Comparison of Extraction Protocols To Determine Differences in Wine

Apr 29, 2014 - Wine-Extractable Tannin and Anthocyanin in Vitis vinifera L. cv. Shiraz and Cabernet Sauvignon Grapes. Keren A. Bindon,* Stella Kassara...
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Comparison of Extraction Protocols To Determine Differences in Wine-Extractable Tannin and Anthocyanin in Vitis vinifera L. cv. Shiraz and Cabernet Sauvignon Grapes Keren A. Bindon,* Stella Kassara, Wieslawa U. Cynkar, Ella M. C. Robinson, Neil Scrimgeour, and Paul A. Smith The Australian Wine Research Institute, P.O. Box 197, Glen Osmond, SA 5064, Australia S Supporting Information *

ABSTRACT: Cabernet Sauvignon and Shiraz grapes were sourced from different regions within Australia, and microvinified with a skin contact period of 6 days. Grape samples were extracted using two protocols: a 15% v/v ethanol, 10 g/L tartaric acid extract of gently crushed berries (wine-like, WL) and a 50% v/v ethanol, pH 2 extract of grape berry homogenate. It was found that in WL extracts, grape tannin and anthocyanin concentrations were strongly related to wine tannin, anthocyanin and color density achieved during the skin contact period. No relationship was observed for grape tannin concentration analyzed in homogenate extracts and wine tannin, but a strong, positive relationship was found for anthocyanin concentration. When the data obtained from homogenate extraction was treated separately by grape variety, a stronger relationship between grape and wine tannin concentration was observed. Tannin compositional analysis in wines indicated that higher tannin concentrations were due to the extraction of tannin of higher molecular mass during fermentation, most likely from grape skins. KEYWORDS: anthocyanin, tannin, color, grape, wine, extractability, (−)-epigallocatechin, (−)-epicatechin-3-O-gallate, prodelphinidin, molecular mass, methyl cellulose, phloroglucinolysis

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has been found to be weak7 or absent.6 Some studies have explored the relationship between only grape skin tannin concentration and wine tannin concentration.8−12 These have shown a strong relationship, in which higher grape skin tannin concentrations were correlated with higher wine tannin concentrations. Interestingly, a cocorrelation between higher grape skin tannin concentration, grape anthocyanin concentration and wine color density was a common observation by these authors.8−12 Color, as well as anthocyanin and tannin concentrations in wine, have been shown to be positively associated with market value grade, and they are therefore useful as objective markers for wine quality.13,14 Higher wine tannin, anthocyanin and polymeric pigment concentrations have been shown to be strongly correlated with wine color density and overall quality score as defined by a sensory panel.10 Yet, despite this clear indication that the strategic management of wine tannin and anthocyanin is important, a rapid analytical method to predict wine phenolic profile from grapes has not yet been developed. Various factors may have hindered progress in this area. Foremost, rapid, high-throughput analysis methods which are specific for tannin have only recently been developed.15−18 Another important consideration is that most analytical methods aim to achieve as close to an exhaustive extraction as possible, and this is evident in techniques applied for grape tannin and anthocyanin.15,16 Wine, on the other hand,

INTRODUCTION In the production of red wines, it is crucial to optimize the extraction of grape-derived phenolic compounds, primarily anthocyanin and condensed tannin (proanthocyanidin) in order to ensure the development of stable wine color and desirable textural properties. Tannins extracted during winemaking potentially originate from grape skins, seeds and pulp1 while in most commercially-produced grape varieties, anthocyanins occur only in the grape skins. The extraction of phenolics from these respective grape components during fermentation differs significantly, with anthocyanin and skin tannin being extracted early,2,3 and seed tannin being extracted later. While a significant amount of tannin is present in grape pulp, this appears to remain nonextractable during fermentation, potentially due to a strong association with pulp cell wall constituents.1 Interactions between tannin and cell wall constituents may also occur in grape skins (and potentially seeds), which limits tannin extraction into the wine.1 Apart from exerting a limiting effect on tannin extractability, cell wall constituents from grape and yeast cells which become suspended during fermentation can bind, and remove tannin and anthocyanin as these settle to form wine lees.1,4,5 There are only a limited number of published studies which have directly explored the relationship between grape phenolic concentration and wine composition. Studies which have attempted to draw correlations between an exhaustive extract of homogenized grape samples (50% v/v ethanol) and the corresponding wines6,7 have shown positive relationships for grape anthocyanin analysis and wine color indices. However, for tannin analysis in 50% v/v ethanol extracts of whole-grape homogenates, the relationship between grape and wine tannin © 2014 American Chemical Society

