Spectrophotometric Methods for the Analysis of Polysaccharide Levels in Winemaking Products

Transcription

1 Spectrphtetric Methds fr the Analysis f Plysaccharide Levels in Wineaking Prducts I. SEGARRA ~, C. LAO 2, E. LOPEZ-TAMAMES a*, and M. C. DE LA TORRE-BORONAT" The plysaccharide cntent in 27 white wineaking saples was exained using tw clrietric ethds (phenl and -hydrxydiphenyl reagents, respectively). Ttal plysaccharide (TPS), acid plysaccharide (APS), and neutral plysaccharide (NPS) fractins were deterined. The advantages f these ethds were denstrated: linearity, precisin, accuracy, and selectivity were studied. The levels f TPS, APS, and NPS depended n varietal characteristics, harvest, and wineaking prcess. KEY WORDS: plysaccharides, acid plysaccharides, neutral plysaccharides, spectrphtetry The plysaccharides (PS) fund in wineaking prducts (grapes, juices, and wines) have tw surces: in usts, they ce fr the grape and can be divided int neutral plysaccharides (NPS) and acid plysaccharides (APS); in wine, they are released by yeast during ferentatin. Wine plysaccharides have lwer cncentratin than that f grape juice due t the actin f endgenus pectlytic enyes and t the effect f the ethanl increase. Must and wine plysaccharides have an iprtant influence n a nuber f stages f the wineaking prcess: grape juice extractin, ust racking, ferentatin, and wine filtratin (1,4,11,15,27). These cpunds cntribute t the rganleptic prperties f the prduct, as they stabilie flavr, clr, and fa (17,26). Table 1 shws a brief review f the literature n extractin, separatin, and quantificatin ethds used fr deterining different plysaccharide fractins in grapes, juices and wines. Fr quantitative PS deterinatin in a very cplex atrix f substances such as ust and wine saples, previus extractin is necessary. The ethds st cnly used fr extractin are dialysis (1,4,5,10) and precipitatin with ethanl (8,9,11,14,15,16,24,28). Quantitative deterinatin is ainly by clrietric ethds, but chratgraphic techniques can als be used fr a individually analysis f PS: gas chratgraphy (4,6) and sie exclusin chratgraphy with refractin index (10). Hwever, the chratgraphy is used as a preparative separatin technique (1,5,9,10,19) fr PS fractins r fr the PS structural deterinatin. The st frequently used clrietric ethds fr APS deterinatins are the carbale ethd (8,9,10,18,22) and the - hydrxydiphenyl ethd (1,4,5,15,18,22). NPS are usually deterined by the rcinl ethd (1,5,23) and the 1,a.4Nutrici6n y Bratlgia, Facultad de Faracia, Universidad de Barcelna, Avda. Jan XXlII s/n, Barcelna. Spain; 2E.U. Plit~cnica de Manresa, Universidad Plitecnica de Catalufia, Avda. de les Bases de Manresa 61-73, Manresa, Spain. *Crrespnding authr. Acknwledgents: This wrk was ade pssible by financial assistance fr the Cisi6n Interinisterial de Ciencia y Tecnlgfa, Prjet ALI C02-01 (Spain). We are grateful t Freixenet S.A. winery fr their cllabratin. Manuscript subitted fr publicatin 31 Octber 1994 Cpyright 1995 by the Aerican Sciety fr Enlgy and Viticulture. All rights reserved. phenl ethd (4,8,9,11,14,15). The ain shrtcing f these ethds is the lw reactin specificity f the chrgen reagent; phenl is nt selective fr NPS deterinatin but als reacts with APS, and s it is used by se authrs fr deterining ttal plysaccharides (TPS) (4,11,14). The rcinl ethd is nt selective fr NPS (due t prtein and plyurnid interferences), and the carbale ethd is nt selective fr APS (due t NPS interferences). T btain re accurate values fr bth kinds fps (APS and NPS), se authrs (1,5,8,9) use crrectin frulas. These frulas, hwever, are nt usually described. Only