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Effect of Wine pH and Bottle Closure on Tannins

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Eect of Wine pH and Bottle Closure on Tannins Jacqui M. McRae,* Stella Kassara, James A. Kennedy, # Elizabeth J. Waters, and Paul A. Smith The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia ABSTRACT: The impact of wine pH and closure type on color, tannin concentration, and composition was investigated. A single vintage of Cabernet Sauvignon wine was divided into three batches, the pH was adjusted to 3.2, 3.5 or 3.8, and the wines were bottled under screw caps with either SaranTin (ST) or Saranex (Sx) liners. After 24 months, the tannin concentration, tannin percent yield (relating to the proportion of acid-labile interavan bonds), and the mean degree of polymerization (mDp) had decreased signicantly, all of which can contribute to the softening of wine astringency with aging. The higher pH wines contained less percent ()-epicatechin 3-O-gallate subunits, whereas the Sx pH 3.2 wines were signicantly lower in percent yield and mDp than the other wines. Overall, the tannin structure and wine color of the lower pH wines (pH 3.2) bottled under Sx screw caps changed more rapidly with aging than those of the higher pH wines (pH 3.8) bottled under ST screw caps. KEYWORDS: screw-cap liners, tannin composition, wine aging, wine pH, wine tannin INTRODUCTION As red wines age they change in physicochemical properties, particularly those associated with color and astringency. Anecdotal evidence suggests that red wines often decrease in astringency with aging, and this has been corroborated by red wine aging trials 1 and trials involving sensory analysis of vertical series. 2 Hypotheses for the change in red wine astringency with wine aging have been based on analysis of vertical series or on model wine studies and have been ascribed variously to the reduction in tannin concentration due to precipitation, 3 the reduction in tannin size through depolymerization, 2,4 and a reduction in protein binding of aged wine tannins due to a change in tannin structure over time. 1,5 Research into the eect of aging on tannins has been limited to analysis of total phenolic concentrations 1 and analyses of small polyphenol monomers or oligomers. 6,7 To our knowledge, there have been no trials specically monitoring the actual changes in tannin concentration and composition in red wines over time, and therefore the overall eects of aging on wine tannins remain unknown. The color of young red wines is generally a deep purple associated with high concentrations of monomeric anthocyanins, particularly malvidin-3-glucoside, 8 as well as some derived anthocyanins. 9 Red wine aging trials have indicated that with aging, wine color changes to a red-orange due to the decrease in anthocyanin concentrations and the increase in pigmented polymer concentrations from condensa- tion reactions between anthocyanins and proanthocyani- dins 10,11 and, to some extent, the formation of pyranoantho- cyanins. 8,1214 The impact of oxygen and pH on anthocyanin concentrations has been explored previously, 1,6,15 although not in the context of the impacts of pigmented polymer formation on tannin composition. The rates of changes in wine physicochemical properties are dependent upon many factors including the level of oxygen exposure and wine pH. 6 Oxygen can oxidize ethanol to acetaldehyde, which reacts readily with avan-3-ols to increase polymerization 6 and alter the structures of the polymers relative to direct condensation reactions. 16 Studies on wine micro- oxygenation (MOX) have indicated that the rate of decline in anthocyanin concentration is more rapid when the wine is exposed to more oxygen during winemaking. 13,15 MOX has also been shown to stabilize wine color by promoting the formation of pyranoanthocyanins 17 and pigmented polymers. 18 Oxygen ingress via closures, sometimes referred to as nano-oxygen- ation, can aect wine mouthfeel, with greater rates of oxygen ingress reducing astringency intensity after 42 months of aging. 1 Screw caps currently dominate the Australian wine market and can provide dierent rates of oxygen ingress. Saran Tin (ST) closures are highly impervious to oxygen (up to 0.00043 mg/L/day), whereas the multiple layers of the Saranex (Sx) closures allow slight oxygen ingress (up to 0.0273 mg/L/ day). 19,20 Variations in the level of oxygen entering the wine through the closure have been shown to inuence the avor and aroma of red wines and, in some cases, low levels of oxygen, preserving positive fruity aromas 21 and enhancing color stability in rose ́ wines, 22 although insucient oxygen ingress can potentially promote reductive aromas. 20 The impact of dierent screw-cap closures on wine color as well as tannin concentrations and compositions in red wine with aging has not been investigated. The more acidic media of lower pH wines increase the reaction kinetics for many of these reactions, and therefore the concentrations of anthocyanins and small polyphenols decrease more rapidly than in wines of higher pH. 6,23 More acidic wines have also been shown to exacerbate the impact of oxygen exposure on astringency, 1 further suggesting some more rapid reactions in wines with a lower pH. The impact of pH on wine tannin structure has not been explored with wine aging. This project investigated the impacts of wine aging, wine pH, and dierent screw-cap closures on wine color, tannin concen- tration, and composition in a single-vintage Cabernet Sauvignon over 24 months of bottle aging. Received: August 20, 2013 Revised: October 1, 2013 Accepted: November 6, 2013 Published: November 6, 2013 Article pubs.acs.org/JAFC © 2013 American Chemical Society 11618 dx.doi.org/10.1021/jf403704f | J. Agric. Food Chem. 2013, 61, 1161811627
Transcript

Effect of Wine pH and Bottle Closure on TanninsJacqui M. McRae,* Stella Kassara, James A. Kennedy,# Elizabeth J. Waters,⊥ and Paul A. Smith

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia

ABSTRACT: The impact of wine pH and closure type on color, tannin concentration, and composition was investigated. Asingle vintage of Cabernet Sauvignon wine was divided into three batches, the pH was adjusted to 3.2, 3.5 or 3.8, and the wineswere bottled under screw caps with either SaranTin (ST) or Saranex (Sx) liners. After 24 months, the tannin concentration,tannin percent yield (relating to the proportion of acid-labile interflavan bonds), and the mean degree of polymerization (mDp)had decreased significantly, all of which can contribute to the softening of wine astringency with aging. The higher pH winescontained less percent (−)-epicatechin 3-O-gallate subunits, whereas the Sx pH 3.2 wines were significantly lower in percent yieldand mDp than the other wines. Overall, the tannin structure and wine color of the lower pH wines (pH 3.2) bottled under Sxscrew caps changed more rapidly with aging than those of the higher pH wines (pH 3.8) bottled under ST screw caps.

KEYWORDS: screw-cap liners, tannin composition, wine aging, wine pH, wine tannin

■ INTRODUCTION

As red wines age they change in physicochemical properties,particularly those associated with color and astringency.Anecdotal evidence suggests that red wines often decrease inastringency with aging, and this has been corroborated by redwine aging trials1 and trials involving sensory analysis of verticalseries.2 Hypotheses for the change in red wine astringency withwine aging have been based on analysis of vertical series or onmodel wine studies and have been ascribed variously to thereduction in tannin concentration due to precipitation,3 thereduction in tannin size through depolymerization,2,4 and areduction in protein binding of aged wine tannins due to achange in tannin structure over time.1,5 Research into the effectof aging on tannins has been limited to analysis of totalphenolic concentrations1 and analyses of small polyphenolmonomers or oligomers.6,7 To our knowledge, there have beenno trials specifically monitoring the actual changes in tanninconcentration and composition in red wines over time, andtherefore the overall effects of aging on wine tannins remainunknown. The color of young red wines is generally a deeppurple associated with high concentrations of monomericanthocyanins, particularly malvidin-3-glucoside,8 as well assome derived anthocyanins.9 Red wine aging trials haveindicated that with aging, wine color changes to a red-orangedue to the decrease in anthocyanin concentrations and theincrease in pigmented polymer concentrations from condensa-tion reactions between anthocyanins and proanthocyani-dins10,11 and, to some extent, the formation of pyranoantho-cyanins.8,12−14 The impact of oxygen and pH on anthocyaninconcentrations has been explored previously,1,6,15 although notin the context of the impacts of pigmented polymer formationon tannin composition.The rates of changes in wine physicochemical properties are

dependent upon many factors including the level of oxygenexposure and wine pH.6 Oxygen can oxidize ethanol toacetaldehyde, which reacts readily with flavan-3-ols to increasepolymerization6 and alter the structures of the polymers relativeto direct condensation reactions.16 Studies on wine micro-oxygenation (MOX) have indicated that the rate of decline in

