Fractionation of Red Wine Polymeric Pigments by Protein Precipitation

Chapter 17

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Fractionation of Red Wine Polymeric Pigments by Protein Precipitation and Bisulfite Bleaching Douglas O. Adams, James F. Harbertson, and Edward A. Picciotto Department of Viticulture and Enology, One Shields Avenue, University of California, Davis, CA 95616

Bovine serum albumin (BSA) precipitates tannins from red wine and also removes some of the red pigments. The pigments that bind to BSA are not released from the precipitate by washing and they are stable in the presence of bisulfite. Together these observations suggest that the pigments removed from wine by BSA precipitation are polymeric pigments. The pigments removed from wines by BSA do not account for all of the polymeric pigments in the wine. After removal of the precipitated pigments by centrifugation the supernatantfractionstill contains pigments that are stable to bisulfite bleaching. Thus, protein precipitationfractionatesthe polymeric pigments into two distinguishable classes; large polymeric pigments (LPP) that precipitate along with the tannins, and small polymeric pigments (SPP) that do not. The number that best expresses the relative amounts of the two classes of polymeric pigment distinguished by protein precipitation is the LPP/SPP ratio. This ratio was found to be highly variable in 454 commercial red wines and could vary by more than a factor of 20 even in wines from a single variety (Cabernet Sauvignon). Composition of must and conditions during fermentation favor formation of LPP compared to SPP, and during barrel aging LPP is also preferentially formed compared to SPP.

© 2004 American Chemical Society In Red Wine Color; Waterhouse, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction For the past few years we have been using a protein precipitation assay developed by Hagerman and Butler to study tannin development in skins and seeds of grape berries during ripening (1,2). The procedure is shown in the polygon in Figure 1 and uses bovine serum albumin (BSA) as the protein in the precipitation step. We scaled the analysis down and adapted it for use with grape extracts, and also used it to measure tannin in wines. In doing so we always encountered a 510 nm absorbance when the protein/tannin pellet was resuspended in TEA/SDS buffer prior to the addition of ferric chloride. The background absorbance was observed in wine samples and skin extracts where anthocyanins are present along with tannins, but was absent from seed extracts. This background absorbance had to be subtracted from the final absorbance obtained after ferric chloride addition, and we soon recognized that the background absorbance was generally larger in wines than in grape extracts. Because of the well known phenomena of polymeric pigment formation during winemaking (3) we set out to determine if this background absorbance could be used as a direct measure of polymeric pigments in wine and grape extracts. In the course of investigating this possibility we found that protein precipitation fractionates the polymeric pigments into two classes, those that precipitate with BSA and those that do not. Because our previous studies indicated that procyanidin dimers and trimers do not precipitate with BSA whereas higher oligomeric forms do (4\ we have designated the polymeric pigments that precipitate with protein as large polymeric pigments (LPP) and those that do not as small polymeric pigments (SPP).

Materials and Methods Bovine serum albumin (BSA, Fraction V powder), sodium dodecyl sulfate (SDS; lauryl sulfate, sodium salt), triethanolamine (TEA), ferric chloride hexahydrate, potassium metabisulfite and (+)-catechin were purchased from Sigma, St. Louis MO, as were all of the reagents used for preparing buffers.

Analysis of Polymeric Pigments The Hagerman and Butler method for tannin analysis was combined with bisulfite bleaching of monomeric anthocyanins to give estimates of polymeric

In Red Wine Color; Waterhouse, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