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January 16, 2014 April 27, 2014 April 29, 2014 April 29, 2014 dx.doi.org/10.1021/jf5002777 | J. Agric. Food Chem. 2014, 62, 4558−4570

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transferred to a plastic bag equipped with a resealable zipper and gently crushed by hand. Expressed juice was collected through a 0.5 mm sieve, and then centrifuged at 1730g for 5 min. Juice °Brix was determined using a digital refractometer. TA and pH were determined using a pH meter and combination electrode. The TA was determined by titrating with 0.33 M sodium hydroxide solution to a pH end-point of 8.2, and expressed in g/L of tartaric acid equivalents. From each destemmed grape berry sample, triplicate 50 g (±0.5 g) and 200 g batches were sampled and the weight recorded, and these were then transferred to plastic bags equipped with a resealable zipper or in screw-cap polypropylene containers, respectively. The triplicate 50 and 200 g samples were stored at 4 °C and processed fresh within 3 h, as described below. For certain grape samples, further triplicate batches of 50 and 200 g were collected as described above, but they were immediately frozen at −20 °C. These grape batches were stored for either 24 h, 1 month or 3 months in order to determine the effect of freezing on anthocyanin and tannin concentration measured by different analytical methodologies. Finally, triplicate 1 kg samples were collected from each destemmed grape batch and transferred to sealed plastic bags. Care was taken to minimize grape crushing, and samples were stored at 4 °C overnight, before being processed for microvinification, as described below. Extraction of Whole Berry Samples under Wine-like Conditions. To simulate the extraction of tannin and anthocyanin from gently crushed grapes under wine-like conditions, a WL extraction protocol in model wine solution was developed. The extraction solvent was prepared containing 400 mL/L absolute ethanol and 10 g/L tartaric acid, pH 3.4. Frozen 50 g berry samples were brought to room temperature by placing them sealed, within a 25 °C water bath for 1 h. Defrosted or fresh triplicate 50 g berry samples, retained in a plastic bag equipped with a resealable zipper, were gently crushed by hand. Crushed grape berries and juice were transferred to a 70 mL polypropylene jar, and 15 mL of extraction solvent was added, to each sample. This addition was based on an approximate juice yield of 0.5 mL per g of fruit, in order to extract in 15% v/v ethanol. Samples were sealed under screw cap and extracted on a platform shaker at 25 °C in the dark for 40 h. After extraction, samples were pressed by hand through a 0.5 mm sieve and the volume of recovered extract recorded. For the sample set studied, the percentage standard deviation of the average recovered extraction volume was 7%. The grape skin and seed residue were recovered and re-extracted in 30 mL of 50% v/v aqueous ethanol pH 2 (with HCl) for a further 18 h. The 50% ethanol extract was recovered as described above, but the volume was not recorded. The WL and 50% ethanol extracts were centrifuged at 1730g for 5 min, and thereafter aliquots were removed for the analysis of tannin and anthocyanin as described below. Optimization of Extraction Volume and Time for Model Wine Extracts. Before the main experiment was commenced, the effect of extraction volume and the length of the extraction on tannin concentration per unit berry fresh mass were investigated for a single Cabernet Sauvignon grape sample obtained from a vineyard located at the Waite Campus, University of Adelaide. Grape samples were prepared as outlined above, but the extraction volume per 50 g sample was adjusted between 35 and 75 mL, maintaining the ethanol concentration at 15% v/v. Extractions were performed in triplicate, and tannin and anthocyanin concentrations were monitored at five time points over a 64 h period and then analyzed as outlined below (Supporting Information S2). Extraction of Grape Berry Homogenates. Homogenate extractions were performed according to a published protocol.5,15,16 The selection of a 200 g grape sample for homogenization was required for the type of homogenizer used, which was a Retsch Grindomix GM200 (Retsch GmbH & Co., Germany). Frozen 200 g berry samples were defrosted by placing them at 4 °C for 18 h. Both fresh and defrosted triplicate 200 g samples were homogenized cold ( 0.9). The calculated root-mean-square error (RMSE) of prediction of wine tannin concentration from the WL assays was 0.18 g/L for the volume-corrected corrected assay, and 0.15 g/L for the uncorrected assay. This is of relevance, since in 4564