anthocyanin concentration is more rapid when the wine isexposed to more oxygen during winemaking.13,15 MOX has alsobeen shown to stabilize wine color by promoting the formationof pyranoanthocyanins17 and pigmented polymers.18 Oxygeningress via closures, sometimes referred to as “nano-oxygen-ation”, can affect wine mouthfeel, with greater rates of oxygeningress reducing astringency intensity after 42 months ofaging.1 Screw caps currently dominate the Australian winemarket and can provide different rates of oxygen ingress. SaranTin (ST) closures are highly impervious to oxygen (up to0.00043 mg/L/day), whereas the multiple layers of the Saranex(Sx) closures allow slight oxygen ingress (up to 0.0273 mg/L/day).19,20 Variations in the level of oxygen entering the winethrough the closure have been shown to influence the flavorand aroma of red wines and, in some cases, low levels ofoxygen, preserving positive fruity aromas21 and enhancing colorstability in rose wines,22 although insufficient oxygen ingresscan potentially promote reductive aromas.20 The impact ofdifferent screw-cap closures on wine color as well as tanninconcentrations and compositions in red wine with aging has notbeen investigated.The more acidic media of lower pH wines increase the

reaction kinetics for many of these reactions, and therefore theconcentrations of anthocyanins and small polyphenols decreasemore rapidly than in wines of higher pH.6,23 More acidic wineshave also been shown to exacerbate the impact of oxygenexposure on astringency,1 further suggesting some more rapidreactions in wines with a lower pH. The impact of pH on winetannin structure has not been explored with wine aging. Thisproject investigated the impacts of wine aging, wine pH, anddifferent screw-cap closures on wine color, tannin concen-tration, and composition in a single-vintage CabernetSauvignon over 24 months of bottle aging.

Received: August 20, 2013Revised: October 1, 2013Accepted: November 6, 2013Published: November 6, 2013

Article

pubs.acs.org/JAFC

© 2013 American Chemical Society 11618 dx.doi.org/10.1021/jf403704f | J. Agric. Food Chem. 2013, 61, 11618−11627

■ MATERIALS AND METHODSChemicals. All solvents used were of high-performance liquid

chromatography (HPLC) grade, all chemicals were of analyticalreagent grade, and water was obtained from a Milli-Q purificationsystem. Acetic acid (100%), acetonitrile, ethanol, formic acid (98−100%), HCl (32%), and H2SO4 (95−98%) were all purchased fromMerck Australia (Kilsyth, VIC, Australia). Acetaldehyde, ammoniumsulfate, ascorbic acid, lithium chloride (LiCl), methyl cellulosepolymer, N,N-dimethylformamide (DMF), potassium metabisulfite,phloroglucinol, sodium acetate, sodium chloride (NaCl), sodiumhydroxide (NaOH), and tartaric acid were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).Wines. Cabernet Sauvignon (Clare Valley, South Australia) wines

were prepared according to standard winemaking practices for bothprimary and secondary fermentations, with a final alcohol concen-tration of 14% v/v ethanol. After cold stabilization, the wine wasdivided into three batches (26 L each), and the pH was adjusted toeither 3.2, 3.5, or 3.8. H2SO4 was used to reduce the pH to 3.2 (18 M,36.7 mL, giving a final concentration of 0.025 M H2SO4 in wine) andto pH 3.5 (1.8 M, 9.75 mL). NaOH was used to give pH 3.8 (1.25 M,241.4 mL, giving a final concentration of 0.012 M NaOH in wine).The differences in the final concentrations of the acid and baseadditions were <10-fold and therefore considered insufficient tosignificantly alter the final ionic strength of the wines.24 Potassiummetabisulfite solution (5%, 13.7 mL) was added to each batch prior tobottling to give an equal addition of 15 ppm of SO2. The free and totalSO2 concentrations (FSO2 and TSO2, respectively) of each batch weremeasured by titration:25 pH 3.2, FSO2 = 40 mg/L, TSO2 = 83 mg/L;pH 3.5, FSO2 = 38 mg/L, TSO2 = 82 mg/L; pH 3.8, FSO2 = 42 mg/L,TSO2 = 85 mg/L. Wines were filtered using Ewkip Z6 polishing gradepad and sterile membrane filters and bottled in 750 mL colorlessbottles and stored at approximately 15 °C away from light.Differentiation in the level of nano-oxygenation was achieved withtwo different screw-cap closures: Saran Tin (ST), allowing minimaloxygen ingress (up to 0.00043 mg/L/day), and Saranex (Sx), allowingslightly greater oxygen ingress (up to 0.0273 mg/L/day).20 Wine andbottles were sparged with N2 prior to bottling, and levels of dissolvedoxygen (DO) and headspace oxygen (HSO) were monitored intriplicate samples (at the start, middle, and end of each batch) forwines at 3 × pH and 2 × closure type using oxyluminescence (PST 3and PST 6 sensors) via a PreSens meter (Nomasens oxygen analyzer,Nomacorc, SA).1 DO levels prior to bottling were 0.33 ± 0.01 mg/L,and these concentrations increased to 0.40 ± 0.04 mg/L after bottlingfor all wines. Headspace volumes were consistently 6.4 mL, and initialHSO was 0.87 ± 0.32 mg/L. Total package oxygen (TPO) levels forall samples were 1.49 ± 0.40 mg/L.For the wine and tannin analyses, a total of nine bottles of wine

were analyzed immediately post bottling, with triplicate wines at 3 ×pH (the impact of the different screw caps was considered negligible atthis stage), and 18 bottles of wine were analyzed at the 6 and 24month time points (i.e., triplicate wines at 3 × pH and 2 × closuretype). Results pertaining to the general trends of wine aging used themean and standard deviation of nine wines at bottling and all 18 winesat the other time points. Results relating to the impacts of wine pH orclosure type used the mean and standard deviation of the triplicateresults for wines at each variable. When no difference was observed forresults from wines of different closure type but the same pH, the meanand standard deviation of all six wines of the same pH was used tohighlight trends pertaining to wine pH alone.Wine Composition. Wines at each pH and of each closure type

were analyzed for tannin concentration and composition, wine color,anthocyanin concentration, and acetaldehyde concentration at 0, 6,and 24 months post bottling (from triplicate samples). Tanninconcentration was measured using the methyl cellulose precipitable(MCP) tannin assay.26,27 Briefly, polymer solution (H2O/0.04%methyl cellulose) or H2O for a control (300 μL) was reacted with thewine sample (25 μL) in a 96-well plate for 3 min (shaken using aplatform shaker for 1 min and settled for 2 min). Saturated ammoniumsulfate solution (200 μL) was then added, and each reaction was

diluted to 1 mL with H2O, shaken for 1 min, settled for 10 min, andcentrifuged at 2000 rpm for 5 min. The tannin concentration wascalculated on the basis of the 280 nm absorbance of the reactionmixture supernatant compared with the control per sample aspreviously described.26,27

Wine color was analyzed using the modified Somers colormeasurements as previously reported.26,27 These analyses gave thewine color density (WCD, the combined absorbance of the wine at420 nm and at 520 nm, referred to as A420 and A520, respectively), thehue (A420/A520), and the SO2 nonbleachable pigments (A520:sulfite, A520after reaction with a buffer solution containing 0.375% w/v sodiummetabisulfite, 0.5% w/v tartaric acid in 12% v/v EtOH). The totalanthocyanin concentrations of the wine samples were determined bycomparing the A520 after reaction with 1 M HCl solution and theA520:sulfite with a malvidin-3-glucoside (M3G) standard curve (to giveconcentration in mg/L M3G equivalents), and total phenolics weremeasured as the absorbance at 280 nm after reaction with 1 M HCl(A280). M3G concentrations were determined using high-performanceliquid chromatography (HPLC) as previously described.26−28

For the acetaldehyde concentration analysis, wine samples (10 mL)were spiked with internal standard (100 μL) containing d4-acetaldehyde and d7-acetoin in a sealed vial prior to SPME GC-MSanalysis using an Agilent 6890N gas chromatograph equipped with aGerstel MPS2 multipurpose sampler and coupled to an Agilent 5973mass selective detector. An SPME fiber was exposed to each sample at35 °C for 10 min and then injected into a split/splitless inlet fittedwith an SPME inlet liner (0.75 mm i.d.), and the sample was allowedto desorb for 10 min (during which the inlet was held at 220 °C insplitless mode). Separation was achieved with a Restek Stabilwax-DAcolumn (30 m × 0.180 mm, 0.18 μm film thickness) using helium(ultrahigh purity) as the carrier graph with a linear velocity of 43 cm/sand a flow rate of 1.4 mL/min in constant flow mode. The oventemperature was held at 40 °C for 4 min, increased to 90 °C at 5 °C/min, then heated at 40 °C/min to 240 °C, and held for 5 min. Themass spectrometer quadrupole temperature was set at 150 °C, thesource was set at 230 °C, and the transfer line was held at 250 °C.Positive ion electron impact spectra at 70 eV were recorded inselective ion monitoring (SIM) mode (relative EM volts).