Duburdieu et al. (8) describe the Mntreuil-Spickque frulas, which allw the use f the carbale ethd t analye APS and the phenl ethd fr NPS. In this study, the phenl ethd was chsen fr the deterinatin f TPS, since phenl reacts with NPS and APS, althugh the sensitivity fr APS is 2.5 ties lwer than fr NPS. The results btained fr APS with - hydrxydiphenyl reagent -- a highly selective ethd fr NPS deterinatin -- allw crrectin f the TPS btained with the phenl ethd, using a siple frula. The clrietric ethds used here are described in detail: linearity, repeatability, reprducibility, liit f detectin and liit f quantitatin, and selectivity. The extractin ethd was ptiied by varying teperature and ethanl prprtin, and by exaining the recvery and the selectivity fr prteins and plyphenls. The spectrphtetric ethds were used t quantify the TPS cntent and the fractins (APS and NPS) in 27 wineaking prducts. The influences f natural variables (variety and harvest) r technical variables (racking, ferentatin and filtratin) n the plysaccharide levels were deterined. Materials and Methds Reagents and slutins: All reagents were analytical grade unless therwise stated. -Hydrxydiphenyl reagent: a slutin (0.15%, w/v) f -hydrxydiphenyl (biphenyl-2-l Merck Ref: ) in 0.5% (w/v) sdiu hydrxide was stred in a brwn glass cntainer wrapped in aluinu fil and kept at 4 C. 564

2 Table 1. Bibligraphic review f subjects, kinds f saple analyed, plysaccharide levels and extractin, separatin and quantitatin ethds used t deterine plysaccharides in wineaking prducts. Authr Ussegli-Tasset, 1976(23) Rbertsn, 1979 (18) Subject Glucidic cllids Quantitatin Study f APS ethds Saple and levels* (g/l) 1 white ust & 2 red usts: TPS 1 white wine & 2 red wines: TPS Riesling grapes Extractin level Ethanl precipitatin Ethanl precipitatin and Prgressive extractin: Water, Oxalate and Alkali Separatin ethd Quantitative ethd -Gravietry -Carbal --Hydrxydiphenyl 3.,, :3 i. < c~ J < 4b, ~ 4:=, ii, O ~ tj1 Duburdieu and Rib6reau-Gayn, 1980 (9) Duburdieu et al, 1981 (8) Ollivier et al., 1987 (15) Brilluet, 1987 (4) Belleville et al, 1991 (1) Guillux-Benatier and Feuillat, 1993 (11 ) Gnal and Mingue, 1993(10) Glucidic cllid cpsitin TPS, APS, NPS quantitatin during grape aturatin TPS quantitatin in usts Structural analysis f NPS in varietal usts Structural analysis f TPS in crss-flw filtered wines Influence f ust clarificatin in TPS cntent PS cllid study Mancini and Benini, TPS levels in different 1993 (14) varietal and vineyard wines Puey, 1994 (16) Influence f NPS in fa stability 1 Seilln ust Sauvignn grapes: TPS, APS and NPS 8 white usts: TPS 7 red usts: TPS Red wine 7 white usts: TPS 6 white wines AOC Pened~s: TPS 64 red wines: TPS 13 white usts AOC Pened~s: NPS 8 white wines AOC Penedes: NPS Ethanl precipitatin (1:5, 2 ties) Ethanl precipitatin (1:5) Ethanl precipitatin (1:5, 18 hr and dialysis 24 hr) Dialysis Dialysis Ethanl precipitatin (1:5, 2 ties) Dialysis Ethanl precipitatin (1:5, 2 ties, 4 -C) Ethanl (precipitatin 1:5, 2 hr) preparative IEC a preparative SEC b preparative SEC preparative SEC -Phenl fr NPS -Carbal fr APS Crrectin fr utual interferences -Phenl fr NPS -Carbal fr APS Crrectin fr utual interferences -Phenl fr NPS --Hydrxydiphenyl fr APS -Phenl fr TPS --Hydrxydiphenyl fr APS -GLC c fr NPS -Orcinl fr NPS fractins --hydrxydiphenyl fr APS fractins Crrectin fr utual interferences -Phenl fr TPS -Carbal fr APS fractins -RP detectin fr PS fractins -Phenl fr TPS withut crrectin -GLC fr NPS "13 O r- -< r) > 0 0 I > i * Maxiu and iniu levels a IEC: In Exchange Chratgraphy b SEC: Sie Exclusin Chratgraphy c GLC: Gas Liquid Chratgraphy RI: Refractin Index > I cn

