Use of Alkaline Phosphatase for the Analysis of Tannins in Grapes and Red Wines

Transcription

1 Use f Alkaline Phsphatase fr the Analysis f Tannins in Grapes and Red Wines DOUGLAS O. ADAMS ~* and JAMES F. HARBERTSON 2 Tannins are the mst abundant class f phenlics in grape berries and are the predminant determinants f astringency in red wines. We have adapted a micrtiter plate assay that was first described fr persimmn tannin, s that it can be used fr analysis f tannins in grapes and wines. Our mdificatin incrprates a high salt wash step that is thught t remve nn-specifically bund alkaline phsphatase enzyme that is used t detect tannin in the analysis. Applicatin f the plate binding assay t fractins cllected frm a nrmal phase HPLC separatin f seed tannins indicates that the assay nly detects tannins having mre than three flavan- 3-l subunits. The standard assay uses bvine serum albumin (BSA) as the prtein bund t the micrtiter plate, but we have fund that casein r gelatin can be substituted fr BSA in the assay. Results shw that the micrtiter plate assay can be used t mnitr extractin f tannin frm grape skins and seeds int a mdel wine slutin. We have develped a new prtein precipitatin assay fr grape and wine tannin that is based n the tannin's ability t c-precipitate alkaline phsphatase and BSA frm a mixture f the tw prteins. The tanninprtein precipitate is pelleted by centrifugatin and washed t remve residual unprecipitated alkaline phsphatase. The tannin-prtein cmplex is then disslved in a 1 M diethanlamine buffer ph 9.4 and the amunt f alkaline phsphatase activity in the dissciated precipitate is determined by additin f p- nitrphenylphsphate substrate. The amunt f alkaline phsphatase activity in the redisslved pellet was shwn t be prprtinal t the amunt f seed tannin used t frm the precipitate. Because the slutin assay is easy t perfrm and requires nly a spectrphtmeter, it shuld be suitable fr use even in small winery labratries. KEY WORDS: tannin, alkaline phsphatase, wine, prtein precipitatin, grapes The cndensed tannins f grapes are plymeric flavan-3-ls that are primarily fund in the skin and the cells surrunding the testa f the seed [18]. Their assciatin with bitterness and astringency in several kinds f fruit and grains is well knwn, thus it is nt surprising that they have been the subject f a great deal f sensry research [2]. The cndensed tannins f grape berries are extracted frm skins and seeds during alchlic fermentatin and are thught t be the primary determinants f astringency in red wines [3]. The analysis f tannins in grapes and wines presents a frmidable challenge because f the large number f unique chemical structures that can result when the mnmeric subunits, catechin and epicatechin, cmbine t frm the cndensed tannins. Fr example, if we cnsider nly plymers with C4-C8 interflavan linkages between the mnmeric units, there culd be 124 unique chemical species in a mixture f tannins cntaining nly dimers up t hexamers. In grape seed tannin, the degree f plymerizatin was studied by thilysis, and the average number f mnmeric units was reprted t range frm 2.3 t 15.1 [13]. Such a mixture cntaining nly plymers with C4-C8 bnds culd cn- 1Assciate Prfessr and 2Graduate Student, Department f Viticulture and Enlgy, University f Califrnia, Davis, CA *Crrespnding authr [ <dadams@ucdavis.edu>; Fax ]. This research was cnducted in the Department f Viticulture & Enlgy, University f Califrnia, Davis, CA Acknwledgements: This wrk was supprted by a grant frm the American Vineyard Fundatin. Manuscript submitted fr publicatin 23 September 1998; revised 15 March Cpyright 1999 by the American Sciety fr Enlgy and Viticulture. All rights reserved. 