Tannin Isolation and Fractionation. Tannin was isolated fromeach wine at each sample point using Toyopearl media as previouslydescribed.29 Briefly, Toyopearl HW-40F size exclusion medium(Optigen Scientific Pty Ltd., Port Adelaide, SA, Australia) in a glasscolumn (50 × 450 mm) was equilibrated with H2O/0.1% v/v formicacid wine prior to loading wine (600 mL). The column was thenwashed with H2O/0.1% v/v formic acid (2 L) followed by 1:1 MeOH/H2O with 0.1% v/v formic acid (approximately 6 L). Tannin waseluted with 2:1 acetone/H2O with 0.1% v/v formic acid (1 L). Thesolvent was removed by rotary evaporator (30 °C) followed by freeze-drying. Tannin samples were stored under nitrogen at −80 °C.

To further measure changes in tannin structure, tannin was alsoisolated and fractionated with solid phase extraction using OASIS HLBSPE cartridges (Waters) as previously described.26,30 Briefly, cartridgeswere activated with MeOH and equilibrated with H2O prior to loading1 mL of wine. After the cartridges were dried with N2 and then washedwith acetonitrile containing 5% v/v 0.01 M HCl (40 mL), the firsttannin fraction (F2) was eluted with MeOH containing 0.1% v/vformic acid (5 mL) and the second tannin fraction (F3) with formicacid (0.3 mL) followed by MeOH containing 5% v/v H2O (2.7 mL).Tannin fractions were dried under nitrogen at 30 °C and dissolved ineither model wine (14% v/v EtOH,1 mL) for quantification by MCPanalysis, as described above, or MeOH (100 μL) for characterizing thetannin structure, as described below.

Tannin Characterization. Isolated tannin and tannin fractionswere characterized using gel permeation chromatography (GPC) foraverage tannin molecular mass31 and using depolymerization reactionswith phloroglucinol for subunit composition.32 For GPC analysis,tannin samples (10 g/L MeOH) were diluted 1:4 with DMF andanalyzed using a series of two columns (PLgel, 300 × 7.5 mm, 5 μm,500 Å then 103 Å, Polymer Laboratories, USA), with an isocraticmobile phase of DMF solution (DMF/0.15 M LiCl/10% acetic acid).

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf403704f | J. Agric. Food Chem. 2013, 61, 11618−1162711619

Retention times were compared to a standard curve of fractionatedpreveraison grape skin tannins as previously described.26,33 Theaverage molecular weight was deemed to be the retention time at theelution of 50% of the tannin peak area, relative to the standard curve.For the depolymerization reactions, tannin samples (25 μL, 10 g/L inMeOH) were reacted 1:1 with phloroglucinol solution (phloroglucinol(100 g/L) in MeOH with 20 g/L ascorbic acid and 0.2 N HCl) at 50°C for 20 min, prior to the addition of sodium acetate solution (70mM, 150 μL). Reaction products were analyzed using HPLC aspreviously described34 to identify and quantify subunits. These resultsgave the mean degree of polymerization (mDp, proportion ofextension to terminal subunits), the percent of (−)-epigallocatechinsubunits in the polymer, the percent of (−)-epicatechin 3-O-gallatesubunits (% ECG), and the percent yield of the reaction, which wascalculated by subtracting the total concentration of individual subunitsfrom the concentration of tannin used in the reaction.32

Statistical Analysis. All significance tests were conducted usingGraphPad Prism statistics software. Student’s t test was used forcomparing differences in wine composition with aging by analyzing themeans and standard deviations of all wines at each time point(regardless of pH or closure type). These results were incorporatedinto Tables 1−3. ANOVAs and Tukey analyses were used forcomparing the triplicate results from wines of different pH value andclosure type. Results were incorporated into Tables 4 and 5.

■ RESULTS AND DISCUSSIONGeneral Aging-Related Trends Associated with Wine

Tannin and Color. The color and tannins of the single-vintageCabernet Sauvignon (CAS) wines bottled at a range of winepH and under two types of screw-cap closures were monitoredfor 24 months. Changes were observed as a consequence ofwine aging, and in some cases these changes were influenced bywine pH and/or closure type. In this section, results arehighlighted that indicate general trends in aging, particularly fortannin concentration and composition, which have notpreviously been studied in detail. Wine and tannin parametersthat varied significantly with pH and/or closure type arediscussed in greater detail in later sections.To compare the general age-related trends in wine tannins,

the results from each of the 18 bottles of wine at each samplepoint were averaged, and the means and standard deviations ofall samples are reported in Table 1. Wine tannin concentrationschanged substantially over the 24 months of wine aging. Tanninconcentrations decreased significantly as measured using theMCP assay (Table 1), and this correlated well with thegravimetric recovery (R2 = 0.913). The decline in concentrationin this experiment suggested that the tannin in these wines mayhave precipitated or degraded into small oligomers aspreviously suggested.3,4 Tannin concentration has been directlylinked to astringency intensity35,36 and, therefore, the reported

decrease in wine astringency with aging 2 may be due, at least inpart, to less tannin in the wine.The structure of the wine tannins also changed substantially

with wine aging, most notably in the percent yield (proportionof acid-labile bonds), molecular masses (MM) as measured at50% elution by GPC, color incorporation, and subunitcomposition (Table 1). Most notably, the percent yield oftannin decreased significantly over the 24 months. The percentyield is calculated from the concentrations of cleaved catechin,epicatechin epigallocatechin, and epicatechin gallate subunitsafter depolymerization reactions and compared to the originalconcentration of tannin in the reaction. Reactions that occur aswines age will reduce the percent yield of wine tanninsincluding oxidation, intramolecular bond formation,37 and A-type linkages or ether linkages involving the B-ring,38 as well asthe incorporation of anthocyanins via either direct A-T or morecomplex interactions.39−41 The formation of pigmented tanninswas observed in this instance as a significant increase in thecolored proportion of wine tannin, as measured by the GPCpeak area at 520 nm as a percentage of peak area at 280 nm,over the 24 months post bottling (Table 1). This wasconsistent with previous reports of a greater “degree ofredness” in more aged red wine tannin compared with youngerwine tannin.29 The reduction in percent yield and the increasein tannin color incorporation were pH- and closure-dependentas described in the later section.The MM of the wine tannins as measured by GPC decreased

slightly yet significantly after 24 months of aging, althoughthere were no appreciable differences in size distributions(Table 1). The mDp as determined using depolymerizationreactions also decreased slightly with aging. These results werepH-dependent as discussed later. Measuring the MM of winetannins using either method has limitations. A decrease incalculated mDp alone can be indicative of oxidation andstructural rearrangements that result in a reduction in theproportion of the polymer chain that can be cleaved (percentyield), rather than an actual decrease in overall MM,37,42 as hasbeen observed in previous analyses of vertical series.26 Thiswould lead to a disproportionate number of terminal subunitscompared with extension subunits (those that react withphloroglucinol upon cleavage during the depolymerizationreactions), reducing the determined mDp. Conformationalchanges in the tannin, such as polymer branching, may induce adifferent GPC response when measured at the sameconcentration. In this instance, the different CAS wine tanninsshowed no differences in GPC peak area, and the MMs asmeasured using both phloroglucinolysis and GPC indicated

Table 1. Tannin Concentration (Measured Using the MCP Tannin Assay) and Composition over 24 Months of Wine Aginga

at bottlingb 6 monthsc 24 monthsc

tannin concentration (g/L EC equiv) 1.86 ± 0.02a 1.69 ± 0.14a 1.29 ± 0.07b% yieldd 55.3 ± 3.3a 43.9 ± 4.8b*g 37.6 ± 4.4c*% (−)-epigallocatechin subunitsd 27.5 ± 0.8 27.5 ± 0.5 26.8 ± 0.6% (−)-epicatechin-3-O-gallate subunitsd 7.4 ± 0.1a 6.8 ± 0.3b* 6.1 ± 0.6c*mean degree of polymerization (mDp)d 10.0 ± 0.1a 10.4 ± 0.5a 9.1 ± 0.7bMM (g/mol)e 2854 ± 62a 2729 ± 144ab 2598 ± 134b*g

