Article

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Factors Affecting Skin Extractability in Ripening Grapes Keren A. Bindon,*,† S. Hadi Madani,§ Phillip Pendleton,§,# Paul A. Smith,† and James A. Kennedy†,⊥

† The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia § Ian Wark Research Institute, University of South Australia, Mawson Lakes, SA 5095, Australia # School of Pharmacy and Medical Science, University of South Australia, G.P.O. Box 2471, Adelaide, SA 5001, Australia ⊥ Department of Viticulture and Enology, California State UniversityFresno, 2360 East Barstow Avenue, MS VR89, Fresno, California 93740-8003, United States

*S Supporting Information

ABSTRACT: The acetone-extractable (70% v/v) skin tannin content of Vitis vinifera L. cv. Cabernet Sauvignon grapes was found to increase during late-stage ripening. Conversely, skin tannin content determined following ethanol extraction (10, 20, and 50% v/v) did not consistently reflect this trend. The results indicated that a fraction of tannin became less extractable in aqueous ethanol during ripening. Skin walls were observed to become more porous during ripening, which may facilitate the sequestering of tannin as an adsorbed fraction within cell walls. For ethanol extracts, tannin molecular mass increased with advancing ripeness, even when extractable tannin content was constant, but this effect was negligible in acetone extracts. Reconstitution experiments with isolated skin tannin and material indicated that the selectivity of tannin adsorption by cell walls changed as tannin concentration increased. Tannin concentration, tannin molecular mass, and cell wall porosity are discussed as factors that may influence skin tannin extractability. KEYWORDS: tannin, anthocyanin, adsorption, ripening, skin, cell wall, porosity, BET, microscopy, molecular mass, gel permeation chromatography

■ INTRODUCTION Due to these conflicting results, it is evident that additional Generally, skin from red grapes are more readily research is required, in particular as new analytical methods become available. Distinguishing extractable tannin from total extracted during vinification than seed tannins, regardless of tannin content is also an important consideration in studies that maceration time.1,2 In wines, later harvest dates are associated have implications for winemaking. Because existing research with an increase in total wine tannin concentration, as well as into the changes in grape skin tannin during ripening has an increase in the molar proportion of 3,4 primarily employed exhaustive extraction techniques such as (epigallocatechin), which is derived from grape skins. As aqueous acetone or methanol, a pertinent question is the extent such, skin tannin concentration may serve as a useful marker for to which these methods relate to vinification conditions. phenolic maturity in ripening grapes. Reports on changes in Extraction conditions that exist during vinification reach only grape tannin quantity during ripening have used a diverse range between 10 and 16% v/v ethanol concentration in commercial fi of extraction, puri cation, and analytical methodologies. practice. In a study comparing the effectiveness of extraction Furthermore, to account for changes in berry weight during solvents for skin tannin,17 it was found that acetone was more ripening, it is necessary to report grape phenolic data effective than ethanol and that acetone-extracted tannin had a normalized as “content per berry” rather than simply as higher average molecular mass. It was also found that varying concentration (per gram berry or skin fresh weight). A review the ratio of water to solvent significantly affected the amount of studies that report tannin content or include berry mass and composition of the tannin extracted. The point was raised information revealed that skin tannin has been found to that aqueous ethanol may more accurately mimic the increase with the progression of ripening when analyzed using conditions of extraction during vinification. − adaptations of Porter’s assay5 9 and by the monitoring of Various studies have attempted to observe differences in tannin absorbance properties in the UV range via normal phase tannin extraction in ripening grapes using aqueous solutions, HPLC10 or methyl cellulose precipitation.3 Nevertheless, some either buffered18,19 or using dilute, acidified ethanol.5,7,20 It is authors10 have suggested that these increases in UV absorbance evident that differences exist in the partitioning of grape tannins may not originate directly from flavan-3-ol incorporation to the between soluble and cell wall-bound fractions, that the nature tannin polymer but could result from other structural of these fractions is significantly different, and that they change modifications. Furthermore, a study using selective precip- during ripening. A single study investigating the subcellular itation of skin tannin with protein11 found no change in content during ripening. Variable results have been reported using the Received: November 10, 2013 12 13 phloroglucinolysis method and showed either no change, Revised: January 15, 2014 variable,14 decreased,15 or increased16 skin tannin content per Accepted: January 17, 2014 berry in the late stages of ripening. Published: January 17, 2014

