Heat-Unstable Prteins in Wine.. Characterizatin and Remval by Bentnite Fining and Heat Treatment JUNN-CHN HSU ~ and DVD. HETHERBELL 2 The effect f bentnite fining n the ttal prteins and the heat-unstable (80 C, 6 h; 4 C, 12 h) prteins in GewLirztraminer, White Riesling, Sauvignn blanc, and Sylvaner wines was investigated. Prtein mlecular weights (MW), iselectric pints (pl), and glycprteins were determined by using lithium ddecyl sulfate plyacrylamide gel electrphresis (LDS PGE) and tw-dimensinal iselectric fcusing-lithium ddecyl sulfate (F-LDS) electrphretic techniques with silver staining as well as prtein bltting fr glycprtein detectin. Relative cncentratins f prteins in stained gels were determined by laser scanning densitmetry. Bentnite fining tends t remve the higher pl (5.8-8.0) and intermediate MW (32 000-45 000) prtein fractins first. Hwever, these represent nly a small prprtin f the sluble prteins. n general, it is necessary t remve the lwer pl (4.1-5.8), lwer MW (12 600 and 20 000-30 000) fractins, which cntain glycprteins and represent the majr cmpnent f the prteins, t prtein stabilize wines t heat testing. Prtein fractins with MW f 60 000 t 65 000 and having a wide range f pl (4.1-8.0) were highly resistant t remval by bentnite fining and remained in prtein-stabilized wine. n additin, trace amunts f fractins with MW f 28 000 may remain in GewiJrztraminer and White Riesling wines and 25 000 in Sauvignn blanc wines. Unstable prteins precipitated by heat tests were recvered and analyzed. Prtein fractins with MW f greater than 14 000 were mre heat sensitive than lwer MW fractins. The heat-precipitated prteins fund in sediments were mainly f lw MW (< 30 000) and primarily glycprteins. t is cncluded that the prtein fractins f lwer MW (12 600 and 20 000-30 000) and lwer pl (4.1-5.8) and glycprteins are the majr and mst imprtant fractins cntributing t prtein instability in wines. lthugh there have been numerus investigatins n grape juice and wine prteins (9), the nature f the prteins respnsible fr wine turbidity remains unclear. Prtein instability des nt crrelate well with the ttal prtein cntent, and there appears t be cnflicting infrmatin in the literature as t which prteins (prtein fractins) are respnsible fr haze and sediment frmatin. Kch and Sajak (10), using paper electrphresis, shwed that grapes and wines cntained tw majr prtein fractins, bth being decreased by heat treatment and additin f bentnite. Mretti and Berg (15) assciated specific prtein fractins with wine turbidity and cncluded that nly a part f the prtein mixture was respnsible fr prtein stability rather than ttal prtein cntent. Bayly and Berg (3) further classified prtein fractins accrding t their heat stability and cncluded that remval f prtein fractins by additin f bentnite des nt ccur in equal prprtins but remves the highly charged prtein mlecules first. Millies (14), using silica sl/gelatin fining and ultrafiltratin t fractinate the wine prteins, claimed that the prtein fractins with MW f less than 10 000 d nt take part in turbidity frmatin, fractins between MW 10 000 and 30 000 are nly partly invlved, and thse with MW f greater than 30 000 are the main unstable prteins. Mesrb et al. (13) indicated that the prtein cluding is mainly caused by the prtein fractin with lwer p and lwer MW. Recently, using gel electrphresis and iselectric fcusing, Heatherbell et al. (8) and Ngaba and Heatherbell (16) 1Graduate Student and 2ssciate Prfessr, Department f Fd Science and Technlgy, Oregn State University, Crvallis, OR 97331. The authrs thank the merican Vineyard Fundatin and the Oregn Table Wine Research dvisry Bard fr financial supprt given; and B. T Watsn, Jr., fr assistance in prviding wine samples fr this study. Technical Paper N. 7892 frm the Oregn gricultural Experiment Statin. This research cnducted at Oregn State University, Crvallis, OR. Manuscript submitted fr publicatin 21 May 1986. Cpyright 1987 by the merican Sciety fr Enlgy and