% colored (520:280)e,f 6.1 ± 0.3a 6.7 ± 0.4b 7.6 ± 0.4caResults in the same row that are significantly different (p < 0.05) are indicated with different letters. bResults are given as the mean of nine winesamples (triplicate wines at 3 × pH) ± one standard deviation. cResults are given as the mean of the 18 wine samples (triplicate wines at 3 × pH and2 × closure) ± one standard deviation. dCalculated using phloroglucinolysis. eAverage molecular mass as determined using gel permeationchromatography (GPC) at 50% tannin elution. fGPC peak area at 520 nm as a percentage of peak area at 280 nm. gAn asterisk denotes results thatwere influenced by pH and/or closure type after 24 months.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf403704f | J. Agric. Food Chem. 2013, 61, 11618−1162711620

that the overall tannin MM decreased with wine aging over 24months. This may be due to the cleavage of interflavan bonds inthe mildly acidic wine medium43 or potentially from someprecipitation. Smaller tannins have both been associated withsofter astringency,29,44 and pigmented tannins have been ratedas less astringent than noncolored tannins.45 Therefore, thedecrease in tannin MM and increase in color incorporation intotannin with wine aging may also contribute to the reduction inperceived astringency. The proportion of (−)-epicatechin-3-O-gallate moieties, subunits that are found more prominently ingrape seeds than skins,33,46 also decreased significantly over 24months of aging. These reductions were pH- and closure-dependent as described in the later section. The proportions ofthe grape skin tannin-like subunits, (−)-epigallocatechinmoieties, were unaffected. Tannin with (−)-epicatechin-3-O-gallate subunits are reportedly coarser compared with thosecontaining more (−)-epigallocatechin subunits,47 and grapeskin tannin concentrations have been positively associated withwine quality in young red wines.48 Thus, the reduction in theproportion of grape seed tannin-like subunits relative to thegrape skin-like tannin subunits may also contribute to the softerastringency of aged wine.To delve deeper into changes in tannin compositions, wine

tannin from each sample was separated into two fractions, F2and F3, using solid phase extraction (SPE),26,30 and theconcentrations and compositions of each fraction were analyzed(Table 2). F3 tannins were consistently more abundant andsignificantly larger than the F2 tannins. At bottling, theproportion of F2 tannin accounted for only 9.8 ± 1.2% w/wof the wine tannin, and this proportion increased significantly

to 23.4 ± 2.6% w/w after 24 months of aging, which was similarto the ratios observed in CAS wine of ≥2 years old.26 Tanninfractions separated using liquid−liquid fractionation with waterand n-butanol have produced fractions with characteristicssimilar to those of F2 and F3.26,29 Sensory analysis of theseliquid−liquid fractions indicated that the more hydrophilic F3-like tannins were more astringent compared to F2-like tanninfractions that were more bitter. Therefore, the increase in theproportion of F2 tannin with wine aging, and consequentdecrease in the proportion of F3 tannin, may contribute to adecrease in overall astringency. The mDp and percent yield forboth fractions decreased significantly after 24 months (Table 2)as was observed for the total wine tannin (Table 1). Thereduction in percent (−)-epicatechin-3-O-gallate subunits wasmore pronounced in the F2 tannins, whereas the percent(−)-epigallocatechin subunits remained relatively constant inboth fractions. The impact of closure type and pH on F2 andF3 tannins is discussed in the later section.Wine color was measured using the modified Somers color

assay27 to give wine color density (WCD), hue, total phenolics,the amount of SO2 nonbleachable pigments, and totalanthocyanins (Table 3). WCD decreased significantly overthe 24 months of aging, and hue increased significantly, whichwas consistent with previous studies on red wine aging.1,26

Total phenolics concentrations decreased in proportion withthe decline in tannin as measured using the MCP tannin assay,further confirming the decrease in tannin concentration. Theamount of total phenolics directly influences the proportion ofSO2 nonbleachable pigments (% NB pigments), as wine tanninincludes pigmented polymers. By correcting for the decrease in

Table 2. Characteristics of Tannin Fractions, F2 and F3, over 24 Months of Wine Aging As Measured Using the MCP TanninAssay (% Total Tannin) and Phloroglucinolysisa

F2 F3

bottlingb 24 monthsc bottlingb 24 monthsc

% total tannin 9.8 ± 1.2a 23.4 ± 2.6b 90.2 ± 1.2c 76.6 ± 2.6dmDp 5.3 ± 0.3a 3.4 ± 0.2b 10.8 ± 0.3c 7.9 ± 0.6d*d

% yield 55.3 ± 7.8a 28.6 ± 6.0b 55.4 ± 3.1a 36.4 ± 4.6c*% (−)-epigallocatechin subunits 14.8 ± 2.6a 17.8 ± 2.5a 27.0 ± 0.3b 25.0 ± 1.1c% (−)-epicatechin-3-O-gallate subunits 7.0 ± 0.7a 2.7 ± 0.3b 7.8 ± 0.3a 5.7 ± 0.6cd

aResults in the same row that are significantly different (p < 0.05) are indicated with different letters. bResults are given as the mean of nine winesamples (triplicate wines at 3 × pH) ± one standard deviation. cResults are given as the mean of the 18 wine samples (triplicate wines at 3 × pH and2 × closure) ± one standard deviation. dAn asterisk denotes results that were influenced by pH and/or closure type after 24 months.

Table 3. Red Wine Color (Determined Using the Modified Somers Color Assay), Acetaldehyde, and SO2 Concentrations over24 Months of Wine Aginga

at bottlingb 6 monthsc 24 monthsc

WCD (AU) 10.9 ± 0.8a 10.3 ± 0.4a 8.2 ± 0.2bhue (AU) 0.60 ± 0.02a 0.63 ± 0.03a 0.71 ± 0.01btotal phenolics (AU) 42.0 ± 0.4a 43.5 ± 1.6a 36.7 ± 0.8bNB pigments (AU)d 2.73 ± 0.04a 2.86 ± 0.05b 2.71 ± 0.06a% NB pigments (% TP)e 6.50 ± 0.06a 6.57 ± 0.18a 7.39 ± 0.23b*g

total anthocyanins (mg/L M3G equiv)f 385.5 ± 4.7a 368.2 ± 26.4a 219.3 ± 23.7b*acetaldehyde (mg/L) 4.73 ± 0.42a 1.44 ± 0.69b ndh

free SO2 (mg/L) 42.0 ± 4.3a 28.1 ± 2.1b 18.3 ± 2.9cctotal SO2 (mg/L) 84.3 ± 2.6a 64.0 ± 3.4b 53.8 ± 3.3c

aResults in the same row that are significantly different (p < 0.05) are indicated with different letters. bResults are given as the mean of nine winesamples (triplicate wines at 3 × pH) ± one standard deviation. cResults are given as the mean of the 18 wine samples (triplicate wines at 3 × pH and2 × closure) ± one standard deviation. dSO2 nonbleachable pigments.

eCalculated as a percent of the total phenolics (%TP). fTotal anthocyaninconcentrations calculated as malvidin 3-glucoside equivalents. gAn asterisk denotes results that were influenced by pH and/or closure type after 24months. hNot determined for these samples.

