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HEAT UNSTABLE WINE PROTEINS AND THEIR INTERACTIONS WITH WINE POLYSACCHARIDES
by
Elizabeth Joy Waters, BSc
A thesis submitted in fulfillment of the requirements for the degree of
Doctor of Philosophy
Department of Plant Science The [Jniversity of Adelaide
The Australian Wine Research Institute
July I99L 1
Declaration
I hereby declare that ttris thesis contains no material which has been accepted for the awa¡d of any other degree or diploma at any university. To the best of my knowledge and belief, no material described herein has been previously published or written by any other person except when due reference is made in the æxt.
If accepæd for the awa¡d of Doctor of Philosophy, this thesis wilt be available for loan or photocopy
Elizabeth Joy Waters ü
Abstract
Unstable grape juice and wine proteins can cause cloudiness or deposits in wine after bottling resulting in adverse effects on consumer acceptance. This is of concern to white wine producers and frning with bentonite to remove protein is an established winery practice. However, such methods are inconvenient, non-specific and can result in extensive flavour loss. In this study, proteins responsible for hazp, have been isolated and characterized, and their interaction with wine polysaccharides has been explored. An examination of the use of peptidases to remove such proæins was also undertaken.
To get a reliable estimate of the concentration of grapo juice and wine proteins it was necessary to determine their amino acid composition. The commonly used Bradford dye- binding assay grossly underestimates the protein concentration by 50 to 807o and in addition, grape and wine phenolic compounds were found to strongly interfere.
Using a chromogenic protein substrate it was established that fungal, animal and plant peptidases were active in grape juice and wine. However, at typical wine making temperatur€s (10-25oC), wine proteins were incompletely hydrolysed and material with Mt greater than 10,000 was still measurable. Thus potentially unstable proteins were still present in enzyme-treated juice after 7 days at25oC and in wine after 4 months at 15oC. In the case of the wine, both the control and treated wines showed decreases in protein content and bentonite requirements for stabilization, suggesting that the effects seen may have been due to the activity of endogenous grape peptidases.
An initial fractionation of the proteins of a heat-unstable wine was obtained by a combination of salting out with (NH¿)ZSO4 and ultrafiltration. The least soluble fraction [4, precipitated with 607o saturation of (NII¿)ZSO4] was dominaæd by a proæin band of M¡ 32,000, the fraction salted out with 65 to 707o saturation of (NH4)2SO+ tBl had a major protein of M¡ 24,000, while the most soluble material [C, supernatant at707o saturation of (NH¿)zSO+ I comprised proteins of Mr 26,000 and 24,000 [E] together with a carbohydrate-rich fraction [D].
A micro heat test applied to the fractions showed that fractions B and E caused the most haze. The carbohydrate-nch component [D] was the most thermo-stable and could reduce the haze forming potential of E. The major proteins in fractions B and E were further purified by anion exchange chromatography to homogeneity, confirming that, although both proteins were important to wine haze, the l\4 24,000 protein gave more haze than the ìvlr 32,000 protein. lll
The stabilizing effect of the carbohydrate-rich fraction [D] was found to apply to all wine proteins in addition to a standard protein, bovine serum albumin. The protection became greater, reducing the visible haziness induced by heating, as the ratio of added carbohydraæ-rich fraction to protein was increased The relationship between the extent of protection and the concentration of added carbohydrate-rich fraction was not linear, and haze decrease diminished at high polysaccharide concentration. It was shown that haze was notreduced by preventing protein precipiøtion but by reducing the particle size of the heat-induce dhazn.
The component in the carbohydrate-rich fraction which was responsible for haze protection were isolated by a combination of lectin affinity chromatography, anion exchange chromatography, and cation exchange chromatography. It was a macromolecule containing carbohydrate (96Vo), which was predominantly mannose (787o), and protein (47o). The amno acid composition of the protein component was dominated by the hydroxyl bearing amino acids, serine (317o) and threonine (I37o). iv
Acknowledgements
I sincerely thank my two supervisors, Dr Patrick V/illiams and Dr William Wallace, for their advice and encou¡agemenl I also gratefully acknowledge the Director of the Australian \Mine Research Institute, Prof. Terry Lee, and the Council of that Instituæ for granting permission and time to undertake the study. The Australian Grape and Wine Research Council is thanked for financial support.
I wish to thank staff of the Deparnnent of Plant Science, The University of Adelaide, in particular Dr Max Tate for his help with sugar analyses, his inærest and infectious enthusiasm and many stimulating and helpful discussions. Ms Hilary Phillips, Ms Jenny Guerin and Mr Richard Batt are also thanked for their friendship and assistance with the Pharmacia FPLC system.
My colleagues of the Australian Wine Research Institute are thanked for their friendship and help, in particular, Mr Vassilios Marinos for the gas chromatographic analyses and Mr Holger Gockowiak for assistance with ami¡s acid analysis. The donation of proanthocyanidin grape seed extract from Dr Christopher Somers is also acknowledged
David Hewitt of the School of Chemical Technology, The University of South Australia and ttrat School a¡e ttranked for the use of the Malvern Autosizer particle sizing apparatus.
Penfolds Wines, Nuriootpa and Lindemans Wines, Karadoc are acknowledged for their donation of grape juice and wine. Mr Blair Duncan, of Penfolds Wines is thanked for helpful discussions.
Finally,I wish to thank my husband, Chris, for his loving support, understanding and encouragement. v
Publications
Part of the work described in this thesis of Elizabeth Joy Waters (formerly Modra) has been published:
1. Modra, E. J.; Williams, P. J. Are proteases active in wines and juices? Ausr. Grape grower Winemaker 1988, 292, 42-46.
2. Modra, E. J.; Williams, P. J.; Lee, T. H.; Wallace, W. Effect of commercial peptidases on must and wine. Proceedings,4emc Symposium Internatìottal d'Oerwlogíe: Actualités Oenologiques ; 15-17 June 1989; Ribéreau-Gayon, P.; Lonvaud, A.,Fds., Dunod: Paris, 1990; pp2l7-22L.
3. Waters, E. J.; Watlace, W.; Williams P. J. Peptidases in winemaking. Proceedíngs, SeventhAwtralianWine IndwtryTechnical Conference; 13-17 August 1989; \[il[ams, P. J.; Davidson, D.; I-ee, T. H., Eds.; Australian Industrial: Adelaide, SA, 1990: pp186- tgr.
4. Waters, E. J.; Watlace, W'.; Williams P. J. Heat haze characteristics of fractionated wine proteins. Anr. I. Ernl. Vitic.l99l,42, 123-127 . vr
TABLE OF CONTENTS page
Declaration i
Abstract ü
Acknowledgements iv
Publications v
Table of conænts vi
List of Figures xüi
List of Tables xvüi
Abbreviations ixx
1 INTRODUCTION AND GENERAL LITERATURE REVIEW 1 IWINE 1.1 CLARITY OF 1
1.2 PROTEINS OF GRAPES, ruICES AND WINES 2
7.2.lEnzyme protein 3
l.2.lJ Peptidases 3
1.2.L.2 Polyphenoloxidases 4
1.2.1.3 Invertases 5
1.2.I.4 Glycosidases 6
I.2.2 Evidence for glycoproteins 7
1.2.3 Factors affecting protein concentration 8
l.2.4Heat instability 9
1.3 PROTEIN AGGREGATION 9
1.3.1 Beer chill haze 10
1.4 FINING FOR WINE CLARIFICATON 11
1.4.1 Bentonite fining for proæin removal T2
1.4.2 Other methods of protein removal t3 vll
page
1.5 POLYSACCHARIDES OF GRAPES, JTTICES AND WINES L4 1.5.1 Pectins t4
1.5.2 Neutral polysaccharides 15
1.5.3 Yeast-derived polysaccharides t9
1.5.4 Polysaccharides from other sources 22
1.6 AIMS OF THIS STUDY 22
2 ESTIMATION OF GRAPE JUICE AND WINE PROTEIN
CONCENTRATION 24
2.1 INTRODUCTION AND LITERATURE REVIEV/ 24
2.2 EXPERIMENTAL 27
2.2.1 Materials 27
2.2.2UItafiltration 27
2.2.3 IJtgh Performance Liquid Chromarography (HPLC) 28
2.2.4 Bradford protein assay 28
2.2.5 Acid hydrolysis of purified grape proteins 29
2.3 RESULTS 30
2.3.1 UV absorbance 30
2.3.2 T\e Bradford as say 33
2.3.2.1Effect of monomeric phenolic compounds 33
2.3.2.2 Effect of polymeric phenolic compounds 33
2.3.2.3 Effect of bound polymeric phenolic compounds 33
2.3.2.4 Effect of ultrafiltration 37
2.3.2.5 Time course of dye-binding 4l
2.3.3 Acid hydrolysis 44
2.3.3.1 Total grape protein 44
2.3.3.2 Purified wine protein 44
2.4 DISCUSSION 47 vrll
page 3 REMOVAL OF GRAPE JUICE AND WINE PROTEIN
BY PEPTIDASE TREATMENT 50
3.1 INTRODUCTION AND LITERATURE REVIEV/ 50
3.2 EXPERIMENTAL 52
3.2.1 Materials 52
3.2.2General Methods 54
3.2.2.t Heat Test 54
3.2.2.2 Bentonite fining 54
3.2.3 Assay of peptidase activity with casein 54
3.2.4 Assay of peptidase activity with HPA 55
3.2.5 Preparation of peptidase inhibitors 55
3.3 RESULTS 56
3.3.1 Peptidase activity of the commercial enzymes in buffer 56
3.3.1.1 Effect of temperature s6
3.3.L.2 Effect of glucose, fructose and ethanol 56
3.3.2 Peptidase activity on HPA in juice and wine 62
3.3.3 Peptidase activity on grape proteins 66
3.3.3.I In buffer 66
3.3.3.2In juice 66
3.3.3.3In wine 69
3.3.4 Inhibition of peptidase activity 74
3.4 DISCUSSION 77 lx
page
4 IDENTIFICATION OF UNSTABLE PROTEIN 82
4.1 INTRODUCTION AND LITERATURE REVIEW 82
4.2 EXPERIMENTAL 85
4.2.1 Materials 85
4.2.2 General methods 86
4.2.2.I Sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS PAGE) 86
4.2.2.1 J Gel polymerization 86
4.2.2.I.2 S ample preparation 87
4.2.2.1.3 Electrophoretic conditions 87
4.2.2.L.4 Staining 87
4.2.2.1.5 Glycoconjugate detection with enzyme
immunoassay 88
4.2.2.1.5.L Digoxigenin (DIG) labelling 88
4.2.2.r.5.2 SDS PAGE 88
4.2.2.I.5.3 Transfer to nitrocellulose paper 88
4.2.2.I.5.4 Glycoprotein detection 89
4.2.2.2 Infrared analysis 90
4.2.2.3 Ultraf iltration 90
4.2.2.4 Micromethod for haze porential 90
4.2.3 Isolation of wine haze 91 4.2.4 Isolation of wine protein 9l
4.2.4.L Gel permeation HPLC 91
4.2.4.2 Ammonium sulphate fractionation 9L
4.2.4.3 Anion exchange chromatography 92 x
page
4.3 RESULTS 93
4.3.1 Cha¡acterisation of wine haze 93
4.3.L.1Characterization of protein 93
4.3.I.2lnfra¡ed Spectroscopy 93
4.3.I.3 Solubility properties 93
4.3.2 Wine fractions separated by gel perrneation 97
4.3.2.I Characterization of protein 97
4.3.2.2lnfrared Spectroscopy 97
4.3.2.3 Micromethod for haze potential I02 4.3.2.4 Haze potential t02
4.3.3 v/ine fractions separated by ammonium sulphate precipitation 105
4.3.3.1Characterization of protein 105
4.3.3.2 Evidence for glycoproteins 107
4.3.3.3 Amino acid composition 111 4.3.3.4Protein quantification Il2 4.3.3.5 Haze potential ll2
4.3.4 Purified wine proteins isolated by anion exchange chromatography 115
4.3.4.1Chromatographic separation 115
4.3.4.2 Amino acidcomposition 115
4.3.4.3 Haze potential 119
4.4 DISCUSSION 122 x1
page
5 IJAZE PROTECTIVE POLYSACCHARIDES 125
5.1 INTRODUCTION AND LMERATURE REVIEW 125
5.2 EXPERIMENTAL 127
5.2.1Materials 127
5.2.2General methods t27
5.2.2.1 Carbohydrate quantification t27
5.2.2.1.1 Total neutral sugars t27
5.2.2.I.2 Acid hydrolysis and monomer analysis r28
5.2.2.2 Micromethod for haze potential r28
5.2.2.3 Particle size measurement t29
5.2.3 Purifrcation t29 t. 5.2.3.IConcanavalin A affinity chromatography t29
5.2.3.2 Anion exchange chromatography t29
5.2.3.3 Cation exchange chromatography 130
5.3 RESULTS 131
5.3.1 Carbohydrate-rich fractions isolated by ammonium sulphate
fractionation L3L
5.3.1.1 Cha¡acterization of protein and polysaccharide by
SDS PAGE 131
5.3.1.2 Amino acid composition 131
5.3.1.3 Haze potential 131
5.3.2 Identification of haze-minimizing factor t34
5.3.2.1Effect on haze potential of wine proteins t34
