvidin-3- glucoside. In one set the ph was 4.1 (original wine ph), and in the other set the ph was adjusted to 3.1 with H 2 SO 4. Seed extract: Cabernet Sauvignon grape seeds were extracted for 24 hr with acetone:water (70:30) under N 2 atmosphere. After filtering the solids, the acetone was evaporated under reduced pressure at 35 C and the aqueous solution was then freeze-dried. The concentration of tannin was determined by normal-phase HPLC according to Kennedy and Waterhouse (2000) using the gradient described below. An epicatechin standard was used to quantify tannin. By weight, 61% of the seed extract was tannin. The remaining material included flavan-3-ol monomers and gallic

3 Incorporation of Malvidin-3-Glucoside 141 acid. This material was added to the wine so that the concentration of tannin added was 1 g/l. Wine was stored under the same conditions as the control. Copigmentation: Same as control with chlorogenic acid added for a final concentration of 10 mm. Storage was under the same conditions as the control. Analysis. Isoamyl alcohol extraction. Liquid-liquid extraction was performed according to Somers (1971) using smaller volumes. Wine sample (200 µl) was extracted four times with 1 ml of isoamyl alcohol previously equilibrated with a saturated solution of potassium bitartrate. The isoamyl alcohol fraction, containing anthocyanin monomers, was counted by adding 10 ml of Ultima Gold cocktail. Spectrophotometric calculations of the aqueous phase were done by adding model wine solution (13 mm potassium bitartrate, 14% ethanol water solution, adjusted to ph 3.6). A second reading was obtained after addition of 10 µl 6 N HCl to a final ph value of 1.5. After color calculation, 500 µl 0.12 N NaOH was added to neutralize the acid and 10 ml of Ultima Gold cocktail was added to measure tritium incorporation. Radioactivity was determined using a Packard Tri-Carb liquid scintillation analyzer (model 2000; Meriden, CT) by the external standard method and tsie as the quench-indicating parameter. A series of quenched standards was prepared with a wine sample following the isoamyl alcohol procedure and adding a known amount of tritiated water (L Annunziata and Kessler 1998). Reversed-phase HPLC. Quantification of malvidin-3-glucoside during fermentation, wine, and skin extract samples with a standard was conducted with a Hewlett-Packard 1100 MSD instrument (Palo Alto, CA) and a Luna C18 column (5 µm particle size, 250 x 4.6 mm) (Phenomenex, Torrance, CA). Mobile phases were (A) water with 2.5 ml/l trifluoroacetic acid and (B) acetonitrile:water (80:20) with 2.5 ml/l trifluoroacetic acid. The flow rate was 0.8 ml/min and linear gradients were from 25% to 50% B in 20 min, from 50% to 80% in 0.5 min, and 100% B in 2 min. The column was reequilibrated with 25% B for 7 min. Peak area was calculated at 520 nm and malvidin-3-glucoside was identified by retention time and by mass spectrometry [M + = 493] with electrospray-atmospheric pressure ionization source in positive mode. Gas temperature was 350 C, drying gas was 10 L/ min, nebulizer pressure was 60 psi, and capillary voltage was 4000 V. Normal-phase HPLC. Chromatography was a modification of a published method (Kennedy and Waterhouse 2000). Ethanol contained in 200 µl of wine was evaporated with a stream of nitrogen. The remaining aqueous solution was added to a C18 solid-phase extraction cartridge (100 mg; Supelco, St. Louis, MO) previously conditioned with 2 ml of methanol followed by 2 ml of water. The stationary phase was then washed with water. The cartridge was allowed to dry before adding methanol to elute the retained material. The collected material was dissolved in methanol: methylene chloride (1:1) to a 1 ml final volume and injected in a Hewlett-Packard 1090 model coupled with a photodiode array detector. The column was a Luna Silica (particle size 5 µm, 250 mm x 4.6 mm) (Phenomenex, Torrance, CA), protected with a guard column (4 mm x 3mm) containing the same material. Injection volume was 250 µl; eluent was monitored at 280 and 520 nm. This method uses a binary gradient with mobile phases: (A) methanol:formic acid:water (97:2:1) and (B) methylene chloride:methanol:formic acid: water (83:14:2:1), both containing 20 mm heptanesulfonic acid. The flow rate was 0.75 ml/min and linear gradients were from 0% to 24% A in 30 min, 24% to 100% in 5 min, and 100% A for 10 min. The column was re-equilibrated with 100% B for 10 min. Each sample was injected three times and eluent was collected with a fraction collector model 202 (Gilson, Middleton, WI), collecting fractions every 5 min. Methylene chloride and some of the methanol contained in each fraction were evaporated, 10 ml of Scintiverse E was added, and the sample counted. Radioactivity was calculated as described in the isoamyl alcohol extraction section. A series of quenched standards was prepared with a wine sample fractionated by chromatography as indicated above and adding a known amount of tritiated water. Results and Discussion Fermentation experiment. Malvidin-3-glucoside and the other anthocyanins reached a maximum after day 3 and then decreased throughout the rest of the fermentation (Figure 1). Based on acetone extraction of the skins, the maximum concentration measured (78 hr) was only 58% of the total amount of malvidin-3-glucoside available for extraction. The concentration of anthocyanins during fermentation has been previously well documented (Castellari et al. 1998, Mayen et al. 1994, Somers and Evans 1979). Extraction (maceration) is responsible for the initial rise in pigment concentration, but after reaching a maximum, it quickly starts to decrease. The assumption has been that anthocyanins condense with polymers resulting in polymer pigments during fermentation. This conclusion is solely based on the observations made from wine aging. Figure 1 Concentration of malvidin-3-glucoside (mg/l) and radioactivity (µci/l) during fermentation, after press, and aging for 10 months.