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total phenolics, the % NB pigments increased over the 24months (Table 3), aligning well with the proportion of color inwine tannin as measured using GPC (Table 1). Theconcentrations of total anthocyanins were reduced to aroundhalf of the original concentrations within 24 months of wineaging with the formation of more stable pigments,49,50 and thisdecrease was pH-dependent, as described in the later section.The rate of pigmented tannin formation is enhanced by theincorporation of fermentation and oxidation products, such asacetaldehyde, into the polymer.51,52 Acetaldehyde concen-trations decreased significantly with wine aging, and the rate ofdecline was pH- and closure-dependent at 6 months, asdiscussed in detail in the later section. Sulfur dioxide (SO2)concentrations were initially adjusted to minimize the differ-ences in free and total SO2 concentrations induced by creatingwines of different pH values. The level of both free and totalSO2 decreased significantly over the 24 months of aging withno significant variation due to pH or closure type. These valueswere similar to those obtained for Shiraz wines with low O2exposure after 360 days,53 highlighting the low rate of oxygeningress of these samples.Impact of Closure Type on Wine Oxygen Concen-

trations. To minimize the amount of HSO in the wines, boththe headspace and screw-cap closure were sparged withnitrogen before the bottles were sealed, and so the HSO that

was measured after bottling was mainly due to oxygen ingressthrough the closure. Both HSO and DO were monitored over24 months of aging (Figure 1). HSO declined continually frombottling over the 2 year trial. Initial variations in HSO betweenSx and ST were not statistically significant, and within 2months, all wines contained similar HSO levels of 0.15 ± 0.04mg/L (Figure 1a). After 5 months, significant differences inHSO levels for the two screw-cap closures were revealed usingthe more sensitive PST6 sensor, with 0.020 ± 0.001 and 0.007± 0.001 mg/L for the Sx and ST wines, respectively (Figure1b). HSO levels remained fairly constant for both closures from9 months onward, with 0.012 ± 0.002 mg/L for the Sx winesand no detectable HSO for the ST wines.Wine DO levels did not vary with closure or pH but did

show some fluctuations across the 24 months (Figure 1c,d).The DO initially decreased in the first 4 weeks post bottling(Figure 1c) and then began to gradually increase from 0.011 ±0.001 mg/L to peak at 0.017 ± 0.002 mg/L at 4 months ofaging (Figure 1d). This may have been due to the rate ofoxygen absorption into the wine being greater than the rate ofconsumption over this time.53 The DO of all wines continuedto decline after 4 months to 0.0003 ± 0.0002 mg/L at 12months and contained no detectable DO by 24 months. Thetotal package oxygen (TPO) for all wines showed similaroxygen concentrations for both closure types in the first 12

Figure 1. In-bottle oxygen concentrations across the aging trial. Initial readings (higher O2 concentrations) were taken using the PST3 sensor for (a)headspace oxygen (HSO), (c) dissolved oxygen (DO), and (e) total package oxygen (TPO). Later readings (lower O2 concentrations) weremeasured using the PST6 sensor for (b) HSO, (d) DO, and (f) TPO. Results are given as the mean of nine wine samples (triplicate wines at 3 ×pH) ± one standard deviation.

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weeks after bottling (Figure 1e), and from 5 months onward(Figure 1f), Sx wines were exposed to significantly higheroxygen concentrations than the ST wines (0.03 ± 0.00 and 0.02± 0.001 mg/L, respectively).Impact of Closure Type and pH on Tannin

Composition and Wine Color. The single-vintage CASwine was divided into three batches just prior to bottling, andthe pH was adjusted to 3.2, 3.5, or 3.8 using either NaOH orH2SO4. Each batch was bottled under two different screw-capclosures: Sx, to allow slight oxygen ingress, and ST, to minimizeoxygen exposure. Analysis of the wine immediately postbottling indicated that the pH adjustment did not cause arapid change in tannin or color, because all samples producedsimilar characteristics.In the first 6 months post bottling, both wine pH and closure

type began to affect wine tannin structures and color attributes(Figure 2). The percent yields (proportions of acid-labileinterflavan bonds) of the Sx wine tannins were significantlylower than those of the ST wine tannins (Figure 2d), and thiscorresponded to lower acetaldehyde and lower anthocyaninconcentrations in these wines. Anthocyanins are not observedas cleaved subunits using the phloroglucinol assay32,34 andtherefore are not accounted for in the percent yield calculations.Thus, the incorporation of anthocyanins into the tanninstructure, forming pigmented tannin, will lower the percentyield. Acetaldehyde-modified oligomers can also be incorpo-rated into the wine tannin, inducing structural rearrangementreactions that also reduce the tannin percent yield.16,54

The Sx wines contained fewer anthocyanins than the STwines (Figure 2a), further indicating that polymerizationreactions had incorporated some anthocyanins into thepigmented tannin structure. The decrease in anthocyaninconcentrations is also associated with the formation ofpyranoanthocyanins as well as the degradation of anthocyanins,as has been previously noted for more stable pigments, such as

indicating that oxygen exposure can facilitate the degradation ofanthocyanins and may enhance formation of more stablepigments, including pigmented tannins and, as previouslyreported for micro-oxygenation (MOX) trials.6 Wine pH alsoinfluenced the concentration of anthocyanins in the Sx wines,with lower anthocyanin concentration in the Sx pH 3.2 winescompared to the Sx pH 3.8 wines. This effect was not observedin the ST wines, highlighting that a combination of smallamounts of oxygen and low pH promotes pigmented tanninformation.49,55 Wine hue began to increase in some wines after6 months of aging (Figure 2b). This change was pH-dependent,although the effect was different for the different closure wines.In the Sx wines, hues were slightly greater for pH 3.8 winesthan for pH 3.2 wines, whereas the reverse was the case for theST wines (Figure 2b), highlighting the impact of low oxygenexposure on wine color. The change in hue may be indicative ofoxidized polyphenol formation and the development of yellowpigments56 increasing the 420 nm absorbance compared with520 nm absorbance.Acetaldehyde can be consumed in wine by reactions with

flavan-3-ol monomers to form ethylidene-linked flavanololigomers and reactions with anthocyanins to form derivedpigments such as pyranoanthocyanins.11,54,57,58 The reductionin acetaldehyde concentrations in all wines after 6 months(Figure 2c) suggested that the rate of consumption was greaterthan the rate of formation. Acetaldehyde is produced as afermentation product as well as from the oxidation of ethanol.11

Increasing the oxygen concentration of wine can thereforeincrease the acetaldehyde concentration, but this response hasbeen shown to be variable59 and can depend on theconcentration of phenolics in the wine.15 In the presence ofoxygen and a catalyst, o-diphenol-containing flavanols willundergo one- and two-electron oxidation to form reactivemolecules that facilitate polyphenol polymerization. In thisexperiment, slight oxygen ingress in the Sx wines resulted in

Figure 2. Wine and tannin characteristics influenced by pH and closure type after 6 months bottle aging: (a) total anthocyanins (mg/L malvidin 3-glucoside equivalents); (b) wine hue; (c) acetaldehyde concentrations (mg/L) for the wines bottled under SaranTin (ST) or Saranex (Sx) after 6months of aging at pH 3.2, 3.8, and 3.8 and at bottling for comparison; (d) tannin percent yield (related to the proportion of acid-labile interflavanbonds). Results shown as means ± one standard deviation of nine wines at bottling (triplicate wines at 3 × pH) and triplicate wines for each variableat 24 months.

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lower acetaldehyde concentrations than in the ST wines,presumably due to the greater rate of consumption comparedwith formation.51 Wine pH influenced the amount ofacetaldehyde in the ST wines with lower concentrationspresent in the pH 3.5 and 3.8 wines. This effect was notobserved in the Sx wines, potentially due to greater reactionrates in polymer formation, mitigating the impact of wine pHon acetaldehyde.After 24 months of bottle aging, wine pH variation produced

significant differences in total anthocyanin concentrations andNB pigments (Table 4), as well as tannin MM, percent yield,and percent galloylation, with closure type predominantlyinfluencing tannin structure at pH 3.2 (Figure 3). Thedifference between total anthocyanin concentrations in thepH 3.2 and 3.8 wines increased after 24 months of agingcompared with 6 months as more NB pigments formed atlower pH (Table 4). M3G concentrations decreased to lessthan half of the original concentration at bottling in the pH 3.8wine and to almost a fourth in the pH 3.2 wine after 24 months

(Figure 3a), and other anthocyanins showed similar patternsover aging and wine pH (data not shown). The influence ofwine pH on NB pigments and anthocyanins did not result insignificant pH-dependent differences in overall WCD or hue,although it may have affected the tannin structure by varyingthe extent of anthocyanin incorporation.Wine tannin structure was influenced by both pH and

closure type, particularly for the percent yield, MM, andproportion of (−)-epicatechin-3-O-gallate subunits (Figure 3).The pH 3.2 wines formed tannins with significantly lowerpercent yields after 24 months of aging than the pH 3.5 and 3.8wines (Figure 3b). The percent yields of the pH 3.2 Sx tanninswere also significantly lower than the ST tannins at the samepH, demonstrating that low oxygen ingress at lower pH canaffect tannin conformation. Wines bottled under Sx closuresdemonstrated initial rapid decrease in percent yield (within 6months) along with a significant reduction in anthocyanins atpH 3.2 (Figure 2a,d), whereas ST wines demonstrated a moregradual decline in percent yield over the 24 months of aging