5.3.2.2 Haze and supernatant protein and polysaccharide
composition 134
5.3.2.3 Haze particle size t34 xrl
page
5.3.3 Purification of haze-minimizing factor 140
5.3.3. 1 Affinity chromatography 140
5.3.3.1.1 Effect on protein haze potential 140
5.3.3.2 Anion exchange chromatography 143
5.3.3.2.1Effect on protein haze potential t43
5.3.3.3 Cation exchange chromatography 147
5.3.3.3.1 Effect on protÊin haze potential t47
5.3.4 Properties of the haze protective factor 151
5.3.4.1 Composition 151
5.3.4.2 UV Spectrum 151
5.4 DISCT.]SSION 156
6 CONCLUSIONS 160
References r62 )o11
LIST OF FIGURES
Figure page
1.1 Structure of the repeating unit of a grape juice a¡abinan 16
I.2 Possible pafüal structure of grape arabinogalactan protein carbohydrate components (arbitrary chain length) L7
I.3 Possible partial structure of wine arabinogalactan (arbitrary chain length) 18
7.4 Partial structtue of yeast stnrctural mannoprotein carbohydrate components 20
1.5 Partial structure of yeast ß-glucan (arbitrary chain length) 2L
1.6 Structure of the repeating unttof Botryrts cinerea Fglucan 23
L.7 Structure of the repeating wrjt of Pediococcus ß-glucan 23
2.1 Gel permeation separation of juice protoins 3l
2.2 Influence of grape juice and caffeic acid on the reaction of the Bradford dye-binding reagent with BSA. 34
2.3 Spectra of grape seed extract, tannic acid and BSA after rraction with the Bradfordreagent. 35
2.4 Effect of tannic acid on the reaction of BSA with the Bradford reagent. 36
2.5 Visible profiles of the retentates from ultrafiltered juices afær reaction of the retentates with the Bradford reagent. 38
2.6 Visible profiles of ultrafiltered juices after reaction with the Bradford reagent. 39
2.7 The effect of time on the colour yield using the Bradford roagent. 42 xlv
Figure page
2.8 The effect of time on the colour yield using the modiFred Bradford rcagent
3.1 Peptidase activity of the commercial preparations
3.2 The effect of æmperature on pepúdase activity
3.3 The effect of incubation time at 80oC on peptidase activity
3.4 Effect of glucose on peptidase activity
3.5 Effect of fructose on peptidase activity
3.6 The effect of ethanol concentration on peptidase activity
3.7 Endogenous peptidase activity of grape juice
3.8 Influence of æmperature and enryme concentration on the activity of peptidases in juice and wine
3.9 Effect of Bromelain Eeamont on isolated grape protein
3.10 Effect of VinozymP treaunent on wine protein
3.11 Change in wine protein content withVínozymP treatnent
3.I2 Effect of incubation of wine protein at 15oC
4.I Electrophoretic separation of the proteins in Gordo wine and haze
4.2 Infrared spectrum of Gordo wine haze
4.3 Solubility of wine haze
4.4 Preparative HPLC separation of proteins in Gordo wine XV
Figure page
4.5 Elecrophoretic separation of proteins in Gordo wine and Gordo wine fractions 99
4.6 Infrared spectrum of Gordo wine ultrafiltration rctentate 100
4.7 Infra¡ed spectrum of lst Fraction 101
4.8 Infrared spectrum of 2nd Fraction 103
4.9 Influence of the concentration of BSA and ovalbumin on heat-induced haz,ein wine. r04
4.10 Influence of protein concentration on the heat induced haze of wine protein fractions in the wine 104
4.ll SDS PAGE of (NIIa)2SO4 fractions of wine. 106
4.12 Principle of gþoconjugate detection 108
4.13 SDS PAGE of DIG - conjugated (NlI¿)zSO+ fractions of wine 109
'Western 4.I4 blot of DIG - conjugated proteins transferred to nitrocellulose 110
4.I5 The 7o molar composition of amino acids detected in (NH4)2SO4 fractions of wine 113
4.16 Influence of concenüation of G\üI4)2SO4 wine protein fractions on heat-induced haze in wine. LT4
4.17 Separation of wine proteins by anion exchange chromatography 116
4. 18 Elecuophoretic analysis of FPlC-purified proteins It7
4.I9 Tlrc Vo molar composition of amino acids detected in (NIIa)2SO+ precipitated protein, FPLC starting material, and FpLC- purified proteins 118 xvl
Figure page
4.2O Influence of concentration of FPlC-purified proteins on heat-induced haze in wine. t2l
5.1 The 7o molar composition of amino acids detected in fractions C and D isolated from wine bV (NH¿)ZSO4 precipitation t32
5.2 Influence of the protein concentration of wine fractions on the heat induced haze in wine r33
5.3 The effect on wine protein haze fomration of increasing concentration of carbohydrate-rich fraction D 135
5.4 SDS PAGE of wine hazes and supernatants 136
5.5 SDS PAGE of haze and supernatants r37
5.6 The effect of the concentration of carbohydrate-rich fraction D on haze particle size 138
5.7 The relationship between haze particle size and visible haziness r39
5.8 SDS PAGE of fractions frompreparative Concanavalin A chromatography t4l
5.9 The effect on the haze poæntial of proteins in wine of the Concanavalin A retained fraction L42
5.10 Separation of Con A retained fraction by anion exchange chromatography r44
5.11 SDS PAGE of anion exchange chromatography fractions t45
5.12 The effect on the haze potentiat of BSA of wine fractions isolated by anion exchange chromatography 146 xvü
Figure page
5.13 Separation of the major UV-absorbing fraction from anion exchange chromatography by cation exchange chromatography 148
5.I4 SDS PAGE of cation exchange chromatography fractions r49
5.15 The effect of cation exchange chromatography fractions on the haze potential of proæins in wine 150
5,16 Purification of the haze protective factor t52
5.I7 (fV spectra of the haze protective factor r54
5. 1 8 Second order derivative spectrum of the haze protective factor t54 xvru
LIST OF TABLES
Table page
2.1 Comparison of the Bradford assay with UV absorption for the estimation of grape juice protein 32
2.2 Effect of ulnafiltration on protein quantification by the Bradford assay 40
2.3 Protein quantification by amino acid composition and Bradford assay 45
2.4 C-oncentration of proteins in (NrI¿)zSo4 fractions as determined by the Bradford dye-binding assay and from amino acid composition 46
3.1 Characteristics of the enzymes used in the proteolytic studies 53
3.2 Effect of peptidase treatment for 7 days on Gordo juice proteins 68
3.3 Concentration of bentonite needed to heat stabilize wine afær periods of peptidase ûeatment 73
3.4 Effect of inhibitors on peptidase activity in buffer 75
3.5 Effect of inhibitors on peptidase activity in wine 76
4.L Protein concentration and yield from anion exchange chromatography t20
5.1 Composition of the haze limiting factor 153 lxx
Abbreviations
7o percent AA change in absorbance
A abso¡bance A*a* maximum absorbance ala alanine arg arginine asn asparagine asx aspartic acid or asparagine (undefined) AUFS absorbance units full scale BSA bovine semm albumin oc degree Celsius cm centi (10-2¡ meEe cm-1 wavenumber c\{c carbox¡rmethylcellulo se pCMPS p-Chloromercuriphenolsulfonic acid Con A Concanavalin A d dalton DIG digoxigenin et al. and others e.8. for example g gram g acceleration due to gravity gal galactose glx glutamic acid or glutamine (undefined) glv glycine Gordo Muscat Gordo Blanco (synonym Muscat of Alexandria) his histidine HPA Hide Powder Azu¡e HPLC high performance liquid chromatography ile isoleucine i.e. that is KDa kilo (103¡ dalton KPa kilo (103) Pascal L lite leu leucine XX lys lysine M molar 2-ME 2-mercaptoethanol met methionine mg milli (10-3) gram tlg micro (1ùo) gram min minute mL mitli (1ù3) ttre FL micro (10-6) titre mM milli (tO-l¡ molar pM micro (10ø¡ molar Ilìm milli (10-3) meüe pm micro (10ø¡ meue M relative molecular mass mV milli (10-3) vols Nz nitrogen gas nla not available nd not determined nm nano (10-9¡ metre oP 1,1O-phenanthroline phe phenylalanine pI isoelectric point PMSF phenylmethylsulfonylfl uoride PPO polyphenol oxidase r correlation coefficient rpm revolutions per minuæ s second Sauv. Blanc Sauvignon Blanc SDS sodium dodecyl sulphate SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis ser serine TCA trichloroacetic acid TEMED N,N,N'/V'-tetramethylethylene-diamine thr threonine TLCK N-a-p-Tosyl-L-lysine chloromethyl ketone TPCK N-Tosyl-L-phenylalanine chloromethyl kerone
Tris Tris (hydroxymettryl) - aminomethane trp tryptophan XXi tyr tyrosine I.IV Ultraviolet V volt v/v volume by volume Wks weeks wlv weight by volume A'TE ¡NSTITUÌÉ TDRARY 1
Chapter 1
INTRODUCTION AND GENERAL LITERATURE REVIEW
I.1 CLARITY OF WINES
The quality of wine is deærmined in most cases by subjective assessment. Several criteria are used: taste, clarity, aroma and the total appearance of the product, including the label. If the wine is cleat, the consumer may forgive a slight defect in taste, but, if even a slight turbidity is found in an otherwise good quality wine, the consumer is dissatisfied and will ask for a replacement bottle without even tasting the wine. V/ine clarity is therefore very important because it is this property that makes the first impression on the consumer.
There are two main factors which contribute to wine turbidity: microbial contamination and chemical instabitity. Microbial turbidity is generally caused by yeasts and lactic acid bacteria. Chemical instability can be due to metal ions, tarrate anions or gmpe proteins.
The metal ion turbidities are commonly due to excesses of Fe++ (iron haze or "casse") accompanied by oxidation of the wines. Such instability often occurs following aeration during the bottting process of a wine containing high quantities of iron. The problem of "white casss" is due to ferric phosphate and "blue casse" in red wines is due to formation of insoluble complexes of ferric ions and polyphenols. Over-enthusiastic copper sulphate addition to remove hydrogen sulphide from wines can result in residual copper in wines which forms hazes under reducing conditions. Excluding copper, excesses of metals in wines are almost always due to contamination during the wine making process. For this reason, modern wine making equipment is produced from stainless steel, plastic and glass where possible (Ribéreau-Gayon et aL l9l7, Amerine et al. 1980. Peynaud lgS4,Rankine 1989).
Tartrate instability is due to both potassium hydrogen tartrate and calcium tarEate. Both of these compounds are of gape origin and are supersaturated in wines. Over time, these components precipitate and form crystalline deposits. If this occurs in the bottle, the consumer regards them suspiciously, as perhaps broken glass. For this reason it is important to stabilize wines against tarrate instability. Considerable research effort has been devoted to the problem of potassium hydrogen tartrate instability. The slow precipitation of potassium 2
hydrogen tartrate can be hastened by chilling the wine and seeding with potassium hydrogen tartrate crystals. Crystallisation is inhibited by grape colloids and polyphenols, which appe¿ìr to act by binding to potential crystal nucleation sites ('Wucherpfennig & Dietrich 1989, Gaillard et al. 1990, Piracci & Garafolo 1990). Artificial colloids such as carboxymethylcellulose (CMC) have also been used to prevent crystallization and addition of this material imparts potassium tartrate stability to the wine for a certain time period (Wucherpfennig & Dietrich 1989). Stabilization of calcium tartrate is not so easily achieved a¡rd research on this problem is still in its infancy. Calcium tÍìrtrate is much less soluble in wine but also crystallizes slower than potassium tartrato. It crystallisation can also be prevented by CMC (Stocke & Goertges 1989).
Protein instability is the topic of this study. Grape proteins can precipitate to give an amorphous sediment or flocculate and produce a suspended and unattractive haze. Ngaba- Mbiakop (1981) suggested a general mechanism forhazæ,formation in wine in which protein acts as a support to which other constituents become attached, causing co-precipitation. However, we lack adequate basic information about grape juice and wine proteins to test this hypothesis.
