Measuring the Molecular Dimensions of Wine Tannins: Comparison

In this study, we determined the relative dimensions of wine tannin samples .... on the SAXS/WAXS beamline at the Australian Synchrotron (Melbourne, A...
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Measuring the Molecular Dimensions of Wine Tannins: Comparison of Small-Angle X‑ray Scattering, Gel-Permeation Chromatography and Mean Degree of Polymerization Jacqui M. McRae,*,† Nigel Kirby,‡ Haydyn D. T. Mertens,‡,§ Stella Kassara,† and Paul A. Smith† †

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, South Australia 5064, Australia The Australian Synchrotron, 800 Blackburn Rd, Clayton, Victoria 3168, Australia

‡

S Supporting Information *

ABSTRACT: The molecular size of wine tannins can influence astringency, and yet it has been unclear as to whether the standard methods for determining average tannin molecular weight (MW), including gel-permeation chromatography (GPC) and depolymerization reactions, are actually related to the size of the tannin in wine-like conditions. Small-angle X-ray scattering (SAXS) was therefore used to determine the molecular sizes and corresponding MWs of wine tannin samples from 3 and 7 year old Cabernet Sauvignon wine in a variety of wine-like matrixes: 5−15% and 100% ethanol; 0−200 mM NaCl and pH 3.0−4.0, and compared to those measured using the standard methods. The SAXS results indicated that the tannin samples from the older wine were larger than those of the younger wine and that wine composition did not greatly impact on tannin molecular size. The average tannin MWs as determined by GPC correlated strongly with the SAXS results, suggesting that this method does give a good indication of tannin molecular size in wine-like conditions. The MW as determined from the depolymerization reactions did not correlate as strongly with the SAXS results. To our knowledge, SAXS measurements have not previously been attempted for wine tannins. KEYWORDS: depolymerization, GPC, SAXS, tannin molecular weight, wine matrix, wine tannin

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INTRODUCTION Tannins from foods, including apples, grapes, and wine, consist largely of “condensed tannins” which comprise mixtures of polymers with flavan-3-ol subunits.1 The molecular weight (MW) of condensed tannins has been shown to be an important driver of wine astringency,2,3 with greater astringency reported for tannins with higher MWs.4,5 MW can be estimated using two main standard methods: depolymerization reactions, which give the mean degree of polymerization (mDP), or gelpermeation chromatography (GPC), which gives an average molecular weight based on relative hydrodynamic volume. Depolymerization reactions sever the bonds between flavan-3ol subunits and attach an electrophile, such as thioglycolic acid6 or phloroglucinol,7,8 to the polymer extension subunits, thus allowing the differentiation between extension and terminal subunits using HPLC. Calculating the relative proportion of extension and terminal subunits enables an estimation of the mean number of subunits per polymer in the tannin polymer mixture, giving the mDP. The tannin MW can then be calculated based on the mDP and subunit composition. GPC analysis gives an average MW of the tannin sample by comparing the retention time of the elution of 50% of the tannin sample with that of a calibration curve.9 Tannin polymers are differentiated based on their relative hydrodynamic volume, and thus relative retention time, compared with a calibration curve consisting of polymers with known MWs. Generally preveraison grape skin tannin is used for the calibration curve for measuring wine tannins.10 Both methods for assessing tannin MW are widely used for characterizing wine tannins, although both have limitations. © XXXX American Chemical Society

The MW as determined by mDP is limited by the proportion of the tannin that can be characterized using depolymerization reactions (referred to as the “percent yield” of the reaction). It is therefore most effective for preveraison grape tannins, which can be readily depolymerized into subunits and therefore have a higher percent yield. During winemaking, the tannins extracted from the grapes are rapidly oxidized by chemical and biochemical reactions and structurally altered by the incorporation of fermentation products including acetaldehyde,11 dramatically reducing the proportion of acid-labile interflavan bonds and thus lowering the percent yield.6,12,13 The experimentally determined mDP for wine tannins may therefore not be representative of the actual MW of the tannin. This is particularly the case for tannins from aged wines, where the percent yield can be less than 10% of the total wine tannin.14 The result is that a gradual decrease in mDPdetermined MW is observed in wine tannin with wine aging as a result of the number of cleaved extension subunits being reduced due to the increase in modified interflavan linkages and intramolecular bonding, which leads to an overestimation of the relative proportion of terminal subunits.15 Measuring the MW using GPC relies on comparing the retention time of a tannin sample to that of a calibration curve. The calibration curve used to determine wine tannin MW is generally based on preveraison grape skin tannin that has been Received: November 25, 2013 Revised: June 12, 2014 Accepted: June 24, 2014