© 2014 American Chemical Society 1130 dx.doi.org/10.1021/jf4050606 | J. Agric. Food Chem. 2014, 62, 1130−1141 Journal of Agricultural and Food Chemistry Article partitioning of skin tannin19 compared extraction in buffered molecular mass distribution. Tannin and anthocyanin solutions solution (pH 7.5) to tannin remaining bound to the residue were combined with isolated cell walls to determine the effect following extraction, presumed to be cell wall fragments. Both of concentration on their respective adsorption affinities. In the buffer-soluble extract, designated the vacuolar fraction, and addition, scanning electron microscopy (SEM) and nitrogen the cell wall-bound fraction were further extracted using adsorption isotherms were used to characterize changes in cell acidified methanol.19 The experiment showed that the mean wall surface area (porosity) during ripening. degree of polymerization (mDP) of tannin within the did not change during grape development, yet the mDP of ■ MATERIALS AND METHODS tannins associated with the cell wall was higher and tended to Instrumentation. An Agilent model 1100 HPLC (Agilent 19 increase at the end of maturation. However, in said study, the Technologies Australia Pty Ltd., Melbourne, Australia) was used mDP of tannins isolated from cell walls was markedly lower with Chemstation software for chromatographic analyses. For SEM, a than observations from studies on the same cultivar using 70% Philips XL30 field emission scanning electron microscope (Philips, v/v acetone extraction.10,14,16 This may indicate that a greater Eindhoven, The Netherlands) was used. fraction of tannin is desorbed from the grape cell wall using Grape Sample Preparation and Extraction. Vitis vinifera L. cv. acetone. This phenomenon has been shown definitively for Cabernet Sauvignon grape samples were obtained from a commercial vineyard in the Langhorne Creek growing region of South Australia tannin extracted from apple using methanol or aqueous (Pernod Ricard Australia, Orlando Wines) at different commercial acetone, in which acetone extraction yielded tannin of higher 22 ripeness stages in the 2010 season. Berry samples were collected from mDP. Furthermore, in that work, aqueous acetone was shown three rows distributed within the vineyard block to obtain a to completely desorb high mDP tannin bound to cell wall representative sample, pooled, and processed fresh. A 200 berry material, in a fashion similar to the application of urea. It is subsample was processed fresh, and the juice centrifuged at 1730g for therefore evident that further research is needed to better 5 min and analyzed for total soluble solids (°Brix) using a digital 3 understand the factors which influence partitioning of tannin to refractometer. This data has been published previously. Triplicate extractable and nonextractable fractions. subsamples of 100 berries were prepared for cell wall isolation, and a Recent work using model studies has demonstrated the high further 36 subsamples of 10 berries each were prepared for phenolic ffi fi extraction experiments. a nity of puri ed grape cell wall material for tannin and The 100-berry samples were weighed, and skins were then highlighted that these interactions may limit tannin extraction separated from flesh and seed while kept on ice. Recovered skin during vinification. This may explain some of the significant material was weighed to determine the proportion of fresh skin variability that is observed in wine-extractable tannin between material to total berry mass, frozen in liquid nitrogen, and stored at − grape cultivars and grape ripeness stages.16,23 26 For grape skin −80 °C until used for the isolation of cell wall material. Cell wall tannin from red grapes, highly polymerized tannins had a low material was isolated from frozen grape skins as described previously.25 affinity for skin cell walls compared with tannin of intermediate For the purpose of comparing ripening-related changes in cell walls, molecular mass.16 However, it was found that with the two early-stage (preveraison and veraison) cell wall samples were retained from a previous study.25 progression of ripening, changes in skin cell wall composition The 10-berry samples were peeled, and the skins were rinsed with facilitated an enhanced adsorption of this high mDP tannin 25 ice-cold 30 mM aqueous citric acid, gently blotted with a paper towel, fraction. Structural changes in skin cell wall polysaccharides weighed, and immediately transferred to 30 mL of extraction solvent. were found to occur early in grape development, before or close Four different extraction solvents were used, 10, 20, or 50% v/v to veraison, and as such the altered adsorption properties of cell ethanol and 70% v/v acetone, each containing aqueous 0.01% v/v wall material for tannin during late-stage ripening could not be trifluoroacetic acid (TFA). To minimize sample degradation and directly linked to changes in polysaccharide composition. prevent any partial extraction during the rinsing step, a maximum Rather, it was noted that the specific enhancement of high number of 10 fresh berries could be processed per experiment. The molecular mass tannin adsorption was consistent with increases ratio of solid material to solvent was 1:15, according to the optimal conditions described by Verries and co-workers,21 to ensure that in cell wall porosity. Theoretically, an enhanced porosity could extraction efficiency was not influenced by saturation of the extraction enable penetration and encapsulation of high molecular mass medium, and the concentrations of tannin and anthocyanin within tannins within the cell wall structure and has been observed to extracts did not exceed 1 and 0.6 g/L, respectively. Extractions were in 27−29 contribute to cell wall−tannin interactions in apple. In triplicate, performed at 25 °C in the dark, under nitrogen in sealed 50 ripening grapes, multiple factors may influence tannin mL centrifuge tubes. To ensure continuous contact of skins and extractability: interactions with cell walls, cell integrity, tannin solvent, centrifuge tubes were placed flat and mixed gently using a concentration, tannin molecular mass, tannin structural platform shaker. All extractions were performed over 64 h to keep the modification, and interaction with other phenolics, including conditions consistent between experiments. The extraction time was self-association.30 As reviewed here, these factors can change selected to optimize tannin extraction in dilute ethanol solutions (10 and 20% v/v), which was maximal at 64 h, whereas tannin extraction simultaneously during grape ripening, rendering the system containing 50% v/v ethanol or 70% v/v acetone reached a plateau at highly complex. 42 h (Supporting Information Figure S1). Following extraction, 50 mL The work presented in the current study has sought to build tubes containing residual skin solids were centrifuged at 1730g for 5 on previous research using model experiments, to observe how min, and aliquots of the supernatant were directly analyzed as differences at a cellular level might affect skin tannin described below. extractability during ripening. Extraction of grape skins has Analysis of Skin Tannin and Anthocyanin. A 500 μL aliquot of been carried out in various concentrations of aqueous ethanol 70% v/v acetone extract was dried under a stream of nitrogen and to determine whether differences in tannin extractability could reconsituted in the same volume of aqueous ethanol (20% v/v). Aqueous ethanol extracts were directly analyzed in duplicate. Tannin be related to ripening stage. In certain samples, ethanolic “ ” and anthocyanin concentrations in aqueous ethanolic extracts were extraction was compared to a harsh or total extraction in 70% determined in a 96-well plate format using the high-throughput v/v acetone. Whole extracts were subjected to gel permeation method described previously.31 Tannin concentration was expressed as chromatography (GPC) to observe whether changes in tannin (−)-epicatechin equivalents using an external standard curve (Sigma- extractability were associated with differences in tannin Aldrich, St. Louis, MO, USA). To standardize tannin analysis across

