Evaluation of Direct Phloroglucinolysis and Colorimetric

Article pubs.acs.org/JAFC

Evaluation of Direct Phloroglucinolysis and Colorimetric Depolymerization Assays and Their Applicability for Determining Condensed Tannins in Grape Marc Josh L. Hixson,† Keren A. Bindon,† and Paul A. Smith*,†,§ †

The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, Adelaide, South Australia 5064, Australia Flinders Centre for Marine Bioproducts Development (CMBD) and Department of Medical Biotechnology, School of Medicine, Flinders University, Bedford Park, South Australia 5042, Australia

§

ABSTRACT: To determine the optimum methods for determining condensed tannin (CT) content in grape marc, butanolhydrochloric acid assays and phloroglucinolysis were adapted for use, applied to a range of grape marc types, and the methods compared. Porter’s assay (butanol-HCl) was found to give unreliable results due to nonlinear color responses to grape skin and seed tannin concentrations, whereas the modification to include acetone (Grabber’s assay) overcame this. Differences between skin and seed tannin responses highlighted the need to adequately select the correct grape tannin standard, and the formation of pH-dependent color was accounted for through acidification of blank samples. For phloroglucinolysis, the inability to remove highly bound tannins from cell wall material was highlighted, although a measure of tannins remaining post-phloroglucinolysis (Grabber’s assay) showed a trend with the level of exposure to oxidative storage or processing conditions. The comparison of CT concentrations from phloroglucinolysis and Grabber’s assay gave poor correlation coefficients. KEYWORDS: butanol-HCl, phloroglucinolysis, condensed tannins, proanthocyanidin, grape marc

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INTRODUCTION

The limitations of the butanol-HCl method have been widely cited with respect to the requirement for an adequate CT standard sourced from the same material as is being analyzed, as well as the extent of color production being dependent on tannin size and composition or from small alterations in the solvent and water content of the assay reagent.24−28 Often this analysis is linked with fractionation of CT from fiber to understand the nature or how it is complexed with other feed components.29 Recently, similar methods have been used in determining tannin concentration in grape marc for animalrelated studies;30,31 although this does provide a simple and rapid method of analysis that can be performed in most laboratories, it provides no information on the subunit composition of the CT. Phloroglucinolysis is widely used and effective for the assessment of CT in grape extracts and wine and allows for compositional determination through “trapping” of the CT extension units using phloroglucinol and preventing oxidation to anthocyanidins, which provides information on the degree of polymerization (mDP), subunit composition, and extent of oxidation as well as overall tannin content.17,20 Whereas phloroglucinolysis has been widely applied to soluble tannins,17−22,32 the use of a method for soluble tannins is of limited advantage for grape marc as the majority of the readily extractable tannins from grapes are removed from the solids during winemaking.

Condensed tannins (CTs, syn. proanthocyanidins) have farreaching applications from astringency and color stability1−3 to human health4 but are also a promising class of compounds with respect to reducing emissions from ruminant animals. The concentration of tannins in a ruminant feed ration has been linked with reductions in methane emissions.5 However, inconsistencies in correlating tannin concentration to function alone6,7 have resulted in more recent interest in understanding the role of tannin structure and subunit composition with respect to function and efficacy.8−13 One potential tanniferous feed supplement is grape marc (syn. grape pomace), a solid byproduct of winemaking.14−16 Whereas wine tannin and extractable grape tannin have been extensively studied for concentration and composition using a number of methods,13,17−22 there is little information on the composition of tannins remaining in grape marc.13 Traditionally, the analysis of forages and livestock feed additives for condensed tannin content has consisted of a colorimetric assay facilitated by acidic depolymerization of condensed tannin and allowing for autoxidation of tannin extension units to colored anthocyanidins. Early methods utilized butanol and hydrochloric acid mixtures,23 although improvements in autoxidation reproducibility through the addition of ferric iron salts, commonly referred to as Porter’s assay,24 have seen it become a widely used tool in determining condensed tannin concentrations directly from plant fibers. More recently, the addition of acetone to the assay reagent has facilitated complete dissociation of condensed tannins with plant cell wall material,25 referred to in the text as Grabber’s assay. © 2015 American Chemical Society

Received: Revised: Accepted: Published: 9954

August 27, 2015 October 29, 2015 November 2, 2015 November 2, 2015 DOI: 10.1021/acs.jafc.5b04207 J. Agric. Food Chem. 2015, 63, 9954−9962

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Journal of Agricultural and Food Chemistry