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grape-based measures were strongly related to wine anthocyanin concentration (R2 ≥ 0.9) and wine color density (R2 > 0.85) (Table 3, Figure 1B, D). A weaker relationship was found for the grape-based measures of anthocyanin and nonbleachable wine pigments, with WL and homogenate extracts giving R2 = 0.7 (Table 3) and 0.65, respectively. Rather, a stronger relationship was found for wine tannin concentration and nonbleachable pigments (R2 > 0.85). Nonbleachable pigment and wine tannin concentration also strongly contributed to wine color density, as evidenced by similarly high R2 values. Since wine color was analyzed in young wines, a significant proportion of anthocyanin was still in the monomeric form (Table 2), and it had not yet undergone the reactions associated with aging which significantly increase nonbleachable pigments.34 Hence, the greatest contribution of nonbleachable pigments in young wines was potentially from anthocyanin-tannin condensation products (polymeric pigments) formed during fermentation35 as opposed to smaller, anthocyanin-derived pigments. Since both anthocyanin and tannin are required for the formation of stable wine color, their analysis in WL extracts may be a good predictor of wine color density, as well as nonbleachable pigments. Multiple linear regression analysis revealed an improvement of the R2 of prediction for both wine color density and nonbleachable pigments to 0.87 when both WL tannin and anthocyanin concentration measures were used (data not shown). A further important consideration was how basic grape compositional differences in terms of either ripeness or berry weight were related to measured color and tannin in grapes or wine. It was found that the degree of grape sugar ripeness (as juice °Brix) was related (R2 > 0.7) (Table 3) to uncorrected WL tannin concentration, wine tannin concentration, wine color density and nonbleachable pigments. Interestingly, juice °Brix had a weaker relationship with concentrations of corrected WL tannin, grape anthocyanin and wine anthocyanin (Supporting Information S5). No relationship was found for juice °Brix and the recovered volume of WL extracts, so the strong relationship to uncorrected WL tannin concentration is not accounted for simply by changes in juice volume. A further important observation was that no correlation was found between berry weight and any measure of grape or wine tannin and color (data not shown). This is of particular relevance for grapes of the Shiraz variety,36 since shrivel in the later stages of ripening may increase the relative concentration of tannin and sugar per unit fresh mass. As discussed previously, both the corrected and uncorrected measures of grape WL tannin concentration were strongly related to wine tannin concentration, but the R2 value was slightly higher for the uncorrected measure (0.94 compared to 0.91). Therefore, the correlation between wine tannin and °Brix may simply indicate that increases in grape ripeness may have been associated with increased extractability of tannin. Since all ferments were pressed off skins before the completion of alcoholic fermentation, and wines made from higher °Brix grapes had high residual sugar levels, ethanol concentration was unlikely to have influenced tannin extraction. The relationship between grape ripeness (°Brix) and tannin concentration in grapes is unclear, and reports in the literature are variable, showing that tannin can increase, remain constant or decrease during ripening.26,37−43 While our results do not demonstrate a causative effect, the current data indicate that changes in grape composition associated with ripening may