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Table 1. Tannin and Anthocyanin Content Per Berry at Different Total Soluble Solids Level Determined by Extraction in a Aqueous Ethanol

tannin anthocyanin extraction solvent total soluble solids (°Brix) mg/berry mg/g skin FW mg/berry mg/g skin FW 10% ethanol 22 0.50 ± 0.10g 2.55 ± 0.52e 0.94 ± 0.04g 4.75 ± 0.19g 23 0.70 ± 0.01fg 3.56 ± 0.03de 1.07 ± 0.02fg 5.47 ± 0.08fg 24 0.81 ± 0.05f 4.07 ± 0.26d 1.17 ± 0.01fg 5.86 ± 0.04fg 26 0.91 ± 0.05ef 4.36 ± 0.28d 1.25 ± 0.09ef 5.98 ± 0.45f

20% ethanol 22 0.74 ± 0.06f 3.72 ± 0.31d 1.09 ± 0.09fg 5.49 ± 0.46fg 23 1.25 ± 0.06cd 6.38 ± 0.32c 1.58 ± 0.13d 8.06 ± 0.64de 24 1.13 ± 0.04de 5.65 ± 0.18c 1.44 ± 0.02d 7.21 ± 0.08e 26 1.38 ± 0.05c 6.59 ± 0.25c 1.66 ± 0.05cd 7.96 ± 0.26de

50% ethanol 22 1.29 ± 0.11cd 6.54 ± 0.55c 1.67 ± 0.13cd 8.42 ± 0.68cd 23 2.01 ± 0.10b 10.27 ± 0.51b 1.93 ± 0.11b 9.86 ± 0.57b 24 1.95 ± 0.07b 9.75 ± 0.35b 1.90 ± 0.06bc 9.51 ± 0.31bc 26 2.64 ± 0.11a 12.63 ± 0.54a 2.39 ± 0.09a 11.45 ± 0.44a aData are the mean ± SE. Data compared by ANOVA, n = 36, P < 0.001, followed by post-hoc Student’s t test; different letters indicate statistically significant differences between means. FW, fresh weight. the staggered time points at which grape sampling and extraction took vortexed and then incubated for 1 h at 27 °C with shaking and place, a purified commercial seed extract (Tarac Technologies, thereafter centrifuged at 16000g for 20 min. For each experiment a Nuriootpa, Australia) was included for each 96-well plate assayed. standard blank of the respective tannin solution without cell wall Data were expressed on a skin weight and per berry basis. Berry weight material was included. Supernatants were analyzed by GPC. The loss data have been previously published.25 of tannin in supernatants through interaction with cell wall material Gel Permeation Chromatography. An aliquot of each skin was determined by difference in GPC peak area, calibrated for the extract was dried under a stream of nitrogen and recovered in N,N- respective tannin preparations used in the study, as described dimethylformamide. Samples were centrifuged at 16000g for 5 min previously.26 Adsorption isotherms were fitted using the Langmuir and directly analyzed by GPC as describedin ref 32. Preveraison skin equation,35 modeling the quantity of tannin bound (mg/g) as a tannin fractions of known molecular mass (by phloroglucinolysis) function of the concentration of tannin remaining in solution, at were used as standards for calibration.23 For calibration, a second- theoretical equilibrium. Curve fit R2 values of 0.99 were obtained for order polynomial was fitted with the tannin elution time at 50% for both tannin types tested, and these results were used to derive the ffi each standard. apparent a nity constant (KL) and the maximum adsorption capacity Cell Wall Surface Area. The cell wall material samples were Nmax of cell wall material for each tannin type, where N represents the mounted on specimen stubs with carbon tabs and coated with 5 nm of mass of tannin bound (mg) per gram of cell wall material. platinum in a Cressington 208HR sputter coater (Cressington Binding Reaction of Monoglucoside Anthocyanins with Cell Scientific Instruments Ltd., Watford, UK). Samples were examined Wall Material. Shiraz grapes at commercial ripeness (24 °Brix) were in a Philips XL30 field emission scanning electron microscope partially homogenized with a blender. Crude methanolic extracts (50% operated at an accelerating voltage of 10 kV. Specific surface areas of v/v) were prepared from the grape slurry and then concentrated under dry cell wall isolates were determined using an automated manometric reduced pressure at 30 °C. A Toyopearl (660 mL bed volume) column gas adsorption apparatus33 providing data collection for samples at 77 equilibrated with 0.1% v/v TFA was prepared, and aqueous extracts K and subatmospheric pressures. An accurate liquid nitrogen level were loaded in 100 mL batches. Water-soluble nonadsorbent control of up to ±0.2 mm was achieved and maintained throughout compounds were eluted in 2 L of the loading solvent. The each measurement by a custom-built liquid nitrogen delivery system.34 monoglucoside anthocyanin fraction was eluted with 1.5 L of 30% Ultrahigh-purity grade gaseous helium (>99.9%) and ultrahigh-purity v/v methanol containing 0.1% v/v TFA. The column was then washed grade gaseous nitrogen (>99.9%) (both from BOC Gases, Australia) with 2 L of 2:1 v/v acetone/water and re-equilibrated with 0.1% v/v were used for dead-volume and nitrogen adsorption measurements at TFA. The eluate was concentrated under reduced pressure at 30 °C, 77 K, respectively. Duplicate analyses were performed at staggered frozen at −80 °C, and then lyophilized, yielding ≅200 mg of dry points in the construction of nitrogen adsorption isotherms to confirm material, which was stored under nitrogen at −80 °C until used. the reproducibility of measurements. Extract purity was verified by HPLC,31 and the sample was free of Binding Reaction of Tannin and Cell Wall Material. Skins acetylated or coumaroylated anthocyanins, derived pigments, and prepared fresh from commercially ripe Cabernet Sauvignon grape polymeric material. The extract was found to have trace quantities of samples were extracted for 18 h in either 70% v/v acetone or 15% v/v early-eluting material at 280 nm, but this was below the detection limit ethanol containing 0.01% v/v TFA. Tannin was purified from extracts for quantification, indicating minimal contribution of non-anthocyanin according to a published method.24 Tannin isolates were characterized phenolics. Anthocyanin concentration was determined as the sum of by phloroglucinolysis,12 which yielded mDP values of 11 units for the individual 3-O-monoglucoside anthocyanins, as malvidin-3-glucoside 15% v/v ethanol extract and 33 units for the 70% v/v acetone extract. units using a commercial standard (Polyphenols Laboratories, The absence of monomeric phenolics, in particular anthocyanin, from Norway). Anthocyanin was added to a solution of 12% v/v ethanol the tannin fractions was confirmed by GPC analysis32 (negligible containing 0.01% v/v TFA at increasing concentration from 0.1 and 2 material eluting later than 16.3 min). Tannin solutions were prepared g/L either alone or combined with a 1.25 g/L solution of the 33 mDP in 12% v/v ethanol containing 0.01% v/v TFA. Tannin concentrations tannin. These were combined in 1 mL aliquots with 10 mg of a skin were from a minimum of 0.25 g/L and up to a maximum of 10 g/L. cell wall preparation that corresponded to the most ripe harvest date in These were combined in 1 mL aliquots with 10 mg of a skin cell wall the current study, in 1.5 mL centrifuge tubes. Cell wall solutions were preparation, which corresponded to the most ripe harvest date in the vortexed and then incubated for 1 h at 27 °C with shaking and current study, in 1.5 mL centrifuge tubes. Cell wall solutions were thereafter centrifuged at 16000g for 20 min. Methyl cellulose