Table 1. Phloroglucinolysis Results for 2010 Shiraz Marc and Constituents Using Heating Block or Water Bath Reaction Conditions and for Isolated Skin Tannin and Seed Tannin Standards (Determined from HPLC Peak Areas after Phloroglucinolysis)a heating block phloroglucinolysis total CT (g/kg) LEM (g/kg) mDP molecular mass (g/mol) cis/trans ratio (molar %) prodelphinidin (molar %) galloylation (molar %) terminal subunits (molar %) C EC ECG extension subunits (molar %) EGC-P C-P E-P ECG-P

water bath phloroglucinolysis

tannin standards

Shiraz marc

marc skin

marc seed

Shiraz marc

marc skin

marc seed

skin tannin

seed tannin

60.2 27.9 10.8 3360 19.4 14.9 12.2

56.3 39.0 27.8 8390 51.3 26.1 5.2

63.3 15.2 6.9 2230 12.1 1.7 20.4

18.7 18.6 11.7 3650 23.9 11.7 13.2

13.1 25.1 25.2 7600 56.1 22.8 5.3

31.2 11.3 7.6 2460 12.9 0.0 21.2

722.6 170.7 13.5 4040 20.8 25.9 3.5

858.2 115.3 6.2 1990 6.4 2.9 20.3

3.9 3.9 1.5

1.5 1.5 0.6

6.1 6.1 2.2

3.5 3.5 1.6

1.8 1.8 0.5

5.4 5.4 2.3

3.5 3.5 0.4

4.4 4.4 7.5

14.9 1.0 64.1 10.7

26.1 0.4 65.3 4.6

1.7 1.5 64.2 18.2

11.7 0.5 67.5 11.7

22.8 0.0 68.4 4.8

0.0 1.8 66.2 19.0

25.9 1.1 62.5 3.1

2.9 9.1 59.0 12.8

a

CT, condensed tannin; LEM, late-eluting material, epicatechin equivalences; mDP, mean degree of polymerization; C, catechin terminal subunit; EC, epicatechin terminal subunit; ECG, epicatechin gallate subunit; EGC-P, epigallocatechin phloroglucinol adduct; C-P, catechin phloroglucinol adduct; E-P, epicatechin phloroglucinol adduct; ECG-P, epicatechin gallate phloroglucinol adduct. compacted to remove excess air, sealed with a zip-tie, and stored at ambient conditions for 5 months. All remaining samples were stored at 4 °C until required. For analysis whole marc was lyophilized and ground using a coffee grinder (to pass a 1 mm sieve); marc constituents (skin and white grape seed) were separated by hand into seed and skin while wet and then prepared in the same manner as whole marc. All tannin concentrations are expressed as grams per kilogram of CT in freeze-dried dry matter (g/kg DM). Direct Phloroglucinolysis. Marc fibers were analyzed in triplicate on the basis of the method of Bindon et al. with modifications.13 The content of monomeric flavan-3-ols was determined using a single blank extraction (in methanol). In a 1.5 mL screw-cap eppendorf tube, 25 mg (±1.00 mg) of material was reacted with 1 mL of phloroglucinol solution (50 g/L in methanol) in a heating block (25 min, 50 °C) with stirring using a vortex mixer every 5 min. The reaction was cooled in ice−water and allowed to settle. Supernatant (400 μL) was collected, neutralized with sodium acetate (1.2 mL, 77 mM), shaken, and centrifuged, and the resulting solution was analyzed by HPLC using the same conditions as previously described.13 The total tanninderived material (CT) was determined as the sum of all flavan-3-ol subunits resulting from depolymerization. Late-eluting material (LEM), which was used as a measure of cross-linking between flavan-3-ol subunits and a proxy for oxidative exposure, was quantified in epicatechin equivalences.20 Tannin composition variables were determined using molar subunit ratios: mean degree of polymerization (mDP) from the ratio of total to terminal subunits; the cis/trans ratio from 2,3-cis-based to 2,3-trans-based subunits; the percentage galloylation (%Gall) from the occurrence of epicatechin gallate terminal and extension subunits; and the percentage of prodelphinidin subunits (%PD) from the occurrence of epigallocatechin terminal and extension subunits. Preparation of Post-phloroglucinolysis Fibers. From the initial reaction, 400 μL of reaction supernatant was used for phloroglucinolysis, and 300 μL of the phloroglucinolysis reaction solution was discarded. The remaining 300 μL of supernatant and fibers was neutralized by shaking (vortex mixer) with sodium acetate solution (900 μL, 77 mM), centrifuging (10000 rpm, 5 min), and discarding the supernatant. The fibers were washed with methanol (2 × 1 mL) and then water (1 × 1 mL) using a procedure similar to neutralization (shake, centrifuge, discard supernatant), ensuring no

Currently there is no routine analysis for determining condensed tannin composition directly from solid grape fibers and no indication as to the relative tannin concentrations obtained from phloroglucinolysis and butanol-HCl assays. As such, the current study investigated the application of both methods to the analysis of condensed tannins in grape marc and assessed the speed and ease of implementation of the colorimetric assay relative to the more labor intensive phloroglucinolysis method, which delivers a greater extent of information. The applicability and comparability of these common methods for determining tannin chemistry in grape marc will be assessed, along with the development of protocols that can be used in wine research laboratories and elsewhere.