Viticulture. ll rights reserved. determined the MW and p f heat-stable and unstable prteins present in Oregn and Washingtn wines and investigated their remval by bentnite fining, ultrafiltratin, and prtease enzymes. Prtein fractins detected had a MW range f 16 000 t 90 000 daltns and p frm 4.5 t 8.0. With prgressive bentnite fining, it was nt until the lwer MW (ca 16 000-25 000 daltns) and higher p (5.6-8.0) fractins were remved by bentnite fining that the wines became prtein stabilized t heat testing. mre recent reprt by Lee (12) fr ustralian wines claimed that the majr prtein fractins f wine have MW f 40 000 t 200 000 daltns and p f 4.8 t 5.7 and "these fractins must be largely remved frm wine befre stability... is cnferred." n this study, we further investigated the nature f the unstable prteins and their remval by bentnite fining and heat treatment by applying imprved sensitive techniques (9) fr the detectin and characterizatin f prteins and glycprteins. Materials and Methds Preparatin f wines: Gewtirztraminer, White Riesling, Sauvignn blanc, and Sylvaner grapes frm the Oregn State University experimental vineyards were harvested and prcessed int wines by cnventinal prcedures in the Oregn State University experimental winery. Yung wines (after tw rackings) were sampled fr this study. Bentnite fining: Fr determinatin f the amunt f bentnite required t stabilize wines t heat testing, each wine sample was fined with 5-80 g bentnite per hl wine and thrughly mixed. Bentnite (sdium bentnite, Vlclay) was added as a smth aqueus 3% suspensin (1 ml bentnite slutin when added t 100 ml wine, crrespnding t 30 g bentnite per hl r 2.5 lb bentnite per 1000 gal wine). The wines were held at 11
12 -- HET-UNSTBLE PROTENS.. rm temperature fr at least 48 hurs and then filtered thrugh 0.45-#m Millipre membrane befre analysis. Filtered wines were prepared fr gel electrphresis as previusly described (9). Cncentratin f sluble prtein and ttal phenls were determined as previusly described (9). Heat stability test: The heat stability f the wines was determined by the prcedure recmmended by Pcck and Rankine (17). Fifty milliliters f filtered wine was transferred int 65-mL bttles. Bttles were sealed with screw caps and heated in a water bath (80 C) fr six hurs, held at 4 C fr 12 hurs, and allwed t warm t rm temperature. Frmatin f turbidity was measured by visual bservatin under a strng beam f light and by a Hunter mdel D25P-2 clr difference meter (Hunter ssciates Lab., nc.) perated in the transmittance mde fr the transmissin haze reading: percent haze = Y(rrangement )//Y(rrangement ) }< 100 Filtered distilled water was used as the blank giving a reading f 2 units. Preparatin f haze/sediment frm heat stability test: Fifty milliliters f unfined wine was heated at 80 C in a water bath fr six hurs and then stred at 4 C fr 12 hurs. Prtein haze/sediment was cllected by centrifuging at 12 100 g fr 20 minutes at 4 C in a Srvall RC-5 centrifuge. The precipitate was cllected and suspended in 2 ml sample buffer fr gel electrphresis (9). The supernatant was filtered thrugh 0.45-#m Millipre filter membrane, and the filtrate was prepared fr gel electrphresis as previusly described (9). Gel electrphresis: Lithium ddecyl sulfate plyacrylamide gel eletrphresis (LDS PGE), tw-dimensinal iselectric fcusing-lds PGE (F-LDS PGE), silver staining, laser scanning, prtein bltting, and glycprtein detectin were perfrmed as previusly described (9). Carbxymethyl cellulse cncentratin f wine prteins: n sme instances fr tw-dimensinal F-LDS PGE, it was desirable t cncentrate prteins. Prteins were cncentrated using a mdificatin f the prcedure described by Calbichem-Behring (1) using carbxymethyl cellulse (CMC) (quacide, Calbichem, La Jlla, C). Samples f 20 t 30 ml were placed in dialysis tubing and then packed in CMC fr tw t three hurs until up t 10-fld cncentratin was achieved. Results and Discussins Bentnite fining: The effect f bentnite fining n the prtein stability (as determined by heat test), sluble prtein, and ttal phenls f Gewtirztraminer, White Riesling, Sauvignn blanc, and Sylvaner wines was investigated (Fig. 1). There was a gd agreement between visible bservatins and Hunter haze readings fllwing heat testing. Whereas heat-induced haze frmatin prgressively decreased with bentnite fining and prtein reductin, bentnite additin had n effect n ttal phenl (p > 0.05). n these fur wines, prtein cncentratin f ca 5 mg/l (mean = 5.4 + 1.6 mg/l) cincided with wine stability (Fig. 1). This ccurred irrespective f JJ a. ~ uj 60 50 40 30... 