1132 dx.doi.org/10.1021/jf4050606 | J. Agric. Food Chem. 2014, 62, 1130−1141 Journal of Agricultural and Food Chemistry Article precipitation31 was selected to analyze tannin concentration in the proportion of tannin extractable in 10% v/v ethanol supernatants. This was due to the potential interference of a high remained constant during ripening. However, at 20 and 50% v/ anthocyanin concentration in the phloroglucinolysis and GPC fi v ethanol, it was evident that with the progression of ripening a methods, which would require an additional sample puri cation fraction of skin tannin became nonextractable at 24 and 26 step. Anthocyanin was analyzed as the sum of individual 3-O- ° ° monoglucosides, quantified by HPLC.31 Anthocyanin adsorption Brix in comparison with the 23 Brix sample. As a more isotherms were fitted using the Langmuir equation as described for hydrophobic solvent, ethanol disrupts hydrophobic interac- tannin adsorption experiments, above. tions, potentially between tannin and cell walls, protein, or other skin phenolics,36 thereby facilitating greater extraction. It ■ RESULTS AND DISCUSSION is evident that a portion of grape skin tannin remained readily extractable in aqueous ethanol, independent of the total tannin Skin Tannin and Anthocyanin Extraction in Aqueous concentration. Ethanol. The extraction of both skin tannin and anthocyanin To characterize the size distribution of fractions extractable increased with the addition of ethanol to the solvent (Table 1). under the different solvent conditions, extracts were subjected For extraction using dilute, 10% v/v ethanol, significant to GPC analysis (Figure 2). The cumulative molecular mass increases in both tannin and anthocyanin content were evident distribution based on unpurified extracts represents both only between the first sampling point (22 °Brix) and the final polymeric material and smaller molecular mass phenolics. For sampling point (26 °Brix). When 20% v/v ethanol was used as all of the GPC distribution data, quantitatively greater the extraction solvent, extractable tannin and anthocyanin differences in molecular mass between treatments were evident content increased from the first to the second sampling point at later elution by GPC (70% elution), which corresponds to and thereafter remained constant. When ethanol concentration the polymeric fraction (mDP ≅ 3−15). This is consistent with was increased to 50% v/v, it was evident that there was an the observed increases in tannin concentration with increasing increase in the extraction of both skin tannin and anthocyanin ethanol concentration in the solvent (Table 1). For all ethanolic with advancing ripeness. However, for the transition from 23 to extracts, the distribution was shifted toward a higher molecular 24 °Brix, there was no increase in either anthocyanin or tannin mass average with advancing ripeness (Figure 2A−C). For 70% extracted in 50% v/v ethanol from the skin samples. v/v acetone extracts, the curves were more closely overlaid Partitioning of Tannin to a Nonextractable Fraction. (Figure 2D), indicating that higher total grape tannin content In the previous experiment it was reported that no differences resulted in increases across the entire molecular mass were found in the extraction of tannin in dilute ethanol (10 and distribution. Two effects are evident: first, that higher molecular 20% v/v) between the 23, 24, and 26 °Brix samples. Extraction mass tannins were increasingly extracted as solvent polarity in 70% v/v acetone was also performed on samples from these decreased and, second, that at the latest grape sampling point, a ripeness stages and is hereafter referred to as “total” tannin, fraction of intermediate molecular mass tannin was more because this acetone/water ratio presents a more exhaustive extractable in ethanol. This finding points to greater complexity 17 extraction for tannin than the ethanol-based solvents. in the factors that drive tannin extractability of during ripening Nevertheless, we note that some residual high molecular and raises questions as to whether there are (a) changes in the 24,25 mass tannin remains nonextractable in 70% v/v acetone, interaction of tannin with skin cell walls with advancing which was not accounted for in the current sample set. ripeness; (b) changes in tannin concentration that affect the It was found that during late-stage ripening, there was a selectivity of tannin adsorption by cell walls as a function of continuous increase in total skin tannin content (Figure 1). tannin molecular mass; and/or (c) changes in the composition When the tannin content extracted at different ethanol of other phenolics, namely, anthocyanins, that affect the concentrations was expressed as a percentage of total tannin, interaction of tannin with cell walls. These questions are further addressed below through cell wall characterization and model adsorption experiments. Analysis of Grape Cell Wall Surface Characteristics with Advancing Ripeness. Previous research on the tannin adsorption properties of skin cell walls from the same grape sample series presented in the current study demonstrated that the selective adsorption of high molecular mass tannin increased in the late stages of ripening.25 The analysis of cell wall polysaccharides indicated that major compositional changes occurred around veraison, with a preveraison loss of galacturonic acid (galacturonan) followed by losses in arabinogalactan I, consistent with published research.37 However, compositional changes in cell wall polysaccharides in late-stage ripening were found to be minor and could not be used to qualify the changes in cell wall binding properties for tannin. These cell wall samples25 were further characterized by SEM and for surface area using nitrogen adsorption isotherms. It is evident from scanning electron micrographs that Figure 1. Ripening-related changes in tannin extracted using an increasing concentration of aqueous ethanol as a proportion (%) of preveraison grape cell walls display a regular, relatively smooth ± (Figure 3A) surface structure with defined pit fields (Figure total tannin extractable in aqueous acetone (data are the mean SE; fi fi data compared by ANOVA, n = 27, P < 0.001, followed by post-hoc 3B). The presence of de ned pit elds was not detectable in Student’s t test; different letters indicate statistically significant cell walls imaged at veraison (Figure 3C), which is consistent differences between means). with losses in homogalacturonan-rich regions of the cell wall,38