a commercial application of the WL extraction protocol, the estimate of juice volume or recovered extract volume would be tedious and time-consuming, and possibly hinder its uptake. The current results indicate that this volume correction step may not be necessary in order to compare compositional differences between grape samples. The relationship between grape tannin concentration determined using the WL tannin protocol and wine tannin concentration is shown graphically (Figure 1A), and it demonstrates the distribution of samples by grape variety, maturity and regional origin, as discussed previously. Re-extraction of the WL extract residues with 50% v/v ethanol increased the estimate of total grape tannin concentration by 52% (±13%) but reduced the strength of the relationship with wine tannin slightly to give an R2 value of 0.86 (Supporting Information S5). With sample homogenization and extraction in 50% v/v ethanol, the grape tannin concentration was on average 204% (±92%) higher than that determined in the WL extracts. Interestingly, a poor relationship was observed between grape tannin concentration determined from homogenate extracts and wine tannin concentration (R2 = 0.46 for total sample) (Figure 1C, Supporting Information S5). A “tannin extractability” estimate was calculated as follows: [WL tannin ×100] ÷ [homogenate tannin]. This measure expresses the extractable grape tannin concentration as a proportion of total grape tannin, and we considered that this might correlate with wine tannin concentration. However, no relationship was found with the “tannin extractability” estimate and wine tannin concentration (data not shown, see Supporting Information S5). From the scatter-plot shown in Figure 1C, a separation of tannin data by grape variety could be observed, which indicates that there may be different responses for grape tannin concentration determined in homogenate extracts for the Shiraz and Cabernet Sauvignon varieties. Indeed, separation of the samples by variety brought an improvement in the R2 values for grape tannin determined in homogenate extracts and wine tannin following linear regression, and this was better modeled for Shiraz (R2 > 0.9) than for Cabernet Sauvignon (R2 = 0.75). It is likely that at high ethanol concentration (50% v/v) and with homogenization, a significantly increased seed tannin was extracted than can be expected with WL extraction. Since seed tannin is not readily extracted during the early stages of fermentation2,19 (Supporting Information S6), it is unlikely that seed tannin was significantly extracted in the wines, and this will be discussed in more detail at a later stage. The WL extraction protocol is therefore more accurate in predicting the concentration of grape tannin that is extractable during primary fermentation, in this case during a set 6-day skin contact period. Differences in the concentration and proportional distribution of tannin within the grape berry (skin versus seed) may account for somewhat higher concentrations of homogenateextract grape tannin in the Cabernet Sauvignon samples, possibly reflecting a higher seed tannin concentration. The relatively strong relationship found for homogenate-extracted tannin concentration and wine tannin concentration for the respective grape varieties treated as distinct sample sets shows that this extraction protocol may still have relevance for the prediction of wine-extractable tannin, but only when the calibration is determined for a given variety. For grape anthocyanin, the homogenate extraction, corrected WL (Table 3) and uncorrected WL protocols (Supporting Information S5) were strongly correlated to one another. Both 4565

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Table 4. Tannin Compositional Properties Determined by Phloroglucinol Analysis for Selected High-Tannin and Low-Tannin Shiraz and Cabernet Sauvignon Wine Samples Obtained from a Variety of Regions within South Australiak

a

Analysis of a subsample of wines designated HIGH or LOW in tannin concentration based on methyl cellulose precipitation analysis. bSignificant differences are indicated by different superscripted letters alongside the mean and were determined by one-way ANOVA, followed by a post-hoc unpaired Student’s t-test, ns = not significant. cTannin concentration determined by methyl cellulose precipitation as epicatechin equivalents. d Tannin concentration as the sum of flavan-3-ol subunits by phloroglucinolysis expressed as epicatechin equivalents. eMass conversion is the recovery of tannin as its component subunits by phloroglucinolysis, as a proportion of the total tannin concentration by methyl cellulose precipitation. fMolecular mass as determined by phloroglucinolysis. gMean degree of polymerization in epicatechin units. hMolar proportion of extension (−)-epigallocatechin subunits. iMolar proportion of (−)-epigallocatechin subunits of extension (EXT) subunits only. jMolar proportion of terminal and extension (−)-epicatechin-3-O-gallate subunits. kANOVA, n = 16, different letters after the mean within a column indicate significant differences determined by a post-hoc Student’s t-test.