4 142 Zimman and Waterhouse The procedure to add 3 H-malvidin-3-glucoside ensured a proper distribution of the radioactive material into all the liquid before contact with the solids and did not attempt to reproduce any winemaking practice. The first sample that measured radioactivity was obtained before pouring the liquid back into the flask containing the solids, and it was used to calculate the recovery of the added radioactivity in following samples. After 24 hr, the concentration of radioactive material in solution decreased by 52%, whereas the concentration of malvidin-3-glucoside only decreased by 15%. From that point to the end of the fermentation the concentration of pigment and radioactivity continued to decrease. Finally, 10 months of aging caused a decline in malvidin-3-glucoside from 239 to 92 mg/l, but the radioactivity per unit volume did not change. The initial rapid decrease of the labeled 3 H-malvidin-3- glucoside a day after the addition suggested a rapid equilibration of the new malvidin between solution and an association with the solids. Further work is required to identify what reversible interaction is responsible for precluding anthocyanins from the solution at this stage of the fermentation. The second process observed during fermentation was slower and indicated a steady decrease of malvidin-3- glucoside in solution, while there was also a small decrease in the amount of radioactivity. It is difficult to assess the cause of tritium decrease, though it may be related to losses by precipitation of adsorptive materials, such as yeast lees (Vasserot et al. 1997). The last stage (albeit at 5 C), which showed a sharp decrease in malvidin-3-glucoside yet no change in radioactivity, showed that the loss of anthocyanins during early aging was due not to precipitation but to chemical reactions to new forms. Aging experiment. The total amount of tritium in solution was measured 8 months after the addition of 3 H- malvidin-3-glucoside (10 months after the end of fermentation) (Table 1). Under the control conditions, the concentration of the radioactivity dropped by 37%, while at 35 C the losses were 54% and at 5 C the losses were only 14%. While this first experiment did not measure radioactivity in any residues or precipitates, it is expected that the losses were due to precipitation of polymer, an event accelerated by higher temperatures. Seed addition and the highest ph were significantly higher than the control by 19% and 13%, respectively. Chlorogenic acid addition and ph 3.1 had values similar to the control experiment. Anthocyanin and their presumed reaction products in solution can be divided into two groups (Figure 2) based on their partitioning between water and isoamyl alcohol, the latter removing most of the monomeric anthocyanins, but also some other materials. Measurements by reversedphase HPLC indicated that only 1.5% of malvidin-3-glucoside remained in the water fraction after four isoamyl alcohol extractions. Comparison of the amount of radioactivity partitioned into the aqueous fraction after 8 months of aging also indicated differences caused by the variables studied. Seed addition, which had 19% more radioactivity overall, had 30% more radioactivity in this aqueous fraction than that found in the control. That would be expected if the addition increased the amount of tannin reaction products, as these are not effectively removed by the extraction. The chlorogenic acid treatment also had a higher concentration, but the 11% increase was small. Surprisingly, there were no differences observed among the three ph experiments studied. These results, together with the small differences in total radioactivity, suggest that acid may not be involved in the rate-limiting step in the pigmentation of tannin. Interestingly, the temperature extremes of 5 C and 35 C had similar values in the aqueous fraction and both had 30% less radioactivity than the control. However, the drastic differences in total radioactivity and other data imply that this similarity was coincidental. In the case of the isoamyl alcohol fractions, the 5 C treatment clearly had the highest amount of