30 0:: Ll - z :D 20 0 30 20 0 30 20 0 0 600 prtein --- haze - 500 _~ ~ phenl - 400 " = = ~ 300? - - 0 2O0 B 400 300-2_00 00 (.9 E, i 0..j - 0 C 0-300 z Ll -200 ~r" D t3 - e e e = e ~ 1200 1, i -'0 0 0 20 30 40 50 60 70 80 BENTONTE (g/hl) Fig. 1. Effect f bentnite fining n prtein stability and cncentratin f sluble prtein and phenl in wines: - GewUrztraminer (ph 3.67, 12.2% ethanl), B - White Riesling (ph 3.15, 11.7% ethanl), C - Sauvignn blanc (ph 3.20, 11.9% ethanl), D - Sylvaner (ph 3.20, 11.1% ethanl). BSE - bvine serum albumin equivalent, GE - gallic acid equivalent. rrw (,--) represents bentnite treatment required t prtein stabilize wine as determined by visual assessment. 1Determined as Hunter haze frmatin r by visible bservatin fllwing heat test. the initial cncentratin f prtein in the wines which ranged frm 19 t 44 mg/l (Fig. 1). Hwever, it shuld be nted that higher cncentratins f prteins have been bserved in wine prtein stabilized by bentnite fining bth in the Department f Fd Science and Technlgy, Oregn State University, Crvallis, (cmmnly 10-20 mg/l) and in the literature (12,17). Relatively large amunts f bentnite were required t remve the prteins which are mst resistant t remval by bentnite (Fig. 1). These fractins, which cntribute t persistant residual haze, are present in small amunts, as lw as 1 t 2 mg/l as previusly reprted (8). n additin, lw cncentratins f phenlics may be cntributing t persistent residual haze in juices and wines, either independently r in assciatin with the prteins (7,8,18,19). T btain further infrmatin abut the remval f individual prtein fractins frm wines by bentnite fining, the prtein fractins were separated and their MW determined by using LDS PGE. lthugh there were differences in the electrgrams f these fur wines,
HET-UNSTBLE PROTENS.. m 13 L Unfined Wine ++++ 11 --- d i! t B 0g/hL Bentnite 45 t~ t- O Q. n," 0 " ~ l t C 50g/hL Bentnite ++,, i ~j~,~1~j,= ~., a 70g/hL Bentnite 14 Fig. 2. LDS PGE f GewfJrztraminer wine prteins in wine fined with different levels f bentnite: - 0 g/hl, B - 20 g/hl, C - 50 g/hl, D - 70 g/hl, E - 80 g/hl (heat stable). 100 #L f each sample was applied t gel. Mlecular weights (K = 1000 daltns) f standards are given n left side f gel. bentnite fining had a similar effect n the remval f the prtein fractins present. Prtein fractins with MW f 12 600 and between 20000 and 30000 are the last fractins t be remved t prtein stabilize wine t heat testing. Fr instance, the last fractins t be remved by bentnite fining in bth the Gewtirztraminer and White Riesling wines were the 12 600 and 25 000 fractins; in Sauvignn blanc wines, they were the 12 600 and 28 000 fractins. typical electrgram fr Gewtirztraminer wine is shwn in Figure 2 (wine in Fig. 1). Bentnite fining remved intermediate MW fractins (32 000-45 000) first, befre remving the lwer MW fractins (11 200-25 000). Hwever, the fractins in the higher MW range f 60 000 t 65 000 were highly resistant t remval by bentnite fining. The same pattern f results was als btained fr White Riesling and Sauvignn blanc wines. The densitmetric patterns f the stained gel are shwn in Figure 3. These results indicate that the lw MW fractins (12 600 and 25 000) are imprtant t prtein instability because their remval by bentnite fining cincides with prtein stabilizatin f wines t heat testing (Fig. 2 (E), 3 (E)). n cntrast, prtein fractins with MW