247 tain unique structures. When we cnsider that C6-C8 bnds as well as C4-C8 interflavan bnds are pssible and that sme f the plymers may cntain gallyllated units, the number f unique chemical species that might be fund in the tannin fractin f wines culd be many times greater than This chemical hetergeneity suggests that analysis f individual cmpnents f wine tannins will require analytical methds having far greater reslutin than thse currently available. In the absence f a methd t analyze individual tannins, several assay prcedures have been described that rely n the prperty frm which this class f plant plyphenls gt their name, i.e. their ability t crsslink r precipitate prteins in the "tanning" f animal hides [10]. Hagerman et al. have recently presented evidence indicating that prcyanidins precipitate prteins frm slutin by frming hydrgen-bnded crss links between prtein mlecules [7]. Hagerman and Butler described a prcedure in which tannin is added t a prtein slutin; the resultant precipitate is then slubilized in a basic buffer, and the amunt f tannin assayed at 510 nm after the additin f ferric chlride [4]. Asquith and Butler cmbined bvine serum albumin (BSA) with Remazl brilliant blue R t btain a dyelabeled prtein which culd be used fr tannin assay [1]. After precipitatin f the blue prtein with tannin, the precipitate was slubilized and the absrbance read at 590 nm. Thus, the frmer assay measures the amunt f tannin precipitated and the latter gives a measure f the amunt f prtein precipitated by a given amunt f tannin. These tw appraches were cmbined by

2 ~ 248- ADAMS and HARBERTSON Makkar et al. wh described a methd fr analysis f bth the tannin and the prtein incrprated int the tannin-prtein precipitate [11]. This prcedure was develped specifically t measure the tannin t prtein rati which they used t btain infrmatin abut the degree f tannin plymerizatin in the sample. In 1991, Ittah described an assay prcedure fr persimmn tannins that als relied n their ability t crsslink prteins [8,9]. Ittah's analysis was carried ut in a 96-well plystyrene micrtiter plate in which the wells were first cated with a layer f BSA prtein. Tannin was bund t the BSA-cated wells and alkaline phsphatase was added after the unbund tannins had been washed away. The amunt f alkaline phsphatase in each well was assayed by additin f p- nitrphenylphsphate substrate and was shwn t be prprtinal t the amunt f persimmn tannin in the test slutin. This assay ffered several advantages ver ther methds, the greatest being the ability t analyze large numbers f samples. The bjective f the research reprted here was t adapt the micrtiter plate prcedure fr use with grape and wine samples. We have shwn that the methd described by Ittah can be used fr analysis f tannins in grapes and wines, and in the curse f this wrk we have develped a new prtein precipitatin assay based n alkaline phsphatase activity. Materials and Methds Bvine serum albumin (BSA, Fractin V pwder)pnitrphenyl phsphate disdium hexahydrate (pnpp), alkaline phsphatase (APase) and all materials used fr buffer preparatin were purchased frm Sigma, St. Luis, MO The plystyrene micrtiter plates used in the plate binding assay were btained frm Rainin. Chardnnay seed tannin was extracted as described by Prieur et al. [13] except that seeds were nt hmgenized prir t extractin. A single lt f extract was prepared and used thrughut these experiments. Plate binding assay: A phsphate buffered saline (PBS) slutin cntaining 1.44 g/l Na2HPO4, 0.24 g/l f KH2PO4, 8 g/l NaC1, and 0.2 g/l KC1, adjusted t ph 7.4 r 6.8 with HC1 was used fr plate washing and diluting samples. Wine samples were diluted with PBS ph 6.8 t give a tannin level in the range f 2 t 20 ~tg/ml, and a standard curve using Chardnnay seed tannin was run in triplicate n each plate. The seed tannin slutins were prepared by disslving 2 mg in 10 ml f a mdel wine slutin cntaining 5 g/l ptassium bitartrate, 12% ethanl v/v adjusted t ph 3.3 with HC1. This mixture was further diluted with PBS ph 6.8 t give the final tannin slutins that were applied t the micrtiter plate fr the standard curve. The plate binding assay was carried ut accrding t the methd described by Ittah [8] with mdificatins as described belw. Micrtiter plates were cated with BSA by intrducing 200 pl f a 15 mm Na2CO 3 buffer (ph 9.6) cntaining 0.2% BSA int each well and incubating the plate vernight at 4 C. After incubatin, the plates were washed