oxidation48 or during wine aging.47 This consequently affects wine tannin concentration as determined by subunit yield. It was evident that tannin mass conversion was similar by variety for both low- and high-tannin wines (Table 4), and was higher in Cabernet Sauvignon wines than in Shiraz wines. Varietal differences in tannin mass conversion may not necessarily be incurred during winemaking, and they can be found in tannin extracts from grape.1,26,45,46 The differences in phloroglucinolysis yield between high- and low-tannin wines may therefore be a varietal effect. Varietal differences in tannin composition were also observed for the degree of tannin galloylation ((−)-epicatechin-O-gallate), which was significantly higher for Shiraz wines. Since seed tannins contain proportionally higher levels of epicatechin gallate than skin tannin,40,44 this could be considered to reflect a greater extraction of seed tannin in Shiraz wines. However, this is unlikely to be the case, since our work to date with Shiraz grapes has shown minimal extraction of seed tannin during a similar skin contact period to that used in the current study (Supporting Information S6). Furthermore, the degree of galloylation in skin tannin can be up to 10% in Shiraz grapes,1,44,47 but it is generally low in ripe Cabernet Sauvignon grapes26,43,49 (also see Supporting Information S7). Tannin molecular mass, which gives similar information to the mean degree of polymerization (mDP) increased as wine tannin concentration increased, and this was associated with a minor increase in the proportion of prodelphinidin ((−)-epigallocatechin) of total tannin subunits. However, when prodelphinidin was expressed as a proportion of extension

influence the extraction of grape tannin into wine during fermentation, and this warrants ongoing investigation. Compositional Differences between High- and LowTannin Wines. A subset of wine samples were analyzed for subunit compositional analysis following solid-phase extraction14 and phloroglucinolysis24 to determine whether differences in wine tannin concentration observed using the MCP tannin assay could be (1) confirmed using an alternative method of quantification and (2) associated with changes in tannin subunit composition or molecular mass. A subset of wine samples from both grape varieties studied were selected, having high- or low-tannin concentration based on the MCP assay. The Shiraz wines had higher tannin concentration, as measured by MCP, than Cabernet Sauvignon wines (Table 4). However, when tannin concentration was determined by phloroglucinolysis for these samples, high-tannin Shiraz and Cabernet Sauvignon wines showed similar concentrations based on subunit yield. On the other hand, for the low-tannin samples, the Cabernet Sauvignon wines had a somewhat higher subunit yield by phloroglucinolysis than Shiraz wines. Nevertheless, it was evident that differences in high and low wine tannin concentrations determined using the MCP tannin assay were confirmed by the phloroglucinolysis assay. The differences in the phloroglucinolysis yield point to varietal differences in the extent to which the tannin could be cleaved to its component subunits by acid-catalyzed depolymerization (tannin mass conversion). It is well-known that wine tannin mass conversion can vary by grape variety, ripeness1,26,38,44−47 and decreases during winemaking,1,45 following 4566

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Figure 2. Effect of a 3 month freezing period at −20 °C on grape composition determined by one-way ANOVA (n = 124) showing the distribution of samples as box and whisker plots (mean, maximum, minimum): (A) tannin concentration determined following wine-like extraction; (B) tannin concentration determined following homogenate extraction; (C) anthocyanin concentration determined following wine-like extraction; (D) anthocyanin concentration determined following homogenate extraction.