277 pigments in grape-skin extracts and wines (Figure /). A model wine consisting of 12% aqueous ethanol (v/v) containing 5 g/L potassium bitartrate (pH 3.3) was used to dilute wines or aqueous extracts prior to analysis. Assay of polymeric pigments by bisulfite bleaching, and precipitation of tannins and polymeric pigments were both conducted in a buffer containing 200 mM acetic acid and 170 mM NaCl (pH 4.9). The tannin precipitation reaction was carried out in this buffer by including bovine serum albumin (BSA) to give a final protein concentration of 1 mg/mL. Analysis of polymeric pigments in parallel with tannin in wines or grape extracts required two 1.5 mL microfiige tubes for each sample. The first tube was made up by adding 1 mL of the acetic acid/NaCl buffer to the tube and then adding 500 of the diluted skin extract or wine. One mL of the mixture was transferred to a cuvette and the absorbance at 520 nm was determined (reading A) . Then 80 of 0.36M potassium metabisulfite was added, and the absorbance at 520 nm was re-determined after a 10 minute incubation (reading B) . From this tube the absorbance due to monomeric anthocyanin could be determined (A-B) where reading Β represents the total amount of polymeric pigment (SPP+LPP). The second tube contained lmL of the acetic acid/NaCl buffer along with BSA (lmg/mL) into which 500 μL of the diluted skin extract or wine was added. The mixture was allowed to stand at room temperature for 15 minutes with slow agitation, after which the sample was centrifiiged for 5 minutes at 13,500g to pellet the tannin-protein precipitate. One mL of the supernatant was transferred to a cuvette, then 80 μL of 0.36M potassium metabisulfite was added. After a 10 minute incubation the absorbance was determined at 520 nm (reading C). This absorbance represents polymeric pigment that did not precipitate along with the tannin and protein (SPP), and this value was used to calculate the amount of polymeric pigment that precipitated with the tannin and protein (B-C).

Background Absorbance in the Analysis of Tannin The tannin-protein pellet from the second tube described above was washed with 250 μί^ of the acetic acid /NaCl buffer to remove residual monomeric anthocyanins. The precipitate was re-centrifuged for 1 minute at 13,500g and the wash solution was discarded. Then 875 \iL of a buffer containing 5% TEA (v/v) and 5% SDS (w/v) was added and the tube was allowed to stand at room temperature for 10 minutes. This buffer dissolves the precipitate containing


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BSA Precipitation/Bisulfite Bleaching Assay . Wine . Dilute into Buffer pH4.9 Read at 520 nm (Total Figments) AddSO, Read at 520 nm (LPP + SPP)

Dilute into Buffer pH 4.9 Containing BSA Protein

J

Centrifuge 13,500g Supernatant - Add SO2 Read at 520 nm (SPP)

Pellet - Disolve in TEA/SDS Buffer

Add Ferric Chloride Read at 510 nm (Tannin)

Figure 1. Parallel Determination of Tannins and Polymeric Pigments. The procedure shown in the rectangle (left) is a Somers assay performed at pH 4.9 and provides a measure of total pigments and total polymeric pigments (LPP + SPP) at that pH. The procedure shown in the polygon (right) is the Hagerman Butler assay for tannin. © 2003 American Society for Enology and Viticulture. AJEV54:301-306


279 tannin, protein and any polymeric pigment that precipitated with the tannin and protein. After incubation the tube was vortexed to dissolve any of the remaining precipitate and the absorbance at 510 nm was measured after allowing the solution to stand at room temperature for 10 minutes. This is the background absorbance in the tannin assay we described previously (7). For tannin analysis 125 μΐ, of 10 mM ferric chloride was added and the absorbance at 510 was re determined after 10 minutes.


Berry Extraction Cabernet Sauvignon berries were collected near Oakville California from eight-year-old vines planted on 110 Richter rootstock. Pinot noir fruit was obtained from the Carneros region of Southern Napa Countyfromtwo six-yearold vineyards (Dijon 115) planted on 3309 rootstock. Syrah berries used to determine how polymeric pigments changed as fruit ripened were gathered from a five-year-old vineyard near Esparto California planted on 110 Richter rootstock. Sample collection was conducted as described previously (/). Briefly, three twenty-berry samples were collected, put into plastic bags and transported to the laboratory on ice. The samples were weighed, and the skins were removed and extracted for polymeric pigment analysis. Berries were sliced in half with a razor blade and skin was carefully collected from each berry-half using a small metal spatula. Skins from the twenty-berry sample were put into a 125 mL Erlenmeyer flask containing 20 mL of 70% aqueous acetone (v/v). The flasks were sealed with a rubber serum cap and extracted overnight with gentle shaking (100 rpm). After overnight incubation the extraction solution was filtered and the acetone was removed at 38° C using a rotary evaporator at reduced pressure. The residual aqueous extract was adjusted to 10 mL with deionized water and frozen at -20° C until used for analysis.

Wines for Total Polymeric Pigment Comparison Twelve commercial 1998 red wines that were part of a different study were used to study the correlation between polymeric pigments as determined by the method of Somers and Evans, and the polymeric pigments (background absorbance) in the protein-tannin precipitate. The twelve commercial wines were from nine different wineries; nine of the wines were Cabernet Sauvignon, two were Merlot and one was Petite Sirah.