fur times with PBS ph 7.4. After wash- ing, 100 pl f each sample cntaining 0 t 20 pg/ml f tannin in PBS ph 6.8 was intrduced and incubated fr ne hur at rm temperature. After incubatin, the plate was washed fur times with PBS ph 7.4. Fllwing the washes, 100 ~tl f alkaline phsphatase (AP, 0.5 U/ ml ) in PBS ph 7.4 was pipetted int each well, and the plate was incubated fr 15 minutes at rm temperature; then the plate was washed twice with PBS ph 7.4. Then, 200 ~tl f a PBS slutin ph 7.4 cntaining 80 g/l f NaC1 was put int each well, and the plate was allwed t stand fr 10 minutes, after which the plate was washed twice with PBS ph 7.4. The amunt f AP bund t the plate was determined by adding 100 pl f 1 M diethanlamine buffer ph 9.4 cntaining 0.5 mg/ml pnpp and 0.5 mm MgC12 and fllwing the change in absrbance at 405 nm every 30 secnds fr up t 15 minutes in a BiRad mdel 450 micrplate reader. In sme experiments, casein r gelatin was substituted fr BSA as the prtein bund t the micrtiter plate. Plates were cated as described abve except that either 0.2% casein in 15 mm Na2CO3, r 0.2% gelatin in water was used in place f the BSA. Extractin f skins and seeds in a mdel wine slutin: Cabernet Sauvignn clusters were btained at harvest frm the Department f Viticulture and Enlgy Experimental Vineyard at Oakville, Califrnia. The clusters were transprted t the labratry n ice, and 20 berries were randmly selected frm the clusters. The skins and seeds frm the 20 berries were separated, weighed and placed int separate Erlenmeyer flasks cntaining 20 ml f the mdel wine slutin described abve. A 1-mL sample was taken immediately and then replaced with 1 ml f mdel wine slutin. The flasks were then sealed with parafilm and incubated at rm temperature with gentle shaking (75 RPM). A 1-mL sample was remved frm each flask every 24 hurs fr tannin analysis, and an equal vlume f fresh mdel wine slutin was added t cmpensate fr the sample vlume remved. HPLC fractinatin and fractin analysis: Seed tannins were separated by nrmal-phase HPLC as described by Rigaud et al. [15] and fractins f the eluent were cllected by hand at ne-minute intervals during the 70-minute run. The slvent was remved under a stream f nitrgen and the residue was disslved in PBS ph 6.8. Fractins were then analyzed using the plate binding assay. Slutin assay fr tannins: The starting buffer fr the slutin assay cnsisted f 0.2M acetic acid cntaining 0.17 M NaC1 adjusted t ph 4.9 with NaOH. BSA was disslved in the starting buffer t give a final cncentratin f 1 mg/ml and then AP was added t give an activity f 0.5 U/mL. Seed tannin used fr the standard curve was disslved in the mdel wine slutin described abve t give final cncentratins frm 20 t 320 ~tg/ml. Wine sampleswere diluted using the mdel wine slutin t give tannin levels in the same range. The assay was cnducted in a 1.5-mL micrfuge tube by adding 0.5 ml f tannin r diluted wine t 1 ml

3 TANNIN ANALYSIS m 249 f the BSA-AP mixture and then allwing the slutin t incubate at rm temperature fr 30 minutes with slw agitatin. After incubatin, the precipitate was pelleted by centrifugatin fr five minutes at g in a benchtp micrfuge. The supernatant was pured ff, the pellet was carefully washed with 0.5 ml f the sdium acetate starting buffer, and the precipitate was re-pelleted by centrifugatin fr ne minute. After the wash-supernatant was discarded, 0.5 ml f 1 M diethanlamine buffer ph 9.4 cntaining 0.5 mm MgC12 was added t the tube. After a 10-minute incubatin the amunt f AP activity in the precipitate was determined. Individual samples were vrtexed t cmpletely disslve the pellet and then immediately assayed in a 1.5-mL cuvette by cmbining 0.25 ml f the redisslved pellet slutin with 0.75 ml f diethanlamine buffer cntaining pnpp t give a final cncentratin f 0.5 mg/ ml pnpp, and recrding the change in absrbance at 405 nm fr ne minute in a Shimadzu UV-160 spectrphtmeter immediately after adding the pnpp substrate. Results Plate binding assay: Figure 1 shws the absrbance change in three individual wells cntaining different levels f