3 566 LOPEZ-TAMAMES et al Sulfuric acid~sdiu tetrabrate slutin: sdiu tetrabrate ( M) in cncentrated sulfuric acid. Phenl reagent: 5% (w/v) in duble distilled water and was stred in a brwn glass cntainer and kept at 4 C. Ply-D-galacturnic acid ethyl ester slu. tin: a slutin (500 g/l) f ply-d-galacturnic acid ethyl ester f pectin fr apples (Fluka Ref:76282) in duble distilled water (slutin A). Galacturnic acid slutin: I g/l f D-a-galacturnic acid (TCI Ref. G0010) in duble distilled water (slutin B). Galactse slutin: lg/l f D-(+)-galactse (TCI Ref.G0008) in duble distilled water (slutin C). Galacturnic acid and arabinse slutins: tw different ixtures with galacturnic acid and L-(+)- arabinse (Siga Ref. A3256) in duble distilled water were prepared: galacturnic acid (1 g/l) and arabinse (1 g/l) ( slutin D) and galacturnic acid (1 g/l) and arabinse (2 g/l) (slutin E). Galacturnic acid, galactse and arabinse slutin" a slutin cntaining galacturnic acid (lg/ L), galactse (1 g/l) and arabinse (2 g/l) in duble distilled water (slutin F). Bvine seru albuin slutin: a slutin (100 g/l) f albuin fractin V (fr bvine bld) (Merck Ref:12018) in duble-distilled water. PS extractin ethd. Extractin cnditins: T select the best cnditins fr PS precipitatin with ethanl fr thse prpsed by ther authrs (8,9,11,14,15,16,24,28), 12 aliquts f 4 L f ust saple were precipitated fr 18 hurs with tw different ethanl prprtins and at different teperatures. The precipitates were dried inside an ven at 50 C and then weighed. Tw prprtins f ust:ethanl (1:3 and 1:5) and tw teperatures (22 C and 4 C) were cnsidered, each assay being perfred in triplicate. Table 2 shws the eans and standard deviatins f the carbhydrate cllids expressed in g/l. Prcedure: Five illiliters f ethanl (96%, v/v) and 50 pl HC1 (1 N) were added t 1 L f centrifuged saple (2500 X g, 15 in). After 18 hurs at 22 C, the tubes were centrifuged (1800 X g, 20 in), after which the supernatant was discarded and the pellet was washed in ethanl (96%, v/v), three ties fr wine saples and fur ties fr ust. The precipitate was dried in a strea f nitrgen. This extractin was perfred fur ties: tw extracts were used fr TPS deterinatin and the ther tw fr the APS deterinatin. Finally, the precipitates were disslved in 5 L r 4 L f water, depending n the type f PS and n the type f saple in the phenl ethd and in the - hydrxydiphenyl ethd 4 L r 1 L fr ust r wine, respectively. Recvery: Slutin A was used as a standard. The APS was analyed by the -hydrxydiphenyl ethd in the standard slutin, in the precipitate f a wine and in the precipitate f the standard slutin added t the wine in a prprtin 1:1 (v/v). These assays were carried ut 5 ties (Table 3). Selectivity assay: A selectivity assay (Table 4) f the ethanl precipitatin fr prteins and plyphenls was perfred. The plyphenlic and prtein cntents in ust and wine saples and in the PS precipitate were analyed in triplicate by the Flin-Cicalteau (21) and Bradfrd ethds (3), respectively. -Hydrxydiphenyl ethd. Prcedure: The Bluenkrant ethd (6) was fllwed as described by Rbertsn (18), but instead f -hydrxydiphenyl slutin as a chrgen reagent, -hydrxydiphenyl slutin was used fr the APS deterinatin. Linearity: The linear relatinship between absrbance and APS cncentratin was studied using 10 standard curves (slutin B) at different cncentratins: 10, 20, 40, 60, 80, and 100 g/l f galacturnic acid (Table 5). Repeatability: Repeatability was expressed as the cefficient f variatin btained with eight wine aliquts under nral perating cnditins (Table 5). Slutin A