1.2 PROTEINS OF GRAPES, JUICES AND WINES
The heterogeneous nature of grape and wine proteins was first reported by Koch & Sajak (1959). Soluble grape and wine proteins were separated into two major fractions by paper electrophoresis. Since then, numerous separation techniques based on size and charge have been used to fractionate grape and wine proteins. Up to 16 fractions were identified by electrophoresis (Bayly & Berg 1967, Radola & Richter 1972), while with isoelectric focussing, 20 fractions have been detected (Radola & Richter 1972, Anelli 1977, Ough & Anelli 1979). In a two dimensional separation based on lithium dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focussing, more than 25 fractions wero identified (Hsu & Heatherbell 1987a). Proteins in grapes, juice and wine have a reported relative molecular mass (lvfr) range of about 11,000 to 65,000, with the majority having low lM¡,i.e.20,000 to 30,000 (Görg et aI. 198I. Hsu & Heatherbell 1987a, Correa et al. 1988, Nakanishi et a|.1989, Polo et aI.1989) and low isoelectric point (pI), i.e. 3.5 to 6.0 (tlsu & Heatherbell 1987a, Correa et aI. 1988, Moio & Addeo 1989, Polo er al. 1989). Some of these proteins have been classified as enzymes and or as grycoproteins. 3
1.2.1 Exzyrvrn pRorErN
1.2.I.1 Peptidases
The most signif,rcant class of enzymes relevant to this study are the peptidases. These are tho class of enzymes which act on the peptide bond, and the correct terrn recornmended by the ruPAC-ruB is peptide hydrolyase. It has been suggested for historical reasons thar prorease and peptidase be used synonymously instead of peptide hydrolyase (Barrett & McDonald 1986); and the ærm peptidase is used in this study. Peptidase classification and nomenclature has been rccently reviewed (Storey & Wagner 1986) and a brief explanation appears here for clarity. There are two types of peptidases: the endopeptidases which cleave internal peptide bonds and are sometimes referred to as proteinases, and the exopeptidases which cleave terminal-end peptide bonds. The endopeptidases are conveniently classified according to four mechanistic sets; three depending on the amino acid in the enzymes active site: serine, cysteine, and aspartic acid endopeptidases, and the fourth containing a metal ion in its active site: metalloendopeptidases. Exopeptidases are most easily classified by their substrate specificity. Those specific for the cr-NH2 terminus are either aminopeptidases which rsmove amino acids sequentially, or dipeptidyl peptidases, which remove dipeptides sequentially from peptides and proteins. The enzymes acting at the ü-COOH terminus are either carboxypeptidases, which remove amino acids sequentially, or peptidyl dipeptidases, which remove dipeptides sequentially. Dipeptidases convert dipeptides to amino acids, while tripeptidases act on tripeptides to produce a dipeptide and an amino acid (Storey & Wagner 1986).
Peptidases have been detected in grapes andjuices (Feuillat et al.1980. Feuillat & Ferrari 1982). A comprehensive study of Riesling grape exopeptidases resulted in the detection and pa¡-tial purification of two forms of aminopeptidase, a carboxypeptidases and a dipeptidase (Pallavicini & Dal Belin Peruffo L977). Using synthetic substrates, it was found that the aminopeptidase preferentially released hydrophobic amino acid residues. Optimum activity was in the pH range of 7.2 to 7.4 and was stimulated by Mg++ but grossly inhibited by Hg** and to a lesser extent by a number of other bivalent cations. Carboxypeptidase activity was optimum at pH 4.8 and was promoted by aromatic amino acid residues in the carboxy position of the peptide. Bivalent cations had little effect on this enzyme. The grape dipeptidase activity however, was completely inhibited by Hg++ and Cu+ and enhanced by Mn++, Ca++ and Co++. Maximal activity was at pH 8.2 and was promoted by aromatic amino acid residues and glycine on the carboxyl side of the peptide bond. A later study of the 4
aminopeptidase (Pallavicini ¿r al. L980) showed that there were two isoforms of this enzyme with pI 4.4 and 5.2. The maximal activity was at 45oC, but the thermal stability was low; only a trace of activiry was left after 30 min at 55oC.
A grape endopeptidase has been partially characterised (Cordonnier & Dugal 1968). Of the total proteolytic activity, SOTo was in the pulp therefore during the wine making process, most of the endopeptidase would be eliminated in the cellular debris of the lees. The enzyme had good thermal stability (50Vo actlity retained after 15 min at 70oC) and was activated by SO2, a preservative used in wine making. The enzyme showed maximum activity atplF.2 and 55oC on denatured haemoglobin. At present, it is not clear if any of these natural peptidases have activity on grape proteins in wine, or whether this would effect heat hazing potential.
L.2.I.2 Polyphenoloxidases
Grapes also contain polyphenoloxidase (PPO), the enzyme associated with undesirable browning reactions in the grape and other fruits and vegetables (Radola & Richter 1972, Wissermann & Lee 1980, Mayer 1987, Nakanishi ¿r al.1987). Browning occurs rapidly in the damaged berry, or after crushing fresh grapes for juice and wine production, and changes the colour and aroma of the juice, diminishing the quality of the final product (Traverso-Rueda & Singieton 1973). Hence the enzyme has been the subject of many investigations.
Levels of PPO differ between varieties (Traverso-Rueda & Singleton 1973, Wissermann & Lee 1980, Sapis et al.1983a), but general trends can be seen in PPO activiry during be.ry maturation. Sapis et al. (I983a) tabulated PPO levels in the developing berries of Grenache, Clairette, Carignane and Ugni blanc. Activity was found to decrease rapidly during the green stage then increase until the end of véraison. Following véraison, during the ripening stage, activity regularly declined until harvest. Other studies of a number of different varieties cultivated in the USA and Spain gave similar results (Traverso-Rueda & Singleton 1973, Wissermann &l-ee 1980, Valero et a\.1989).
In the intact berry, PPO is located in the chloroplasts (Lerner et at. 1972, Mayer 1987), possibly as an integral membrane protein (Sánchez-Ferrer et al. L989). When the berry is crushed or bruised, the enzyme is released into the juice and its active site undergoes a conformational change, induced by the acid shock and the presence of 02 (Lerner et aI. 5
l972,I3dron et a|.1978). This activates the enzyme and greatly accelerates its reaction rate (Lerner et al. L972).
PPO in the Japanese grcpe variety, Koshu, had a I\rlr of approximately 40,000 with optimal activity at pH 6.0 and 25oC (Nakamura et al. L983). In contrast, PPO from Muscadine grapes had a Mr of approximately 20,000 (Laminkanra 1988) while that from the Spanish grape variety Monastrell, had temperature and pH optimum within the ranges 20 to 40oC and 3.5 to 5.0 respectively (Sánchez-Ferrer et al. L988).
Stability of the Koshu enzyme ìüas poor; there was a loss of activity at temperatures gfeater than 30oC and pH below 6.0 (Nakamura et a|.1983). PPO from Semillon, Clairette and Carignane grapos was completely inhibited by SO2, and largely inhibited, although reversibly, by ethanol (Kidron et aI.1978). PPO from the Spanish grape variety, Airen was found to be inhibited by several aliphatic alcohols which are formed during grape fermentation (Valero et al. 1990). PPO activity can also be inhibited by honey. The responsible component appeared to be a small peptide of approximate ìvI¡ 600 (Oszmianski & Lee 1990).
In the pressed juice, the bulk of PPO activity is in the particulate mattor (Harel & Mayer 1971, Sapis et al. 1983b, Yokotsuka et ø/. 1988). Activity is therefore maximal during crushing, and then declines as vinification progresses (Wisserrnann & Lee 1980, Yalero et al.1989) provided that the particulate matter is removed as soon as possible (Yokotsuka er al. t988).
L2.1.3 Invertases
Invertase has been purified from Ohanez, Delaware and Semillon grapes, and because this enzyme has highest activity at acid pH, it may be responsible for the inversion of sucrose added to juice (Arnold 1965, Arnold 1966,Ishikawa et al. 1989, Nakanishi & Yokotsuka 1990a). Inhibition studies demonstrated that the active site in this enzyme contained a thiol group (Ishikawa et al. 1989). Levels of the enzyme varied significantly between the varieties, but all had pH optima in the range 3 to 4 and were stable in the range p}J2 to7. The optimum temperature for each was at 75oC but the enzymes were stable up to 7@C. The enzymes were also stable at ethanol levels of tíVo and SO2 levels up to 400mElL. The stability of the enzyme was such that enough persisted in the wine for active enzyme to be purified from this source. On average,30Vo of the activity shown in juice was present in wine (Nakanishi ¿r aI.l99I). 6
The enzyme from Semillon grapes was purified and had Mr of 65,000 and pI of 4.2.That from Semillon juice and wine was also found to have M, of 65,000 and pI of 3.9 (Nakanishi et al. t99I). The similarity in Mt and pI, in addition to the known I\4 of yeast invertase (M¡ 270,000, Ballou L982) indicates that the purified enzyme was of grape origin and not from yeast The amino acid compositions of the Semillon enzyme and that from Chardonnay, Sylvaner and Riesling wines (Nakanishi & Yokotsuka 1990b) were dominated by aspartic acid and/or asparagine, glycine and leucine, with a strong contribution from glutamic acid and/or glutamine, threonine and serine. The enzyme was a glycoprotein, containing approximatety 337o sugar. This explains the retention of Delaware invertase on a lectin affinity column (lectins are proteins which bind carbohydrates and components containing carbohydrate residues) (Ishikawa et aI. L989).
1.2.1.4 Glycosidases
Glycosidases ¿ue particularly relevant to wine making since they have the potential to release volatile aroma compounds from involatile flavour precursors and thus increase the flavour and aroma of wine (rWilliams et al. 1987). ß-glucosidase, q-arabinosidase and cr- rhamnosidase, have been detected in grapes from 10 varieties (Gunata et aI.1989), and the former two enzymes, in addition to ß-galactosidase and cr-glucosidase, were shown to be present in both the fruit and leaves of Muscat Gordo Blanco (Gordo, syn. Muscat of Alexandria)(Aryan et al. 1987).
The ß-glucosidase from Gordo grapes was partially purified and had pH optimum in the range 5.0 to 5.5. The enzyme was sensitive to sugar and, to a lesser extent, ethanol, which limited its application in wine making. These results were confirmed by Lecas et al. (I99L), who purified a ß-glucosidase from Gordo $apes. The enzyme activity was present in two forms with M¡ of 98,000 and 50,000. As shown previously, the pH optimum was pH 5.0, but the enzyme had weak stability at grape juice and wine pH. The optimum temperature was 45oC and the activity was sensitive to ethanol (loss of 60Vo activity at 107o ethanol). Inhibitor studies suggested a thiol group in the active site and because the activity rapidly decreased during clarification, it was considered that the activity of this enzyme was negligible during fermentation (Großman et al.l99O).
Grapes also contain pectinases, methylesterases and polygalacturonases (for a review, see Amerine et al. t980). However, the bulk of such enzyme activity in juice comes from 7
exogenous enzyme preparations routinely added during crushing to aid in juice extraction and clarification (Amerine et al. 1980).
I.2.2 F,vIDENCE FoR GLYcoPRoTEINS
Glycoproteins have been isolated from many plants and algae and are found in the vacuole, extracellular matrix, the cell wall, and are associated with membrane systems (Golgi, endoplasmic reticulum, tonoplast and plasma membrane). A well characterised example is extensin, a cell wall structural protein found throughout the plant kingdom. It is rich in the amino acid hydroxy¡lroline, in addition to serine, threonine, valine, lysine and tyrosine residues, with the carbohydrate component dominated by arabinose and galactose (Cooper er al.1987).
There is some evidence that grape proteins follow the general tend amongst plant proteins and are complexed with polysaccharides. Most Rieslaner wine protein fractions, sepamted on polyacrylamide gels, stained positively with the periodic acid-Schiff reagent indicating a carbohydrate component (Görg et al.l98l). The same technique also demonstrated the presence of sugar in most protein fractions in other studies (Radola & Richter 1972, Yokotsuka et al. l99l). However, it was suggested that much of the material responding positively to the stain was pectic in nature and had not been completely removed by the protein isolation technique. Incomplete removal of non covalentþ bound carbohydrate may also explain why carbohydrate co-purified with protein on chromatofocussing (Paetzotd et al. 1990). Such an artefact would suggest incorrectly that all juice proteins were glycoproteins. Hsu & Heatherbell (1987a) with a sensitive stain utilizing the lectin, Concanavalin A, detected only three glycoprotein fractions of Mr 13000, 25000 and 28000 in Gewürztraminer wine. Only one grape eîzyme, invertase, has been reported as a glycoprotein (Arnold L965,Ishikawa et al.l9S9,Nakanishi & Yokotsuka 1990a).
More circumstantial evidence is provided by the reported amino acid composition of grape and wine proteins. Analysis of total proteins from 20 different grape varieties showed that all have high levels of serine and threonine (Anelli 1977, Yokotsuka et ø1. 1977, Ough & Anelli t979, Villettaz et al. 1982). These residues can be involved in O-glycosidic linkages in glycoproteins (Beeley 1985). Such high levels of these hydroxyl bearing amino acids in grape proteins suggests that linkages between protein and carbohydrate may be present. 8
1.2.3 FACToRS AFFECTING PROTEIN CONCENTRATION
Accurately measuring protein concentration in grape juice and wine has proved to be very difficult. Reports in the literature vary by several orders of magnitu de, i.e. as low as 1.5 mg/L (Yokotsuka et aI. 1977), to as high as 840 mg/L (Somers & Ziemelis 1973a). As discussed in Chapter 2, some of this variability can be attributed to the use of protein determination assays which were inappropriate for grape juice and wine analysis.
Despite the limitations of protein determination methods, variations in protein content of grape juice and wine have been documented. Some of the factors which account for such va¡iation are discussed below.
The protein content of grape juice depends on the state of marurity of the berry at hawest; the protein level increasing as the berry matures. This was extensively studied by Luis (1983) and Murphey et aI. (1989b). Viticultural practices are important, for example, overcropping of the vine produces berries with low protein contenr (Ough & Anelli 1979). Fungal infection is also a factor, grapes infected with Botrytis cinerea having a lower protein content than healthy grapes. It is proposed that the latter results from proreolysis due to the combined action of extracellular endopeptidases of B. cinerea, arrd gape endopeptidases liberated as a result of cellular degradation caused by B. cinere¿ infection (Cordonnier & Dugal 1963). Protein content also appears to be geneticatty programmed (Koch & Sajak 1959) because the juice from some cultivars generally contain morc protein than others, for example Traminer and Gordo (I-uis 1983).