tritium compared to the other treatments, with more than twice the concentration of tritium compared to the control. The other experiments had almost equal amounts of radioactivity. Similarities in the isoamyl alcohol fractions may be caused by the fact that this fraction also contains anthocyanin derivatives. Therefore, small differences in anthocyanin concentrations would not be measureable. But the higher levels of radioactivity in the 5 C treatment clearly showed that the wine had much higher concentrations of unreacted anthocyanins. When the ph of the aqueous fraction was shifted by addition of HCl, all the absorbances increased (Figure 3). For most samples the increase was ~23%, but the 5 C sample showed a much higher increase of 94% and the 35 C sample showed a small increase of only 13%, indicating that this anthocyanin-free material is ph responsive in some cases but not in others and that there is a diversity of structures in pigmented tannin. Thus, some of the material was possibly malvidin-3-glucoside, such as that recovered by thiolysis as found by Remy et al. (2000). Table 1 Sum of radioactivity found in aqueous and isoamyl alcohol fractions (µci/l) in wines aged under different conditions after 8 months aging. Initial amount: 38 µci/l. Control Seed Chlorogenic ph 3.6; 20 C ph 4.1 ph 3.1 addition acid addition 5 C 35 C 24.1 ± 1.1d a 27.2 ± 1.7bc 24.1 ± 1d 28.6 ± 0.6b 25.8 ± 1.5cd 32.8 ± 2.4a 17.3 ± 1.1e a Means followed by the same letter are not significantly different (LSD, 0.05).

5 Incorporation of Malvidin-3-Glucoside 143 Normal-phase chromatography with an ion-pair reagent offers a different approach to understanding the composition of the radioactive products formed during aging. In this system, anthocyanins (including malvidin-3-glucoside) elute in fraction A (10 to 15 min) or before, and the pigmented polymers elute afterward (fractions B to F), approximately in size order. A cacao seed extract standard provides a reference to the size of the material eluted in each fraction. Cacao tannins are composed of epicatechin units only, and oligomers elute as: dimer in fraction B; trimer in fraction C; tetramer, pentamer, and hexamer in fraction D; and heptamer and octamer in fraction E. For simplicity, fractions B to E are referred to as oligomer fractions and F as a polymer fraction. The substitution of epicatechin by another flavonoid (such as an anthocyanin or epicatechin gallate) as one of the units of the oligomer is expected to increase the retention time (Kennedy and Waterhouse 2000). In addition, the high values of radioactivity and absorbance in fraction F are caused by a jump in the gradient, which favors the remaining material to be eluted rapidly. The peak area of each collected fraction is measured in absorbance units at 520 nm from the chromatogram. Area values are converted to equivalents of malvidin-3-glucoside chloride. Four months after adding 3H-malvidin-3-glucoside, wine aged at 5 C had incorporated small amounts of 3H-labeled material in all oligomer fractions, as well as in the polymer fraction F. Nevertheless, most radioactivity was as anthocyanin seen in fraction A (Figure 4A). The high error bars in fractions A and B of the 5 C experiment at 7 months are consequences of late elution of 3H-malvidin-3-glucoside in one of the replications. Experiments incubated at higher temperatures incorporated more radioactivity into oligomer and polymer fractions and greatly decreased the anthocyanin fraction. Three additional months of aging did not produce large differences in the oligomer and polymer fractions at 5 C, but at 20 C and 35 C all fractions decreased, with the A Figure 2 Radioactivity (µci/l) in aqueous (x-axis) and organic phase (y-axis) under different conditions after 8 months of aging. B Figure 3 Absorbance at 520 nm of the aqueous fraction at ph 3.6 (lower value) and ph 1.5 (upper value) in wines aged under different conditions for 8 months. Error bars for x-axis are not shown. Figure 4 Radioactivity (A) in µci/l and absorbance (B) in mg/l malvidin3-glucoside equivalents of wine fractionated by normal-phase HPLC during aging at different temperatures.