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fractionated using Sephadex LH-20 gel,10 and the MW of each tannin fraction is determined based on depolymerization reactions. Estimating the MW of wine tannins using GPC requires the assumption that the hydrodynamic volumes of grape skin tannins equate to those of wine tannins at the same MW and that there is no interaction between the tannins and the gel media. The correlation between the results from GPC and mDP calculations can also be limited14 as a consequence of low percent yield or differences in hydrodynamic volumes between the tannin sample and the grape tannins of the calibration curve. Polymers can expand or contract in different solvents16 and tannins in particular can be more condensed in water than in organic solvents and may change conformation in different hydroalcoholic solutions.13 The hydrodynamic volumes of tannins in different wine-like conditions (pH 3.0−4.0, 10−15% aqueous ethanol) may therefore be very different to those measured using the GPC conditions. Given that wine tannin size can directly impact astringency,2,3 it is important to determine if the relative tannin sizes determined using GPC, such as between aged and young wines tannins, are actually representative of relative tannin size in wine. For this reason, the MWs of a selection of tannins were measured using smallangle X-ray scattering (SAXS) with synchrotron radiation in a range of wine-like conditions and compared with those estimated using GPC and mDP. SAXS can be used to measure particle size and shape in a range of solutions17,18 and has been previously used to measure tannins in aqueous ethanol solutions.13 The size of macromolecules can be determined for a large range of particle sizes, from less than 1 kDa to several MDa and, although routinely used for model reconstruction from monodisperse systems of biomolecules (e.g., purified proteins),17 the technique can be applied to the study of polydispersed systems. Previous experiments have measured changes in the MW of polydisperse grape and apple tannins with oxidation by comparing the SAXS forward scattering intensities with mDP,6,12 highlighting the potential for using this technique for wine tannins. SAXS also provides information on the conformation of tannins in solutions,19 providing an added advantage over GPC or mDP analysis. Wine astringency is known to decrease in intensity with aging.20,21 The underlying cause for this in unclear, and may relate to changes in tannin MW or conformation as no agerelated trends have been observed in tannin concentration.14 Studies that investigate tannin structure in aged wines have reported conflicting results, depending on the method used for analysis. Analyses comparing the mDP of tannins from younger and older wines have suggested that aged wine tannins are smaller than young wine tannins,22,23 while GPC studies have reported that aged wine tannins are generally larger than younger wine tannins.14,24 The difference in results produced from these two methods as well as the potential changes in the molecular dimensions of tannins with wine aging highlights the value in measuring aged and young wine tannins in wine-like conditions. In this study, we determined the relative dimensions of wine tannin samples isolated from a 3 and a 7 year old Cabernet Sauvignon wine using SAXS in a range of wine-like matrixes, including different ethanol concentrations, pH, and ionic strengths. These results were then compared with those of the same wine tannin samples measured using GPC and mDP to assess the usefulness of each method for estimating the relative size of tannins in red wines. Results are also discussed

with respect to the potential impact of tannin shape on wine astringency with aging.

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MATERIALS AND METHODS

Chemicals. All solvents used were high-performance liquid chromatography (HPLC) grade, all chemicals were analytical reagent grade, and water was obtained from a Milli-Q purification system. Acetic acid (100%), acetonitrile, ethanol, formic acid (98−100%), and HCl (32%), were all purchased from Merck Australia (Kilsyth, VIC, Australia). Ascorbic acid, lithium chloride (LiCl), N,N-dimethylformamide (DMF), phloroglucinol, sodium acetate, tartaric acid, and sodium chloride (NaCl) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Tannin Isolation and Fractionation. Tannins were isolated from two Australian Coonawarra Cabernet Sauvignon wines, aged 3 and 7 years, using Toyopearl HW-40F, as previously described.4 Both isolated tannin samples were then fractionated using liquid−liquid separation with butanol and water as reported previously4 to give six tannin samples: total tannin samples from each vintage (TT3 and TT7 for the 3 and 7 year old wine tannins, respectively), aqueous fractions from each vintage (Aq3 and Aq7) and butanol-soluble fractions from each vintage (Bu3 and Bu7). The aqueous fractions were more abundant than the butanol-soluble fractions for both vintages, with ratios of 3.6:1 (Aq/Bu) for the 3 year old tannin samples and 2.9:1 (Aq/Bu) for the7 year old tannin samples. Relative water-solubility of the tannin samples were determined using octanol/water assays as previously described.14 Tannin Molecular Weight Measurements Using Gel Permeation Chromatography and Depolymerization Reactions. Standard laboratory methods for determining average tannin molecular weight (MW) for each sample were performed using depolymerization reactions with phloroglucinol (phloroglucinolysis)7 and gel permeation chromatography (GPC).9 Briefly, for phloroglucinolysis, tannin solutions (25 μL, 10 g/L tannin in methanol) were added to phloroglucinol solution (25 μL, 100 g/L phloroglucinol in methanol with ascorbic acid (20 g/L) and 0.2 N HCl) to give final concentrations of 5 g/L tannin. Reactions were performed at 50 °C for 25 min, after which sodium acetate solution (70 mM, 150 μL) was added to neutralize the reaction. The cleaved subunits were analyzed using HPLC as previously described7 to give the mean degree of polymerization (mDP, calculated from the proportion of extension to terminal subunits), percent yield (proportion of the tannin weight that was cleaved by acid hydrolysis), percent epigallocatechin subunits (% GC), and percent epicatechin gallate subunits (%ECG). The MW for each sample (MWmDP) was calculated based on the mDP and subunit composition. GPC was performed as described previously9 using a series of two PLgel columns (500 and 103 Å) with an isocratic solvent system of DMF solution (0.15 M LiCl, 1% v/v acetic acid, 5% v/v water in DMF). The retention time of 50% tannin elution of each sample (2 g/L tannin in 1:4 methanol/DMF) was compared with a calibration curve of preveraison grape skin tannins fractions25 to give an average tannin MW (MWGPC). Tannins are a mixture of polymers and thus the wine tannin samples were polydispersed with MW ranges within the 10−90 percentile range of 900 to 5900 g mol−1 for TT3 and from 1100 to 5800 g mol−1 for TT7, as measured using GPC.9 The tannin subfractions were less polydispersed than the total tannin fractions with MW ranges from 1000 to 5000 g mol−1 for the butanol subfractions and from 1500 to 6000 g mol−1 for the aqueous subfractions. Sample Preparation for SAXS Analysis. The tannin samples were prepared in 100% ethanol and in model wine solutions for smallangle X-ray scattering (SAXS) analysis. Model wine composition varied depending on the parameters investigated. For the experiments assessing the impacts of different ethanol concentrations, the model wine contained 2% v/v ethanol at pH 3.5 (adjusted with tartaric acid to a final concentration of 0.043 mM). The ethanol concentration was adjusted after tannin addition to give the final concentration range of 5, 10, 12, and 15% v/v ethanol in model wine. For experiments assessing the impacts of pH, the model wine contained 2% v/v ethanol B