Precipitate A Precipitate wine Table 3. APS recvery f the ethanlic extractin, using the -hydrxydiphenyl ethd. Precipitate wine:slutin A (1:1 v:v) APS cntent Recvery g galacturnic % acid/l av ± Sn-1 av Sn-1 (n = 5) (n = 5) 360 _ _ _+ 3 40_ _ _+ 5 Table 2. PS precipitate quantity, expressed in g/l, btained fr a ust saple (4 L) and different ethanl prprtins at tw different teperatures, ver 18 hurs. Saple Prprtin Teperature Abient 4- C av ± SD av ± SD Must 1 n=3 n=3 Must 2 Must:Ethanl 1: _ _ 0.61 Wine 1 Must:Ethanl 1: _ 0.06 Table 4. Prtein cntent deterined by Bradfrd ethd, expressed in g/l and as percentage, in tw usts and ne wine, befre and after the ethanlic extractin. Prteins Prteins int Precipitatin g/l precipitate f prteins n = 3 g/l % n= _ _ _ _ _ _

4 POLYSACCHARIDES IN WINEMAKING~ 567 Table 5. Validatin results f the study f the -hydrxydiphenyl and phenl sulfuric ethds. -Hydrxydiphenyl ethd Linearity ~ g/l (n = 10) Repeatibility ~ n = 8 3.8% LD 0.65 g/l LQ ~ 2.2 g/l a Crrelatin cefficient (r) f absrbance/cncentratin Cefficient f variatin c LD: Liit f detectin d LQ: Liit f quantitatin Saples :: Table 6. TPS levels (g galactse/l), APS (g galacturnic acid/l), and NPS f the 27 saples analyed.. TPS. ~.. JPg... CWcV 91 : 325:~:9 :~ ~:~;: : :~ :'2~7~: '::... JX 92 : 5~ ~ ~ : 2~:,~ JP92... ~i~ RbV ~ ] ~ 21~ RX 92 : : :3:::::::::::::::/:::::::::::::3::4::::::: RP 92 ::::::::::::::::::::::::::::::::::::::::::::::::::::::: cwx ;... JX 93 ~ ~... ~i~... JP 93 ~!~ ~ ~0 ~ i~ ~::/::::,~:::::::::::::::::::::::::::::::::::: i!':: ~::: :~: ~:~i RX ~8~... ~i~... RP93 8~ ~ ~i~ CWV 93 : ili:i i i i: i'i :!iiiii::i :::::::::::::::::::::::::::::::::::::::::::::::::::: CWX 93 CWP 93 ~ ~ ~ ~ i~ i i i i:: : : i l! :: FWaV ~ ~ ~i~ FVV Cefficients f variatin (%)av+sd 1.9 _+ 1.0 (n=27) a j. Juice "V: Viura b R: Racked ust f X: Xarel.l CW: Cludy wine g P: Parellada d FW: Filtered wine. 2.5_+ 2.0 Phenl ethd h 91" 1991 i92:1992 J93: % 1.36 g/l 4.45 g/l NPS = TPS- APS O O Reprducibility: The precisin was calculated as the ean f the cefficients f variatin f each f the saples, fr the three replicate easures (Table 6). Liit f detectin (LD) and liit f quantificatin (LQ): Liits were calculated fr seven blanks using the Lng and Winefrdner (13) frulas fr the I.U.P.A.C. definitins: LD and LQ = K.SD 8 + ta.sd where K= 3 fr LD and 10 fr L.Q; SD B = standard deviatin f the blank absrbances; = average slpe f the calibratin curves; t a = tw-tailed Student "t" distributin fr a = ; and SD = standard deviatin f slpe calibratin curves. Selectivity: T establish the selectivity f the - hydrxydiphenyl ethd fr APS cpared with NPS, the calibratin curves were perfred in duplicate with each f the slutins B, D, E, and F. The range studied was 10 t 100 g/l fr each cpund, with standard cncentratins f 10, 20, 40, 60, 80, and 100 g/l (Fig. 1). Phenl ethd. Prcedure: As described by Dubis (7), phenl was used as chrgen reagent and TPS (APS + NPS) were quantified. Linearity: The linear relatinship between absrbance and NPS cncentratin was studied using 10 standard curves perfred with the slutin C at different cncentratins: 10, 20, 40, 60, 80, and 100 g/l f galactse (Table 5). Repeatability, reprducibility, LD, and LQ: Deterinatins fr the phenl ethd were by the sae prcedure as fr -hydrxydiphenyl ethd (Table 5). Selectivity: T establish the selectivity f the phenl ethd fr NPS cpared with APS, the calibratin curves were perfred in triplicate with each f the slutins B, C, and D. The range studied was 10 t 100 e I3, t~ x} < galacturnic acid (sl. B) [] galacturnic acid:arabinse 1:1 (sl. D) il galacturnic acid:arabinse 1:2 (sl. E) ii 8/ galacturnic acid:galacturnic:arabinse ii ' i 10 i 20 i g/l Fig. 1. Calibratin curves btained with different standard slutins (B, D, E, and F slutins) t establish the selectivity f -hydrxyphenyl ethd. *