6 144 Zimman and Waterhouse exception of F at 20 C. The absorbance of the fractions before collection and the values for the wine before it was spiked with tritiated malvidin (time 0) are shown in Figure 4B. Higher temperatures and longer aging clearly had an effect on the anthocyanin fraction reducing the absorbance values. Different trends are observed in oligomer fractions. In fractions B and C, the experiment aged at 5 C had similar values as time 0, but temperature and aging decreased the absorbance. In fractions D, E, and F, there were samples where absorbance was higher than the time 0 value. For instance, in fraction D the experiment stored at 5 C had higher values than the starting wine after 4 and 7 months of aging. In fraction E only 35 C was lower than time 0, and in fraction F all samples were higher than the starting wine. Differences among the ph experiments after 4 months (Figure 5A) were not noticeable in fractions C to F, indicating that similar amounts were present. In contrast, a trend was observed for the absorbance, where lower ph produces oligomers and polymers with higher absorbances in HPLC solvent (Figure 5B). Interestingly, the addition of seed tannins resulted in smaller amounts of radioactivity incorporated into the polymer fraction (Figure 6A). Nevertheless, the absorbance was similar to the control (Figure 6B). Chlorogenic acid additions did not show any difference in amounts incorporated or absorbance measured. Amounts of anthocyanin incorporated into water-soluble tannin structures can be calculated based on the specific activity of malvidin-3-glucoside in wine and the number of counts observed in the aqueous fraction or in the oligomer and polymer fractions by HPLC. The amounts in the aqueous fraction after isoamyl extraction were between 153 mg/ L (35 C) and 319 mg/l (seed addition) at 8 months; however, if isoamyl alcohol removes some of the products, then chromatographic analysis gives a better estimate of incorporation. For the chromatographic separation after 7 months of aging, the addition of fractions B to F accounted for 273 mg/l (35 C) to 484 mg/l (seed addition). Therefore, 41% (35 C) to 73% (seed addition) of malvidin-3-glucoside is in- A A B B Figure 5 Radioactivity (A) in µci/l and absorbance (B) in mg/l malvidin3-glucoside equivalents of wine fractionated by normal-phase HPLC in experiments with different ph (after 4 months). Figure 6 Radioactivity (A) in µci/l and absorbance (B) in mg/l malvidin3-glucoside equivalents of wine fractionated by normal-phase HPLC in experiments with seed and chlorogenic acid additions and the control (after 4 months).

7 Incorporation of Malvidin-3-Glucoside 145 corporated into tannins after the addition of radioactive malvidin-3-glucoside. At 4 months the amounts were between 564 mg/l (85%) in the control and 252 mg/l (38%) in the 5 C treatment. Although oxygen was not included as one of the variables in the aging experiments, it appears to affect the amount of radioactivity found in solution. All results presented here are from experiments opened for analysis during the course of the aging process. Another set of samples was left unopened for approximately the same period of time (8 months) and analyzed by the isoamyl alcohol extraction procedure. There was no replication for the unopened set, but all of the treatments had values of radioactivity in solution much higher than their exposed counterparts, except for the experiment kept at 5 C that had only 1.4 µci/l more in solution (4%). The increases of radioactivity in solution for the unexposed experiments compared to the exposed experiments ranged from 16% (seed addition) to 54% (35 C). The increase in the aqueous fraction ranged from 16% (seed addition) to 89% (35 C). Thus, oxygen exposure clearly had a dramatic effect on the reactions observed and in the future should be directly controlled. Conclusions Radioactive tracers have demonstrated that malvidin-3- glucoside losses during fermentation are likely due to substantial adsorption to the solids. During aging, malvidin-3- glucoside reacts with other substances in wine to yield new higher molecular weight products. A substantial proportion is lost to precipitation, even early in the aging process, and accelerated aging gives rise to substantial losses of the original pigment to apparent precipitation, presumably of the high molecular weight forms. In the future, any precipitate needs close scrutiny. The chemical form of these adducts also changes, with the anthocyanin-like property of increasing absorption in response to lower ph but losing this property at higher aging temperatures. In all cases, the average light absorbance of the resulting pigment was less than the original anthocyanin. These results suggest that more than one form of anthocyanin tannin bonds are present and that the forms that are present change with time. Literature Cited Asenstorfer, R.E., Y. Hayasaka, and G.P. Jones Isolation and structures of oligomeric wine pigments by bisulfite-mediated ionexchange chromatography. J. Agric. Food Chem. 49: Bakker, J., and C.F. Timberlake Isolation, identification, and characterization of new color-stable anthocyanins occurring in some red wines. J. Agric. Food Chem. 45: Benabdeljalil, C., V. Cheynier, H. Fulcrand, A. Hakiki, M. Mosaddak, and M. Moutounet Evidence of new pigments resulting from reaction between anthocyanins and yeast metabolites. Sci. 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