seed tannin that were mnitred fr six minutes during a plate binding assay. Values fr standard curves were btained by running at least triplicates f each cncentratin, and then averaging the reactin rate calculated fr the individual wells. Figure 2 shws a typical standard curve btained by averaging triplicates f each cncentratin. In rder t btain cnsistent results frm identical samples run n different plates, we fund that it was necessary t have a standard curve n each plate, even if multiple plates were run n the same day. Figure 3 shws a cmparisn f BSA, casein, and gelatin as the prtein cated nt the micrtiter plate. The useful range f tannin that can be assayed is different fr each f the three prteins, but fr sme applica- E 1.0- c- u') (D r c-~ 0 c'~ <1: tjg/ml 8 I.Jg/mL 16 pg/ml J I 50 I I I 1 I Time (Secnds) Fig. 1. Change in absrbance at 405 nm representing alkaline phsphatase activity in three individual plate wells having different levels f tannin" (0) 4 ~g/ml; (11) 8 ~g/ml; (A) 16 pg/ml ,&.-,.. x 25- "ID t- O ~ D I f I I I 1 I Tannin (flg/ml) Fig. 2. Standard curve fr the plate binding assay cnstructed using triplicate samples f Chardnnay seed tannin at 4, 8, and 16 pg/ml. Absrbance changes were measured at 405 nm. Data pints represent the mean f triplicate samples and the errr bars represent the standard deviatin. tins the infrmatin btained with prteins cmmnly used fr fining might be mre useful than BSA. Figure 3 shws that casein and gelatin can be substituted fr BSA in the plate binding assay. We fund that catechin and epicatechin alne, r dimers f these flavan-3-ls did nt give a reactin in the plate binding assay [Harbertsn and Adams, unpublished]. The questin then arse as t the minimum number f flavan-3-l subunits required fr a tannin t give a psitive reactin in the assay. In rder t address this questin, we cllected fractins frm an elutin prfile f seed tannin separated by a nrmal phase HPLC methd [15]. Figure 4 shws the elutin prfile and the amunt f tannin detected in each fractin by the plate binding assay. Frm these data, we estimate that the plate binding assay will nt detect tannins having less than fur flavan-3-l subunits, since fractins cntaining the lwer mlecular weight tannins shwed n respnse. Hwever, the amunt f material 40- x 30- -(3 t- O 20- O9 a O I I I Tannin (~g/ml) BSA Casein Gelatin Fig. 3. Respnse f the plate binding assay when using either BSA (O), casein (m) r gelatin (A) as the prtein initially bund t the plate. Absrbance changes were measured at 405 nm. Data pints represent the mean f triplicate samples and the errr bars represent the standard deviatin. I 50

4 ~ 250 m ADAMS and HARBERTSON _.J c~ E Seeds --i-- Skins 600 5OO ~ 4OO -~ 3 2OO IO0 0 I! I I I Jill Time (minutes) Fig. 4. Elutin prfile f a seed tannin preparatin frm a nrmal phase HPLC separatin. Fractins were cllected at 1 minute intervals during the run and the plate binding assay was used t determine which fractins shwed activity in the assay. Bar graph insert shws the relative activity in the regin f the elutin prfile where binding was detected. The area where activity was first detected is apprximately where tetramers elute in this separatin [J. A. Kennedy and A. L. Waterhuse, persnal cmmunicatin]. in the higher plymeric fractins was greater than that in the nes f lwer mlecular weight, s we cmbined several f the lw mlecular weight fractins t give the same A2s nm as nes frm the higher mlecular weight regin f the elutin prfile. Even when this was dne, n reactin was seen with the lw mlecular weight fractins (data nt shwn). Thus it seems that the plate binding assay des nt give a respnse with tannins having fewer than fur subunits. Extractin f skins and seeds with mdel wine slutins is cmmnly used t indicate ptential phenlic extractin during winemaking. We wished t determine if the plate binding assay culd be incrprated int this prcedure t estimate tannin extractin frm skins and seeds int the mdel wine slutin. Figure 5 shws extractin f tannin frm seeds and skins f Cabernet Sauvignn int mdel wine slutin during a five-day