5 568- LOPEZ-TAMAMES et al : 0.8 J~ "- 0.6 J~ < galactse (sl. C) [] galacturnic acid (sl. B) qalactse:galacturnic acid 1:1 (sl D) 4~ g/l Fig. 2. Calibratin curves btained with different standard slutins (B, C, and D slutins) t establish the selectivity f phenl sulfuric ethd. g/l fr each cpund, with standard cncentratins f 10, 20, 40, 60, 80, and 100 g/l (Fig. 2). Sapling: Must saples were cllected fr three varieties f A.O.C. Pened~s: Viura (V), Xarel.l (X), and Parellada (P), during three harvests: 1991 (91), 1992 (92), and 1993 (93). These usts were btained in a winery with a pneuatic press f kg capacity and were sulfited at a level 60 t 100 g SO/L: sulfitedjuice saples (J). The latter were then left t settled fr 24 hurs, and the supernatant was decanted ff: racked ust saples (R). Ferentatin tk place in 1000-L tanks. The yeasts used were Saccharyces bayanus in 1991 and Saccharyces cerevisiae in 1992 and After ferentatin the crrespnding cludy wine saples (CW) were btained. Later, they were filtered with a candle filter with a 0.45-/t cellulse ebrane: filtered wine saples (FW). Statistical prcedures: Significant differences between saples fr each plysaccharide fractin were assessed with a ultiple analysis f variance (MANOVA) using Statgraphics 4.2 prgra. The kind f saple [usts (M) and wines (W)], variety [Viura (V), Xarel.l (X), and Parellada (P)] and harvest (1991, 1992, and 1993) were cnsidered as qualitative variables. Results and Discussin PS extractin ethd: The higher quantity f PS precipitate was btained with ethanl additin in 1:5 (v/ v) prprtin (Table 2),fr 18 hurs at 22 C. This prprtin and teperature were chsen as extractin cnditins. The recvery results (Table 3) shw the reliability f the extractin under these cnditins. With the selectivity assay, we shw that the plyphenls were extracted in very lw aunts (< 0.01%) and are thus insignificant when the ethanlic extractin is perfred in white usts r wines. The prtein results btained fr tw usts and ne wine and their precipitates are shwn in Table 4. Accrding t these results, the PS precipitate with ethanl cntains apprxiately 34% f the prteins, but these prteins crrespnd t 1% r 2% f the dry weight f the precipitate. Thibault (21) shwed that the prteins did nt cause interference with the -hydrxydiphenyl when they are in a range < 2 g/l, and we als bserved that they did nt react with the phenl reagent; prtein cncentratins belw 100 g/l d nt interfere in the spectrphtetric respnse. Fr these reasns the prtein extracted did nt interfere with the clrietric ethds used in this study. a-hydrxydiphenyl ethd: The crrelatin cefficient btained fr the calibratin curves prepared with a slutin B is (p < 0.001) (Table 5). The absrbance/cncentratin rati is, therefre, cnsidered t be linear fr the range f cncentratins studied (0-100 g/l). The cefficients f variatin are 3.8% fr repeatability and 2.5% _+ 2.0% (Table 5) fr reprducibility. These precisin values are cnsidered satisfactry, since they are belw the values described by Hrwit (12) as axiu intralabratry variability (3.2% - 4.2%) fr 500 g/l cncentratin. The liit f detectin (LD= 0.65 g/l) and the liit f quantitatin (LQ = 2.2 g/l) shw that the ethd has acceptable sensitivity. The selectivity f -hydrxydiphenyl fr APS cpared with NPS is denstrated when the spectrphtetric respnse is siilar fr the different calibratin curves btained with the slutins B, D, E, and