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MATERIALS AND METHODS

Tannin Standards. Skin and seed tannin standards were prepared separately as previously described.33 Briefly, pre-veraison Tannat grapes were manually separated into skin and seed and extracted with 70% v/v aqueous acetone. Acetone was removed in vacuo, and the remaining aqueous extracts were diluted with methanol and trifluoroacetic acid (TFA) (to approximately 45% MeOH containing 0.01% v/v TFA) and loaded onto a Toyopearl column (5 cm × 30 cm, 40 mL/min) that had been equilibrated with 1:1 MeOH/H2O containing 0.01% v/v TFA. The column was washed with 1:1 MeOH/ H2O containing 0.01% v/v TFA (8 L, 5.7 column volumes) at 40 mL/ min and then the tannin eluted using 2:1 acetone/H2O containing 0.01% v/v TFA (4.4 L) at 40 mL/min. The acetone was removed under vacuum using a rotary evaporator (water bath at 30 °C), the aqueous residue was removed by freeze-drying, and the resulting powder was analyzed by phloroglucinolysis for CT content (purity shown as mass conversion) and subunit composition using HPLC.17 Grape Marc. Grape marc samples were taken from a number of stages across marc production and processing. Fresh marc was obtained directly from the winery press or from the winery marc pile (produced that day), red seeds were from the bottom of a fermenter, and stems were from the destemmer. Processed marc was sampled at a marc processing facility from either the predistillation pile (approximately 5−7 days ensiled) or from steam-distilled pile or obtained as a dried marc meal. One sample (40 kg) was placed into a plastic bag, 9955

DOI: 10.1021/acs.jafc.5b04207 J. Agric. Food Chem. 2015, 63, 9954−9962

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Figure 1. Calibration curves for butanol−hydrochloric acid assays: (a) Porter assay skin and seed tannin calibrations 0−100 g/kg with nonlinear regression; (b) Porter assay skin and seed tannin calibrations 0−250 g/kg with linear regression; (c) Grabber assay skin and seed tannin calibrations 0−250 g/kg with linear regression; (d) Grabber assay skin and seed tannin calibrations 0−225 g/kg with linear regression; (e) Grabber assay skin tannin calibration 0−250 g/kg for both sample-based absorbance maximum and standard measurement 550 nm absorbance. marc fiber was lost. The fiber and residual water were freeze-dried and analyzed using Grabber’s assay, as described below. Traditional Butanol-HCl Assay (Porter’s Assay). The assay reagent was prepared by dissolving ammonium iron sulfate dodecahydrate (400 mg/L) in water (33 mL/L) and concentrated HCl (50 mL/L) and making up to volume with butan-1-ol. For calibrations, 5.7 mL of assay reagent was added to a 20 mL screw-cap test tube followed by an aliquot of skin or seed tannin standard (10 mg/mL in methanol) added to align with g/kg DM equivalences of a 10 mg marc sample (i.e., 1 mg of tannin standard for 100 g/kg DM equivalent) and made up to 6 mL total volume with methanol. For samples containing marc, the marc was first added to the test tube and then the assay reagent. The assays were performed in a shaking water bath at 85 °C for 1 h before being removed and cooled in ice−water. Acetone−Butanol-HCl Assay (Grabber’s Assay). Tannin standards (10 mg/mL) were prepared in 70% v/v aqueous acetone. The assay reagent was prepared by dissolving ammonium iron sulfate dodecahydrate (420 mg/L) in concentrated HCl (53 mL/L), adding acetone (525 mL/L), and making up to volume with butan-1-ol. For calibrations, 5.7 mL of assay reagent was added to a 20 mL pressure vessel with a 300 μL addition of 70% aqueous acetone, containing tannin standard and/or blank addition for desired concentration, giving final volumes of 400 mg/L of ammonium iron sulfate dodecahydrate, 5% HCl (v/v), 65 mL/L water (including that contained in concentrated HCl), and 500 mL/L acetone, in butanol. The assay was performed in a shaking water bath at 70 °C for 2.5 h.