280 For comparing polymeric pigments in fruit and the resulting wine we collected three 20-berry samples from picking bins and extracted skins in 70 % aqueous acetone as described above. We measured polymeric pigments in the grape skin extracts and then analyzed the resulting wines 90 days after pressing.


Results In order to determine if the background absorbance in our tannin assay could be used as a direct measurement of polymeric pigment, we compared the background absorbance to the polymeric pigment values measured by the Somers method in a set of wines. Figure 2 shows the correlation between the background absorbance and polymeric pigments as determined by the Somers assay, and clearly demonstrates that the measurements are poorly correlated.

•• · • m

·•

/: •

• • 0.0

9

•

• •

Trial 1 Trial 2

τ

τ

0.1

0.2

—ι

1

ι— • ι

I

0.4 0.5 0.6 0.7 Background Absorbance 0.3

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Figure 2. Absence of a correlation between the background absorbance in the tannin assay and the amount ofpolymeric pigment in 12 wines as determined in the standard Somers assay at wine pH. The analysis was performed twice about eight months apart. © 2003 American Society for Enology and Viticulture. AJEV 54:301-306 Absorbance measurements indicated that the reason for the poor correlation was that some of the polymeric pigments were not precipitated by



281 protein and we set out to measure this fraction of polymeric pigments directly. We did this by first precipitating tannin and the accompanying polymeric pigments with BSA under the conditions of the Hagerman Butler tannin assay (pH 4.9, acetate buffer) so as to obtain maximum precipitation of protein along with tannin and polymeric pigment. After removal of the precipitate by centrifugation, we used bisulfite to bleach any monomeric anthocyanins remaining in the supernatant. The residual absorbance at 520nm represents polymeric pigment in the sense that it does not bleach with bisulfite. The absorbance due to the monomeric anthocyanins was taken to be the amount of 520 nm absorbance that was bleached with bisulfite. The results of one experiment to directly observe SPP is shown in Figure 3.

Figure 3. Percentage of the total color contributed by the three classes of pigments found in a Syrah wine shortly after pressing. MP, monomeric pigments (anthocyanins); SPP, small polymeric pigments; LPP, large polymeric pigments. The wine was a Syrah obtained soon after pressing and we could attribute over 30 percent of the total color at pH 4.9 to polymeric pigments that did not precipitate with protein. When the wines shown in Figure 2 were re-examined and the amount of LPP and SPP were added together, the correlation with polymeric pigments by the Somers assay was found to be quite good (Figure 4).



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LPP +S P P

Figure 4. Correlation between the amount ofpolymeric pigment as determined in the Somers assay at wine pH and the sum ofLPP and SPP as determined by combined protein precipitation and bisulfite bleaching. © 2003 American Society for Enology and Viticulture. AJEV 54:301-306 This is not surprising since the way in which total pigments and total polymeric pigments (LPP +SPP) are determined is nothing more than a Somers assay at pH 4.9 (Figure 1). From these results (Figures 3,4) we concluded that protein precipitationfractionatespolymeric pigments into two classes, those that precipitate with protein and those that do not. Our next objective was to determine if polymeric pigments were present in fruit during ripening. The results obtained with Syrah are shown in Figure 5. Since analysis of the grape extracts was conducted at pH 4.9 the contribution of monomeric anthocyanins to the total color is minimized and thus is greatly underestimated in Figure 5. However, at this pH the polymeric pigments are most easily observed because they change very little in absorbance with pH, unlike the monomeric anthocyanins (3,5). The data indicate that fruit at harvest may contain small amounts of polymeric pigment and that most of it is SPP. After finding that protein precipitationfractionatedpolymeric pigments, we recognized that the ratio of large to small polymeric pigments must be different in the wines used in Figure 2 and Figure 4, otherwise a good correlation would have been observed in both cases. Recognizing that the ratio of LPP/SPP


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was variable in wines prompted us to measure the ratio in fruit at harvest and compare that with the ratio in the resulting wine.