The fermentation process decreases the protein content of the juice. Ferenczy (1966) found that this decrease was in the order of 15 to757o, with the greatest loss in fermentations conducted at high temperatures. Protein loss during fermentation has been attributed to the change in solubility of many wine components, thus precipitating prorein (Luis 1983). Recent research has shown that the decrease in protein concentration from juice to wine is a function of wine pH. Protein retention was greatest at pH 3.2 and. markedly decreased at higher or lower fermentation pH values (Murphey et al. 1989b).
Protein content is decreased further by frning agents such as bentonite, or by precipitation during aging and storage. The loss of protein during bottle aging has not been well investigated, but it is thorrght to be due to a combination of pH change due to acid decomposition, exposure to temperature extremes and compositional changes d,ue to wine blending (Ferenczy 1966). 9
I.2.4 IJøIT INSTABILITY
A number of tests a¡e used to examine for the presence of "unstable" or haze-forming proteins in grape juice or wine, and they have been reviewed by Ngaba-Mbiakop (1981). Unstable protein is presumed to be present if it can be precipitated. Precipitation can be induced by heating or by chemical means.
In Australia, a heat test is commonly used and this involves holding the juice or wine at 80oC for 6 hours and then at 4oC for 24 hours. The occu:rence of ahaze or sediment is taken to imply that the juice or wine is unstable and is likely to form ahaze during long term srorage at lower temperatures. Ngaba-Mbiakop (1981) found that this rest correlated well with the longer test suggested for Californian wines (2 days at 50oC) (Berg & Akiyoshi 1961). More recent work has shown that the 80oC test correlated well with long term storage in the cellar (2 years) (Flores et aI. 1990). It appears that wine protein instability is a consequence of protein denaturation. The wine industry's specific problem of protein aggregation is therefore a general phenomenon in many other foods and beverages.
1.3 PROTEIN AGGREGATION
Protein aggegation reactions a¡e involved in the manufacture of a range of foods and beverages. The coagulation and gelation of proteins often controls the success of cooked foods and these reactions a¡e also necessary to the structure of many foods (Schmidt 1981, Gossett et al. L984).
Denaturation has been defined as the process in which protein goes from an ordered to disordered state without the rupture of peptide bonds. Foods such as fish and milk may be denatured and their proteins destabilized during freezing and frozen storage. Denaturation usually involves loss of biological activity and significant changes in physical or functional properties such as solubility (deMan 1990). The denaturation of proteins in food is not always undesirabla, €.9. the blanching of vegetables before freezing denatures enzymes involved in undesirable browning reactions.
The process of heat denaturation of globular proteins is as follows: dissociation into subunits, the rupture of hydrophobic bonds followed by irreversible denaturation and aggegation through the exposed hydrophobic regions. The third phase is the formation of 10
bigger aggregates. Atthough aggegated because of heat denaturation, not all proteins show turbidity, e.g.hen egg ovalbumin, under appropriate conditions, forms transparent gels as does gelatin (Gossett et al.1984).
Aggregation is a general term for protein-protein interactions to give higher M, complexes. Its occurrence is governed by a balance between repulsive and attractive forces. Gelation is the orderly aggregation of proteins which arc not necessarily denatured., to give a three dimensional network, which is capable of immobilizing or trapping very large amounts of water. More random aggegation may be def,rned as protein coagulation. Coagulation often produces turbidity and is usually thermally irreversible. The coagulated protein may settle out of solution (Gossett et al.1984). Heaçinduced gelation is generally a two step process, 1) unfolding or dissociation, and 2) aggregation leading to gel formation. Step 2) musr be slower than 1) to achieve a highly ordered gel matrix (Schmidt 1981).
The mechanism of gel fonnation can be conceived as a break of existing disulphide bridges in proteins during heating and reformation of disulphide bonds between proteins. Other non covalent bonds involved are H bonds, ionic bonds between charged amino acid sidechains and hydrophobic interactions.
1.3.1 BEER cHILL l¿Azr,
Beer chill haze is considered to be due to an aggregated protein-polyphenolic complex (Savage & Thompson t972, Righetti & Bosisio 1981, Asano et aI. lgSz).Its composition was shown to be approximaæly 507o protein ,407o carbohydrate and l}Vo phenolic (Dadic & Belleau 1980). The haze-forming proteins in beer originate from the barley storage protein hordein, and contain carbohydrate (Asano et al.l9B2). In model solutions, haze-forming proteins react with the phenolic compound, catechin, and produce haze. Haze formation in this system was inhibited by hydrogen bond acceptors and non-polar solvents but not ionic bond acceptors, implying that H bonding and/or hydrophobic interactions were responsible for haze-formation (Asano et al.lgSZ).
Model studies have shown that, among the phenolics found in beer, proanthocyanidins had a specific affinity for beer haze-forming proteins (Asano et aI. 1984). Improved beer stability was obtained with beers brewed from proanthocyanidin-free barley, demonstrating the role of tlrese phenolics in beer har.e.Haze could be induced in such beers by adding back various phenolics (Delcour et al. 1984). Proteins purified from these beers also complexed with 11
proanthocyanidins in model solutions, and proanthocyanidin affinity was greatest with those proteins containing high proline levels (Outtrup 1989). Proline content of haze forming proteins purified from normal beers also correlated well with the amount of catechin bound (Asano et aI. 1982). Beers brewed from proanthocyanidin-free malt contained more proteins with higher proline than from normal beer. The reason suggested for this is that high proline containing proteins would have been precipitated by proanthocyanidins in the normal beer during the brewing process, hence were not present (Outtrup 1989).
High proline content itself is not the only factor and hydrophobic amino acids are also important Although these amino acids do not interact with proanthocyanidins, they function in the hydrophobic inæractions necessary for protein aggregation (Outtrup 1939). It has also been suggested that free radicals generated during beer storage attack proanthocyanidins to produce oxidation products in the beer. These oxidants had high affinity for beer proteins and were involved in haze formation (Kaneda et aI.1990).
Protein haze formation in beer can be prevented by peptidase treatment. Peptidases such as papain, and also bromelain, ficin and bacterial peptidases, have been used in the brewing industry for 80 years (Hebert et aI. 1978). Although it has been shown that papain hydrolyzes the proteins involved in beer haze formation, the enzyme may also have a non- enzymatic role. Inactivated papain can also prevent haze formation. It was suggested that inactivated enzyme upset the equilib¡ium between beer proteins and proanthocyanidins, and/or acted as a protective colloid, holding haze particles in solution (Fukal et al. 1987).
1.4 FINING FOR WINE CLARIFICATION
Wines tend to clarify themselves naturally with aging and settling. Howqver, such clarif,rcation is usually not sufficient for the wines to be marketable, hence modern wine making accelerates and extends this process with frning agents. These are substa¡ces that aid in the precipitation of material that would otherwise remain in the wine and cause haziness and turbidity. Such treatment ensures physical stability of the bottled product under unfavourable storage conditions. L2
1.4.1 BpNToNrrE FTNTNG FoR pRorErN REMovaL
Bentonite is the most commonly used fining agent in Australia for protein removal (Miller er al. 1985), being first introduced to wine making n 1934 by Saywell in California (Saywell 1934).It is a montmorillonite clay i.e. an hydrated aluminium silicate with some aluminium ions replaced by sodium, calcium and magnesium ions. Bentonite exists as layers of atoms approximately 1nm thick by approximately 500nm wide. These plates are separated in solution and present a large negatively charged surface area; approximately 750 square metres per gram for the sodium bentonite form.
A suspension in water is prepared and then dosed into the juice or wine at a level indicated by laboratory scale trials. For best results, the suspension should be smooth, with all bentonite fully hydrated, and must be rapidty mixed into the juice or wine (Rankine 1963). This presents certain logistic difficulties, for example, a fining rate of 0.5g/L involves the preparation and addition of 5L of a LÙVo suspension to 1000L of juice and wine.
In addition to these technical difficulties, bentonite treatment can result in the costly loss of up to l0Vo of the wine volume as bentonite lees (Rankine 1984). The treatment can also strip wine of flavour (Mitler et al. 1985). Because there is no economically viable alternative, bentonite is widely used and its ability to remove proteins by adsorption is well documented (Bayly & Berg 1967, Danilatos & Sotiropoulos 1968, Somers & Ziemelis 1973b, Görg et al.l98I, Ngaba-Mbiakop 1981, Tyson et aI. L981, Heatherbell et a\.1984, Dubourdieu ¿t al.1986, Hsu & Heatherbell 1987b).
Absorption of protein by bentonite is primarily a cation exchange process. Proteins with pI above the pH of wine have an overall positive charge and can by adsorbed onto bentonite by exchanging with the sodium, calcium and magnesium ions. Hydrogen bonding and anion exchange at the ends of the bentonite plates can also be involved (Tettenhoßt et al. 1962). The extent to which adsorption will occur depends on rhe capacity of the bentonite and. the composition of the juice and wine. The absorption of the standard protein, bovine serum albumin (BSA) by bentonite in model wine solutions was srudied (Blade & Boulton 1988). It was found that the protein was rapidly bound to the bentonite within 30 seconds of mixing. The adsorption was independent of temperature but varied with protein content, pH and ethanol content. Studies with wine proteins have indicated that proteins with higher pI (4.1 to 8.0) and Mt in the range 32,000 to 45,000 were mosr easily removed (i.e. required Iow additions of bentonite for their removal). Proteins with low pI (4.1 to 5.8) and M. in the L3
range 10,000 to 30,000 wero removed next and their removal corresponded to stabilization of the wine to heat. Proteins with higher Mr (60,000 to 65,000) were difficult to remove with bentonite treatment QIsu & Heatherbell 1987b). It is not clear what effect inæractions of proteins with other wine macromolecules, phenolics and polysaccharides, would have on protein adsorption but it is not unreasonable to expect them to decrease bentonite efficiency (Blade & Boulton 1988).
A variation on the bentonite procedure is the use of a column of synthetic hydrated alumino- silicate manufactured by W. R. Grace & Co. (Welsh & Parent 1985). The column process, rather than a batch process, has the advantage of creating no wine lees, but presents problems of regeneration.
I.4.2 OI¡¡ER METHoDS oF PRoTEIN REMovAL
Ultrafilration can substantially reduce the protein and polysaccharide content of the treated wine (Gnekow et a|.1983, Heatherbell et al. 1984, Hsu ¿f al. t987, Flores et al. 7988,Peri et aL 1988, Flores et al.1990). This technique removes compounds with M¡ greater than a nominal value by physical exclusion. It is possible to protein stabilize juices and wines by passage through membranes with M¡ cutoffs of 10,000 thus eliminating the need for bentonite fining. However with some juices and wines, small amounts of protein passing through the membranes, especially after increasing process time, can cause instability problems (Hsu & Heatherbell I987a, Flores et al. 1988, Flores et al. 1990). Further disadvantages of ultrafiltration of juices and wines are the loss by absorption to the membrane of flavour and aroma compounds (Miller et al.1985, Voilley et aL t990) and pigments (Peri et a|.1988, Serrano et ø1.l998,Flores et al.1990). In addition, a decrease in "body" or "mouth feel" results from the removal of colloids (Heatherbell & Flores 1988, Konja et al.1988).
Treatment of juice and wine with proteolytic enzymes has been suggested but is not a common wine making practice. This subject is considered in detail in Chapter 3.
A novel method of protein removal using polyphenols has been reported. On the laboratory scale, white wines were passed through a column of immobilized tannic acid derivative (Weetall et al. 1984), or immobilized grape proanthocyanidins (Powers et al. t988). Although these columns successfully stabilized wines passed through them, the columns t4
showed poor protein-binding ability after a relatively small number of cycles, and were difficult to rogenerate.
1.5 POLYSACCHARIDES OF GRAPES, JUICES AND WINES
The carbohydrate polymers of grapes, juice and wine can be divided into two groups: the pectins or acidic polysaccharides, and the neutral polysaccharides. Their total content in grape juices canrange from 133 to 593 mglL (Brillouet 1987).
1.5.1 PncrrNS
The pectins in grapes are complex polymers of galacturonic acid with sidechains of neutral sugars, usually arabinose, rhamnose and galactose (Dubourdieu & Ribéreau-Gayon 1980, Brillouet 1987). Their sfucture comprises "smooth" homogalacturonic areas and "hairy" rhamnogalacturonic areas highly substituted by neutral sugar side-chains. The smooth regions were sensitive to enzymatic attack whereas the haþ regions were resistant (Saulnier et al.1988b).
The structure of the neutral sugar side chains of water soluble pectins from grape berries comprised a central core of a (l->3)Jinked cr-galactose carrying side chains of (1->6)linked ø-galactose which in turn were heavily substituted at the 3 position by single arabinose residues. The water insoluble pectins have neutral side chains which were described as arabinan-like (Saulnier et al. I988a).
Both types of pectins have associated protein. The protein associated with the water soluble pectin contained a high proportion of hydrox¡'proline, serino, alanine and glycine amino acid residues. The suggested linkage of protein to the pectins was to the neutral sugar side chains (Saulnier et al. I988a).