incubatin. The results shw that extractin f tannin frm skin is maximum after ne day, whereas extractin f tannins frm seeds required fur days t reach the I 60- x w 40- c c 20- k I I I I I Extractin time (days) Fig. 5. Extractin f tannin frm seeds and skins f Cabernet Sauvignn berries int a mdel wine slutin. Seeds and skins frm 20 berries were separated, incubated in a mdel wine and samples taken each day fr tannin analysis by the plate binding assay. Data pints represent the mean f triplicate determinatins made n the sample cllected and the errr bars represent the standard deviatin. highest value. At the end f the five-day experiment, abut 85% f the tannin frm the 20-berry sample came frm the seeds and 15% was extracted frm the skins. Of curse this nly represents the amunt that culd be extracted int the mdel wine and nt necessarily the ttal amunt in the tissue at the utset. Nevertheless, it shws that the plate binding assay can be used t estimate tannin levels under these cnditins. We have als used it t estimate the amunt f tannin in finished wines (data nt shwn). Slutin assay: In the curse f adapting the plate binding assay fr use with tannins in grapes and wines, we discvered that the diethanlamine buffer used fr the clrimetric assay f AP activity culd dissciate the prtein-tannin cmplex frmed n the micrtiter plate. This suggested the pssibility f a slutin assay based n the c-precipitatin f AP and BSA, where the AP/ BSA precipitate wuld be pelleted by centrifugatin and E c (1) r" t 0 0 < pg/ml [] 160 pg/ml 320 pg/ml I I I I I I Time (Secnds) Fig. 6. Typical curves btained f alkaline phsphatase activity in the redisslved pellet in the slutin assay using three different levels f seed tannin. (@) 40!ag/mL; (I) 160 pg/ml" (A) 320 pg/ml.

5 TANNIN ANALYSIS- 251,~ 25 ~ x 20- c- O 15- (1) (D 10- D <:1 5- _ 1 I 0 8O 1 I I O 320 Tannin (pg/ml) Fig. 7. Standard curve fr the slutin assay described in the Materials and Methds sectin using five different levels f Chardnnay seed tannin. Data pints represent the means and standard deviatins f triplicate samples. The zer sample illustrates that n alkaline phsphatase activity was precipitated when buffer was used instead f a tannin slutin. the AP activity then determined in the redisslved pellet. Figure 6 shws the AP activity in the disslved pellet when three different cncentratins f seed tannin were used t precipitate BSA and AP frm slutin. This result indicates that the amunt f AP activity in the pellet increases as the amunt f tannin is increased. Figure 7 is a typical standard curve using a range f seed tannin cncentratins, and it reveals that the amunt f AP activity precipitated alng with the BSA prtein is prprtinal t the amunt f tannin in the slutin. This result shws that the slutin assay described abve can be used t determine the amunt f tannin in apprpriately diluted samples. The slutin assay was used t determine the amunt f tannin in a set f experimental wines prduced during the 1997 crush. These wines were selected t represent a range f varieties and were intended t represent a range f tannin levels. Table 1 gives the Table 1. Tannin range in Cabernet Sauvignn and Pint nir. Variety Tannin Level 1 Cabernet Sauvignn Wine Pint nir Wine Units are expressed as mg/l relative t the Chardnnay seed tannin used fr the standard curve, as described in the Materials and Methds sectin. amunt f tannin fund in each f the wines using the slutin assay, and shws that this methd can be used fr wines having a wide range f tannin. Discussin Ittah described a plate binding assay fr persimmn tannin which we have adapted fr use with tannin derived frm grape seeds [8]. Our mdificatin f that riginal methd incrprates a wash with PBS cntaining 80 g/l f NaC1 prir t determining the amunt f AP bund t the micrtiter plate. We fund that this high salt wash was necessary t reduce the well-t-well variability and give stable results fr the standard curves and unknwn samples. We suspect that this high salt wash remves AP that is nt specifically bund t the BSA-tannin cmplex n the plate. Fr a detailed