tannin concentration at 24 h of freezing, the most significant decreases (14% loss) in tannin concentration were found for the grape samples with the highest tannin, while samples with lower tannin were unchanged. After 1 month and 3 months of freezing, losses in measured tannin concentration measured in WL extracts were consistent for both high- and low-tannin grape samples, between 34% and 37%. Unlike the WL extraction protocol, frozen grape samples analyzed using the homogenate extraction procedure showed no significant loss in measured grape tannin concentration (Figure 2B). This indicates that the effect on tannin concentration in WL extracts was not likely to be due to tannin degradation, but rather due to changes in tannin extractability brought about by freezing. This is also supported by the observation that tannin concentration is stable in frozen extracts (and wines) at −20 °C (Supporting Information S2). Speculatively, freezing of grapes may increase the adsorption of tannin by grape cell walls,1 since breakdown of subcellular compartments during freezing and defrosting may occur. In contrast to the effect on WL tannin, no impact of freezing was found for WL anthocyanin extracts (Figure 2C). Similar to a previously published study,50 freezing up to 3 months caused no reduction in measured anthocyanin concentration in homogenate extracts. This indicates that following limited time periods of frozen storage, the homogenization extraction protocol can be used for grape tannin and anthocyanin analysis, but the WL extraction protocol is acceptable only for anthocyanin analysis. Implications of the Results for Commercial Winemaking Practice. The reported results have shown that for fresh grape samples, the application of a WL extraction provides a good indication of tannin and anthocyanin concentrations extractable during fermentation (in this study during a 6-day

units only, this was unchanged by either grape variety or tannin concentration. The proportion of prodelphinidin as a proportion of extension units can range considerably in skin tannins, and it is quite variable by season and grape variety1,26,42−45,49 (Supporting Information S7). For the current wine sample set, therefore, the high values for prodelphinidin as a proportion of tannin extension units, together with a low percent galloylation are strong evidence that the wine tannin extracted during the 6-day skin contact period was primarily derived from the grape skins. This is expected based on published work2 as well as our unpublished data (Supporting Information S6), which indicate that skin tannin extraction predominates within the early skin contact period. Effect of Freezing on the Analysis of Grape Tannin and Anthocyanin. The effect of storing grape samples at low temperature is an important consideration when samples are collected at a faster rate than analytical capacity and throughput allow, such as in a commercial or research context. Previous studies have shown that grape samples can be frozen at −20 °C for up to 3 months without measuring a loss in anthocyanin, when this was determined following a homogenate extraction protocol.50 At the outset of the current study, the effect of freezing grapes on tannin concentration was unknown. The analysis of tannin and anthocyanin in homogenate and WL extracts was performed on a subset of grape samples that had been frozen at −20 °C for 24 h, 1 month and 3 months (Figure 2). Due to the previous observations that anthocyanin concentration declines after 3 months of freezing,50 the storage period was not extended further. It was found that freezing grape samples for 24 h brought about a small reduction in grape tannin concentration measured using the WL extraction protocol, and this decreased further by 1 month and 3 months (Figure 2A). In observing the distribution of measured WL 4567

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region, vineyard, grape variety or management practices, and it would be of great advantage in commercial winemaking.