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Table 1. Characteristics of Wine Tannin Samples As Measured from Depolymerization Reactions and Octanol/Water Assays, As Well As the Forward Scattering Intensities Normalized for Concentration (I(0)/c) at 12% and 100% Ethanol as Determined Using SAXS TT3 TT7 Aq3 Aq7 Bu3 Bu7

mDPa

% yieldb

% GCc

% ECGd

Log Pe

I(0)/c 12%

I(0)/c 100%

9.1 7.7 11.1 9.6 5.8 5.7

33.9 12.0 30.6 15.4 26.0 12.4

27.9 29.6 33.4 34.7 21.8 24.2

3.6 2.1 3.0 1.8 3.5 2.0

−0.61 −0.55 −1.31 −1.34 −0.70 −0.72

0.0078 0.0088 0.0091 0.0130 0.0038 0.0044

0.0188 0.0178 0.0204 0.0237 0.0079 0.0073

a

Mean degree of polymerization. bPercent of the tannin mass hydrolyzed using phloroglucinolysis (proportion of tannin analyzed). cPercent epigallocatechin subunits. dPercent epicatechin gallate subunits. eLog of the octanol/water partition coefficients. for a final concentration of 12% v/v ethanol after tannin addition. The pH was adjusted with tartaric acid at 0.08 mM and 0.01 mM for pH 3.0 and 4.0, respectively. For the ionic strength experiments, the model wine contained 2% v/v ethanol for a final concentration of 12% v/v ethanol after tannin addition. NaCl solution (0.5 M in model wine, pH 3.5) was added to give final concentrations of 25, 50, 100, or 200 mM NaCl. The ionic strength and pH experiments were performed only on the total tannin samples. Blank samples were prepared as the model wine solutions without tannins. Wine tannin solutions were prepared in 100 μL volumes in 96-well plates at a range of concentrations (1, 2, 5, and 10 g/L). The plates were centrifuged (5 min, 2000 rpm), and the supernatant was transferred to clean 96-well plates prior to SAXS analysis. Tannin samples were first dissolved in ethanol to maximize solubility prior to addition into model wine solutions, however precipitation was observed in some solutions after centrifugation. The relative GPC peak areas of tannin solutions before and after centrifugation indicated that precipitation significantly impacted tannin concentration in Bu3, Aq3, and Aq7 in the 10g/L solutions at 5% ethanol, although there was no significant impact on the MW range of these samples. These samples were not included in the MW calculations as described in the next section. SAXS Analysis and Data Processing. Small-angle X-ray scattering (SAXS) experiments were performed on the SAXS/WAXS beamline at the Australian Synchrotron (Melbourne, Australia).26 The incident beam energy was 11.000 ± 0.005 keV (wavelength, λ = 1.0332 Å) with a corresponding scattering vector (q) ranged between 0.015 to 0.7 Å−1, where q = (4π sin θ)/ λ, and 2θ is the scattering angle. Experiments were performed at 25 °C using a Pilatus 1 M detector (DECTRIS, Switzerland). Samples were loaded using an automatic sample loader with a 2 × 96-well plate capacity (approximately 80 μL volume used per sample). Data collection involved recording successive frames (typically 10) of 2 s each per sample, and the multiple frames averaged as appropriate using ScatterBrain Analysis IDL v8.1 software (Australian Synchrotron) and subtracting the scattering recorded from the solvent blanks. Data were placed on absolute intensity scale using water as an intensity standard. The radiation exposure of the samples under these conditions did not result in sample degradation, as indicated by comparing scattering intensities of successive frames. The cleanliness of the capillary was periodically checked during system washing by monitoring intensity changes during water exposures. SAXS data were analyzed using the ATSAS software package27 to give forward scattering intensity, I(0) and radius of gyration, Rg (using PRIMUS28), as well as the distance distribution function P(r) (using GNOM29) for each tannin sample at each concentration. The I(0) and Rg parameters were extracted from the data by the standard Guinier approach (where qRg < 1.3 in all cases):19