F (Fig. 1). Phenl ethd: The crrelatin cefficient between absrbance/cncentratin with different galactse cncentratins is (p < 0.001) (Table 5). The ethd ay be cnsidered t shw acceptable linearity fr NPS in the cncentratin range studied ( g/ L). The repeatability is 2.4% and the reprducibility 1.9% _+ 1.0% (Table 6). These precisin values are cnsidered satisfactry, since they are belw the value accepted as axiu intralabratry variability (4.2% - 5.6%) fr 1000 g/l cncentratin (12). The LD = 1.36 g/l and LQ = 4.45 g/l shw that the sensitivity is acceptable (Table 5). The results btained fr the selectivity study shw that the phenl reagent des nt have the sae respnse fr APS and NPS (Fig. 2). The prprtin f APS cpared with NPS was calculated: the galactse respnse is 2.5 ties higher than the galacturnic acid respnse at the levels ( g/l) studied. Therefre, this value allws t crrect the APS deterinatin with the phenl ethd. The frula used fr crrectin is based n the suitability and selectivity f the APS deterinatin with -hydrxydiphenyl cpared with the NPS. With the phenl ethd, a TPS experiental value is btained (expressed in g/l galactse) because this reagent deterines APS and NPS: The TPS cntent can be expressed as: TPS = NPS + APS Eq. 1 With the data btained in the phenl selectivity study, the TPS cntent culd be expressed as: Experiental TPS = NPS + APS/2.5 Eq. 2 Taking the NPS ter fr Equatin 1 and substituting in equatin (2), gives: Experiental TPS = TPS - APS + APS/2.5 Eq.3

6 POLYSACCHARIDES IN WINEMAKING~ 569 and finally: TPS = experiental TPS APS Eq. 4 Equatin 4 is used t crrect the experiental values and t give the TPS f the saples (Table 6). Equatin 1 is used t btain the NPS cntent f the saples (Table 6). Plysaccharide levels: Table 6 shws the TPS, APS, and NPS results, each deter- ined in triplicate and expressed in g/l. The MANOVA analysis verified that the plysaccharide cntent depends significantly (p < ) n the kind f saple (ust r wine), variety, and harvest. The plysaccharide cntent variatins ang the three harvests cnsidered culd be attributed t cliatic cnditins, since the saples analyed were cllected in the sae gegraphical ne. In 1993, TPS and NPS values were higher than the ther tw years: in 1992 APS values were the lwest. Figure 3 shws the eans and 95% cnfidence fr ean f the TPS, NPS, and APS fr usts (M) and wines (W) f each variety. The TPS, NPS, and APS cntent were lwer in wine than in ust saples. Viura usts have higher TPS, NPS, and APS levels than the ther varieties (p < 0.05). In wines, the inter-variety differences are iniied due t the plysaccharide decrease during the white wineaking prcess and are nt significant ~ 800 E The plysaccharide levels f the white usts and wines btained in this study are higher than thse described in the literature fr white varieties (Table 1). This difference culd be explained by the analytical ethds used, especially fr the plysaccharide fractin extractin and separatin. If this is s, ur values are siilar t thse described by Villeta in 1988 (26). PS cntent during wineaking: The white wineaking prcess decreases the plysaccharide cntent; the PS levels in usts are higher than in their respective wines. This decrease during ferentatin depends n the initial plysaccharide cntent (r = , p < ). The higher the cntent in the initial TPS, the greater the decrease during the ferentatin prcess. This bservatin has been verified in the three harvests and with the three varieties studied. In Table 7, the percentages f APS cpared with TPS is shwn. The ttal average f this rati fr the three varieties and harvests is 38% + 10% fr