Table 4. Red Wine Color Measures (Determined Using the Modified Somers Color Assay) after 6 and 24 Months of Aging ThatWere Significantly Influenced by Wine pHa

months pH 3.2 pH 3.5 pH 3.8

NB pigments (AU) 6 2.88 ± 0.07 2.88 ± 0.03 2.81 ± 0.0224 2.76 ± 0.02 2.73 ± 0.04 2.65 ± 0.05

total phenolics (AU) 6 43.3 ± 0.4 43.9 ± 0.8 43.4 ± 1.424 36.2 ± 1.2 36.6 ± 0.2 37.3 ± 0.4

% NB pigments (% TP)b 6 6.65 ± 0.20a 6.56 ± 0.20b 6.48 ± 0.30c24 7.62 ± 0.22a 7.45 ± 0.12b 7.11 ± 0.11c

total anthocyanins (mg/L M3G equiv)c 6 358.8 ± 3.2a 369.4 ± 7.3b 376.3 ± 5.8c24 192.0 ± 5.0a 218.0 ± 2.1b 247.8 ± 3.9c

aResults in the same row that are significantly different (p < 0.05) are indicated with different letters. Results are given as the mean of six winesamples (triplicate wines at 2 × closures) ± one standard deviation. bCalculated as a percent of the total phenolics (%TP). cTotal anthocyaninconcentrations calculated as malvidin 3-glucoside equivalents.

Figure 3. Wine and tannin compositional characteristics influenced by pH and closure type after 24 months of bottle aging: (a) malvidin 3-glucoside(mg/L); (b) tannin percent yield (related to the proportion of acid-labile interflavan bonds); (c) tannin molecular mass (MM, g/mol); (d) tanninpercent (−)-epicatechin-3-O-gallate subunits (% galloylation) for the wines bottled under SaranTin (ST) or Saranex (Sx) after 24 months of aging atpH 3.2, 3.8, and 3.8 and at bottling for comparison. Results shown as means ± one standard deviation of nine wines at bottling (triplicate wines at 3× pH) and triplicate wines for each variable at 24 months.

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(Figures 2d and 3b). These differences in tannin reactivity dueto closure and pH ultimately led to the observed differences intannin size and percent yield after 24 months, which mayincrease with further aging. Tannin MM as measured by GPCwas relatively consistent across the pH and closure treatmentsexcept for the tannin from the Sx pH 3.2 wines, which wassignificantly smaller than the other wine tannins (2345 ± 25.2g/mol compared with around 2650 g/mol for the other winepH and closure samples after 24 months) (Figure 3c). Thisindicated that the more rapid reactions involving pigmentedtannin formation after 6 months may also produce smallertannins, potentially from the cleavage of interflavan bonds inthe mildly acidic wine medium with slight oxygen ingress.43

The proportion of (−)-epicatechin-3-O-gallate (% ECG)decreased significantly in the pH 3.5 and 3.8 wine tanninsand only slightly in the pH 3.2 wine tannin samples (Figure3d). This was consistent across both closure types andappeared to be independent of percent yield and tannin MM.The same result was also observed in F3 tannin fractions and toa lesser extent in F2 fractions (Table 5). A decline in % ECGhas also been reported with increasing grape seed maturity, andthis has been related to a decrease in percent yield due tooxidation.60 Enzymatic oxidation of pear polyphenols canpotentially lead to polymerization via the galloyl groupinteracting directly with the catechin B-ring,61 and a similarmechanism may be occurring in wines with aging, particularlyat higher wine pH. Tannins with more (−)-epicatechin-3-O-gallate subunits, such as seed tannins, have been associated withcoarse astringent qualities,47 whereas skin tannins have beenpositively associated with wine quality.48 The reduction in theproportion of seed-like tannin subunits with aging at the higherpH without a significant change in the proportion of skin-liketannin subunits may contribute to a softer wine mouthfeel atslightly higher pH.The impact of wine pH and closure type on the F3 tannin

fractions that were isolated from SPE demonstrated the sametrends as the total tannin (Table 5), although no such trendswere observed in the F2 tannins. The mDp of the F3 tanninsfrom the pH 3.2 wines were lower than that of the other wines,especially for those bottled under Sx, and were higher inpercent (−)-epicatechin-3-O-gallate than those fractionsisolated from the higher pH wines regardless of closure type.F3 fractions were dominant in the wine tannins, accounting foraround 76% w/w of the wine tannins after 24 months of aging(Table 2), which is likely to be the reason for the similar trendsobserved in these fractions and the total tannin. Thishydrophilic portion of the wine tannin has also been associated

with greater astringency.29 Therefore, although the F2 tanninswere not influenced by wine pH or closure type, winemakingand storage conditions can still alter the overall tannincomposition and thus influence red wine mouthfeel.In summary, aging red wines in bottles over 24 months

significantly reduced the tannin concentration, tannin MM,tannin percent yield (proportion of acid-labile interflavanbonds), and proportion of grape seed tannin-like subunits((−)-epicatechin-3-O-gallate), all of which can contribute tochanges in wine astringency with aging. Closure type and winepH influenced wine color and tannin structure, and slightdifferences observed after 6 months of aging were indicative ofdifferences in the reaction kinetics of changes in tannincomposition and wine color. Within 6 months post bottling, SxpH 3.2 wines contained lower anthocyanin concentrations andmore NB pigments than ST wines, and after 24 months of wineaging, the anthocyanin concentration was substantially reducedin all wines, particularly those at lower pH. Sx pH 3.2 winescontained tannins with lower mDp and percent yield and agreater proportion of (−)-epicatechin-3-O-gallate subunits.Overall, the tannin structure and wine color of the lower pHwines (pH 3.2) bottled under Sx screw caps changed morerapidly with aging than those of the higher pH wines (pH 3.8)bottled under ST screw caps. Further investigations will revealthe impact of these changes on sensory perception andconsumer preferences. Understanding how different wine-making styles and storage conditions impact tannin composi-tion and thus mouthfeel can enable winemakers to bettermanage the textures of their red wines.

■ AUTHOR INFORMATION

Corresponding Author*(J.M.M.) E-mail: [email protected]. Phone: +6188313 6600. Fax: +61 8 8313 6601.

Present Addresses#(J.A.K.) California State University, 5241 N. Maple Ave.,Fresno, CA 93740, USA.⊥(E.J.W.) Grape and Wine Research and DevelopmentCorporation, P.O. Box 610, Kent Town, SA 5071, Australia.

FundingThe work was conducted at The Australian Wine ResearchInstitute, a member of the Wine Innovation Cluster at theWaite Precinct in Adelaide, and is supported by Australiangrape growers and winemakers through their investment body,the Grape and Wine Research and Development Corporation,with matching funds from the Australian government.

Table 5. Characteristics of Tannin Fractions, F2 and F3, after 24 Months of Aging That Were Significantly Influenced by WinepH or Closure Type (SaranTin (ST) or Saranex (Sx)), As Measured Using Phloroglucinolysisa

tannin fraction characteristic closureb pH 3.2 pH 3.5 pH 3.8

F2 mDpc ST/Sx 3.4 ± 0.2 3.5 ± 0.2 3.4 ± 0.2% yield ST/Sx 30.7 ± 4.7 27.4 ± 3.8 30.5 ± 6.5% (−)-ECGd ST/Sx 3.0 ± 0.2a 2.6 ± 0.1b 2.4 ± 0.3b

F3 mDp ST 7.5 ± 0.2a 8.1 ± 0.2b 8.6 ± 0.1cSx 7.1 ± 0.2a 7.8 ± 0.0b 8.3 ± 0.3c

% yield ST 42.1 ± 3.7 38.5 ± 1.0 41.3 ± 3.5Sx 30.9 ± 2.2a 33.9 ± 3.5ab 38.1 ± 2.1b

% (−)-ECG ST/Sx 6.5 ± 0.1a 5.5 ± 0.1b 5.2 ± 0.1caResults in the same row that are significantly different (p < 0.05) are indicated with different letters. bResults from closures listed as ST/Sx are themean of six wine samples (triplicate wines at 2 × closure) ± one standard deviation. When an individual closure type is given, the results are themean of triplicate wines ± one standard deviation. cMean degree of polymerization. d% (−)-epicatechin-3-O-gallate.

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NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

We express great appreciation to Gemma West of the WICWinemaking Services for wine production, Mark Solomon ofthe AWRI − Metabolomics Facility for acetaldehyde analysis,and the AWRI Commercial Services team for SO2 analysis.