The level of soluble pectins in grapes decreases during berry maturation due to the action of grape pectinases (Robertson et al.198O, Mourgues 1931). Levels of soluble pectins in grape juices ranges from 50 to 340mgll- (Brillouet 1937). High pectin levels in grapes are thought to lower juice yield during crushing and hamper subsequent cla¡ification and filtration, thus pectolytic enzymes are routinely added during crushing (Robertson et al. 1980). This exogonous enzyme treatment combined with the action of grape pectinases and the 15
precipitation of pectins by ethanol means that finished commercial wines have low levels of pectins.
1.5.2 NEUTRAL PoLYSACcHARIDES
The neutal polysaccharides in wines originating from the grape are composed predominantly of galactose and arabinose, with minor amounts of rhamnose, mannose and xylose (Brillouet 1987). An a¡abinan isolated from grape juice was shown to have a (l-5)-linked backbone of cr-L-arabinofuranosyl residues with sidechains of 3-linked cr-L-arabinose (Villettaz & Amado 1981, Villettîz et ø/. 1981) (Figure 1.1).
Arabinogalactans have been reported by a number of workers (Usseglio-Tomasset 1978, Dubourdieu & Ribéreau-Gayon 1980, Mourgues 1981, Villenaz & Amado 1981, Mourgues et al.1984). An arabinogalactan protein isolated from grape berries had Mr of 165,000. Most of the macromolecule (897o) comprised neutral sugars of which arabinose accounted for 4OVo and galactose for 60Vo. The structure of the carbohydrate portion was composed of inner chains of (1->3) linked D-galactose 6-substituted with (1->6) linked D-galactose 3- substituted by terminal cr-I.-arabinosyl units (Figure 1.2). The remainder of the macromolecule was composed of uronic acids (3Vo) and protein (\Vo). The protein appeared to be covalently linked to the carbohydrate portion by O-glycosidic links to the amino acid th¡eonine (Saulnier & Brillouet 1989).
A simila¡ macromolecule has been isolated from wine @riltouet et al. 1990} It accounted for l4Vo of the total wine carbohydrate colloids and had Mrof 178,000. The macromolecule was suggested to be composed of inner chains of (l->3)-linked ß-D-galactose 6-substituted with (1->6)-linked ß-D-galactose 3- and 4-substituted by terminal cr-L-arabinosyl units (Figure 1.3). It also contained uronic acids (5Vo) and protein (3.5Vo). 16
->5)-cr-L-Araf- ( 1 ->5)-a-L-Ara f- ( 1 -> 33 1111 u-L-Araf q-L-Araf
Figure 1.1 Structure of the repeating unit of a grape juice arabinan cr-L-Ara f = ø-L-Arabinofuranose t7
->3)-D-Galp(1 ->3)-D-Galp( 1 ->3)-D-G alp( 1 6 I 1 cr-L-Araf- ( 1 ->3 )-D-Galp 6 I 1 D-Gatp 6 I 1
a-L-Araf-( 1 ->3)-D-G alp 6 I 1
ø-L-Araf-( 1 ->S)-D-Galp 6
1' 1
cr-L-Araf-( 1 ->3)-D-Galp
Figure 1.2 Possible partial structure of grape arabinogalactan protein carbohydrate components (arbitrary chain length) D-GalP = D-Galactopyranose c¡'L- Ar af = a-L-Arabinofuranose 18
->3)- P-rcdp( 1 ->3)-P-D-Gatp( 1 ->3)-p-D-c at p(1->...... 6 f 1 ø-L-Araf- ( 1 ->s)-p-D-G alp 6 I 1 cl-L- Araf- (1 ->3)-p-D-Gdp(4<- 1 )-cr-L-Araf 6 I 1 a-L-Araf-(1 ->3)-P-D-Galp(4<- 1 )-cr-L-Araf 6 I 1 a-L-Ara f-( 1 ->3)-B-D-G alp 6 I 1 p-D-ca¡p 6 I 1 a-L-Araf-(1 ->3)-P-D-Galp(4<-1 )-a-L-Araf 6I 1 cr-L-Araf- (1 ->3)-P-D-Galp(4<-1 )-ø-L-Ara f 6 I 1 cr-L-Ara f- ( 1 ->3)-p-D-G alp 6 I 1 c¡-L-Araf
Figure 1.3 Possible partial structure of wine arabinogalactan (arbitrary chain length) ß-D-Galp = ß-D-Galactopyranose (J'-L- Ar af = cr-L-Arabinofuranose L9
1.5.3 YEAST.DERIvED PoLYSAccHARIDES
In addition to polysaccharides sourced from the grape, the yeasts also contribute to the polysaccharide content of finished wines, mainly with mannose and glucose containing colloids (Usseglio-Tomasset 1976). These macromolecules can have a positive effect on the sensory qualities of wine (Llauberes et al. 1987 , Feuillat et al. t988).
The release of yeast polysaccharides into wine depended on the yeast strain, fermentation temperatue and the duration and conditions of storage over yeast lees. Higher fermentation temperature did not significantly affect colloid release during fermentation, but caused an increase in colloid release at the end of fermentation and during storage on lees. Increasing yeast lees contact resulted in an increase in polysaccharide content and this increase was greatest when the lees were periodically resuspended (Llauberes et al.1987). The release of colloids from yeasts was shown to be maximal after 8 months on lees, having increased from approximately 150mg/L to 500mg/L, and then progressively decreased @euilLat et al. 1988, Feuillat et al. 1989). However srorage on yeast lees was not necessary for colloid release. By the end of fermentation in synthetic medium, yeasts had released about 150 mg/L of polysaccharide material (Saulnier et al. L99l).
A yeast ø-D-mannan found in wine had a highly branched structure comprised of an (1-6)- linked backbone having sidechains attached by (1->2) and (1->3) bonds (Villettaz & Amado 1981). This structure is similar to that of mannan from bakers yeast (Saccharomyces cerevisiae) @revert & Ballou 1985). Bakers yeast mannoprotein has long (1->6)-linked cr-D- mannose chains with short side chains linked by (1->2) and (1->3) bonds. These macromolecules are attached to asparagine in the protein via N-acetyl-D-glucosamine, or directly to serine and threonine via O-glycosidic links (Figure 1.4). Strains differ in length of sidechains and their phosphate content (Ballou 1976).
Colloids isolated from sparkling wines were composed predominantly of mannose (40Vo) and glucose (307o). Their origin is thought to be from the yeast cell wall, and their composition was similar to that of colloids released by yeasts in synthetic media (Feuillat er ¿/. 1988). The major yeast cell wall components are glucan, mannoprotein ("mannan") and chitin (Ballou 1982). Yeast ß-glucan is water insoluble and has a (1->3)-linked backbone with a limited amount of branching via (1->6) links (Figure 1.5). It forms long twisted ribbon-like structures with one side hydrophobic and the other hydrophilic. It has been postulated that these twist together to form a double helix in the cell wall. The mannans or 20
I û-D-Manp(1->6)-c-D-Manp(1->6)-o-DManp(1->4)-p-GNAc-(1->4)-p-GNAc-Asn 2 2 3 I I I 1 1 1 a-DManp cl-DManp a-DMarp 2 2 I I I 1 æDilIrp eDMarp 2 I 1 æDlvlap
a-D-Manp(1 ->3)-o-D-Manp(1 ->2)-cr-DManp(t ->2)-a-DManp>
æDlüarp{l ->2)+Dlvlar¡p(1 ->2)c-DManp> SsfitT æDlt4a¡p(1 ->2)+Dlvlap>
æDlrlblo>
Figure 1.4 Partial structure of yeast structural mannoprotein carbohydrate components a-D-Manp = Cr-D-Mannopyranose ß-GNAo = N-acetyl-ß-D-Glucosamine
Asn = Asparagine ) Ser = Serine ) amino acids in peptide chain Thr = Threonine ) 2T
ftDGlup 1 ,l 6
Figure 1.5 Partial structure of yeast ß-glucan (arbitrary chain length)
ß-D-G I up = ß-D-Glucopyranose 22
mannoproteins comprise hydrolytic enzymes, such as the external invertase and acid phosphatase, sexual agglutinins, and the structural mannoproteins described above (Frevert & Ballou 1985).
1.5.4 PoLYSAccHARIDES FRoM oTHER SoURcES
Grape juice and wine from grapes infected by the fungus, Botrytis cinerea contain high levels of glucans. These cause filtration problems due to their rod-shaped sftucture comprising a backbone of (l-3)-linked ß-D-glucose with single branches of ß-D-glucose attached by (1-6)-linkages at every second residue (Dubourdieu et a|.1981) (Figure 1.6). This macromolecule can function as an energy sourco for the fungus but is only utilised, and the enzyme required for its degradation synthesized, in a glucose depleted medium. In the grape this energy source is not needed, thus the enzyme is not synthesizod and the macromolecule is very stable (Dubourdieu & Canal-Llaubères 1990). B. cínereaß-glucans can be removed from wine by hydrolysis with ß-glucanase containing ß-(1->3) activity (Canal-Llaubères 1989).
Spoilage of wines by Pediococcus species of lactic acid bacteria manifests itself as the defect known as "ropiness". These wines are characteized.by their "oily" pouring properties and have been shown to contain polysaccharides secreted by these bacteria (Lonvaud-Funel & foyeux 1988). One such polysaccharide has been identifred as a ß-D-glucan comprising a trisaccharide repeating unit with a (l->3)-linked backbone and a (1->2)-linked branch of one glucose, with lt4¡ 800,000 (Llaubéres et al.1990) (Figure 1.7).
1.6 AIMS OF THIS STUDY
These \ryere to:
1. Establish a method for the quantification of grape proteins in juice and wine. 2. Explore the possibility of protein removal by peptidases. 3. Determine which grape proteins were responsibre for heat instability. 4. Investigate the role, if any, of glape and wine polysaccharides in protein haze formation.
A separate chapter of this thesis is devoted to each. 23
B-DGlup 1 t 6
>3)-P-D-G|up(1 ->3)-P-D-clup( 1 ->
Figure 1.6 Structure of the repeating unit of Botrytìs cínerea ß-glucan ß-D-Glup = þD-Glucopyranose
->3)-p-D-Glup(1 ->3)-P-D-clup( 1 -> 2 I 1 FÞGlup
Figure 1.7 Structure of the repeating unit of Pedìacoccus ß-glucan ß-D-Glup = &D-Glucopyranose 24
Chapter 2 ESTIMATION OF GRAPE JUICE AND WINE PROTEIN CONCENTRATION
2.1 INTRODUCTION AND LITERATURE REVIEW
Measuring protein concentration in grape juice and wine with accuracy has proved to be very difficult. Estimates in the literature vary by several orders of magnitude, from as low as 1.5mgll- (Yokotsuka et al.1977), to as high as 840mglL (Somers & Ziemelis I973a). Some of this variability can be attributed to the use of protein assays which were inappropriate for grape juice and wine analysis. Many of the methods rely on the precipitation of protein by ethanol, trichloroacetic acid, sodium tungstate or ammonium sulfate, followed by assaying the precipitate for nitrogen content by the Kjeldaht merhod, or protein estimation with colourimetric reagents. If low levels of protein are pressnt, as is the case with grape juice and wine, some of these reagents will not quantitatively precipitate protein (Bensadoun & Weinstein 1976), or other compounds can coprecipitate and thus interfere (Ferenczy 1966).
Ultraviolet (UV) absorption at 280nm is the traditional method for protein deærmination. It cannot be used in juices and wines unless the bulk of the non-proteinacous UV-absorbing material is removed prior to analysis. Using gel permeation chromatography to isolate the protein fraction, and calibration of its A2gg value with the total nitrogen content of the fraction by the Kjedahl method, it was determined that wines had a range of protein contents from 50 to 840m9/L (Somers &ZiemeLis 1973a). However, it was demonstrated that the standard protein, bovine serum albumin (BSA) gave approximately half the peak height of wine proteins. This suggests that wine proteins were complexed with a small amount of phenolic material which was not separaæd from the protein (Mattoo et al.1987) and absorbed strongly in the UV. Gel permeation chromatography for wine protein separation has also been undertaken using HPLC columns @ubourdieu et al.1986, Luis 1983, Tyson,er aL I98l). Protein content was estimated by direct comparison of wine protein peak area at 280nm or225nmto the peak area of BSA at known concentration, with no correction for possible phenolic contaminants. The values obtained ranged from 20 to 752mglL in white juice and 346mglL in a white wine. 25
Quantification of protein by the analysis of the amino acids released on acid hydrolysis is a more accurate method (Allen 1981). However, some amino acids a¡e labile under the conditions used for protein hydrolysis, and others are liberated slowly (Beeley 1985). Yokotsuka et aI. (1977) used this procedure to measure proteins in Japanese grapes. The values reported were low compared to those reported in the literature; 1.5 to 9.0mg/L in grapes, 0.1 to 1.0m9/L in juice and 0.1 to O.4mgll- in wine. This may be a reflection on the inaccuracy of other protein measurements, or the incomplete precipitation of grape proteins by trichloroacetic acid (Bensadoun & Weinstein 1976).
The Coomassie blue dye-binding assay (Bradford 1976) is the most popular procedure for the estimation of protein in many laboratories. For juice and wine, previous studies have suggested that it is more reliable than other methods (Ngaba-Mbiakop 1981, Hsu & Heatherbell 1987) and a number of authors have used this assay to quanrify grape and wine proteins (Ngaba-Mbiakop 1981, Hsu & Heatherbell 1987a, Hsu & Heatherbell 1987b, Hsu ¿f al. 1987, Correa et al. L988. Flores et aI. 1990). However, a recent report showed that wine proteins have a much reduced rate of colour development compared to BSA (Murphey et al. 1989a). Free phenolic compounds did not cause this slow dye binding phenomenon, and it was speculated that it may have been due to polyphenolic compounds already bound to wine proteins. Apart from this study possible interference from grape polyphenolic compounds has not been addressed although it is known that polyphenolics from other plants are an important source of interference.