f 60 000 t 65 000 remained in the bentnitefined prtein-stabilized wines (Fig. 2 (E), 3 (E)). n additin, trace amunts (limit f detectin 10 ng (20)) f 80g/hL Bentnite 14 21 31 45 66 92 116 200 Mlecular Weight (Daltns x 10-3) Fig. 3. Densitmetric scans f electrphretic patterns (Fig. 2) f GewUrztraminer wine prteins in wine fined with different levels f bentnite: - 0 g/hl, B - 20 g/hl, C - 50 g/hl, D - 70 g/hl, E - 80 g/hl. Prtein instability indicated by +. the 28 000-MW fractin was detectable in sme instances. Prtein bltting cmbined with glycprtein staining (9) was used fr the detectin f glycprteins. With this technique, the three prtein fractins with MW f 12 600, 25 000, and 28 000 in Figure 2 were identified as cntaining glycprteins in Gewfirztraminer wine. Only tw fractins with MW f 25 000 and 28 000 were detected in White Riesling wine. Hwever, n glycprteins were detected in the bentnite-stabilized wines. The effect f bentnite fining n wine prteins was further investigated by subjecting wine t tw-dimensinal F-LDS PGE (9). typical result fr a Gew~irztraminer wine is shwn in Figure 4 (wine in Fig. 1) E
14- HET-UNSTBLE PROTENS.. 4.1 5.0 l ph 8.0 4.1 1.! 5.O! ph 6.0! 92K-* 66K--*0. Fig. 4. Tw-dimensinal F-LDS PGE f Gew0rztraminer wine prteins fllwed by silver staining: - unfined wine cncentrated tw-fld befre electrphresis, B - wine fined with 80 g/hl bentnite and cncentrated 10-fld befre electrphresis. Mlecular weights (K = 1000 daltns) f standards are given n left side f each gel. The ph gradient is labeled abve each gel. 14K which shws that the majr prtein fractins in Gewtirztraminer wine are f lw MW (20 000-30 000) and lw p (4.1-5.8). The absence f the majr fractin with MW f 12 600 (Fig. 2) in Figure 4 is nt accunted fr. t is pssible that it may have been lst thrugh dialysis tubing during cncentratin (wine samples applied t gels in Fig. 4 were cncentrated as indicated in an attempt t detect lw cncentratins f sme fractins, particularly in the bentnite-fined sample). selectric fcusing has further separated each f the prtein fractins in Figure 2 (wine ) int several fractins n the basis f their p (Fig. 4 ()). Fr instance, the fractin with MW f 28 000 has been separated in five majr fractins with p frm 4.1 t 5.8. When the same tw-dimensinal technique was applied t bentnite-stabilized wine (wine E, Fig. 2), nne f the fractins knwn t be imprtant cntributrs t heat instability (8) culd be detected (data nt shwn). Hwever, with sufficient cncentratin (ca 10-fld), trace amunts f these fractins culd be demnstrated t remain in the stabilized wine (Fig. 4 (B)). n cntrast, cnsiderable amunts f the prtein fractins with MW f 60 000 t 65 000 which were highly resistant t remval by bentnite fining and had a wide range f p (4.1-8.0), remained in the bentnite-fined wine (Fig. 4 (B)) and were readily detectable withut cncentratin. n interpreting these results, cnsideratin must be given t the extreme sensitivity f the methd; trace amunts f prtein (10 ng) are detectable (20). The fllwing interpretatins are based upn analysis f single strength (uncncentrated) wine. n general, the applicatin f this technique cnfirmed and extended previus studies (8). Bentnite fining remved the intermediate MW (32 000-45 000) (Fig. 2) and higher p (5.8-8.0) (Fig. 4) fractins first. Hwever, these fractins tgether nly cmpse a small prprtin f the sluble prteins present in these wines, and althugh they may cntribute t instability, their remval did nt stabilize wines. t was necessary t remve the lwer p (4.1-5.8) and MW (12 600 and 20 000-30 000) fractins and glycprteins t prtein stabilize wines. Specifically, fr Gewtirztraminer wine, the fractins with MW f 12 600 (cntaining glycprteins) and MW f 25 000 (having p f 4.1-4.8 and cntaining glycprteins) are the last remved by bentnite fining t prtein stabilize wine t heat testing (Fig. 2, 3, 4). n cntrast, the prtein fractins with MW f 60 000 t 65 000 and trace amunts f the 28 000-MW fractin remained in wine