Iexp(q) = I(0)exp( − q2R g 2/3)

since the correlation factor, R2, was greater than 0.99 in most cases30 (Supporting Information (SI) Table S1). R2 values were less than 0.99 for Bu3, Aq3, and Aq7 at 10g/L in 5% ethanol (and at 10% ethanol for Aq7), and therefore these values were excluded from the MW calculations. The MW for each wine tannin sample was calculated by comparing the averaged I(0) as a function of concentration (I(0)/c) at each ethanol concentration, pH, and ionic strength, with the (I(0)/c) values of a calibration curve of preveraison grape skin tannin fractions (MW range 1277 to 8996 g mol −1 as determined from depolymerization reactions) in wine-like conditions (12% v/v ethanol, pH 3.5). These were the same grape tannin samples as used in the GPC calibration.25 This gave the calibration curve for the I(0)/c values: MWk . I / c(g mol−1) = 518 396*(I(0)/c)

(2)

The averaged Rg values of the wine tannins were also compared with those of the grape tannin calibration curve to give a different MW measurement: MWRg(g mol−1) = 268.14*R g

(3)

The MW of wine tannins was also calculated directly from the I(0)/c, independently of the calibration curve, using the equation as per Mylonas et al:31

MWΔρ = [NAI(0)/c]/ΔρM 2

(4) −1

where the Avogadro number, NA = 6.023 × 10 mol . The scattering contrast per mass, ΔρM = [ρM.tannin − (ρsolv υ)]r0, assumes that ρM.tannin is similar to that of a flavan-3-ol monomer (3.19 × 1023 electrons/g calculated from the molecular formula and known molecular weight), and that the wine tannin density is 1.57 g/cm3 as previously reported,12,32 giving the tannin partial specific volume, υ = 0.637 cm3/ g. The solvent density for water, ρsolv = 3.34 × 1023 e/cm3 was used for tannins in aqueous solvents and that of ethanol, ρsolv = 2.68 × 1023 e/ cm3 was used only for those samples in ethanol. The scattering length of an electron, r0 = 2.8179 × 10−13 cm. Information on the conformation of the wine tannin samples was obtained from Kratky plot representations and from the distance distribution functions, P(r), as calculated from the Fourier transformation of the scattering intensity.19 23

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RESULTS AND DISCUSSION Characteristics of Selected Wine Tannins. Wine tannins were isolated from a 3 and a 7 year old Cabernet Sauvignon wine and then separated into two fractions each, one watersoluble (aqueous fraction) and one butanol-soluble (butanol fraction) to give six wine tannin samples. The composition of each tannin sample was determined using phloroglucinolysis and subsequent HPLC analysis of the cleaved subunits (Table 1). This gave the mean degree of polymerization (mDP), proportion of epigallocatechin (%GC), and epicatechin gallate (%ECG) subunits, in addition to the percent yield (proportion of acid-labile interflavan bonds) of the reaction. The aqueous

(1)

The I(0) values were normalized for tannin concentration, c, and subsequently averaged for the molecular weight (MW) calculations. Plots of I(0) vs c (1, 2, 5, and 10 g/L tannin), were generally linear and indicated that the scattering results were concentration-independent C

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fractions from both wines were larger (greater mDP) and contained more epigallocatechin subunits than the butanol fractions. The tannin samples isolated from the older wine contained fewer epicatechin gallate subunits and were less susceptible to the depolymerization reactions, with percent yields around half those of the tannin samples from the younger wine. This is likely to be a consequence of gradual oxidation and structural rearrangement reactions leading to intramolecular bond formation, as has been previously shown with grape and apple tannins,6,12 as well as intermolecular interactions such as the incorporation of pigmented polymers.4 The difference in water-solubility was demonstrated in the octanol/water partition coefficients (Log P) (Table 1). The tannins isolated from the older wine were more hydrophobic than those from the younger wine (−0.61 and −0.55 for TT3 and TT7, respectively), and the total tannin samples were more hydrophobic than the tannin fractions. This potentially suggested that some hydrophobic tannin species were removed from the fractionation process, however the benefit of fractionating the tannins was to enable the analysis of a range of chemically different wine tannins from the same starting wine. The aqueous fractions were more water-soluble than the butanol fractions, as expected. Comparison of Relative Wine Tannin Sizes in Different Matrixes. Wines of different vintages (3 and 7 years old) were selected to compare wine tannin sizes from young and aged wines in different matrixes. Isolated tannins from each wine were separated into two fractions with distinctly different characteristics that were less polydispersed than the total wine tannins. This gave a range of tannin samples for molecular size analysis. Relative wine tannin sizes were determined using SAXS in 100% ethanol, which has been shown to be a good solvent for tannins,16,32 and compared to more wine-like conditions: (5−15% ethanol, pH 3.0−4.0, and 2−200 mM NaCl). The forward scattering intensities of wine tannins in 100% ethanol indicated an absence of pronounced aggregation (Figure 1), which concurs with previous reports for grape and apple tannins.16,32 It must also be noted that the greater I(0)/c values for the tannins is 100% ethanol compared with in the hydroalcoholic solutions (Figure 1 and Table 1) is likely to be influenced by differences in solvent densities (ρsolv = 3.34 × 1023 e/cm3 for water and 2.68 × 1023 e/cm3 for ethanol). This is corrected for in the MW calculations as noted in the next section. The aqueous tannin fractions were consistently larger than butanol fractions, and the aged wine tannins were generally larger than the younger wine tannins. The major advantage of SAXS analysis over other techniques for tannin characterization is the potential to characterize macromolecules in a range of solvents. Polymer mixtures may expand or contract in different solvent conditions,16 suggesting that the tannin characterization methods that calculate average MW from relative hydrodynamic volume, such as GPC, may be solvent-dependent. Wine astringency is known to decrease with increasing ethanol concentrations and with increasing wine pH33 and this may relate to changes in tannin conformation in solution. The forward scattering plots for wine tannins in hydroalcoholic solutions showed aggregation, as indicated by large increases in the scattering intensities at low-q (Figure 1) for all measured tannin samples and at all concentrations. The extent of this scattering varied depending on solvent and sample, with tannin fractions from the older wine showing the greatest aggregation. The TT3 and Aq3 samples only aggregated at 5% ethanol, and the Bu3 sample showed some