Date 1999 Figure 5. Pigments in Syrah Berries During Ripening Determined at pH 4.9 The color contributed by monomers is underrepresented because at this pH very little of the monomeric pigments are in theflavilliumform. © 2003 American Society for Enology and Viticulture. AJEV 54:301-306 The results for four wines made from three different grape varieties are shown in Table I. In these four examples the absolute amount of LPP and SPP both increased (data not shown). However, since LPP increased more than SPP the ratio showed an increase of nearly two fold from fruit to wine in the case of Cabernet Sauvignon, to over seven fold in one of the Pinot noir experiments. Since we found that even a limited set of wines could exhibit a large range of LPP/SPP values we wished to characterize this range in a larger set of finished wines. Table II shows the range and average of LPP/SPP values for 454 commercial bottled wines of Zinfandel, Pinot noir, Cabernet Sauvignon and Syrah, all of various ages and origins.


284 Table I. LPP/SPP Ratio in Fruit of Four Grape Varieties Compared to Wine Made from the Fruit Grape Variety LPP/SPP in Fruit LPP/SPP in Wine Fold Increase Zinfandel 0.37 0.62 1.8 Pinot noir 0.18 3.6 0.64 Cabernet Sauvignon 0.24 1.73 7.2 Syrah 0.20 1.14 5.7


NOTE: The ratio was measured in fruit at harvest and in the resulting wine 90 days after pressing.

Table IL Analysis of the LPP/SPP Ratio in Wines of Four Grape Varieties. Variety Ν Min. Max. Average Std. Dev. Zinfandel 200 0.08 7.19 1.10 0.93 Pinot Noir 134 0.14 0.39 2.20 0.79 Cabernet Sauvignon 87 0.18 0.63 3.93 1.16 Syrah 33 0.28 0.30 1.71 0.85 NOTE: All wines were commercial products. N , number of wines analyzed; Min., minimum ratio; Max, maximum ratio; Std. Dev., standard deviation of the Average. Figure 6 shows the frequency distribution of LPP/SPP values in 85 of the commercial Cabernet Sauvignon wines. The distribution of the Cabernet Sauvignon wines about the mean is fairly normal, but the range of values was surprising, over 20 fold in the set of Cabernet Sauvignon wines (Table II). The large increase in the LPP/SPP ratio we observed between fruit and the resulting wine 90 days after pressing (Table I) prompted us to study the effects of winemaking practices and aging on LPP and SPP levels. Thus far we have found that fermentation temperature is one of the most important factors that influence LPP and SPP formation during winemaking. Ourfirstindication of this came from interrogating a fermentation temperature experiment performed in David Block's lab (Department of Viticulture & Enology, University of California, Davis). In this experiment Cabernet Sauvignonfroma common must-pool was fermented at different temperatures. We measured the amount of LPP, SPP and monomeric pigment (at pH 4.9) in wines from a 17° C and a 27° C fermentation at bottling and again after three years of aging. The results are shown in Figure 7. From these data we can see the effects of both fermentation temperature and bottle aging on LPP and SPP formation. In the year the wines were made (1999) the one from the 27° C fermentation had more



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LPP/SPP Figure 6. Frequency distribution of LPP/SPP ratios in 85 commercial Cabernet Sauvignon wines.

than twice as much LPP as the wine fermented at 17° C, whereas SPP was only about 60% greater in the wine from the 27° C fermentation. This observation is consistent with commercial scale experiments that we have monitored during the past two years (data not shown). The effect of bottle aging on LPP and SPP formation in these two wines can also be seen from the results in Figure 7 by comparing the pigment composition in the wine at bottling with the composition three years later. The data show that LPP was preferentially formed compared to SPP during the three years of bottle aging, and that the amount of LPP increased about three-fold in both wines during this period.

Discussion The routine analysis of polymeric pigments in wine is based on the work of Somers who showed that monomeric anthocyanins are bleached with bisulfite whereas polymeric pigments are not (3,5). Thus, the difference in absorbance readings at 520nm before and after bleaching with bisulfite is widely used as a measure of the amount of polymeric pigment present in red wine. Figure 1 shows the scheme we currently use for the parallel determination of tannin and polymeric pigments in wines and berry extracts. The boxed branch


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on the left is a Somers assay at pH 4.9. This pH was chosen so that the bisulfite bleaching reaction would take place in the same buffer system used in the Hagerman Butler assay for tannin.