which had been prtein stabilized by bentnite fining. Heat treatment: standard heat test (80 C, 6 h; 4 C, 12 h) recmmended by Pcck and Rankine (17) was used t evaluate the heat stability f wine prteins. n previus studies (6), Heatherbell et al. demnstrated that, fr Oregn and Washingtn wines, this ne-day test crrelated well with the lnger five-day test (49 C, 4 days; 5 C, 1 day) prpsed by Berg and kiyshi (4). T assist in the further characterizatin f the heat-unstable prteins, prteins precipitated by the heat test (1-day test) were recvered and analyzed. Heat treatment f wines precipitated ca 50% (15-30 rag/l) f the prteins (Table 1). Similar results were reprted by Kch and Sajak (10) wh claimed ver 60% (20-45 rag/l) Vit-N cntent remained in heat-treated (75 C, 2 min) wine. n cntrast, Pcck and Rankine (17) and Lee (12) claimed that heat treatment f wine at 80 C fr six hurs appeared t cmpletely cagulate the sluble prtein present and remve all prteins frm the ustralian wines tested. lss (p < 0.05) f up t 7.8% f ttal phenl was bserved in heat-treated wine (Table 1) and may be significant in
HET-UNSTBLE PROTENS..- 15 Table 1. Effect f heat treatment a n cncentratin f sluble prtein and phenl in wines. Sample Prtein (mg/l) b Phenl (mg/l) b GewfJrztraminer Befre treatment 59.2 + 0.2 302.1 + 7.7 fter treatment 28.6 + 1.5 282.9 + 5.4 % Remval 51.7 6.4 White Riesling Befre treatment 36.1 + 0.7 371.3 +_ 3.1 fter treatment 17.8 + 0.8 342.3 + 1.3 % Remval 50.7 7.8 a Heat test fr determining prtein stability (6 h. 80 C; 12 h, 4 C). b Mean f triplicate determinatins _+ standard deviatin. g'; C:: O Q.. U) n- Befre Heat Treatment fter Heat Treatment B 20OK- B C O,l-- fj E3 C 116K- 92K- 66K- 45K-- 31K-- 21K- ::: 14K- Fig. 5. LDS PGE f GewLlrztraminer wine prteins in wine subjected t heat test: - befre heat treatment, B - after heat treatment, C - sediment. Mlecular weights (K = 1000 daltns) f standards are given n left side f gel. haze frmatin during heat testing. The cmbinatin f phenlics with prteins in hazes and sediments frm wines (5,10,18), juices (7,11), and beer (2) has been reprted frequently. The MW f prtein fractins, their relative cncentratins, and the presence f glycprteins in initial wine, heat-treated wine, and in recvered sediments were determined (Fig. 5, 6). Prtein fractins with MW f greater than 14 000 were heat sensitive and were remved by heat treatment (Fig. 6 (B)). n cntrast, prtein fractins with 14 21 31 45 66 92 116 200 Mlecular Weight (Daltns x 0-3) Fig. 6. Densitmetric scans f electrphretic patterns (Fig. 5) f Gew(Jrztraminer wine prteins in wine subjected t heat test: - befre heat treatment, B - after heat treatment, C - sediment. MW f lwer than 14 000, including ne majr MW fractin (12 600) cntaining glycprteins, remained in the wine after heat testing (Fig. 6 (B)). n sme wines, trace amunts f fractins f MW 60 000 t 65 000 als remained after heat testing. Prtein hydrlysis/dissciatin may have ccurred during heat treatment, there being an increase in the lw MW fractins (< 30 000) present in the sediment (Fig. 6). t is als pssible that a higher percentage f the larger MW fractins were heat sensitive/denatured than the lwer MW fractins during heat testing. The heat-precipitated prteins fund in sediments were mainly f lw MW (< 30 000) (Fig. 6 (C)) and primarily glycprteins. These findings supprt the cnclusins we btained fr characterizing the nature f the heat-unstable prteins remved by bentnite fining. Cnclusins t is cncluded that the prtein fractins f lwer MW (12 600 and 20 000-30 000) and lwer p (4.1-5.8) and glycprteins are the majr and mst imprtant fractins cntributing t prtein instability in wines. Literature Cited 1. nnymus. quacides. Bilgics Technical nfrmatin, Calbichem-Behring, Divisin f merican Hechst Crpratin, La Jlla, C (1985). 2. san, K., K. Shinagawa, and N. Hashimt. Characterizatin f haze-frming prteins f beer and their rles in chill haze frmatin. J. m. Sc. Brew. Chem. 40:147-54 (1982).