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Figure 2. Cumulative molecular mass distribution of grape skin extracts at different ripeness levels according to their grape juice total soluble solids determined by gel permeation chromatography: (A) extracts in 10% v/v ethanol; (B) extracts in 20% v/v ethanol; (C) extracts in 50% v/v ethanol; (D) extracts in 70% v/v acetone. which confirmed earlier cell wall analysis.25 In the later stages of structure with ripening. These observable changes in the surface ripening, deepening of folds in the cell wall was evident, leading structure of cell walls were of interest because previous to a “wavy” appearance (Figure 3D−F). Initially, we considered observations related to enhanced adsorption of high molecular that this might be a fault of the tissue preparation method, mass tannins at late-stage ripening were consistent with a because lyophilization is noted to cause collapse of the cell wall greater cell wall porosity.25 The working hypothesis is that a structure and a reduction in macroporosity.28 This effect is greater surface area over which tannin binds to cell walls can particularly evident in the case of harsher drying methods.27 occur via hydrogen bonding and hydrophobic interactions However, we note that this phenomenon has been documented within cell wall cavities. In the absence of pores, high molecular previously in fixed tissue from ripening grape skins, using mass tannins may be excluded from the cell wall and have transmission electron microscopy.39 The authors proposed that limited interaction.24 a “wavy” appearance resulted from localized swelling of the cell To quantify the SEM observations, and as a prerequisite for wall. However, it was noted that the appearance of an employing the unripe cell wall fraction as a reference material, undulating cell wall was concomitant with degradation of the specific surface areas were defined for cell wall preparation from middle lamella. Therefore, it is possible that cell wall the ripening series. Although the specific surface area of each conformation was retained through the extraction and drying sample was low, well-defined low-pressure isotherms were steps, and the SEM images may reflect true changes in cell wall obtained with negligible uncertainty (values were approximately

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Figure 3. Scanning electron micrographs (5000× magnification) of isolated cell wall material from unripe and ripening Cabernet Sauvignon grape skins: (A, B) January 15, −11 DAV, preveraison; (C) January 26, DAV, veraison; (D) February 23, 28 DAV, 22 °Brix; (E) March 2, 35 DAV, 23 °Brix; (F) March 17, 50 DAV, 26 °Brix (DAV = days after veraison, bars = 10 μm).

Figure 4. Nitrogen gas adsorption isotherms determined at standard temperature and pressure for isolated cell wall material from unripe and ripening Cabernet Sauvignon grape skins (P = sample cell pressure; Po = nitrogen vapour pressure at 77K; preveraison = January 15, 11 days before veraison; veraison = January 26). the size of the symbols used) (Figure 4), leading to area values penetration of high molecular mass tannin within cell wall with precision suitable for three significant figures, and this data pores could occur.25 This could result in a reduced is presented in Table 2. As expected, the folds seen by SEM extractability of this tannin fraction with ripening, due to represent a roughened surface leading to an increased specific greater encapsulation within pores and, therefore, adsorption by surface area susceptible to nitrogen gas analyses. For the sample cell wall components. The measurable changes in ethanol- series under investigation, two surface area categories were extractable tannin (Table 1) showed that no change in tannin noted in ripening skin cell walls, corresponding to the ripeness extraction to dilute ethanol solution occurred, despite increases grades 22 and 23 °Brix, as distinct from 24 and 26 °Brix, in cell wall porosity. However, when the partitioning of tannin respectively. This conclusion is better defined by reference to extractable in aqueous ethanol was compared with that by 70% the isotherms in Figure 4 than from the subtle, but real, acetone extraction, a stronger relationship with cell wall differences in the specific surface areas extracted from these porosity could be demonstrated (Figure 1). The highest cell isotherms. wall porosity category was for samples collected at 24 and 26 Enhancement of cell wall porosity with the progression of °Brix (Figure 4; Table 2), and the corresponding samples ripening lends support to the hypothesis that greater demonstrated a reduction in ethanol-extractable tannin (in 20