Figure 1. SAXS intensity plots, log I(q)/c vs log q, at each ethanol concentration for (a) TT3; (b) TT7; (c) Aq3; (d) Aq7; (e) Bu3; and (f) Bu7.

aggregation at 15% ethanol. The TT7, Aq7, and Bu7 samples all aggregated in all hydroalcoholic solutions with increasing aggregation at decreasing ethanol concentration. In determining the Rg values for each sample, a shorter q-range was used to obtain a linear fit for the Guinier plots (within the qRg < 1.3 limitation) to minimize the impact of aggregation on this measurement.19 Some precipitation was observed at higher tannin concentrations (10 g/L) in 5% and 10% ethanol, and this was reflected in a decrease in linearity in the I(0) vs c plots (SI Table S1). This was not the case in 12% ethanol, and these values were used for the MW comparisons as discussed in the next section. The differences in aggregation between the aged and younger wine tannins are likely to be due to the greater oxidation of the tannins from the older wine, as observed from the comparatively lower percent yield in the phloroglucinol reactions (Table 1). Oxidized wine tannins reportedly have lower water solubility than “native” grape tannins, and are therefore more prone to aggregation in aqueous solutions.12,32 This potentially contributes to the precipitation of tannins as wines age. The octanol/water partition coefficients (Table 1) also demonstrated that the tannin samples from the older wine were slightly more hydrophobic than the same fractions from the younger wine. The Rg values were relatively consistent across the measured ethanol concentrations range for most tannin samples (Figure 2). Only the Aq7 fraction increased in Rg with a decrease in ethanol concentration which may relate to a change in tannin size, a change in the solvation layer around the tannin in more aqueous systems or, potentially, interference from aggregation.13 Previous research has indicated that astringency intensity decreases with increasing ethanol (up to 15%) in D

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Comparison of Wine Tannin Molecular Weight Using Different Methods. The molecular weight of all wine tannin samples were compared using three different methods: SAXS analysis (from the forward scattering intensities (I(0)/c) and radii of gyration (Rg) values as compared to a standard curve, MWk.I/c and MWRg, respectively, and calculated directly from the I(0)/c, MWΔρ), GPC analysis (MWGPC), and analysis of tannin subunits from depolymerization reactions (MWmDP) (Table 2). The I(0)/c and Rg of the tannin samples from the Table 2. Comparison of Wine Tannin Molecular Weights (MW) in g mol−1 as Calculated Using Each Methoda 12% ethanol MWmDP MWGPC TT3 TT7 Aq3 Aq7 Bu3 Bu7

Figure 2. Impact of ethanol concentration on the radii of gyration (Rg, Å) for each tannin sample. Error bars represent the standard deviation of averaged replicates.

2730 2300 3340 2870 1740 1700

2930 2850 3030 3270 1800 1930

MWΔρb

MWk.I/c

5242 5914 6115 8736 2554 2857

4043 4562 4717 6739 1970 2281

c

100% ethanol MWRgd

MWΔρ

MWRg

4207 4210 4486 5808 3148 3325

6485 6140 7037 8176 2725 2518

4376 4347 4757 5285 3065 3708

a

Depolymerization reactions (MWmDP), gel permeation chromatography (MWGPC) in methanol/DMF, and SAXS (MWk.I/c in 12% ethanol, MWΔρ and MWRg in 12% and 100% ethanol). bTannin MW calculated directly from I(0)/c: MWΔρ = [NAI(0)/c]/ΔρM2. cTannin MW calculated from I(0)/c using the calibration curve: MWk.I/c = 518 396x. dTannin MW calculated from Rg using the calibration curve: MWRg = 268.14x.

model wine solutions containing oliomeric tannins.33 This study has shown that there is no change in tannin size over wine-like ethanol concentrations. The reported changes in astringency intensity with ethanol concentration may relate to changes in tannin aggregation rather than relative tannin size in solution. Wine pH and ionic strength have been shown to impact wine astringency and protein binding33,34 and therefore the impact of these characteristics on the relative sizes of the total tannin fractions was also assessed. The I(0)/c and Rg values of the total tannin samples showed no significant change in tannin dimensions across the ionic strength range from 0 to 200 mM NaCl (Figure 3a). Increases in ionic strength have been shown to augment the aggregation of grape and apple tannins35 potentially due to changes in the solubility of tannins, since ionic strength did not directly alter the wine tannin molecular size. Wine pH also showed negligible impact on wine tannin dimensions with the Rg values remaining fairly consistent across the wine-like pH range (3.0 to 4.0) (Figure 3b). Wine pH has been previously correlated with model wine astringency, with solutions at pH 2.5 and 3.0 producing greater astringency intensity than model wines at 3.5 and 4.0,33 and this is most likely to be a direct consequence of the wine matrix.