16- HET-UNSTBLE PROTENS.. 3. Bayly, F. C., and H. W. Berg. Grape and wine prteins f white wine varietals. m. J. Enl. Vitic. 18:18-32 (1967). 4. Berg, H.W., and M. kiyshi. Determinatin f prtein stability in wine. m. J. Enl. Vitic. 12:107-10 (1961). 5. GOrtges, S. Prblems with prtein stabilizatin in winemaking. Weinwirtschaft 118:931-5 (1982). 6. Heatherbell, D.., P. R. Ngaba, B.T. Watsn, and E. E. Ddd. Determinatin f prtein and investigatin f prtein instability in Pacific Nrthwest wines. Presented at the 32nd nnual Meeting f the merican Sciety f Enlgists, San Dieg, C (June 1981). 7. Heatherbell, D.. Fruit juice clarificatin and fining. Cnfructa 28:192-7 (1984). 8. Heatherbell, D., P. Ngaba, J. Fmbin, B. Watsn, Jr., Z. Garcia, J. Flres, and J. Hsu. Recent develpments in the applica, tin f ultrafiltratin and prtease enzymes t grape juice and wine prcessing. n: Prceedings f the nternatinal Sympsium n Cl Climate Viticulture and Enlgy. D.. Heatherbell, P.B. Lmbard, F. W. Bdyfelt, and S. F. Price (Eds.). pp 418-45. Oregn State University, Crvallis, OR (1985). 9. Hsu, J. C., and D.. Heatherbell. slatin and characterizatin f sluble prteins in grapes, grape juice, and wine. m. J. Enl. Vitic. 38:6-10 (1987). 10. Kch, J., and E. Sajak. review and sme studies n prtein. m. J. Enl. Vitic. 10:114-23 (1959). 11. Lea,. G. H. Tannin and clur in English cider apples. Fluss. Obst. 8:356-61,399-400 (1984). 12. Lee, T. H. Prtein instability: Nature, characterizatin and remval by bentnite. n: Physical Stability f Wine. T. H. Lee (Ed.). pp 23-39. The ustralian Wine Research nstitute, Glen Osmnd, S.., ustralia (1986). 13. Mesrb, B., N. Grinva, and D. Tzakv. Characterizatin f the electrical prperties and f the mlecular weights f the prteins f white wines. Nahrung 27:727-33 (1983). 14. Millies, K. Prtein stabilizatin f wine using silica sl/gelatin fining. Mitteilungsblatt der GDCh-Fachgruppe. Lebensm. Gerich. Chemie. 29:50-3 (1975). 15. Mretti, R. H., and H. W. Berg. Variability amng wines t prtein cluding. m. J. Enl. Vitic. 16:69-78 (1965). 16. Ngaba, P. R., and D.. Heatherbeil. Prtein instability in wine.. Characterizatin f unstable prteins.. The applicatin f prtease enzymes as bentnite substitutes in prtein stabilizatin wine. Presented at the 32nd nnual Meeting f the merican Sciety f Enlgists, San Dieg, C (June 1981). 17. Pcck, K. F., and B. C. Rankine. Heat test fr detecting prtein instability in wine. ust. Wine. Brew. Spirit Rev. 91:42-3 (1973). 18. Smers, T.C. The inevitability f phenlic instability. n: Physical Stability f Wine. T. H. Lee (Ed.). pp 81-90. The ustralian Wine Research nstitute, Glen Osmnd, S.., ustralia (1986). 19. Thmas, B., and R. B. Bultn. Studies f prteinaceus materials in white wines. Presented at the 35th nnual Meeting f merican Sciety fr Enlgy and Viticulture, San Dieg, C (June 1984). 20. Wray, W., T. Bulikas, V. P. Wray, and R. Hancck. Silver staining f prteins in plyacrylamide gels. nal. Bichem. 118:197-203 (1981).