For marc samples, 10 mg of sample was placed into a pressure vessel, followed by 5.7 mL of assay reagent and 300 μL of 70% v/v acetone. The assay was performed in the same manner as described for calibrations, although blank samples containing no acid were acidified directly prior to analysis to account for any pH-dependent interference (anthocyanins). Grape marc fiber remaining post-phloroglucinolysis was weighed prior to colorimetric analysis, and all remaining material was analyzed as above. The final concentration of CT was calculated on the basis of the initial weight used for phloroglucinolysis. Spectroscopy. For initial investigations absorbance was determined using a Varian Cary 300 UV−vis spectrophotometer measuring absorbance between 600 and 450 nm with the absorbance at 550 nm and the absorbance maximum recorded. Subsequent analyses were performed by measuring absorbance at 550 nm only, to increase sample throughput. Absorbance was adjusted to zero using assay reagent containing no tannin. Samples in the same batch were given the same dilution using only butanol to ensure absorbance readings below 1.5 absorbance units. A three-point calibration curve was produced for each set of analyses, at 0, 75, and 150 g/kg when used for marc samples and at 0, 25, and 50 g/kg for post-phloroglucinolysis tannin quantification, reacted in duplicate.

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RESULTS AND DISCUSSION Direct Phloroglucinolysis. Phloroglucinolysis has previously been applied to grape marc fibers,13 although an 9956


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production per unit mass due to a higher ratio of extension subunits (those that produce color) to terminal subunits. For marc samples with low CT content the nonlinear response in the calibrations would provide an overestimation of CT unless a two-phase calibration, which was exponential from 0 to 100 g/kg and linear from 100 to 250 g/kg, was applied. However, to validate a nonlinear calibration curve for each analysis would become too complex, and correct concentrations could not be determined using an exponential calibration without knowing the curvature and extent of response for a given sample set. This issue is reported to have been rectified by the addition of acetone to the assay.25 Furthermore, this improvement also eliminates incomplete recovery of condensed tannins from the cell wall material as noted for Porter’s assay.25,26 Porter’s assay was not used further in this work due to the nonlinear calibration using grape tannins and the inability to ensure complete tannin recovery from cell wall material. Due to the increased volatility of the solvent system in Grabber’s assay, reactions were performed in pressure-rated tubes rather than in screw-capped test tubes. Slight modifications were made to the original method, including using tannin standards dissolved in 70% aqueous acetone rather than individually weighing small amounts of tannin. Additionally, the modification to using 5.7 mL of reagent followed by a 300 μL addition of 70% aqueous acetone into each tube slightly increased the preparation time for the assay, although it allowed for the use of dissolved tannin standards and also the application of marc extracts (performed in 70% acetone), as well as for use directly on marc fibers without requiring different calibrations to account for changing solvent concentrations. For both skin tannin and seed tannin, the calibrations for Grabber’s assay showed good linear responses across the entire range (Figure 1c), although the 250 g/kg samples in each case gave a slight reduction in color production compared with the 225 g/kg samples. Linear response was improved by eliminating the 250 g/kg samples in each calibration curve (Figure 1d). Limiting the concentration range of the assay to 225 g/kg is not expected to be detrimental as concentrations of tannin in grape marc were not seen above these levels in this work. Calibration curves using Grabber’s assay showed a trend similar to those produced using Porter’s assay, with the skin tannin calibration giving a higher color response than seed tannin. However, in this assay the same mass of skin tannin produced approximately 1.43 times more color than seed tannin with slopes of 0.0433 and 0.0302, respectively. The greater color production in Grabber’s assay, as indicated by the greater calibration curve slopes compared with Porter’s assay (1.43 vs 1.17), suggests that not only does the acetonecontaining modification improve recovery of tannins from cell wall material but it also provided improved sensitivity as shown by the greater slope. However, the increased difference in color production between skin and seed tannin calibrations made the choice of a relevant tannin standard more important in the acetone-containing version of the assay. To more accurately determine the most appropriate tannin standard to use for grape marc CT analysis, the color production response of marc seed and skin component should be known. As such, Shiraz marc was manually separated into seed and skin (73% skin, 27% seed, fresh weight distribution) and lyophilized (60% skin, 40% seed, dry weight distribution).