skin contact period). It was found that there was no benefit in correcting tannin or anthocyanin concentration in WL extracts for variation in juice (extract) volume. This means that extractions and analyses could be implemented in situations which demand high sample throughput, such as commercial or research laboratories. A limitation of the WL extraction protocol for tannin analysis, however, was that grape samples could not be frozen. It is therefore recommended that where delays in sample analysis occur, whole grape bunches are sealed and stored short-term at 4 °C before being destemmed. Alternatively, if WL extractions can be immediately conducted on fresh grapes, the extract appears to be stable at −20 °C, enabling the tannin analysis to be performed later (Supporting Information S3). The use of a homogenate extraction protocol was also found to be of value for predicting wine anthocyanin concentration, and when the calibration is adjusted for a particular cultivar, it could be used to predict wine tannin concentration. The benefit found for the use of the homogenate extraction protocol was that samples could be frozen before analysis. A further advantage of homogenate extraction is that this method has been used to develop calibrations for rapid analytical methods such as near-infrared spectroscopy51 and UV−visible spectroscopy.52 The WL and homogenate extraction techniques were shown to be of value in the prediction of tannin and anthocyanin concentrations extractable during the fermentation period. However, it is important to note that in commercial winemaking practice, different winemaking approaches can increase tannin concentration20 beyond that extracted during fermentation. Some studies have shown that skin tannin concentration tends to reach a maximum during primary ferment,2,3 but others have shown that it can increase continuously during both fermentation49,53 and extended maceration.39 On the other hand, seed tannin is poorly extracted during primary fermentation, but a late, rapid increase in extraction is observed after approximately 6 days, suggested to be facilitated by hydration of the seeds rather than an increase in wine ethanol concentration.19,54 Hence, seed tannin extraction could potentially increase until maceration is stopped, but it has been observed that a maximum in total wine tannin concentration is generally reached by 20 days of maceration.39,55 Given this variability in the kinetics of tannin extraction, a key question is whether the magnitude of the effect of a winemaking technique on final wine tannin concentration is in fact dependent upon grape tannin composition and extractability. In collating data from a limited number of studies which have explored the effect of extended maceration on wine tannin extraction,20,39,49,55−57 it was found that a strong positive relationship exists between tannin extracted during fermentation and the maximum tannin extracted following extended maceration (Supporting Information S8). Based on this, it appears viable that in a commercial context, decision-making regarding the optimal winemaking approach for particular grape batches could be enhanced by knowledge of the tannin and anthocyanin extractable during fermentation. For example, for high-tannin grapes, adequate extraction of phenolics may be obtained during a short skin contact period, whereas for low-tannin grapes extending maceration time or applying thermovinification20 may be required. The benchmarking of grape samples for their predicted wine tannin, anthocyanin, and color could facilitate comparisons by season,

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ASSOCIATED CONTENT

* Supporting Information S

S1: Climatic data for the regions studied in the 2012−2013 growing season. S2: Extraction of anthocyanin and tannin from a crushed, whole-berry grape sample under wine-like conditions over 64 h. S3: Tannin concentration determined in fresh and frozen samples of small-scale wines and 15% ethanol extracts. S4: Evolution of wine tannin during small scale fermentation. S5: Correlation table showing linear regression coefficients for grape and wine analytical parameters. S6: Extraction of skin tannin during the fermentation of two red grape varieties. S7: Distribution of galloylation and prodelphinidin in purified skin tannin isolates. S8: Review of wine tannin concentration data taken at the end of primary ferment and the maximum tannin concentration reached following extended maceration (>20 days). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-8-83136190; Fax: +61-8-83136601; E-mail: keren. [email protected]. Funding

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, the Grape and Wine Research and Development Corporation, with matching funds from the Australian Government. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Accolade Wines, Reynella, South Australia for the donation of grape samples. Chris Bevin, formerly of Accolade Wines, coordinated the logistics of vineyard selection, harvesting and transport of grape samples. Bob Dambergs is recognized for conceptualizing and developing the small-scale “French Press” winemaking method. Michael Coode is acknowledged for small-scale winemaking, and Ruchita Shah, for technical support in both the laboratory and winery. Ross Kolouch, Michael McBryde and Jennifer Baldock assisted in the preparation and handling of grape and wine samples. Eric Wilkes and Peter Godden of the AWRI are thanked for proof-reading the manuscript.

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REFERENCES

(1) Bindon, K. A.; Smith, P. A.; Holt, H.; Kennedy, J. A. Interaction between Grape-Derived Proanthocyanidins and Cell Wall Material. 2. Implications for Vinification. J. Agric. Food Chem. 2010, 58, 10736− 10746. (2) Cerpa-Calderon, F. K.; Kennedy, J. A. Berry Integrity and Extraction of Skin and Seed Proanthocyanidins during Red Wine Fermentation. J. Agric. Food Chem. 2008, 56, 9006−9014. (3) Peyrot des Gachons, C.; Kennedy, J. A. Direct Method for Determining Seed and Skin Proanthocyanidin Extraction into Red Wine. J. Agric. Food Chem. 2003, 51, 5877−5881. (4) Guerrero, R.; Smith, P.; Bindon, K. Application of insoluble fibers in the fining of wine phenolics. J. Agric. Food Chem. 2013, 61, 4424− 4432.

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