Figure 7. Pigment composition of Cabernet Sauvignon wines fermented at 17° C (7A) and 27° C (7B). The composition of each wine was determined at bottling and after three years of bottle aging. MP, monomeric pigment; SPP, small polymeric pigment; LPP, large polymeric pigment. Monomeric pigment declined during aging whereas SPP and LPP increased. The acetate buffer is set at pH 4.9 because 4.9 is the pi of BSA and thus affords maximum precipitation of the tannin/protein complexes formed during the precipitation reaction. Coincidentally pH 4.9 is particularly good for measuring absorbance due to polymeric pigments because the anthocyanins have their minimum absorbance at that pH (6). Since any remaining monomeric anthocyanins are bleached with bisulfite, this procedure assures that all of the remaining 520 nm absorbance is due to polymeric pigments (LPP + SPP). The procedure in the polygon in Figure 1 is the Hagerman Butler assay for tannin. The only modification is that we retain the supernatant after centrifugation of the precipitation reaction mixture and bleach the monomeric anthocyanins with bisulfite. The residual absorbance represents pigments that do not precipitate with protein and do not bleach in the presence of bisulfite. We have designated this material "small polymeric pigment" solely to denote these two characteristics. The LPP removed from the sample by protein precipitation



287 is responsible for the background absorbance in the tannin assay. However, since LPP is only a variable fraction of the total polymeric pigment, the background absorbance is poorly correlated with the total polymeric pigment as determined by the Somers assay (Figure 2). When both large and small polymeric pigments are taken into account the correlation is seen to be very good (Figure 4). We recognize that the small polymeric pigment fraction is likely to be a heterogeneous mixture containing anthocyanins as acetaldehyde cross linked reaction products, direct reaction products, and cycloaddition products, in various proportions. The LPP fraction probably contains acetaldehyde cross linked reaction products and direct reaction products, where anthocyanin has reacted to produce a pigmented molecule "large" enough to precipitate with protein; thus the designation "large polymeric pigment". The number that best expresses the relative amounts of the two classes of polymeric pigment distinguished by protein precipitation is LPP/SPP. We chose to express the relative amounts of LPP and SPP as the LPP/SPP ratio rather than SPP/LPP ratio, because we found that LPP usually increases relative to SPP during winemaking and aging (e.g. Figure 7). In the varieties we have studied thus far this ratio is typically very low in acetone extracts of berry skins at harvest, but shows a consistent increase during winemaking and early barrel aging (Table I). Syrah fruit at harvest had little polymeric pigment and most of what was present was SPP (Figure 5). Thus the increase in the LPP/SPP ratio during winemaking and aging is a result of LPP formation rather than a decline in the amount of SPP. This is most easily seen in the bottle aging experiment shown in Figure 7, where the amount of LPP in the wine increased nearly 3 fold in 3 years while the amount of SPP increased by less than 25 percent. We found that commercial wines of four varieties exhibited an extraordinary range of LPP/SPP values (Figure 6 and Table II). This suggests that polymeric pigment populations are quite different even in wines made from the same variety. That is to say, if protein precipitation did nothing more than fractionate similar populations of polymeric pigments, then the ratio of LPP/SPP would be similar among red wines. In fact the LPP/SPP ratio shows remarkable variation even among wines of the same variety (Table II). Winemaking conditions and the age of the wine when assayed clearly are factors in explaining the large range of values. Nevertheless, as we study more examples of polymeric pigment evolution in wines during aging (such as shown in Figure 7) we should be able to identify other variables that contribute to the wide range of LPP/SPP values seen in finished wines.


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References 1.

Harbertson, J. F.; Kennedy, J. Α.; Adams, D.O. Tannin in skins and seeds of Cabernet Sauvignon, Syrah, and Pinot noir berries during ripening Am. J. Enol. Vitic. 2002, 53, 54-59.

2.

Hagerman, A. E.; Butler L. G. Protein precipitation method for the quantitative determination of tannins. J. Agric. Food Chem. 1978, 26, 809812.

3.

Somers, T. C. Polymeric nature of wine pigments. Phytochemistry. 1971, 10, 2175-2186.

4. Adams, D. O.; Harbertson J. F. Use of alkaline phosphatase for analysis of tannins in grapes and red wines. Am. J. Enol. Vitic. 1999, 50, 247-252. 5.

Somers, T. C.; Evans M. E. Spectral evaluation of young red wines: anthocyanin equilibria, total phenolics, free and molecular SO , "chemical age". J. Sci. Food Agric. 1977, 28, 279-287. 2

6.

Cabrita, L.; Fossen T.; Andersen Ο. M. Colour stability of the six common anthocyanidin 3- glucosides in aqueous solutions. Food Chem. 2000, 68,101-107.


Fractionation of Red Wine Polymeric Pigments by Protein Precipitation

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