■ ABBREVIATIONS USED

DMF, N,N-dimethylformamide; DO, dissolved oxygen; GPC,gel permeation chromatography; HSO, headspace oxygen;MCP, methyl cellulose precipitable; mDp, mean degree ofpolymerization; MOX, micro-oxygenation; MM, molecularmass; NB pigments, SO2 nonbleachable pigments; SPE, solid-phase extraction; TPO, total package oxygen; Sx, Saranex liners;ST, SaranTin liners

■ REFERENCES(1) Gambuti, A.; Rinaldi, A.; Ugliano, M.; Moio, L. Evolution ofphenolic compounds and astringency during aging of red wine: effectof oxygen exposure before and after bottling. J. Agric. Food Chem.2013, 61, 1618−1627.(2) Chira, K.; Pacella, N.; Jourdes, M.; Teissedre, P.-L. Chemical andsensory evaluation of Bordeaux wines (Cabernet-Sauvignon andMerlot) and correlation with wine age. Food Chem. 2011, 126,1971−1977.(3) Haslam, E. In Vino Veritas − oligomeric procyanidins and theaging of red wines. Phytochemistry 1980, 19, 2577−2582.(4) Cheynier, V.; Duenas-Paton, M.; Salas, E.; Maury, C.; Souquet, J.M.; Sarni-Manchado, P.; Fulcrand, H. Structure and properties of winepigments and tannins. Am. J. Enol. Vitic. 2006, 57, 298−305.(5) McRae, J. M.; Falconer, R. J.; Kennedy, J. A. Thermodynamics ofgrape and wine tannin interaction with polyproline: implications forred wine astringency. J. Agric. Food Chem. 2010, 58, 12510−12518.(6) Kontoudakis, N.; Gonzalez, E.; Gil, M.; Esteruelas, M.; Fort, F.;Canals, J. M.; Zamora, F. Influence of wine pH on changes in colorand polyphenol composition by micro-oxygenation. J. Agric. FoodChem. 2011, 59, 1974−1984.(7) Monagas, M.; Bartolome, B.; Gomez-Cordoves, C. Evolution ofpolyphenols in red wines from Vitis vinifera L. during aging in thebottle. II. Non-anthocyanin phenolic compounds. Eur. Food Res.Technol. 2005, 220, 331−340.(8) Alcalde-Eon, C.; Escribano-Bailon, M. T.; Santos-Buelga, C.;Rivas-Gonzalo, J. C. Changes in the detailed pigment composition ofred wine during maturity and ageing: a comprehensive study. Paperspresented at the 4th Symposium In Vino Analytica Aveiro, In Vino2005. Anal. Chim. Acta 2006, 563, 238−254.(9) Vivar-Quintana, A. M.; Santos-Buelga, C.; Rivas-Gonzalo, J. C.Anthocyanin-derived pigments and colour of red wines. Anal. Chim.Acta 2002, 458, 147−155.(10) Jurd, L. Review of polyphenol condensation reactions and theirpossible occurrence in the aging of wines. Am. J. Enol. Vitic. 1969, 20,191−195.(11) Monagas, M.; Bartolome, B.; Gomez-Cordoves, C. Updatedknowledge about the presence of phenolic compounds in wine. Crit.Rev. Food Sci. Nutr. 2005, 45, 85−118.(12) Wang, H.; Race, E. J.; Shrikhande, A. J. Anthocyanintransformation in Cabernet Sauvignon wine during aging. J. Agric.Food Chem. 2003, 51, 7989−7994.(13) Monagas, M.; Bartolome, B.; Gomez-Cordoves, C. Evolution ofpolyphenols in red wines from Vitis vinifera L. during aging in thebottle. I. Anthocyanins and pyranoanthocyanins. Eur. Food Res.Technol. 2005, 220, 331−340.(14) Brouillard, R.; George, F.; Fougerousse, A. Polyphenolsproduced during red wine ageing. Biofactors 1997, 6, 403−410.

(15) Cano-Lopez, M.; Pardo-Minguez, F.; Schmauch, G.; Saucier, C.;Teissedre, P.-L.; Lopez-Roca, J. M.; Gomez-Plaza, E. Effect of micro-oxygenation on color and anthocyanin-related compounds of wineswith different phenolic contents. J. Agric. Food Chem. 2008, 56, 5932−5941.(16) Drinkine, J.; Glories, Y.; Saucier, C. (+)-Catechin-aldehydecondensations: competition between acetaldehyde and glyoxylic acid.J. Agric. Food Chem. 2005, 53, 7552−7558.(17) Cejudo-Bastante, M. J.; Perez-Coello, M. S.; Hermosin-Gutierrez, I. Effect of wine micro-oxygenation treatment and storageperiod on colour-related phenolics, volatile composition and sensorycharacteristics. LWT − Food Sci. Technol. 2011, 44, 866−874.(18) Sanchez-Iglesias, M.; Gonzalez-Sanjose, M. L.; Perez-Magarino,S.; Ortega-Heras, M.; Gonzalez-Huerta, C. Effect of micro-oxygenationand wood type on the phenolic composition and color of an aged redwine. J. Agric. Food Chem. 2009, 57, 11498−11509.(19) Macku, C.; Reed, K. Factors affecting wine closure selection.Practical Winery Vineyard J. 2011, Winter.(20) Ugliano, M. Oxygen contribution to wine aroma evolutionduring bottle aging. J. Agric. Food Chem. 2013, 61, 6125−6136.(21) Ugliano, M.; Dieval, J. B.; Vidal, S. Oxygen management duringwine bottle ageing by means of closure selection: current trends andperspectives. Wine Vitic. J. 2012, Sept/Oct, 38−43.(22) Guaita, M.; Petrozziello; Motta, S.; Bonello, F.; Cravero, M. C.;Marulli, C.; Bosso, A. Effect of the closure type on the evolution of thephysical-chemical and sensory characteristics of a Montepulcianod’Abruzzo rose wine. J. Food Sci. 2013, 78, C160−C169.(23) Ivanova, V.; Vojnoski, B.; Stefova, M. Effect of winemakingtreatment and wine aging on phenolic content in Vranec wines. J. FoodSci. Technol.−Mysore 2012, 49, 161−172.(24) Carvalho, E.; Mateus, N.; Plet, B.; Pianet, I.; Dufourc, E.; deFreitas, V. Influence of wine pectic polysaccharides on the interactionsbetween condensed tannins and salivary proteins. J. Agric. Food Chem.2006, 54, 8936−8944.(25) Iland, P.; Bruer, N.; Edwards, G.; Weeks, S.; Wilkes, E. ChemicalAnalysis of Grapes and Wine: Techniques and Concepts; Patrick IlandWine Promotions Pty Ltd: Adelaide, Australia, 2004.(26) McRae, J. M.; Dambergs, R.; Kassara, S.; Parker, M.; Jeffery, D.W.; Herderich, M.; Smith, P. A. Phenolic compositions of 50 and 30year sequences of Australian red wines: the impact of wine age. J. Agric.Food Chem. 2012, 60, 10093−10102.(27) Mercurio, M. D.; Dambergs, R. G.; Herderich, M. J.; Smith, P.A. High throughput analysis of red wine and grape phenolics-adaptation and validation of methyl cellulose precipitable tannin assayand modified somers color assay to a rapid 96 well plate format. J.Agric. Food Chem. 2007, 55, 4651−4657.(28) Cozzolino, D.; Kwiatkowski, M. J.; Parker, M.; Cynkar, W. U.;Dambergs, R. G.; Gishen, M.; Herderich, M. J. Prediction of phenoliccompounds in red wine fermentations by visible and near infraredspectroscopy. Anal. Chim. Acta 2004, 513, 73−80.(29) McRae, J. M.; Schulkin, A.; Kassara, S.; Holt, H.; Smith, P. A.Sensory properties of wine tannin fractions: Implications for in-mouthsensory properties. J. Agric. Food Chem. 2013, 61, 719−727.(30) Jeffery, D. W.; Mercurio, M. D.; Herderich, M. J.; Hayasaka, Y.;Smith, P. A. Rapid isolation of red wine polymeric polyphenols bysolid-phase extraction. J. Agric. Food Chem. 2008, 56, 2571−2580.(31) Kennedy, J. A.; Taylor, A. W. Analysis of proanthocyanidins byhigh-performance gel permeation chromatography. J. Chromatogr., A2003, 995, 99−107.(32) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidincleavage products following acid-catalysis in the presence of excessphloroglucinol. J. Agric. Food Chem. 2001, 49, 1740−1746.(33) Bindon, K.; Smith, P.; Kennedy, J. A. Interaction between grape-derived proanthocyanidins and cell wall material. 1. Effect onproanthocyanidin composition and molecular mass. J. Agric. FoodChem. 2010, 58, 2520−2528.(34) Koerner, J. L.; Hsu, V. L.; Lee, J.; Kennedy, J. A. Determinationof proanthocyanidin A2 content in phenolic polymer isolates by