improved method for tannin recovery and throughput was developed by heating samples in a static heating block with periodic mixing (vortex mixer) instead of heating in a water bath with constant shaking (Table 1). Although tannin concentration differs between reaction conditions, the subunit composition of tannin determined by each method is comparable. Both of the methods for direct phloroglucinolysis were devised so that the reaction and neutralization conditions gave the same reagent proportions and similar analyte concentrations as those used for soluble grape and wine tannins.17 As such, the analytical conditions were identical, and no revision of the chromatographic method or response factors was needed. Furthermore, samples of both a solid and soluble/extracted nature could be analyzed interchangeably. Purified tannins from grape skin and seed were analyzed and gave expected results, with grape skin tannin having higher mDP, the presence of prodelphinidin subunits (%PD) and lower %Gall, compared with seed tannin. This trend was also observed with tannin depolymerized directly from Shiraz marc skin relative to seed. The concept of late-eluting material, or LEM, was first introduced by Aron and Kennedy; LEM is considered to be compositionally rich in flavan-3-ol but resistant to depolymerization, proposed to be due to oxidative exposure.20 As grape marc can be collected as a fresh product, or having undergone processing that may lead to oxidation, the concept of oxidation and the ability to measure oxidation markers is of interest, especially when considering chemical modification of CT. For Shiraz marc, Shiraz skin, and Shiraz seed the trend was for lower LEM values in marc seed and higher concentrations in marc skin. Shiraz marc, from which the two components were derived, had an intermediate LEM concentration, reflecting the contribution of skin and seed CT. Marc seed remained intact until ground for analysis, and as such lower oxidative exposure was expected when compared with marc skin. Butanol-HCl Assays. Tannin standards for the Porter’s assay calibration curves were prepared such that the mass of tannin present in each test tube represented a realistic range for a 10 mg whole marc addition. For example, a 1 mg addition of condensed tannin standard represented a tannin concentration of 100 g/kg DM equivalences of a 10 mg grape marc sample. Initial calibration curves of skin or seed tannin standards of between 0 and 100 g/kg showed good reproducibility between duplicates but both gave poor linearity (R2 values of 0.932 and 0.931, respectively), although improved correlation could be achieved by fitting an exponential regression (R2 values of 0.995 and 0.994 for skin and seed, respectively; Figure 1a). Extending the calibration curves to 250 g/kg of purified tannin achieved a near-linear response overall, but within the lower concentration range remained nonlinear (Figure 1b). This result aligns with the “curvilinear” response noted for this assay using tannins from Lotus corniculatus and Lotus uliginosus.25 Across the entire calibration, the color response from the skin tannin standard was 1.17 times higher than the seed tannin standard (slopes of 0.0256 and 0.0218, respectively; Figure 1b). This is in agreement with the expected difference in color production due to the mean degree of polymerization for each tannin standard and also that skin and seed tannin standards contain different proportions of prodelphinidins and procyanidins, which can give rise to differences in color production. Skin tannin with a higher mDP had a higher potential for color 9957


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indicating some variability relating to the method. The marc samples displayed higher %RSDs, which was likely due to the heterogeneity of the skin/seed distributed within the sample and the low sample mass used. Experimentation into altering the particle size resulted in the same phenomenon with marc skin tissue grinding to a much finer powder than marc seed tissue. As such, an even distribution of tissue types could not be ensured by a greater extent of sample grinding. One solution to address the heterogeneity issue would be to increase the sample mass from 10 mg, although the calibration curves were only linear to 225 mg/kg for a 10 mg sample, or 2.25 mg of tannin in the reaction. To double the sample weight in the same reaction volume would mean limiting the linear range of the calibration curve to around 110 mg/kg, which is not adequate for analyzing whole grape marc samples. Alternatively, the sample weight and reagent volume could both be doubled to give the same concentrations of tannin, but hopefully provide a more representative sample. In this case Grabber’s assay was also required to understand the concentration of tannin remaining in grape marc fibers following direct phloroglucinolysis. Because 10−15 mg of marc remains post-phloroglucinolysis (when 25 mg is used), we preferred to keep the mass within this range. Thus, increasing the sample weight was not a realistic option. Tannin-Remaining Postphloroglucinolysis. Direct phloroglucinolysis gives the ability to understand compositional aspects of grape marc tannins, but a key knowledge gap regarding this method was the extent of depolymerization, or conversely the concentration of tannins not removed from marc cell wall material and quantified. The complete tannin recovery observed in the literature for Grabber’s assay25 allows for a post-phloroglucinolysis analysis of the remaining marc fibers for the presence of highly bound tannins, herein referred to as post-phloroglucinolysis tannin (PPT). From a red marc, the fibers post-phloroglucinolysis were neutralized with sodium acetate and washed with methanol to remove any remaining depolymerized flavan-3-ols that would have already been accounted for during phloroglucinolysis. The fibers were washed with water, lyophilized, and analyzed using the same procedure as described for Grabber’s assay on whole marc. Commonly, 10−15 mg remained after reacting and washing from an initial 25 mg of grape marc that had undergone phloroglucinolysis. This was analyzed, and the concentration of tannin was normalized to the initial weight of grape marc used for the phloroglucinolysis reaction. Phloroglucinolysis of this red marc gave CT and LEM concentrations and %RSDs of 58.2 g/kg ± 0.6% and 23.0 g/kg ± 8.0%, respectively, and the post-phloroglucinolysis assay (PPT) showed 17.9 g/kg ± 2.4% remaining. In comparison to the combined CT, LEM, and PPT concentrations of 99.1 g/kg ± 1.8%, Grabber’s assay on the same marc sample gave 106.1 g/ kg ± 4.4%. The presence of post-phloroglucinolysis tannin confirms that phloroglucinolysis alone is not adequate for understanding the total tannin concentration in grape marc. From this single sample the majority of the tannin was quantified during phloroglucinolysis, but the highly bound tannin was not accessible. To properly understand the amount of tannin in grape marc, the tannin in the sample remaining postphloroglucinolysis must be determined. Comparison of Analytical Method on Grape Marc Tannin. Samples of known grape variety were collected fresh from wineries; samples without varietal information were