SAXS analyses in 12% ethanol were compared with those of a calibration curve of fractionated preveraison grape skin tannins measured in 12% ethanol to give the average tannin MW values, MWk.I/c and MWRg, respectively. The MWRg values were also determined for the wine tannin samples in 100% ethanol for comparison, however differences in the scattering contrasts of ethanol and aqueous solutions prevented the same comparison for MWk.I/c. Instead, MWs were calculated directly from the I(0)/c values to give an independently determined MW of the wine tannins (MWΔρ) that corrected for the solvent contrast. For GPC, the retention times of the wine tannin samples (at 50% elution) in a methanol/DMF solvent system were compared to those of a calibration curve in the same solvent to give the average wine tannin MW (MWGPC). The

Figure 3. Impact of wine matrix on the radii of gyration (Rg, Å) of the total tannin samples from each vintage wine at (a) varying ionic strength, as NaCl concentration (mM); and (b) varying pH. Error bars represent the standard deviation of averaged replicates. E

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Table 3. Correlation Factors (R2) between the Average Wine Tannin Molecular Weights (MW) As Calculated Using Each Methoda 12% ethanol method

12%

100%

MWmDP MWGPC MWΔρ MWk.I/c MWRg MWΔρ MWRg

MWmDP 0.7952 0.6065 0.6018 0.5616 0.8057 0.7122

100% ethanol

MWGPC

MWΔρb

MWk.I/cc

MWRgd

MWΔρ

MWRg

0.7952

0.6065 0. 8857

0.6018 0.8826 0.9998

0.5616 0.8097 0.9736 0.9753

0.8057 0.9866 0.9117 0.9067 0.8474

0.7122 0.9084 0.9208 0.9255 0.9006 0.8894

0. 8857 0.8826 0.8097 0.9866 0.9084

0.9998 0.9736 0.9117 0.9208

0.9753 0.9067 0.9255

0.8474 0.9006

0.8894

a

Depolymerization reactions (MWmDP), gel permeation chromatography (MWGPC) in methanol/DMF, and SAXS (MWk.I/c in 12% ethanol, MWΔρ and MWRg in 12% and 100% ethanol). bTannin MW calculated directly from I(0)/c. cTannin MW calculated from I(0)/c using the calibration curve. d Tannin MW calculated from Rg using the calibration curve.

methods. Further research is required to more accurately determine this value. The extent to which the determined MWs from each method correlated for the wine tannin samples was measured as R2 values from correlation plots (Table 3). The MWGPC and MWmDP were compared with the MWs determined using SAXS in 12% ethanol for an example of a wine-like solution and in 100% ethanol to ensure that the aggregation of tannins in hydroalcoholic solutions did not greatly interfere with the correlations. Correlations were strongest between the MWΔρ and MWk.I/c in 12% ethanol (R2 = 0.9998), providing good support for the MWs derived from the calibration curves, including the MWRg values in 12% ethanol, which also correlated well with the MWΔρ values (R2 = 0.9736). The MWGPC values correlated most strongly with the MWΔρ 100% ethanol (R2 = 0.9866). The strong correlation between the GPC values and those in from both MWΔρ and MWk.I/c in 12% ethanol (R2 > 0.88) demonstrated that wine tannin MWs as determined with GPC do give a good indication of relative tannin molecular size in wine. The MWmDP values had the weakest correlation with the SAXS methods (R2 ≈ 0.6 for MWs determined in 12% ethanol), further highlighting the differences produced from determining tannin MW from average polymer chain length and from relative tannin size. Overall, there was no significant impact of different wine-like parameters on wine tannin dimensions across the measured ethanol concentration, ionic strength, and pH ranges. Further, the strong correlation between the determined tannin MW in wine-like matrixes as compared with GPC emphasizes that GPC is a useful method for comparing the relative MW of tannins in young and aged wines. Estimation of Wine Tannin Conformation in Solution. One of the advantages of analyzing samples with SAXS over other techniques for characterizing wine tannins is the potential to obtain information on the conformation of macromolecules in solution. GPC has the advantage over mDP calculations by producing separation profiles, which indicate the proportions of the tannin broadly distributed around the nominal classes of “high”, “medium”, and “low” MW range, but this does not indicate the shape of the tannin polymer complex. Tannin conformations in solution were estimated for all samples in 100% ethanol, to ensure the complete dissolution of tannin molecules, using a Kratky plot (q2 I(q) vs q) representation of the SAXS data and from the real-space distance distributions, P(r). The Kratky plots (Figure 4) for each sample indicated that the tannins were compact but extended structures.19 Distance distribution functions (Figure 5) indicated that the