the usts and 13% + 5% fr the wines, and this difference is bvius if we cnsider the increase in ethanl cntent (26,27) and the activity f endgenus pectlytic en- Table 7. APS/TPS rati, expressed in percentage, in usts and wines f the three varieties during three harvests. % APS in Musts % APS in wines % APS Viura Xarel. I Parellada > X ~ > X ~ > X a. > X 13_ TPS NPS > x n > x n APS Fig. 3. Means and 95 percent cnfidence fr ean f the levels fr TPS, NPS and APS in usts (M) and wines (W) f the three varieties studied, V: Viura, X: Xarel.l, and P: Parellada. Table 8. Percentage decreases f TPS, NPS, and APS f the three varieties studied during the 1993 wineaking prcess. % Decrease during % Decrease during % Decrease during ferentatin racking filtratin Variety TPS NPS APS TPS NPS APS TPS NPS Viura Xarel.l Parellada av+_sd 65_+2 59_+3 80_+5 26_+2 27_3 24_ _ APS _ 20 yes f grape and yeasts (8,19,24,25) during the ferentatin. This is crrbrated by the data in Table 8, which shw that the percentage decrease fr APS and NPS during the ferentatin f the 1993 crush were 79.5% _+ 4.6% and 58.8% + 2.5%, respectively. On the ther hand, the lwer percentage decrease f NPS during ferentatin culd be accunted fr by the annans and glucannans released fr the yeasts t the ediu (20). The percentage decrease in TPS, NPS, and APS during the racking and filtratin prcess was calculated fr the three varieties f the 1993 crush (Table 8). Racking decreased the plysaccharide levels 26.1% + 1.7%, there being n significant differences between the tw plysaccharide fractins, APS and NPS. During the wine filtratin there was als a decrease. The APS percentage retained in the candle cellulse filter was higher than the NPS percentage, except fr the Xarrel.l

7 570- LOPEZ-TAMAMES et al variety. This cincides with the results btained by ther authrs (27), wh reprt that the APS have a linear lecular structure which bstructs the filters. Cnclusins Reliable and specific analytical ethds were studied t quantify TPS and its tw ajr fractins (APS and NPS) in ust and wine saples. Initially, an extractin with ethanl precipitatin fr a cplex atrix, such as ust and wine saples, was necessary; fr this reasn, the ptial cnditins fr the extractin were cnsidered. Recvery values and selectivity with respect t plyphenls and prteins were established. Fr APS quantitatin, we used the spectrphtetric ethd with -hydrxydiphenyl as chrgen reagent. This ethd was acceptablely accurate, sensitive, and selective fr APS deterinatin. With phenl as chrgen reagent, the TPS were quantified. The NPS cntent was deterined by difference between the tw ethds. The study f the phenl sulfuric ethd prvided a crrectin frula t btain a re exact, reliable, and siple value fr the TPS than thse used by ther authrs. The variety and the harvest played an iprtant rle in the TPS, APS, and NPS cntent in usts: Viura usts and wines had higher TPS, NPS, and APS levels than the ther varieties nes. White wineaking reduced the PS cntent, and this decrease depended n the initial TPS cntent. Ferentatin was the ain prcess that affected the PS level. During ferentatin, there were clear changes in the percentages f APS cpared with TPS; APS cntent in wines was very lw, due t the actin f endgenus pectlytic enyes fr the grape and yeast r/and the increasing ethanl cntent. Wine TPS were cpsed principally by NPS. Mrever, the NPS levels decreased during ferentatin, but this effect culd be cpensated by the yeast PS release. During the wineaking prcess, the racking and wine filtratin als decreased the plysaccharide levels. 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