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Table 2. Cell Wall Surface Area Estimated by Nitrogen molecular mass tannin, which is nonextractable even at Adsorption Using the Brunauer−Emmet−Teller (BET) increased ethanol concentration. Despite this observation, it Isotherm Model was evident that increased total acetone-extractable tannin fi ° sample surface area (m2/g) concentration in the nal sampling date (26 Brix) (Figure 1) resulted in two measurable phenomena: (a) enhanced tannin preripening (control)a preveraison 1.24 extraction in 50% v/v ethanol (Table 1) and (b) increased veraison 1.66 tannin molecular mass in the extract (Figure 2C). These fl ripeningb observations may be in uenced by cell wall porosity but cannot 22 °Brix 2.42 be explained by this effect alone. 23 °Brix 2.34 Effect of Tannin Concentration and Molecular Mass 24 °Brix 2.87 on the Interaction with a Porous Skin Cell Wall Material. 26 °Brix 2.89 Binding isotherms were constructed for two purified tannins of aCell wall samples from unripe grape cell walls in the same low and high average molecular mass (mDP 11 and 33). The developmental series were used as a control for comparison with tannins were prepared using different extraction methods to b ff ripening samples. Cell wall samples isolated from grapes at di erent selectively extract molecular mass classes, 15% v/v ethanol and stages of commercial ripeness as measured by the juice total soluble 70% v/v acetone, and were not subjected to further solids level. fractionation. Therefore, both tannin fractions were heteroge- and 50% v/v ethanol) as a proportion of acetone-extracted neous in terms of their molecular mass distribution (data not tannin. A possible interpretation of this result is that increased shown). In particular, the 70% v/v acetone extract had a porosity in skin cell walls during late-stage ripening may distribution of high (>15000 g/mol), intermediate, and low fl facilitate sequestering of tannins as an adsorbed fraction within molecular mass material, which re ects the broad spectrum the cell wall. On the basis of the published cell wall−tannin present in grape skins in situ. The dilute ethanol extract interaction data for these samples,25 it would be expected that reflected the distribution of wine-extractable skin tannin the adsorbed tannin fraction would consist primarily of high (<15000 g/mol).

Figure 5. Effect of skin tannin concentration and mean degree of polymerization (mDP) on the binding reaction with 10 mg of porous, ripe skin cell wall material (26 °Brix), analyzed by gel permeation chromatography: (A) constructed adsorption isotherms for mDP 33 and mDP 11 tannins with ffi estimated a nity constants (KL) and maximum adsorption capacity (Nmax); (B) percentage of mDP 33 tannin removed by adsorption to cell walls as a function of tannin molecular mass; (C) change in cumulative molecular mass (% elution) following adsorption of mDP 33 tannin; (D) change in cumulative molecular mass (% elution) following addition of mDP 11 tannin.

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Figure 6. Binding reaction of a porous, ripe skin cell wall material (26 °Brix) with monoglucoside anthocyanin, alone or in combination with 1.25 g/ fi ffi L of a puri ed skin tannin extract (mDP 33.1): (A) adsorption isotherms showing estimated a nity constants (KL) and maximum adsorption capacity (Nmax) constructed using the Langmuir equation; (B) change in tannin concentration as a percentage of a 1.25 g/L control (100%) following addition of 10 mg of skin cell wall material with increasing concentration of anthocyanin (mean ± SE). ffi ff The a nity constant (KL) and estimated saturation level more e ectively adsorb higher molecular mass tannin (>15000 (Nmax) derived from Langmuir isotherms (Figure 5A) for g/mol), and this can account in part for the observed tannin adsorption by skin cell walls differed as a function of sequestering of tannin to a nonextractable fraction in riper tannin molecular mass. Adsorption of high molecular mass skins. Increased porosity may facilitate a greater surface area tannin by cell wall material gave higher KL and Nmax values by over which high molecular mass tannin can bind. Hence, it comparison with the lower molecular mass tannin sample. The might be expected that the saturation of cell wall binding sites binding constants calculated from the isotherms would suggest would be reduced in riper skins, concomitant with increased that for skin cell walls, higher molecular mass tannin was bound total tannin concentration. more effectively even at high concentrations. However, an Nevertheless, it is evident that the effect of ripening on analysis of the selectivity of binding showed that this was not tannin extraction is more complex. The ongoing binding of the case. From the molecular mass distribution data obtained intermediate molecular mass tannins even at higher applied via GPC, for high molecular mass tannin applied at low concentrations, together with the high estimated saturation concentration, adsorption in the highest molecular mass range constant, could indicate that secondary interactions occur approached 100% (Figure 5B). As the tannin concentration between surface-bound tannin and tannin in solution. This increased, the selective adsorption of this molecular mass effect has been demonstrated in studies on tannin−cell wall fraction decreased significantly, whereas, the adsorption of the interactions in apple22,27,28 with the strong emphasis made that intermediate molecular mass tannins remained relatively the formation of tannin “multilayers” through noncovalent constant at 60%. This result indicates a concentration- interactions is likely to account for high saturation constants in dependent effect, whereby the averages taken at different the binding reaction. In particular, it was noted that this effect molecular mass cutoff points (% elution) shifted with increasing may favor multilayer formation by tannins of intermediate tannin concentration (Figure 5C): decreased molecular mass at molecular mass27 (±2900 g/mol) due to their increased low concentrations and increased molecular mass at the highest propensity to form aggregates, a phenomenon observed to be tannin concentration. Conversely, for the low molecular mass lower for high molecular mass tannin (±20000 g/mol). This tannin fraction applied, there was less selectivity observed in observation may further explain the reduction in adsorption of binding events, and molecular mass decreased for all tannin high molecular mass tannin at higher applied tannin molecular mass classes, independent of tannin concentration concentrations, whereas the adsorption of intermediate (Figure 5D). These results show that increases in tannin molecular mass tannins remained constant (Figure 5B,D). concentration may have resulted in a higher total amount of From a reinterpretation of the tannin extraction data tannin adsorbed by cell walls, but also reduced adsorption of obtained using aqueous ethanol concentrations, it is evident higher molecular mass tannins. For the current study, the from the GPC cumulative molecular mass distribution that the tannin/cell wall ratio in situ would have been ≅0.2 (2 g/L highest molecular mass tannins were poorly extracted in tannin application) based on published data for cell wall yield.25 aqueous ethanol, even at 50% v/v (Figure 2A−C). Rather, they This means that for even a small change in total skin tannin were extractable only in 70% acetone, as shown by the shift in concentration, the molecular mass of the nonadsorbed tannin the cumulative molecular mass distribution of the extract fraction could shift to a higher average as a result of a changing (Figure 2D), which approached a molecular mass of 30000 g/ selectivity pattern by the cell walls. mol at 100% elution. Therefore, quantitatively, the greatest Althought it cannot be conclusively shown from the current effect of increasing ethanol in the extraction solvent was found data, the loss of selectivity for higher molecular mass material for tannins within the intermediate molecular mass range even at low applied tannin concentrations suggests the early (Figure 2A−C). This suggests that ethanol may selectively saturation of direct binding sites on the cell wall. The saturation extract tannins of intermediate molecular mass, which are constant calculated from the Langmuir equation, first, exceeds potentially associated as multilayers with cell wall constituents. the solubility of tannin in aqueous ethanol (Figure 5A) and, Our previous work using model experiments in grape24 second, fails to account for the selectivity of binding. Previous highlighted this effect, such that small changes in ethanol work25 showed that more porous cell walls (later ripeness) concentration (12−20% v/v) reduce the interaction of tannins