same grape-skin tannin fractions were used for the calibration curves in both the SAXS analysis and GPC, enabling a direct comparison between the results obtained in wine-like conditions with SAXS with those from the more widely used tannin characterization technique. The depolymerization reactions involved acid-catalysis of the tannin samples and HPLC analysis of the cleaved subunits to give the tannin subunit composition and mean degree of polymerization (mDP). These results were then used to calculate the average MW for each sample (MWmDP). The average MWs of all tannin samples as determined using GPC (MWGPC), depolymerization reactions (MWmDp), and with SAXS (MWΔρ, MWk.I/c, and MWRg) are shown in Table 2. In all methods, the aqueous fractions (Aq3 and Aq7) were higher in MW than the total tannin fractions (TT3 and TT7), and the butanol-soluble fractions (Bu3 and Bu7) were much smaller. MWs derived from the SAXS data in 12% v/v ethanol were larger than those calculated from GPC analysis or depolymerization reactions (Table 2) indicating that the MWs of wine tannins in wine may be underestimated by the standard methods, particularly for aged-wine tannins. The solvation layer around the tannins in hydroalcoholic solutions would also have contributed to the relative tannin size as measured using SAXS, increasing the calculated MW.13 The SAXS-determined MWs and MWGPC values indicated that the tannin samples from the older wine were larger or similar in size to the younger wine tannins. The MWmDP values, however, suggested that the older wine tannins were smaller than the younger wine tannins. This may have be due to the lower percent yield of the older tannin samples (Table 1), which meant that only a very small proportion of the tannin was analyzed (≤15%) and this limited the number of extension subunits that could be cleaved by acid-catalysis. Furthermore, the preveraison grape skin tannin calibration curve used in the SAXS and GPC calculations may have also overestimated the average wine tannin MWs due to differences in spatial distribution between the grape tannin fractions and the wine tannins. The MWΔρ values in 12% ethanol were larger than the MWs determined from the calibration curve (MWk.I/c and MWRg), suggesting that any differences in dimensions between the wine tannins and the grape tannins had only a limited impact on the determined wine tannin MWs. The calculations used for the MWΔρ values presumed a tannin density of 1.57 g/ cm3 as previously reported for grape seed tannins;12,32 however, the actual density of wine tannins may be greater given the disparity between these values and those determined with other F

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In summary, the relative molecular dimensions of a range of wine tannins were measured in wine-like conditions using SAXS (I(0)/c and Rg) and the calculated molecular weights (MWs) were compared to the more commonly used methods of GPC (MWGPC) and depolymerization reactions (MWmDP). The MWs determined with SAXS in 12% ethanol correlated well with the MWGPC values, which suggests that GPC can be used to give a good indication of the average MWs of tannins in wines. Variations in wine-like solvent composition, including ethanol concentration, ionic strength, and pH, did not vary the measured tannin dimensions of all samples in solution, although tannin aggregation was influenced by ethanol concentration in the hydroalcoholic solutions. The conformations of the wine tannins resembled elongated ellipsoids in all tested solvent compositions. To our knowledge, SAXS measurements have not previously been attempted for wine tannins. Further research is needed to understand how tannin structures change with wine aging and how tannin aggregation may alter the perceived astringency of red wine.

Figure 4. Kratky plots (q2 I(q) vs q) for each tannin sample in 100% ethanol.

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ASSOCIATED CONTENT

S Supporting Information *

wine tannin samples were not spherical and that the configuration more closely related to elongated ellipsoid structures.19,36 The P(r) for the wine tannin samples in all hydroalcoholic solutions (data not shown) resembled the elongated ellipsoid conformations that were observed in 100% ethanol.

SAXS intensity plots, log I(q)/c vs log q, at each ionic strength for (a) TT3; (b) TT7; and at each pH for (c) TT3; and (d) TT7 (Figure S1); SAXS data for each tannin sample at each ethanol concentration including the forward scattering intensities normalized for concentration (I(0)/c), radii of

Figure 5. Distance distribution functions for each tannin sample in 100% ethanol. (a) TT3; (b) TT7; (c) Aq3; (d) Aq7; (e) Bu3; and (f) Bu7. G