The effect of polymeric phenotc compounds from other plans was examined by Compton & Jones (1985). It was found that a range of polyphenolics complexed with the dye, although the polyphenolics bound the green form of the dye (A** 700nm) rarher than the blue form to which protein binds (A*a* 595nm). Binding of the green form of the dye still presented a serious interference because the absorbance band at 700nm was broad and overlapped the 595nm peak used in the protein assay. The protein contont of a number of plant tissues rich in phenolic compounds was estimated by both the Bradford method and a modifred Lowry assay (Mattoo et al.1937). It was found that the dye-binding method gave grossly erroneous results with a high degree of variation. As a consequence, it was recommended that the Bradford assay should not be used for such samples. In the specific case of beer protein determination, a subcommittee of the American Society of Brewing Chemists (Am. Soc. Brew. Chemists 1988) recommended that the Bradford assay not be considered for entry into the "Methods of Analysis" because the inter-laboratory error was unacceptable due to variation in the commercial kit reagents. In contrast, another study 26 directed at general plant protein determination concluded that the Bradford assay was moro reliable than the Lowry (Lowry et al. l95I) or biuret (Gornall et al. 1949) assays (Robinson 1979).
The mechanism of dye-binding is unclear, but it is thought that the dye binds by electrostatic attraction to basic amino acid side chains, with preference for arginine, lysine and histidine, respectively (Compton & Jones 1985). The electrostatic binding is reported to be enhanced by the close proximity of hydrophobic side chains (TaI et al. 1979). The specificity of the dye for arginine may be a serious problem for estimating grape and wine proteins because the proteins have low levels of this and the other basic amino acids (Anelli 1977, Yokotsuka et al. 1977, Ough & Anelli 1979, Villettaz et al. 1982, Nakanishi ¿r ø/. 1989). This same specificity is also the probable cause of the variability in rhe assay response for different proteins. It has been shown that many proteins had lower colour yields than BSA at the same absolute protein concentration (Read & Northcote 1981). The median value for the colour yield of 15 proteins was 35Vo that of BSA. By doubling the concentration of the dye in the reagent, this value was increased to 817o, however variability still existed and is a source of error of unknown proportions. Beer proteins gave approximately I5Vo of the response given by BSA (Hii & Herwig 1982). A study of beer foam found that when the Bradford assay was applied to beer, the results gave equivalent or better correlation with foam potential than did other measures of total protein @ale & Young 1987). This was attributed to the strong response of the dye reagent to foam proteins, with a weak response to other beer proteins (Siebert & Knudson 1989).
Apart from the differential response to proteins shown by the Bradford assay, it has also been noted that the rate of colour development varied among proteins (Read & Northcote 1981). If readings were taken at the time specif,red in the original paper (Bradford 1976) i.¿. 3 minutes after reagent addition, the protein concentration of some proteins will be underestimated. This was also shown to be the case with wine proteins (Murphey et al. 1989a). Lysozyme is another example of this; after 3 minutes, only 70Vo of the colour had developed (Read & Northcote 1981).
Before this assay can be used for grape and wine protein quantification it will be necessary to esøbtsh:
1. If grape proteins respond to the dye in a simila¡ manner with respect to colou¡ yield and development, to the protein used to calibrate the assay. 2. If grape polyphenolics specifically, and other grape and wine constituents interfere. 27
2.2 EXPERIMENTAL
The methods relating to individual experiments are given in the relevant Figure or Table legend. General experimental details are given in this section.
2.2.L MaTERIALS
Alanine, bovine serum albumin (BSA), Coomassie Brilliant Blue G and ovalbumin were obtained from Sigma Chemical Company (MO, USA). BioRad BioSit Gel Filtration columns and standards were from BioRad Laboratories Pty. Ltd. (NSW, Australia). Caffeic acid and 2-mercaptoethanol were obtained from BDH Ltd. (England). 0.45¡rm membrane filters (4mm unit, nylon) were from Activon Scientific Products Company, (NSV/, Australia). Microcappilary tubes (20UL) and WISP autosampler vial inserts (lml,) were from Edwards Instrument Company (NSW, Australia). Water used was purified by a Milli-Q reagent rüater system (Millipore Pty. Ltd., NSW, Australia).
Tannic acid (May and Baker Australia Pty. Ltd., VIC, Australia) was prepared as follows: the reagent (4.1mg) was dissolved in ethanol (410pL) then diluted into water (3.69mL) to give a stock solution of lgþ, in 1,07o (v/v) ethanol. A solution of proanthocyanidin grape seed extract was prepared by suspending the proparation (10mg) in ethanol (lml,) then diluting into water (9mL), heating at 50oC for 10 mins, then filtering through a 0.45¡rm membrane, giving a stock solution of approximatety 100mglL tannic acid equivalents (by A280nr), in IÙVo (v/v) ethanol. All other reagents were of the highest purity available.
The grape juices used in these experiments were sourced from the 1988 vintage (Penfolds Wines, Nuriootpa). The Muscat Gordo Blanco (Gordo, syn. Muscat of Alexandria) wine was from the 1988 vintage (Lindemans Wines, Karadoc). Both juice and wine were stored at -200c.
2,2.2 UTTRAFILTRATIoN
The juices (1-2mL) were ultrafiltered through a centricon-10 microseparation device (Amicon Corporation, Danvers, MA, YM-10 membrane, 10 000 nominal Mr cut off) by cennifugation (70009, 60-120min) at 5oC. Centricons are designed for use in a centrifuge and the centrifugal force drives solvents and low M¡ solutes through the membrane and into the filrate cup. Macromolecules vvith Mr above the membrane cutoff size are retained and 28
become more concentrated as the sample volume decreases. Desalting was achieved by concontrating the sample and then reconstituting the retontate twice to the original volume with water. The retentates were collected by inverting the microseparation device and centrifuging at 20009 for 5 min.
2.2.3 Hlrcu PERFoRMANcE LTQUTD Cnnovr¿.ToGRAPHY (HPLC)
Protein mixtures were separated on a Bio-Sil TSK 125 G2000SW gel permeation analytical column (600 x 7.5mm) equipped with a Bio-Sit guard column (75 x 7.5mm) at room temperarure using 200mM NaCl as the mobile phase. Solvent delivery was supplied by a ETP Kortec K35M dual piston HPLC pump, automatic injections were made by a Waters V/ISP 7108 unit, and detection was by a V/aters LC Spectrophotometer 481 recorded with a Varian 27}Integrator. For all samples, except M¡ standards, instrument settings were as follows: flow rate, 0.6ml/min; injection volume, 20¡t'L; detector settings, 225nm,0.2 AUFS; integrator attenuation, 4mV, or 280nm, 0.2 AUFS, intergrator attenuation,2mY; chartspeed, 5mm/min.
Calibration for Mr was achieved with Bio-Rad Gel Filtration Standards in water under the analysis conditions recommended by the supplier. The M¡ values of unknown samples were calculated from the regression equation of log Mr vs. elution volume.
Calibration for protein concenffation was made from the peak area of BSA which gave a linear response in the range 4.0 to 500m9/L.
2.2.4 BgIDFORD PROTEIN ASSAY
Unless stated, the Bradford protein assay (Bradford L976) was employed with double the original dye concentration. It was prepared as follows: Coomassie Brilliant Blue G (20mg) was dissolvedins1%o phosphoric acid (10mL) andgíVo ethanol (5mL).This solution was diluted to 100mL wittr Mitli-Q water and filtered prior to use. The protein sample (100UL) was added to the dye reagent (3mL) and the A595nm was measured every 5 min for 60 min or until the absorbance had stabilized. Calibrations were made with aqueous solutions of BSA at 10 - 400¡rg,/ml, which gave a linear response. Blanks tested with each sample consisted of water (100pL) added to the dye reagent (3mL). 29
2,2.5 ACID HYDROLYSIS OF PURIFIED GRAPE PROTEINS
Protein samples, in duplicate, were hydrolysed in 4.6M HCI containing 0.87o vlv 2- mercaptoethanol ("acid hydrolysis mix" Gruen & Nicholls 1972, Ng et al. t987) at 110oC for 24 hours in hermetically sealed, microcapillary tubes as follows (Lui and Boykins 1989): the protein sample (0.2-SFL) was dissolved in acid hydrolysis mix (56pL),27o wlv ethanolamine (internal standard) (16pL) and then water added to give a final volume of 80pL. After thorough mixing, the solution was intoduced into microcapillary tubes (20pL) by capillary flow, then sealed at one end by melting the glass tubing in a Bunsen burner flame. The tubes were placed in appropriately sized test tubes, sealed end at the bottom, and centrifuged at 2000rpm for 30s in a bench centrifuge to transfer the liquid to the bottom of the capillary tube. The tubes were then cut off at approximately 1cm above the meniscus of the tiquid with the aid of a glass cutter and sealed by melting in a Bunsen burner. The sealed tubes were heated at 110oC for 24 hours in an oven.
To dispense the hydrolysed sample, the tube was cut at 5mm above the meniscus of the liquid and inserted with the open end at the bottom of an autopipetter pipette tip (50- 250tú), which was placed in a WISP autosampler glass vial. The glass vial, containing the capillary tube inserted into the pipette tþ, was placed into a test tube and centrifuged at 2000rpm for 1 min in a bench centrifuge to transfer the liquid from the capillary tube to the glass vial. Samples were then neutralized by addition of 0.4M potassium boraæ buffer (pH 10.4) containing 0.46M NaOH (180pL), filtered through a 0.45pm membrane (4mm filter unit) and stored frozen until analyzed.
Amino acid analysis was performed by reverse-phase HPLC using pre-column derivatization with o-phthaldialdehyde (Lookhart et al. t982). The results were not corrected for hydrolysis losses.
The protein concentration of samples was estimated from the sum of all amino acids found after the above analysis minus the water loss that occurs when amino acid are involved in peptide bonds. (The mass of water loss was estimated by multiplying the total moles of amino acids by the M¡ of water). Quantification of BSA by this method gave a 867o recovery. BSA contalns lÙVo (w/v) proline and cysteine (Peters 1985), which were not quantified by this method. If proline and cysteine were omitted from the calculations, the value was 95Vo recovery. 30
2.3 RESULTS
2.3.1 UV ABSoRBANcE
The gel permeation HPLC technique (Dubourdieu et a|.1986), separates protein in juice and wine into three or four peaks. In the separation of Gordo juice shown in Figure 2.1, there are three main protein peaks followed by a large amount of low M¡ material, probably of phenolic composition. The majority of protein was in a partially resolved peak eluting with M¡ of 23,000 and 12,000. There was another protein peak at 160,000 Mr.
Protein was estimated by summing the area of the juice protein peaks and comparing them to that of BSA. The results of such quantification for different grape varieties are shown in Table 2.I, at two wavelengths (225nm and 280nm) and compared to the results obtained with the Bradford dye-binding assay. (This Table was intended to show the differences between protein assays and not the levels of protein one would expect from particular varieties, because the samples were not necessarily representative). The Bradford assay gave the lowest protein values. Much higher values were obtained from absorbance estimates at225nm and 280nm, especially the latter. The Bradford assay values are, on average, lTVo of those obtained by summing peak areas at 280nm, and 27Vo of those obtained by summing peak ateas at 225nn. 31
1 2,000
23,000J
.1,
[o ot c\¡
1 60,000 \b J
5 10 15 20 25 30 35 40 45
Elution volume (mL)
Figure 2.1 Gel permeation separation of juice proteins Chromatogram of a Muscat Gordo Blanco juice. The apparent lvI. of the three major protein peaks are indicated. Calibration and details of chromatography are given in Section 2.2.3. 32
Table 2.1 Comparison of the Bradford Assay with UV Absorption for the estimation of grape juice protein.
JUICE PROTEIN CONTENT (mg/L) VARIETY Bradfort A225 n*b A280 n b
Chardonnay 0 32 35 Sultana 0 3T 45 Ondenc 0 37 48 Ped¡oÆalomino 0 30 51 Colombard ndc 47 56 Muscadelle nd 59 105 Sauv. Blanc 9 87 160 Semillon L7 t23 260 Frontignac 57 359 368
Gordo 1 69 229 410 Gordo 2 67 265 555
a The Bradford protein assay was used except that absorbance meåsures were made 5 minutes after reagent addirion, and thg reagent concained half ttre dye concentration of that given in Section 2.2.4.This procedure was that given in the original paper (Bradford 1976). Calibrations and blanks \ryers run as deõcribed in Section 2.2.4 b protein content was determined from the sum of all proæin peak areas after gel permeation HPLC at the wavelength indicated, with calibration from the peak a¡ea of BSA (Section 2.2.3) c not determined 33
2.3.2 T}Jø, BRADFORD ASSAY
2.3.2.L Effect of monomeric phenolic compounds
The dye-binding cha¡acteristics of BSA were assessed in protein-stripped grape juice and in solutions containing the monomeric phenol, caffeic acid. Grape juice and caffeic acid, without protein addition, did not give a colour yield, indicating that the juice did nor conrain protein and that caffeic acid, a monomeric phenolic compound, did not interfere in the assay. The response to BSA in juice was linear and very similar to the response in water (Figure 2.2). T\e response to BSA in the presence of caffeic acid was slightly enhanced compared to that given in water and juice, but still linear (Figure 2.2).