Subunit composition of each marc component, as well as whole marc was determined (Table 1), and color production was investigated. Similar total dry matter CT concentration was observed for marc skin and marc seed using phloroglucinolysis, although color production of marc skin relative to marc seed in the assay was markedly increased. From 10 mg of whole marc, marc skin, and marc seed, the absorbance readings postreaction were 2.16, 2.88, and 0.99, respectively. It is likely that three factors played a role in the greater color production from skin tannin compared with seed tannin. First, skin material is known to contain tannins of a higher mDP, resulting in a greater proportion of extension subunits (color producing) per unit of tannin mass. Second, the sample preparation of grape marc is such that the milling process reduces the particle size of marc skin more so than seed. As such, the assay response may be increased for skin tannins due to increased surface area and increased extractability leading to greater amounts of tannin. Also, there may be incomplete recovery from larger seed particles. Finally, it was found that the production of color in marc skin reactions occurred immediately upon addition of the acidic reagent, a phenomenon that was not observed for tannin standards. This instantaneous color production was shown to be due to the presence of pH-dependent color species (anthocyanins), which needed to be accounted for with a blank measurement. For all marc samples analyzed, the blank sample was acidified with concentrated HCl directly before being analyzed. This allowed for development of pH-dependent color, but provided limited opportunity for depolymerization of the tannins in the blank. This was an important step in grape marc analysis as the highest correction factor derived for tannin concentration due to alternative colored species for a marc sample was 10.9 g/kg from Pinot noir (sparkling base), based on a skin tannin calibration. As the total CT determined by Grabber’s assay for that sample was 155.2 g/kg, this represents a significant contribution. Additionally, shifts in the absorbance maximum per sample were much greater in Grabber’s assay (544−564 nm) than in Porter’s assay (546−552 nm). The changes in absorbance maximum when using skin, seed, or marc and molar absorptivity can only be attributed to the changes in subunit composition and possibly the extent of oxidation. However, there was very little difference between the calibration curves obtained from measuring absorbance at either 550 nm or at the maximum (Figure 1e). As such, to reduce the analysis time, the absorbance was measured only at 550 nm instead of a scan across the wavelength range. The analysis of whole marc samples using Grabber’s method had the tendency to give large relative standard deviations (% RSD) between triplicate samples. In an effort to understand why, two different grape marc samples were analyzed across seven replicates. The average and %RSD for Chardonnay marc and Shiraz marc were 61.2 g/kg ± 10.0% and 127.7 g/kg ± 9.0%, respectively. The deviation across seven samples suggests that this is a real assay effect and not due to an outlier within the original triplicates, showing that the method does have a degree of inherent variability. To test whether the variability was due to the skin and seed distribution differences within samples or inherent in the method reproducibility, a skin tannin only standard was then analyzed at two concentrations across seven replicates. The 50 and 100 g/kg tannin standards gave concentrations and %RSDs of 45.7 g/kg ± 4.2% and 102.2 g/kg ± 4.9%, respectively, 9958


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Table 2. Tannin Concentrations for Multiple Grape Marc Samples As Determined by Grabber’s Assay (g/kg, Determined Using Grape Skin Tannin Standard), Phloroglucinolysis (CT and LEM, g/kg, Determined from HPLC Peak Areas), and Postphloroglucinolysis Tannin (PPT, g/kg, Determined Using Grape Skin Tannin Standard)a tannin concentration (g/kg) marc sample white, 5 months ensiled red, steam-distilled mixed red/white, steam-distilled Chardonnay, skin only Chardonnay, seed only Chardonnay 1 Chardonnay 2 Shiraz Cabernet Sauvignon, stalk only Pinot Grigio Merlot white, steam-distilled red, ensiled 1 week red, steam-distilled Pinot noir, sparkling base Sauvignon blanc

CT 60.3 6.9 10.0 63.4 126.1 63.4 59.6 75.0 114.6 119.2 62.0 94.7 60.7 62.0 114.0 73.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

LEM 5.7 4.4 4.8 9.7 9.8 10.4 3.8 1.0 13.1 5.1 7.1 7.6 8.8 7.5 5.3 5.0

22.5 7.7 10.2 12.1 22.0 12.0 12.4 30.4 15.4 15.6 25.6 20.3 27.4 22.5 18.9 10.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