1137 dx.doi.org/10.1021/jf4050606 | J. Agric. Food Chem. 2014, 62, 1130−1141 Journal of Agricultural and Food Chemistry Article of intermediate molecular mass with cell wall material, whereas As discussed, the effect of either ripeness stage or increasing a negligible effect of ethanol is exerted on the adsorption of ethanol in the solvent on tannin extraction was mirrored by high molecular mass tannin. Quantitative data from this changes in anthocyanin extraction. Therefore, a pertinent experiment is shown in the Supporting Information (Figure question was whether complexing between anthocyanin and S2). The selective effect of ethanol in driving the tannin−cell tannin could enhance the 280 nm absorbance, leading to wall interaction response may also indicate the contribution of erroneous conclusions regarding changes in tannin concen- hydrophobic interactions, as indicated by Le Bourvellec and co- tration using the methyl cellulose precipitable tannin assay. We workers.28 have noted in previous research using tannins purified from the Interaction of Cell Walls with Tannin and Anthocya- same grape sample set presented in the current study that nin. It was noteworthy from the extraction experiments that anthocyanin incorporation into the tannin polymer caused anthocyanin extraction was limited by ethanol concentration in minor increases in tannin color in immature grapes,16 but from a similar manner to tannin (Table 1). It is known that 22 °Brix onward there was no further color change observed. In anthocyanin can associate with walls,40 although the comparing the methyl cellulose precipitation results from the interaction of monomeric flavan-3-ols is thought to be same purified tannins16 at different ripeness stages (Supporting negligible.22 We have shown previously, using a wine system Information Figure S3), it was found that no change in the containing both tannin and monomeric anthocyanin, that grape methyl cellulose precipitation result was detected for a fixed cell wall material can remove anthocyanin in the presence of concentration of tannin. On the basis of these results, and our tannin.41 What was unknown at the outset of the study is observations that the presence of tannin in combination with whether the interaction of tannin and anthocyanin influences grape cell walls potentially restricts anthocyanin extraction, we binding events with the cell wall. In a model experiment, the conclude that the similarity in anthocyanin and tannin adsorption of anthocyanin by cell walls was significant (29% extraction data between the different grape samples was a removed from a 1 g/L solution) and enhanced by the presence true effect. of tannin (45% removed from a 1 g/L solution). The binding Potential Mechanisms That May Increase Skin Tannin isotherms for this experiment across a range of anthocyanin Concentration and Facilitate Sequestering to Cell Walls. concentrations are shown in Figure 6A. As anthocyanin In grapes, research has shown that the biosynthetic pathway for concentration increased, tannin adsorption by cell wall material the synthesis of flavan-3-ols (precursors to tannin) is active up was not affected (Figure 6B). The estimated saturation constant until the onset of ripening (veraison), and thereafter gene for anthocyanin using the Langmuir equation was lower in the expression is down-regulated by the hormone abscisic acid presence of tannin, but at the concentration range present in (ABA), which signals the onset of veraison.43 Whereas the grapes, the system would be well below saturation. It therefore cessation of gene expression for flavan-3-ol biosynthetic appears that for the conditions of the current study, enzymes prior to veraison may indicate that tannin should anthocyanin did not influence tannin adsorption by cell walls. decline during ripening, a concurrent decrease in monomeric At the same time, skin tannin present in grapes might limit the flavan-3-ols is also observed. The reason for this phenomenon extraction of anthocyanin. has not been addressed in the literature and, speculatively, In general, 50% v/v ethanol is considered a good solvent for flavan-3-ols could become polymerized to tannin during the anthocyanin, correlating well with accepted methods for ripening period if they remain intact within a subcellular anthocyanin analysis, and is useful in the prediction of compartment. A recent paper has suggested that the site of anthocyanin extractability during grape ripening.42 Conversely, tannin polymerization in the grape cell is within a - some degradation of anthocyanin was found in acetone extracts derived structure, the “”.44 These (data not shown). From the results of the extraction originate from thylakoids within the chloroplast and are experiment, it would therefore be expected that the 50% v/v transported within membrane-bound vesicles, which become ethanol extraction reflects true differences in anthocyanin incorporated to the vacuole by invagination of the tonoplast concentration between grape samples, although this is not the membrane. According to the preliminary work by Brillouet and case for tannin. Furthermore, the results indicated that co-workers,44 polymerization could occur within these anthocyanin content increased in riper samples, but that organelles at any point following formation of the tannosome these differences were not reflected in dilute ethanol extracts because these remain separated from cytoplasmic or vacuolar except for the least ripe (22 °Brix) sample, which had the contents by a membrane. An important consideration based on lowest extractable anthocyanin (Table 1). On the basis of the this work44 is that tannin may remain sequestered by a data from the model binding experiment with anthocyanin and membrane at all ripeness stages, meaning that interaction grape cell walls, we propose that the poor extraction of between tannin and cell walls in situ would be improbable and anthocyanin at low ethanol concentration may be due, in part, potentially occur only once grape cell compartments are to a significant interaction with cell walls. Increased disrupted during crushing and fermentation. Nevertheless, in anthocyanin extraction at higher ethanol concentrations may other studies on grape, the partitioning of tannin has been be due to a loss of hydrophobic interactions between observed between vacuolar and cell wall fractions in both grape anthocyanin and cell walls or between anthocyanin and cell skins and seeds, with higher levels of polymerization occurring wall-bound tannin, but does not exclude the contribution of in cell wall-associated fractions.18,19 Some studies have ionic interactions.40 This result showed that in a like manner demonstrated that a fraction of high molecular mass tannin is for skin tannin, anthocyanin extraction could potentially be nonextractable in acetone (<5% of acetone-extractable tannin), restricted by multiple noncovalent interactions. Further to this, and this may possibly be covalently bound to the cell wall.24,25 an important observation toward interpreting the tannin Furthermore, ultrastructural studies of grape skin cells show extraction data was that anthocyanin is unlikely to affect the aggregations of phenolic-rich inclusions in immature grape cell adsorption of tannin by cell walls within the concentration walls, which are no longer evident in late-stage ripening, range occurring in grapes. concomitant with increased invagination of the cell wall.39 If a