2.3.2.2 Effect of polymeric phenolic compounds
Tannic acid a¡rd the grape seed extract bound the dye but these dye complexes had different Arn* from the protein-dye complexes (Figure 2.3). The polymeric phenolics bound both the green (4¡¡¡* at 700nm) and blue (A** at 600nm) forms of the dye, whereas the protein bound only the blue form of the dye. The colour yield at 595nm with 100¡rg/ml tannic acid and 100pg/ml grape seed anthocyanin extract was equivalent to 63 and 89pg/ml- BSA respectively
2.3.2.3 Effect of bound polymeric phenolic compounds
BSA was titrated with tannic acid to give solutions with varying degrees of protein/phenolic complexation, ranging from an excess of protein to an excess of tannic acid. These solutions were then reacted with the Bradford dye-binding reagent and thefu colour yield at 595nm was measured. The results are shown in Figure 2.4. Low concentrations of tannic acid did not significantly effect the Bradford assay results (between points A and B, Figure 2.4). Although colour yield was affected, the effect was not an increase as expected from the previous Section results (2.3.2.2) but a decrease in apparont BSA concentration (I57o-20Vo). This suggests that phenolic compounds bound to protein prevent the Bradford reagent from binding to both themselves and the protein, resulting in a decrease in colour production. An increase in colour yield became apparent only after a th¡eshold concentration of tannic acid was reached (after point B, Figure 2.4). This is interpreted to mean that, at point B, BSA was saturated with tannic acid and subsequent increases in tannic acid concentration (between B and C) resulted in free tannic 34
0.400
0.300
cE ro o) 0.200 rf)
. juice 0.100 o waler
tr caffeic acid (500 mg/L)
0.000 0 40 80 120 1 60 200 BSA (ms/L)
Figure 2.2 Influence of grape juice and caffeic acid on the reaction of the Bradford dye-binding reagent with BSA. The Gordo juice was bentonite fined as described by Rankine (1963), adjusted to pH 3.0, and heated at 90oC for 5 minutes to inactivate residual endogenous grape peptidases (Cordonnier & Dugal 1968, Feuillat et aI. 1980). BSA, in the range 100 to 2000mg/L (10ttl-) was preincubated with either juice or caffeic acid (90¡rL) for 30 min at 25oC, then the Bradford protein assay (Section 2.2.4) was used with the modifications detailed in Table 2.1. The lines of best fit to the results are shown. 35
s90 1 .20
20 ?00 0.92
0.64
610 690 grape seed extract
0.36
tannic acid BSA 0.08
- 0.20 I 500 550 600 650 700 750
nm
Figure 2.3 Spectra of grape seed extract, tannic acid and BSA after reaction with the Bradford reagent. The Bradford protein assay (Section 2.2.4) was employed except protein concentration of the samples was detennined as described in Table 2.1. The visible spectrum of the teaction mixture was determined from 500nm to 750nm against the reagent blank. Samples were tested at 500mg/L, and ma:rimum peaks in nm are reported. 3ó
0.880 c 0.860
0.840 Ec rO q) 0.820 rf)
0.800 A 0.780 B
0.760 0 10 20 30 40 50
Tannic acid (mg/100 mg BSA/L)
Figure 2.4 Effect of tannic acid on the reaction of BSA with the Bradford reagent. BSA (50ttl, 1000m91L) was mixed with tannic acid (0 to 250pL) and saturated ta¡trate buffer, pH 3.0 (200 to 450FL), incubated at room temperature for 30 mins, then aliquots of this mixture (100pL) were assessed for their effect on the Bradford assay (Section 2.2.4). 37
acid in solution which was able to bind the dye. The effect of this was a gross overestimation of protein content. Increasing concentration of grape seed anthocyanin extract to solutions containing BSA showed similar colour yields with the Bradford reagent to that given by tannic acid in Figure 2.4.
2.3.2.4 Effect of ultrafiltration
Ultrafiltration through a membrane with a nominal M¡ cut off of 10,000 was used to separate grape juice phenolics from the proteins before protein quantification. The UV profiles of the juices and their ultrafiltrates demonstrated that ultrafiltration had successfully separated the bulk of the phenolic compounds from the high M¡ material (data not shown). This is because the frltrates retained more than 90Vo of the UV absorbing material, which is predominantly phenolic in nature (Somers & Ziemelis 1985).
The ultrafilter retentates containing the high M¡ material reacted with the dye reagent (Figure 2.5). All the retentates bound the blue form (A,n* at 600nm) of the dye as did the protein standard, BSA, shown in Figure 2.3. However the peaks were much broader than that given by BSA and the Gordo retentate had a slight shoulder ar 680nm.
The Gordo and Shiraz frltrates containing the bulk of the phenolic material also bound the dye reagent (Figure 2.6). Both the blue (Arna* at 600nm) and green (4,n* at 700nm) forms of the dye were observed, as with the polymeric phenolic compounds studied in Section 2.3.2.2. However, the ultrafiltrates bound much more of the green form of the dye than did the polyphenolics or the protein standard BSA (compare figures 2.3 and 2.6). The Shiraz ultrafiltrate also gave a much greater response to the dye reagent than did the Gordo ultraf,rltrate. This result is consistent with the hypothesis that the response seon in grape juice in the absence of protein was due to phenolic material because the Shiraz ultrafiltrate had a higher concentration of total phenolics than the Gordo ultrafrltrate.
Protein quantifications using the retentates and whole juices are shown in Table 2.2. Ultrafiltration reduced the apparent protein values by l3%o for Gordo juice and by 367o lor Semillon juice. 38
630 0.35 6 13 t 680 0.28 I \ 0.21 \ \ \æ
0.14 SEMILLON 640 \ / 0.o7 'sHrRAz
0.00 500 550 600 650 700 750 nm
Figure 2.5 Visible profiles of the retentates from ultrafïltered juices after reaction of the retentates with the Bradford reagent. Juices were ultrafiltered as described in Section 2.2.2, and the high Mr retentates were diluted back to the original volume with water. The Bradford Assay was used as described in Section 2.2.4 andthe maximum peaks reporæd in nm. 39
0.30 710
0.24 610
0.18 SHIRAZ
0.12 750 590 0.06 m
0.00 5oo 550 600 650 700 750 nm
Figure 2.6 Visibte profiles of ultrafiltered juices after reaction with the Bradford reagent.
Juices were ultrafiltered as described in Section2.2.2. The Bradford Assay was used as described in Section 2.2.4 andthe maximum peaks reporæd innm. 40
Table2.2 Effect of ultrafiltration on protein quantification by the Bradford assay
Juice Protein contenta
whole juice UFrctentate
Gordo 105 9l Semillon L29 83
a estimated by the Bradford Assay as described in Figure 2.3 and,expressed as mB/L with reference to BSA. 4I
2.3.2.5 Time course of dye-binding
The time required for the dye to reach maximum binding to the protein and give maximum colour development was examined. It has been reported that wine proteins take, on average, 55 minutes to reach maximum colour development (Murphey et a\.1989a) while aqueous solutions of BSA reach maximum colour development within 5 minuæs (Bradford 1976). The time courses of dye binding to various protein samples examined in this work are shown in Figure 2.7.It was confirmed that with BSA, maximum colour development was attained in 5 minutes and the presence of tannic acid did not alter this. A mixture of BSA and tanniÇ acid was examined because it had been hypothesized that phenolic components bound to protein may be the cause of slow dye-binding (Murphey et al. 1989a). The ratio of both BSA and tannic acid used in this experiment (5mg tannic acid:l0Omg BSA) was chosen to correspond to the ntio (2-2}mg tannic acid:10Omg BSA) at which previous experiments (Section 2.3.2.3, between points A and B; Figure 2.4) had suggested most available tannic acid was bound to protein. Thus it does not appe¿ìr that polyphenolics bound to protein were involved in increasing the time required for maximal dye binding. By contrast, the glycoprotein, ovalbumin, and Gordo juice were slow in reaching maximal colour development.; ovalbumin requiring 50 minutes and Gordo juice not reaching maximum binding even after 55 minuæs.
Increasing the dye concentration in the reagent as suggested by Read & Northcote (1981) in order to reduce the variability of the rcsponse to different proteins, resulted in a decrease in the time required to reach maximal colour development (Figure 2.8). BSA had reached maximal colour development almost immediately (i.e. within the time required to mix the reagent with the sample and place in the spectrophotometer). With ovalbumin, maximal colour was attained after 20 minutes, rather than after 50 minutes with the original reagent formulation, and all juice samples and a juice protein isolate now reached maximal colour development within 30 minutes. 4Z
0.4 /
0.3 \
cE tf) 0.2 \ o, ro F / 0.1
0.0 0 10 20 30 40 50 60 Time (minutes)
200 mg/L BSA in water
200 mg/L BSA in 10 mg/Ltannic acid Gordo juice 200 mg/L ovalbumin
Figure 2.7 The effect of time on the colour yield using the Bradford reagent. The Bradford protein assay (Section 2.2.4) was used with the modifications detailed in Table 2.1. The arrows indicate the point at which maximum colour yield was achieved- 43
o.4 ü
0.3 / - - { - - t - - -t - -- -r -.*.-_a-.-a- - a _ _ * _ _ _^ 'E.- cE t.- E.- lr) 0.2 O) ro ì 'o
0.1 t I 0.0 0 10 20 30 40 50 60
Time (minutes)
€_ Muscadelle juice -....# Sauvignon Blanc juice 100 mg/L BSA .# 1 g/L grape protein isolate -{- 200 mg/L ovalbumin Gordo juice
Figure 2.8 The effect of time on the colour yield using the modiflred Bradford reagent. The Bradford assay was used as described in 2.2.4. The juice high M¡ fraction was isolated by gel filtration (Somers & Ziemelis 1973a). The arrows indicate the point at which maximum colour yield was achieved. 44
2.3.3 ACID HYDROLYSIS
2.3.3.L Total grape protein
Protein quantification by determination of the total concentration of amino acids yielded on acid hydrolysis of total grape and wine proteins was performed on a Frontignac, Sauvignon Blanc and Semillon juice and a Gordo wine. These data were then compared to the values given by the Bradford Assay (Section 2.2.4). The results are shown in Table 2.3.
For all samples except Sauvignon Blanc, the Bradford assay gtossly underestimated the values given from the amino acid composition by a factor of 4 or more.
2.3.3.2 Purified wine protein
Protein concentration data for purified wine protein fractions (A to E), investigated in the later stages of this project are given in Table 2.4. These data were obtained using the Bradford assay (Section 2.2.4) and from quantitative analysis of the amino acids released on acid hydrolysis (Section 2.2.5). The results of these later analysis are discussed here for comparison with the data above (Section 2.3.3.I).
It can be seen from Table 2.4 that the Bradford assay considerably underestimated the protein concentration of all wine protein fractions. The extent of underestimation varied between the fractions, í.¿. fractions B and E gave 507o and2ÙVo,respectively, of the value obtained by amino acid composition analysis. It should be noted that quantification by amino acid composition may be lower than the absolute value because proline, which can account for between 2 and 8Vo of the amino acid composition of wine proteins (Anelli 1977, Yokotsuka et aI. 1977, Ough & Anelli 1979), was not measured. Inclusion of this data would cause the underestimation of protein concentration by the Bradford assay to be even greater. These results highlight the unsuitability of the dye-binding assay for quantification of semi-purifred grape proteins. 45
Table 2.3
Protein quantiflrcation by amino acid composition and Bradford assay
Sample Protein content a Bradfordå Amino acid compositionc
Frontignac. juice 57 253 Sauv.Blanc juice 7 13 Semillon juice 15 143 Gordo wine 50 202
a mglL b Section 2.2.4.
C^- Grape juice protein was isolated by ulrafiltration (Secrion 2.2.2) urd hydrolyzed in 6M HCl, containing
0.l%o (wlv) phenol lor 20 hours at 110oC under N2 in sealed ampoules. Acid was removed from the samples by drying in a vacuum desiccator at room temperature over KOH. The hydrolysis products were then redissolved in water and analyzed for total amino acid content by reaction with ninhydrin (Lee & Takahashi 1966), calibraæd with alanine. Proæin content was calculated by multiplying the moles of amino acids by the average amino acid M¡i.e. 127.4 46
Table2.4
Concentration of proteins in (NH¿)ZSO4 fractions4 as determined by the Bradford dye-binding assay and from amino acid composition
Protein concentrationb
Bradfordc Amino acid compositiond
Fraction A o.34 1.00 Fraction B 0.49 1.00 Fraction C o.26 1.00 FracúonD 0.44 1.00 FractionE 0.20 1.00 BSA 1.10 1.00
a Section 4.2.4.2 b melL c Section 2.2.4 d Protein content in mg/L, calculated as described in Section 2.2.5,by summing the mass of amino acids in hydrolyzed protein. 47 2.4 DISCUSSION
In this investigation, three methods of protein quantification have been assessed; UV absorption, Bradford dye binding procedure and summation of amino acid content.