descriptin f the assay see Ittah [8]. We have extended the range f prteins that can be used in the plate binding assay t include nes that are typically used as fining agents in wine prductin (Fig. 3). It is clear that the range f tannin cncentratins ver which each f these prteins can be used is different, but fr sme applicatins ne prtein might be mre useful than thers. Our results simply shw that casein r gelatin can be used in place f BSA. Analysis f tannin fractins cllected during an HPLC separatin indicates that the plate binding methd des nt detect tannins having less than abut fur flavan-3-l subunits. It seems reasnable that sme minimum size wuld be required t crss-link platebund BSA and AP and thus give a respnse in the plate binding assay, but because f the cmplexity f tannin mixtures the exact minimum size wuld be difficult t determine withut pure samples f defined mlecular weight. Nevertheless, it is clear that the smaller tannins present in seed extracts are nt detected by this methd. This is cnsistent with the very recent reprt f Sarni- Manchad et al. shwing that high mlecular weight grape seed tannins were selectively precipitated by salivary prteins while prcyanidin dimers and trimers remained in slutin [16]. Failure t detect dimers and trimers might seem t be a shrtcming f the methd, but it shuld be recgnized that the amunt f lwer mlecular weight tannins in grape seeds is usually 10% r less f the ttal. Thus, the plate binding assay wuld detect mst f the tannins present in grape and wine samples and wuld clearly detect the larger mlecular weight nes which have been shwn t interact mre strngly with salivary prteins. In recgnizing that the diethanlamine buffer disslved the BSA-tannin-AP cmplex frmed in the plate binding assay, we realized that this might allw develpment f a simple slutin assay based n the amunt f AP precipitated by tannin. Many prtein precipitatin assays fr tannin have been described and their relative merits have been reviewed [6,10]. Likewise, assays have been reprted that rely n tannin's ability t inhibit enzyme activity. Very recently a prtein precipitatin assay fr tannin has been reprted which measures disappearance f a derivatized BSA prtein frm slu-

6 ~ 252 m ADAMS and HARBERTSON tin after tannin is added [14]. The slutin assay described here has sme advantages cmpared t enzyme inhibitin r prtein disappearance methds. First, we have taken advantage f the bservatin that AP can be released frm tannins t prvide an assay where enzyme activity increases with increasing tannin cncentratin rather than decreasing due t inhibitin. Secndly, this slutin assay measures the amunt f AP activity precipitated by tannin in a BSA-tannin-AP mixture, again giving values that increase with increasing tannin cncentratins rather than decreasing as is the case with the derivatized BSA methd cited abve [14]. Althugh the tw analyses described here are based n the same principle, we feel that the slutin assay has several advantages ver the plate-binding assay. Mst imprtantly, it is simple enugh t be perfrmed in mst winery labratries and des nt require a specialized instrument like a micrtiter plate reader. Nevertheless, the plate binding assay has an advantage when analysis f very large numbers f samples is required. The slutin assay is less sensitive than the plate binding assay but it has a much wider useful range (cmpare Fig. 2, 7). Since tannins are the mst abundant class f phenlics in red wines [17], the reduced sensitivity f the slutin assay is nt a disadvantage, and we have fund that all except the very lightest red wines must be diluted t have them in the prper range fr the assay. Fr the plate binding assay and the slutin assay, we used seed tannin prepared by the methd f Prieur et al. [13] as ur "standard tannin" fr the assays. Thus, ur values are all relative t standard curves prepared with a single lt f Chardnnay seed tannin, since cmmercial standards are nt available. Hagerman and Butler have pinted ut the imprtance f selecting a suitable standard, but they als describe the