1138 dx.doi.org/10.1021/jf4050606 | J. Agric. Food Chem. 2014, 62, 1130−1141 Journal of Agricultural and Food Chemistry Article pathway for tannin transport to the cell wall exists, the These observations highlight that skin cell walls may play a mechanism is currently unknown. However, a recent review on significant role in introducing noncovalent interactions with tannin biosynthesis, drawn from work on various plant species, tannin, which in turn limits its extractability. Further research is has suggested that sequestering of tannin at the cell wall could required to move away from a simplistic model of tannin be mediated by vesicle trafficking, where following deposition at extraction, as directly dependent upon tannin concentration, to the cell wall, tannin may be further oxidized and potentially one that builds upon the multiple levels at which noncovalent polymerized by apoplastic polyphenol oxidase.45 Irrespective of interactions between tannin and macromolecules can occur. whether cell wall−tannin interactions occur in situ in the grape Ongoing research is needed to better understand how tannin berry or only following grape crushing as the result of cell forms colloid complexes with insoluble cell walls, as well as disruption, it appears that this phenomenon plays a significant soluble polysaccharide and protein, and how these might affect role in limiting tannin extraction. tannin extractability. Implications for Winemaking Practice. In commercial winemaking, a correlation has been observed between ■ ASSOCIATED CONTENT enhanced skin tannin concentration, tannin molecular mass, *S Supporting Information and allocation grading.46 Furthermore, in a wine sensory S1, tannin extraction kinetics from grape skins in different “quality” study, grapes with enhanced skin tannin and extraction solvents; S2, effect of increasing ethanol concen- anthocyanin were associated with increased wine tannin and tration on the adsorption of high and low molecular mass color, resulting in an improvement in rating by wine judges.47 tannin fractions by cell wall material; S3, comparison of results On the basis of the current data set, it is evident that despite of the phloroglucinolysis and methyl cellulose precipitable increases in grape tannin and anthocyanin content with the tannin assays on purified grape skin tannins from different progression of ripening, delaying grape harvest may not ripeness stages. This material is available free of charge via the necessarily facilitate a higher skin tannin extraction. This Internet at http://pubs.acs.org. would appear to be due to the significant limitation in extractability exerted by the interaction of skin tannin and ■ AUTHOR INFORMATION anthocyanin with cell walls and, secondarily, the possibility that Corresponding Author further tannin increases were sequestered within cell wall pores *(K.A.B.) Phone: +61-8-83136190. Fax: +61-8-83136601. E- and therefore not extractable. The hypothesis that ripening mail: [email protected]. could facilitate an enhancement of tannin extractability under Funding the conditions of ferment (similar to our experiments with The Australian Wine Research Institute, a member of the Wine dilute alcohol) was not strongly supported by our results. Innovation Cluster in Adelaide, is supported by Australian Nevertheless, the molecular mass of tannin extracted from riper grapegrowers and winemakers through their investment body, grapes increased, even when the extractable tannin content was fi ff the Grape and Wine Research and Development Corporation, not signi cantly a ected. Although an increased wine tannin with matching funds from the Australian government. molecular mass may be associated with higher wine quality,46 this may also cause excessive astringency in the wine, Notes fi introducing the requirement for fining. The authors declare no competing nancial interest. In conclusion, the current study has shown that the factors which underpin skin tannin extractability are complex: ■ ACKNOWLEDGMENTS (a) Increases in total skin tannin content with the We thank Pernod Ricard Australia (Orlando Wines) and the progression of ripening were not consistently reflected University of Adelaide for the donation of grape samples. We gratefully acknowledge Lynette Waterhouse of Adelaide by increases in ethanol-extractable skin tannin. Microscopy, The University of Adelaide, for SEM imaging of (b) The observed increase in cell wall porosity with ripening cell wall samples. We also acknowledge Alex Shulkin at the may partly explain this, because the proportion of skin Australian Wine Research Institute for the isolation of grape tannin bound by noncovalent interactions within cell wall anthocyanins. pores appears to increase in a corresponding manner, rendering a fraction of tannin nonextractable. ■ REFERENCES (c) The molecular mass of the ethanol-extractable skin (1) Busse-Valverde, N.; Bautista-Ortin, A. B.; Gomez-Plaza, E.; tannin fraction shifted to a higher molecular mass average Fernandez-Fernandez, J. I.; Gil-Munoz, R. Influence of skin maceration as total skin tannin content increased. This may be due time on the content of red wines. Eur. 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