Journal of Agricultural and Food Chemistry Article

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reversed-phase high-performance liquid chromatography. J. Chroma-togr., A 2009, 1216, 1403−1409.(35) Kennedy, J. A.; Ferrier, J.; Harbertson, J. F.; Gachons, C. P. D.Analysis of tannins in red wine using multiple methods: correlationwith perceived astringency. Am. J. Enol. Vitic. 2006, 57, 481−485.(36) Mercurio, M. D.; Smith, P. A. Tannin quantification in redgrapes and wine: comparison of polysaccharide- and protein-basedtannin precipitation techniques and their ability to model wineastringency. J. Agric. Food Chem. 2008, 56, 5528−5537.(37) Vernhet, A.; Dubascoux, S.; Cabane, B.; Fulcrand, H.;Dubreucq, E.; Poncet-LeGrand, C. Characterization of oxidizedtannins: comparison of depolymerization methods, assymetric flowfield-flow fractionation and small-angle X-ray scattering. Anal. Bioanal.Chem. 2011, 401, 1559−1569.(38) Cheynier, V. Flavonoids in wine. In Flavonoids − Chemistry,Biochemistry and Applications; Andersen, O. M., Markham, K. R., Eds.;CRC Taylor & Francis: Boca Raton, FL, 2006.(39) Es-Safi, N. E.; Cheynier, V. Flavanols and anthocyanins aspotent compounds in the formation of new pigments during storageand aging of red wine. In Red Wine Color: Revealing the Mysteries;Waterhouse, A. L., Kennedy, J. A., Eds.; American Chemical Society:Washington, DC, 2004; pp 143−159.(40) Kennedy, J. A.; Hayasaka, Y. Compositional investigation ofpigmented tannin. In Red Wine Color: Revealing the Mysteries;Waterhouse, A. L., Kennedy, J. A., Eds.; American Chemical Society:Washington, DC, 2004; pp 247−264.(41) Lee, D. F.; Swinney, E. E.; Asenstorfer, R. E.; Jones, G. P.Factors affecting the formation of red wine pigments. In Red WineColor: Revealing the Mysteries; Waterhouse, A. L., Kennedy, J. A., Eds.;American Chemical Society: Washington, DC, 2004; pp 125−142.(42) Poncet-Legrand, C.; Cabane, B.; Bautista-Ortín, A.; Carrillo, S.;Fulcrand, H.; Perez, J.; Vernhet, A. Tannin oxidation: intra- versusintermolecular reactions. Biomacromolecules 2010, 11, 2376−2386.(43) Vidal, S.; Cartalade, D.; Souquet, J.-M.; Fulcrand, H.; Cheynier,V. Changes in proanthocyanidin chain length in winelike modelsolutions. J. Agric. Food Chem. 2002, 50, 2261−2266.(44) Vidal, S.; Francis, L.; Noble, A.; Kwiatkowski, M.; Cheynier, V.;Waters, E. Taste and mouth-feel properties of different types of tannin-like polyphenolic compounds and anthocyanins in wine. Anal. Chim.Acta 2004, 513, 57−65.(45) Weber, F.; Greve, K.; Durner, D.; Fischer, U.; Winterhalter, P.Sensory and chemical characterization of phenolic polymers from redwine obtained by gel permeation chromatography. Am. J. Enol. Vitic.2012, 64, 15−25.(46) Souquet, J.-M.; Labarbe, B.; Guerneve, C. L.; Cheynier, V.;Moutounet, M. Phenolic composition of grape stems. J. Agric. FoodChem. 2000, 48, 1076−1080.(47) Vidal, S.; Francis, L.; Guyot, S.; Marnet, N.; Kwiatkowski, M.;Gawel, R.; Cheynier, V.; Waters, E. J. The mouth-feel properties ofgrape and apple proanthocyanidins in a wine-like medium. J. Sci. FoodAgric. 2003, 83, 564−573.(48) Kassara, S.; Kennedy, J. A. Relationship between red wine gradeand phenolics. 2. Tannin composition and size. J. Agric. Food Chem.2011, 59, 8409−8412.(49) Alcalde-Eon, C.; Escribano-Bailon, M. T.; Santos-Buelga, C.;Rivas-Gonzalo, J. C. Identification of dimeric anthocyanins and newoligomeric pigments in red wine by means of HPLC-DAD-ESI/MSn.J. Mass Spectrom. 2007, 42, 735−748.(50) Remy, S.; Fulcrand, H.; Labarbe, B.; Cheynier, V.; Moutounet,M. First confirmation in red wine of products resulting from directanthocyanin-tannin reactions. J. Sci. Food Agric. 2000, 80, 745−751.(51) Carlton, W. K.; Gump, B.; Fugelsang, K.; Hasson, A. S.Monitoring acetaldehyde concentrations during micro-oxygenation ofred wine by headspace solid-phase microextraction with on-fiberderivatization. J. Agric. Food Chem. 2007, 55, 5620−5625.(52) Atanasova, V.; Fulcrand, H.; Cheynier, W.; Moutounet, M.Effect of oxygenation on polyphenol changes occurring in the courseof wine-making. Anal. Chim. Acta 2002, 458, 15−27.

(53) Ugliano, M.; Dieval, J. B.; Siebert, T. E.; Kwiatkowski, M.;Aagaard, O.; Vidal, S.; Waters, E. J. Oxygen consumption anddevelopment of volatile sulfur compounds during bottle aging of twoshiraz wines. Influence of pre- and postbottling controlled oxygenexposure. J. Agric. Food Chem. 2012, 60, 8561−8570.(54) Drinkine, J.; Lopes, P.; Kennedy, J. A.; Teissedre, P. L.; Saucier,C. Ethylidene-bridged flavan-3-ols in red wine and correlation withwine age. J. Agric. Food Chem. 2007, 55, 6292−6299.(55) Lopes, P.; Richard, T.; Saucier, C.; Teissedre, P.-L.; Monti, J.-P.;Glories, Y. Anthocyanone A: a quinone methide derivative resultingfrom malvidin 3-O-glucoside degradation. J. Agric. Food Chem. 2007,55, 2698−2704.(56) He, J.; Santos-Buelga, C.; Silva, A. M. S.; Mateus, N.; De Freitas,V. Isolation and structural characterization of new anthocyanin-derivedyellow pigments in aged red wines. J. Agric. Food Chem. 2006, 54,9598−9603.(57) Es-Safi, N. E.; Fulcrand, H.; Cheynier, V.; Moutounet, M.Studies on the acetaldehyde-induced condensation of (−)-epicatechinand malvidin 3-O-glucoside in a model solution system. J. Agric. FoodChem. 1999, 47, 2096−2102.(58) Fulcrand, H.; Duenas, M.; Salas, E.; Cheynier, V. Phenolicreactions during winemaking and aging. Am. J. Enol. Vitic. 2006, 57,289−297.(59) Pozo, A. G.-d.; Arozarena, I.; Noriega, M.-J.; Navarro, M.; Casp,A. Short- and long-term effects of micro-oxygenation treatments onthe colour and phenolic composition of a Cabernet Sauvignon wineaged in barrels and/or bottles. Eur. Food Res. Technol. 2010, 231, 589−601.(60) Kennedy, J. A.; Matthews, M. A.; Waterhouse, A. L. Changes ingrape seed polyphenols during fruit ripening. Phytochemistry 2000, 55,77−85.(61) Li, Y.; Tanaka, T.; Kouno, I. Oxidative coupling of the pyrogallolB-ring with a galloyl group during enzymatic oxidation ofepigallocatechin 3-O-gallate. Phytochemistry 2007, 68, 1081−1088.

Journal of Agricultural and Food Chemistry Article

dx.doi.org/10.1021/jf403704f | J. Agric. Food Chem. 2013, 61, 11618−1162711627


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