difficulty f chsing standards if the abslute amunt f tannin is t be determined [6]. They suggest that wrkers use quebrach tannin purified n a Sephadex LH 20 clumn as a relative standard fr tannin assays, but this may nt be practical fr winery labratries having limited time and equipment fr tannin purificatin. We are currently evaluating several pssible tannin preparatins and purificatins in rder t identify ne that will be mst suitable as a standard fr the analysis f grape and wine samples. There are several types f tannin assays based n prtein precipitatin [6,10]. The mst widely used ne measures the amunt f tannin in the tannin-prtein precipitate and wuld still be the methd f chice fr determining the amunt f prtein-precipitable tannin in a sample [4]. Other analyses are based n measuring the amunt f prtein in the tannin-prtein precipitate, and fr this apprach bth dye- and radiactive-labeled BSA have been used [1,5,12]. Several authrs have pinted ut the imprtance f knwing bth the amunt f prtein and tannin in the precipitate, and this rati f prtein t tannin has been termed the tannin's 'specific activity'. It has been prpsed that a tannin's specific activity culd prvide infrmatin abut changes in tannin structure r degree f plymerizatin during tissue develpment r maturatin [11]. Wrk is currently underway in ur labratry t cmbine the well established methd fr measuring tannin in the tannin-prtein precipitate [4] with ur AP precipitatin t give a cnvenient methd fr determining the specific activity f tannins in grape extracts r wines. Literature Cited 1. Asquith, T. N., and L. G. Butler. Use f dye-labeled prtein as spectrphtmetric assay fr prtein precipitants such as tannin. J. Chem. Ecl. 11: (1985). 2. Cliffrd, M. N. Astringency. In: Phytchemistry f Fruit and Vegetables. F. A. Tm&s-Barberan and R. J. Rbins (Eds.) pp Clarendn Press, Oxfrd (1997). 3. Gawel, R. Red wine astringency: a review. Australian Jurnal f Grape and Wine Research 4:74-95 (1998). 4. Hagerman, A. E., and L. G. Butler. Prtein precipitatin methd fr the quantitative determinatin f tannins. J. Agric. Fd Chem. 26: (1978). 5. Hagerman, A. E., and L. G. Butler. Determinatin f prtein in tannin-prtein precipitates. J. Agric. Fd Chem. 28: (1980). 6. Hagerman, A. E., and L. G. Butler. Chsing apprpriate methds and standards fr assaying tannin. J. Chem. Ecl. 15: (1989). 7. Hagerman, A. E., M. E. Rice, and N. T. Ritchard. Mechanisms f prtein precipitatin fr tw tannins, pentagallyl glucse and epicatechin16 (4,ZE8) catechin (prcyanidin). J. Agric. Fd Chem. 46: (1998). 8. Ittah, Y. Titratin f tannin via alkaline phsphatase activity. Anal. Bichem. 192: (1991 ). 9. Ittah, Y. BSA-bund persimmn tannin interactins with ther prteins. J. Agric. Fd Chem. 39: (1991). 10. Makkar, H. P. S. Prtein precipitatin methds fr quantitatin f tannins: a review. J. Agric. Fd Chem. 37: (1989). 11. Makkar, H. P. S., R. K. Dawra, and B. Singh. Determinatin f bth tannin and prtein in a tannin-prtein cmplex. J. Agric. Fd Chem. 36: (1988). 12. McGrath, R. M., and A. Smith. The islatin f large mlecular mass prcyanidins frm grain srghum and their interactin with [14C]methyl bvine serum albumins. Int. J. Bichem. 22: (1990). 13. Prieur, C., J. Rigaud, et al. Oligmeric and plymeric prcyanidins frm grape seeds. Phytchemistry 36: (1994). 14. Ratnavathi, C. V., and R. B. Sashidhar. Micrassay fr the quantitatin f prtein precipitable plyphenls: use f bvine serum albumin-benzidine cnjugate as a prtein prbe. Fd Chem. 61: (1998). 15. Rigaud, J., M. T. Escriban-Bailn, et al. Nrmal-phase high-perfrmance liquid chrmatgraphic separatin f prcyanidins frm caca beans and grape seeds. J. Chrmatgr. 654: (1993). 16. Sarni-Manchad, P., V. Cheynier, and M. Mutunet. Interactins f grape seed tannins with salivary prteins. J. Agric. Fd Chem. 47:42-47 (1999). 17. Singletn, V. L. Tannins and the qualities f wines. In: Plant Plyphenls. R. W. Hemingway and P. E. Laks (Eds.) pp Plenum Press, New Yrk (1992). 18. Thrngate, J. H. III, and V. L. Singletn. Lcalizatin f prcyanidins in grape seeds. Am. J. Enl. Vitic. 45: (1994).