PPT

5.9 1.8 3.8 8.7 8.7 16.4 3.2 2.0 11.3 8.0 6.5 5.8 7.3 5.0 3.7 3.9

16.5 20.1 38.3 6.0 1.6 7.9 7.7 25.0 15.5 6.6 18.6 20.1 22.3 27.4 9.5 10.4

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

8.2 4.7 2.1 4.7 19.5 1.2 5.4 10.4 10.0 2.6 2.4 7.8 5.7 3.4 2.3 5.0

CT + LEM + PPT 99.3 34.7 58.5 81.4 149.8 83.3 79.7 130.0 145.5 141.4 106.2 135.1 110.5 111.9 142.4 95.0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

5.3 3.9 2.8 9.0 9.7a 9.9a 2.0 1.6 10.6a 5.3a 6.1a 7.1a 7.7a 4.3 4.8 3.7a

Grabber’s assay 115.9 48.7 47.2 75.9 125.6 56.1 61.2 127.7 95.9 98.9 71.1 108.0 90.5 102.1 155.2 125.3

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

19.4 7.1 4.5 1.0 3.7b 21.1b 10.0 9.0 7.3b 11.0b 23.3b 5.7b 8.0b 9.5 8.8 7.6b

Expressed as average of triplicates ± RSD. Mean values of CT + LEM + PT and Grabber’s assay for each sample followed by different letters are significantly different (P < 0.05).

a

Figure 2. Comparison of tannin quantification methods: (a) linear regression for Grabber’s assay against the sum of CT and post-phloroglucinolysis tannin (PPT), samples displayed as average (g/kg) ± SD; (b) linear regression for Grabber’s assay against the sum of CT, LEM, and PPT, samples displayed as average (g/kg) ± SD; (c) Bland−Altman plot for Grabber’s assay against the sum of CT and PPT; (d) Bland−Altman plot for Grabber’s assay against the sum of CT, LEM, PPT. Panels a and b show the average and standard deviation for sample triplicates.

collected from processing facilities. The latter had undergone ensiling in piles or had been steam-distilled. A single sample (white marc, 5 months ensiled) was collected and stored under anaerobic conditions for 5 months at 4 °C. For each grape marc sample, tannin content was assessed using Grabber’s assay, phloroglucinolysis, and post-phloroglucinolysis Grabber’s assay (Table 2). The combination of CT, LEM, and PPT for each of

the samples showed a significant deviation (P < 0.05) from the value obtained using Grabber’s assay for half of the samples. To understand how the different methods of quantifying tannins compared, the sequential analyses (phloroglucinolysis and post-phloroglucinolysis analysis) were summed and compared to results obtained for Grabber’s assay (Figure 2). As phloroglucinolysis and post-phloroglucinolysis tannin were determined from the same sample, the concentrations obtained 9959


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Figure 3. Comparison of post-phloroglucinolysis tannin (PPT, g/kg, determined using grape skin tannin standard) and late-eluting material (LEM, g/kg, epicatechin equivalences): (a) linear regression for PPT against LEM, error bars show standard deviation; (b) box and whiskers plot for PPT and LEM content from marc samples with low, medium, and high expected oxidative exposure. Box shows median, 25th and 75th percentiles, and whiskers to maximum and minimum values, and the mean value is marked (+).

unsuitable for use in determining grape marc tannins and should be used only with great caution. The addition of acetone to the assay gave calibrations that were closer to linear and also, on the basis of literature evidence, provides a more complete recovery of tannins from cell wall material;25 however, Grabber’s assay was not without its drawbacks. The increased volatility of solvent mixture used in Grabber’s assay requires the use of pressure vessels, which may limit the accessibility of this method to some laboratories, and also the difference in color production from skin and seed tannin is greater than for Porter’s assay, making selection of standards more important. In this work, skin tannin was chosen due to it being more compositionally similar to that observed in grape marc, but compositional changes, especially when a marc that is over-represented in seed material is analyzed, would need to be considered. From the analysis of grape marc samples and the comparison of tannin methods, it could be noted that the RSD values for Grabber’s assay tended to be higher than in phloroglucinolysis. The requirement of a tannin standard is eliminated in phloroglucinolysis. The biggest benefit of direct phloroglucinolysis of grape marc is the ability to define tannin composition and hence study structure−function relationships. With regard to phloroglucinolysis, the biggest drawback was the incomplete recovery of tannins, although this could be accounted for using a post-phloroglucinolysis analysis. The concentration of acid in the phloroglucinolysis solution is 5 times less than used in the butanol-HCl assay and may contribute to a reduced breakdown of highly bound CT. Incomplete CT recovery could potentially be avoided by modifying the method to mirror the conditions used in Grabber’s assay, with greater concentrations of acid or to contain acetone. However, the procedure was designed so that it could be run using a commonly used, validated tannin method and applied in laboratories where phloroglucinolysis is currently used without alteration or revalidation of the analytical method. However, the instrumental requirements of phloroglucinolysis and the need for a number of standards, which are largely unavailable commercially, limit the accessibility of this method. The use of post-phloroglucinolysis tannin measurement provides another level of information, the amount of “highly bound” tannin. For the samples analyzed following direct phloroglucinolysis, we propose that PPT analysis may be a useful proxy for understanding the extent of oxidation in the grape marc. Increasing concentrations of highly bound tannin