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(4) McRae, J. M.; Schulkin, A.; Kassara, S.; Holt, H.; Smith, P. A. Sensory properties of wine tannin fractions: Implications for in-mouth sensory properties. J. Agric. Food Chem. 2013, 61, 719−727. (5) Vidal, S.; Francis, L.; Guyot, S.; Marnet, N.; Kwiatkowski, M.; Gawel, R.; Cheynier, V.; Waters, E. J. The mouth-feel properties of grape and apple proanthocyanidins in a wine-like medium. J. Sci. Food Agr 2003, 83, 564−573. (6) Vernhet, A.; Dubascoux, S.; Cabane, B.; Fulcrand, H.; Dubreucq, E.; Poncet-LeGrand, C. Characterization of oxidized tannins: Comparison of depolymerization methods, assymetric flow field-flow fractionation and small-angle X-ray scattering. Anal. Bioanal. Chem. 2011, 401, 1559−1569. (7) Koerner, J. L.; Hsu, V. L.; Lee, J.; Kennedy, J. A. Determination of proanthocyanidin A2 content in phenolic polymer isolates by reversedphase high-performance liquid chromatography. J. Chromatogr. A 2009, 1216, 1403−1409. (8) Kennedy, J. A.; Jones, G. P. Analysis of proanthocyanidin cleavage products following acid-catalysis in the presence of excess phloroglucinol. J. Agric. Food Chem. 2001, 49, 1740−1746. (9) Kennedy, J. A.; Taylor, A. W. Analysis of proanthocyanidins by high-performance gel permeation chromatography. J. Chromatogr. A 2003, 995, 99−107. (10) Bindon, K.; Smith, P.; Kennedy, J. A. Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 1. Effect on Proanthocyanidin Composition and Molecular Mass. J. Agric. Food Chem. 2010, 58, 2520−2528. (11) Monagas, M.; Bartolome, B.; Gomez-Cordoves, C. Updated knowledge about the presence of phenolic compounds in wine. Crit. Rev. Food Sci. Nutr. 2005, 45, 85−118. (12) Poncet-Legrand, C.; Cabane, B.; Bautista-Ortín, A.; Carrillo, S.; Fulcrand, H.; Pérez, J.; Vernhet, A. Tannin oxidation: Intra- versus intermolecular reactions. Biomacromolecules 2010, 11, 2376−2386. (13) Zanchi, D.; Konarev, P. V.; Tribet, C.; Baron, A.; Svergun, D. I.; Guyot, S. Rigidity, conformation, and solvation of native and oxidized tannin macromolecules in water−ethanol solution. J. Chem. Phys. 2009, 130, 245103. (14) McRae, J. M.; Dambergs, R.; Kassara, S.; Parker, M.; Jeffery, D. W.; Herderich, M.; Smith, P. A. Phenolic compositions of 50 and 30 year sequences of Australian red wines: The impact of wine age. J. Agric. Food Chem. 2012, 60, 10093−10102. (15) Jorgensen, E. M.; Marin, A. B.; Kennedy, J. A. Analysis of the oxidative degradation of proanthocyanidins under basic conditions. J. Agric. Food Chem. 2004, 52, 2292−2296. (16) Beaucage, G. Small-angle scattering from polymeric mass fractals of arbitrary mass-fractal dimension. J. Appl. Crystallogr. 1996, 29, 134− 146. (17) Petoukhov, M. V.; Svergun, D. I. Applications of small-angle Xray scattering to biomacromolecular solutions. Int. J. Biochem. Cell Biol. 2013, 45, 429−437. (18) Pérez, J.; Yoshinori, N. Advances in X-ray scattering: From solution SAXS to achievements with coherent beams. Curr. Opin. Struct. Biol. 2012, 22, 670−678. (19) Mertens, H. D. T.; Svergun, D. I. Structural characterization of proteins and complexes using small-angle X-ray solution scattering. J. Struct. Biol. 2010, 172, 128−141. (20) Lattey, K.; Bramley, B.; Francis, I. L. Consumer acceptability, sensory properties and expert quality judgements of Australian Cabernet Sauvignon and Shiraz wines. Aust. J. Grape Wine Res. 2010, 16, 189−202. (21) Herderich, M. J.; Smith, P. A. Analysis of grape and wine tannins: Methods, applications and challenges. Aust. J. Grape Wine Res. 2005, 11, 1−10. (22) Chira, K.; Pacella, N.; Jourdes, M.; Teissedre, P.-L. Chemical and sensory evaluation of Bordeaux wines (Cabernet-Sauvignon and Merlot) and correlation with wine age. Food Chem. 2011, 126, 1971− 1977. (23) Cheynier, V.; Duenas-Paton, M.; Salas, E.; Maury, C.; Souquet, J. M.; Sarni-Manchado, P.; Fulcrand, H. Structure and properties of wine pigments and tannins. Am. J. Enol. Vitic. 2006, 57, 298−305.

gyration (Rg), calculated molecular weight (MW) values, maximum particle length (Dmax) and correlations for I(0) vs c (R2) (Table S1); SAXS data for the total tannin samples (TT3 and TT7) at each ionic strength including the forward scattering intensities normalized for concentration (I(0)/c), radii of gyration (Rg), calculated molecular weight (MW) values, maximum particle length (Dmax) and correlations for I(0) vs c (R2) (Table S2); SAXS data for the total tannin samples (TT3 and TT7) at each pH including the forward scattering intensities normalized for concentration (I(0)/c), radii of gyration (Rg), calculated molecular weight (MW) values, maximum particle length (Dmax) and correlations for I(0) vs c (R2) (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61 8313 6600; e-mail: [email protected]. Present Address

§ European Molecular Biology Laboratory-Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia and at The Australian Wine Research Institute, a member of the Wine Innovation Cluster at the Waite Precinct in Adelaide, South Australia, and is supported by Australian grape growers and winemakers through their investment body, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government.

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ABBREVIATIONS Aq3, aqueous tannin subfraction from the 3 year old wine; Aq7, aqueous tannin subfraction from the 7 year old wine; Bu3, butanol-soluble tannin subfraction from the 3 year old wine; Bu7, butanol-soluble tannin subfraction from the 7 year old wine; GPC, gel-permeation chromatography; mDP, mean degree of polymerization; MW, molecular weight; MWGPC, MW determined using GPC; MWk.I/c, MW derived from the I(0)/c values compared to the calibration curve; MWΔρ, MW calculated directly from the I(0)/c values; MWmDP, MW determined from the mDP; MWRg, MW derived from the Rg values compared to the calibration curve; Rg, radius of gyration; SAXS, small-angle X-ray scattering; SEC, size exclusion chromatography; TT3, total tannin sample from the 3 year old wine; TT7, total tannin sample from the 7 year old wine

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