for each replicate were combined and then the standard deviation was determined. In an effort to understand how LEM influences the colorimetric assay, the tannin concentrations were summed in two ways. Figure 2a shows the sum of CT and post-phloroglucinolysis tannin, whereas Figure 2b includes LEM. Regardless of whether LEM was included in the comparison, the two methods for determining total tannin concentration (Grabber’s assay or sequential phloroglucinolysis followed by PPT) did not correlate well. Although the linear correlation was improved when LEM was included (Figure 2b), the bias between methods, as can be observed in the Bland−Altman plots for each comparison, the average was closer to zero when LEM was not included (Figure 2c,d). The Bland−Altman plots also highlight the variation that exists between the two methods, with 95% agreement lines at +44.8 and −37.0 (bias of 3.9 ± 40.9 g/kg) and +28.7 and −55.6 (bias of −13.5 ± 42.1 g/kg), respectively. Due to the poor correlation between the two methods for quantifying CT in grape marc, it cannot be determined whether the inclusion of LEM improves correlation, and, as such, the extent to which LEM is accounted for in Grabber’s assay. The hypothesis of the authors was that grape marc parcels that had undergone processing such as maceration, storage in piles without compacting to remove excess air, or steam distillation were expected to have increased exposure to oxidative conditions. From the above analysis (Table 2) it was evident that the amount of highly bound tannin increased as expected exposure to oxidative conditions increased (e.g., fresh to macerated or ensiled to steam-distilled). As such, the two CT types (LEM and PPT) were compared (Figure 3a) as these are most likely to account for oxidized components, but they showed no correlation. Furthermore, with increasing oxidative exposure, a clear trend with PPT was apparent, but there was no obvious trend toward higher amounts of LEM (Figure 3b), and it is likely that oxidation conditions promote subunit cross-linking that rendered the tannins inaccessible to phloroglucinolysis. On the basis of the increase in PPT with oxidative exposure, we hereby suggest that the extent of highly bound tannins in grape marc may be a good marker for exposure to oxidative conditions. Additionally, both LEM and PPT should be considered when the overall tannin content of marc is assessed, providing additional information on the nature of the tannins. Concluding Remarks. The lack of linearity in the calibrations for Porter’s assay renders this assay largely 9960


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correlated to marc parcels that have undergone processes that are expected to be oxidative, such as maceration, storing in piles without compacting to remove excess air, steam distillation, or combinations of these. Those samples that were considered to be fresh (seeds, fresh white marc) possessed the lowest amount of highly bound tannin. Furthermore, this suggests that the increased interaction between tannins and cell wall material, possibly through the formation of covalent linkages, is a product of exposure to oxidative conditions. At this stage, the LEM and highly bound tannin fractions cannot be assessed for flavan-3-ol composition. However, development of analytical techniques for this purpose is an area that could provide greater insight into compositional profiles of grape marc tannins and provide better links between CT structure and function.

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AUTHOR INFORMATION

Corresponding Author

*(P.A.S.) Phone: +61-8-8313-6600. Fax: +61-8-8313-6601. Email: [email protected]. Funding

This work is part of the National Livestock Methane Program (NLMP), supported by funding from the Australian government Department of Agriculture as part of its Carbon Farming Futures, Filling the Research Gap Program, and managed by Meat & Livestock Australia. The Australian Wine Research Institute, a member of the Wine Innovation Cluster in Adelaide, is supported by Australian grape growers and winemakers through their investment body, Wine Australia, with matching funds from the Australian government. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Dr. Eric Wilkes for his contribution in preparing the manuscript. ABBREVIATIONS USED HCl, hydrochloric acid; CT, condensed tannin; UV−vis, ultraviolet−visible spectroscopy; mDP, mean degree of polymerization; %Gall, percentage galloylation; %PD, percentage of prodelphinidin; LEM, late-eluting material; PPT, postphloroglucinolysis tannin; HPLC, high-performance liquid chromatography; DM, dry matter; TFA, trifluoroacetic acid; MeOH, methanol; RSD, relative standard deviation

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REFERENCES

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