Measurement of absorption of protein fractions separated from other wine components by gel permeation showed that at 280nm the estimate was higher than at 225nm. This suggests, as previously demonstrated (Somers & Zíemells 1973a), that phenolics were present in the protein peaks, the phenolic compounds contributing to the absorbance at 280nm and thus inflating the apparent protein concentration. The results also showed that the UV absorption methods gave much greater values than did the Bradford method. This was the case even when the same protein standard was used to calibrate both the methods. Earlier work (Somers & Ziemelis 1973a) showed that BSA only gave 50Vo of the peak height of wine proteins on gel permeation because the wine proteins were complexed with phenolics. Phenolic complexation can only partly explain the overestimation of protein concentration seen in this study because it was not 507o but greater than 350Vo. This indicates that other factors in addition to complexed phenolics must be involved. One possible explanation for the overestimation is that wine proteins respond poorly to the Bradford dye reagent.
Previous studies (Ngaba-Mbiakop 1981, Hsu & Heatherbell 1987a) had indicated that in juice and wine, the Bradford assay was more reliable than other methods. Possible interference from juice and wine phenolic compounds, however, had not previously been addressed, although polyphenolics from other plants have been noted as a source of interference in this assay (Compton & Jones 1985, Mattoo et ai,1987). In the current study, caffeic acid, a monomeric phenolic, had only a small stimulatory effect on the colour yield of BSA with the dye-binding assay (Figure 2.2). Grape polymeric phenolics and tannic acid, which is representative of some grape polymeric phenolic fractions, were tested at levels close to those naturally present in white juice. Both these samples reacted with the green form of the dye (A*a* 700nm) as previously reported (Compton & Jones 1985). The binding of the dye to the potyphenolics also produced sufficient absorption at 595nm (blue region and A** with proteins) to be considered an interference.
The extent of interference from the polyphenolics was also influenced by the presence of proteins in the test solution. At low concenfations both tannic acid and grape seed anthocyanins did not increase the 4595 produced by the dye-binding reaction with BSA although, on their own, these phenolics were capable of binding to the dye. However, after 48 a th¡eshold phenolic concentration was reached, BSA was presumably saturated with polymeric phenolic compounds, hence a proportion would exist as free species in solution and produce a signif,rcant interference. Ultrafiltration of juice through a membrane with a cutoff of approximately 10000 M, was successful in removing the bulk of the interfering phenolics and the remaining retentate had an improved ratio of blue to gleen form of the dye.
Ultrafiltration was also adopted by Murphey et al. (1989a) in an attempt to d.etermine the cause of slow colour development of wine proteins after reaction with the dye reagent. The phenomenon of slow colour development was also examined here and the results confirm previous findings (Murphey et al. I989a) that free polymeric phenolics were not involved. These authors proposed that slow colour development was caused by phenolics already bound to protein. In the present study, complexation of tannic acid with BSA did not affect the rate of colour development and other proteins, e.g. ovalbumin, in the absence of bound phenolic compounds, showed slow development. These results suggest that the rate of colour development may be due in part to some inherent property of the proteins, rather than a consequence of bound polyphenolics. In practical terms, this differential rate of colour development need not a problem if a dye reagent containing twice the original dye concentration is used and the time for maximum absorbance to be attained is determined for each protein under study.
To assess the accuracy of any assay, it is necessary to compare the results with an absolute method. In this study we have compared the results obtained with the Bradford assay to those obtained from the amino acid composition. Total grape protein was hydrolyzed by the taditional procedure and the amount of released amino acids estimated by the ninhydrin reagent. The results obtained in preliminary experiments (Section 2.3.3.1) for proteins from four grape varieties were all much higher than those given by the Bradford. assay. These findings were confirmed with the experiments involving purif,red proteins which are described in Chapter 4. Purified wine proteins were hydrolyzed by a method which is reported to give excellent recovery of the labile amino acids, tryptophan, tyrosine and serine (Lui & Boykins 1989). Thus the Bradford assay considerably underestimated the protein content of the purifred wine protein fractions, and the extent of underestimation was variable.
An explanation for this may be found from the mechanism of dye-binding. As discussed in Section 2.l,it is known that the dye binds to basic amino acid groups, especially arginine (Tal et al. 1980, Compton & Jones 1985). Since the purified wine protein fracrions 49
contained a low concentration of each of the basic amino acids (Figure 4.15), a lower response would be expected than with BSA where the basic amino acids accourr-for lTVo of the total amino acid content. If the extent of underestimation was correlated to the basic amino acid content, the assay could still be used after recalibration. However, no such consistency was observd, e.g., fraction E, whose basic amino acids accounted for l2%o of. total amino acids, had a lower colour yield than fraction A with less basic amino acids (87o), and fraction B with LÙvo basic amino acids gave the greatest rþsponse.
These results lead one to conclude that the Bradford dye-binding assay is unsuitable for quantification of purified grape and wine proteins. For such samples, acid hydrolysis of the purif,red protein and then quantification by the sum of the masses of the amino acids liberated is a reliable method. Such a procedure is unfortunately not satisfactory for the winemaker who wishes ûo routinely measure total protein levels in juice or wine because it is complex and requires sophisticaæd equipment. The study here with the Bradford assay may provide enough information about its problems to allow the assay to be used, with due caution, in such circumstances (i.e. minimise phenolic interference by ultrafiltration and allow the dye complex to reach maximum colour development), remembering that protein values could be underestimated by as much as five fold. 50
Chapter 3
REMOVAL OF GRAPE JUICE AND WINE PROTEIN BY PEPTIDASE TREATMENT
3.1 INTRODUCTION
The use of enzyme preparations in commercial food processing is an increasingly popular practice. Exogenous enzymos offer a number of advantages to the food technologist. These include enhanced reaction ratos and specificity and the ability to conduct reactions under mild conditions, usually close to ambient temperature and pressure. Perhaps the most important advantage that enzyme treatment imparts is being perceived by the consumor as a "natural" additive, rather than a chemical or a¡tificial treatment. However enzymes are not commonly used in oenology despite numerous potential applications. Zamorani (1989) offered the following reasons for their low utilization; 1. The "classic" wine industry is based on traditional methods. 2.The reaction conditions are not favourable for most enzymes, juice and wine having low pH and containing polyphenolic material, plus ethanol in the latær. 3. There are legal restrictions.
The only enzymes which are routinely used in wine making are pectinase preparations. These de-esterify and depolymerise poctins and pectic acids which are a physical hindrance to juice extraction. Pectinases are added during grape crushing to increase free-run juice volume and to decrease draining and pressing times, resulting in cost and quality benefits. The juice obtained is also of increased clarity compared to untreated juice because pectins can act as protective colloids, holding insoluble matter in solution (Canal-Llaubères 1989, Rankine 1989, Canal-Llaubères 1990). Pectinases also aid in colour extraction from red grapes (Canal-Llaubères 1990). Other enzymes which have application in oenology are glycosidases for flavour release (Canal-Llaubères 1990) and ß-glucanases to hydrolyse the ß-glucans produced by Botrytis cinerea, which cause filration difficulties (Canal-Llaubères 1989).
This study concerns the potential application of peptidases (proteases) in oenology. These are the class of enzymes which act on the peptide bond, and their classification and nomenclature was discussed in Section L2.t.L. Peptidase ffeatment has the potential to 51
replace bentonite for protein removal by the enzymatic degradation of grape proteins into smaller peptides or their component amino acids.
However, peptidase treaftnent of wine is not a common winemaking practice. Studies with endopeptidase preparations have demonstrated that, when applied to juices and wines at high concentrations, and at temperatures in excess of 30oC, the treated juices and wines showed reduced quantities of protein (Rokhlenko et al. 1980, Heatherbell et al. 1984, Lagace & Bisson 1990). This elevated temperature requirement precludes the use of such enzymes in premium wine making where juices and wines are generally hetd below 15oC. The work of Ngaba-Mbiakop (1981) appea$ to be the only study made with peptidases under conditions approaching those typically used in wine making, and at 14oC, the enzymes had little or no effect. Other workers concluded that grape proteins must be denatured by heat before the protein can be degraded (Feuillat & Ferrari lgSZ). Further, it has been suggested that grape proteins are resistant to proteolysis because of association with other macromolecules present in the fruit (Heatherbell et al. 1984). However, there has been no unequivocal demonstration that exogenous peptidases are even active in juice and wine, nor has it been proved that the reduction in protein content discussed above was not simply due to the activities of endogenous grape enzymes, or to other factors. Natural peptidases have been detected in grapes and juices (Section L.2.l.D although it is not clear from the literature if endogenous peptidases have activity on grape proteins in wine, nor whether such activity would effect heat hazing potential.
The present study was undertaken to test the activity of proteolytic enzyme preparations unde¡ typical wine making conditions. Five enzyme preparations from diverse sources: fungal, plant-derived and animal, were chosen. These enzymes are representative of peptidases active at acid pH, and some are commercially available and have potential application in the wine industry. Evidence is presented that peptidase preparations were active in juice and wine on model protein substrates and the effects of peptidase treatment on juice and wine proteins are reported. In addition, the possibility of inhibiting rhe peptidases with various compounds was assessed, with the view to controlling the extent of the enzyme reaction under investigation. Such control could be particularly relevant during juice and wine heat stability testing where samples are held under conditions conducive to enzyme activity (i.e. high temperatures for long time periods). 52
3.2 EXPERIMENTAL
The methods relating to individual experiments are given in the relevant Figure or Table legend. General experimental details are given in this section.
3.2.1 Ma.TERIALS
The exogenous enzymes tested (Table 3.1) were Pepsin and Bromelain (Sigma Chemical Company; MO, USA), and three commercial preparations: Boerozym (Boehringer Ingelhiem Pty Ltd, NSW Ausralia), Rohapect VR Super (Röhm Gmbh Chemische Fabrik; Darnstadt, Germany) andVirnzymP (Novo Laboratories Pty Ltd; NSW, Australia).
All peptidase inhibitors were supplied by Sigma Chemical Company (MO, USA) except iodoacetic acid, which was from BDH Ltd. @ngland).
Hide Powder Azure (HPA) was purchased from Sigma Chemical Company (MO, USA). Casein (Hammersten) was purchased from BDH Ltd. @ngland). The Bentotest @r Jakob, Fritz Merkel, Geisenheim Rheim, Germany) and sodium bentonite (Volclay) was purchased from Industrial Supplies, SA, Australia. W'ater used was purified by a Milli-Q reagent water system (Millipore Pty. Ltd., NS'W, Australia). All other reagents were of the highest purity available.
Muscat Gordo Blanco (Gordo, syn. Muscat of Alexandria) juice and wine and other 'Wines, varieties of juice were sourced from the 1988 and 1989 vintages (Penfolds Nuriootpa). These juice and wine were stored at -20oC. 1988 Chenin Blanc wine (Brown Bros, Victoria) used in the inhibition studies was purchased from the local retail liqour outlet. 53
Table 3.1
Characteristics of the enzymes used in the proteolytic studies.
NAME ACTIVE EC No. SOURCE ACTIVITY COMPONENT units/g protein
Bromelain bromelain 3.4.22.4 pineapple 21004
Pepsin pepsm 3.4.23.r plg LO6b stomach mucosa
Boerozym paparn 3.4.22.2 latex of nla chymopapain 3.4.22.6 papaya
,d Rolnpect wa nla fungal 2400c VR Super
Vinozym P nla nla fungal nla
4 one unit will release 1.0mg amino nitrogen from gelatin in 2o min at pH 4.5 at 45oc b one unit will produce a change in A2gg of 0.001 in I min at 37oC, measured as TCA-soluble products using haemoglobin as substraúe c one unit will release l¡rmole tyrosine equivalents in I min at 37oC, measured as TCA-soluble products using haemoglobin as substrate d n/unoravailable 54
3.2.2 GnNERAL MBruons
3.2.2.L Heat Test
The method of Pocock & Rankine (1973) was followed. The juice or wine to be analysed was frltered through a 0.45¡rm membrane, sparged with nitrogen gas and then incubated at 80oC for 6 hours, and 4oC for 16 hours. The resultanthaze was assessed by eye.
3.2.2.2 Bentonite fining
The method of Rankine (1963) was followed. A 5Vo bentonite suspension was prepared by adding sodium bentonite powder (5g) to boiling water (100mL) and heating for 5 min with stirring to break up the lumps. After cooling, the suspension was left to swell overnight. The weight of the suspension was adjusted with water to give 1009. The bentonite suspension (0 to 60pL) was added to juice and wine (lml-), mixed by vortex for 30 seconds (Blade & Boulton 1988), centrifuged (10,0009, 10 min), and the supernarant filtered through a 0.45pm membrane before the Heat Test (Section3.2.z.l).
3.2.3 ASSAY oF PEPTIDASE ACTIVITY WITH CASEIN
Casein was prepared as a 17o solution in 0.1M sodium citrate buffer (pH 3.0) in the following manner: Casein (1g) was added to sodium citraæ buffer (100mL) and the weight recorded. The solution was boiled until all the casein dissolved, then cooled and water added until the initial weight was reached.
Al%o casein solution (lml-) and enzyme solution (aO0mg/L in 0.lM sodium citrate buffer, pH 3.0) (250Ff) was added to 0.1M sodium ciüate buffer (pH 3.0) (750¡rL), containing either 0-25Vo (w/v) glucose,0-20Vo (w/v) fructose, or O-I57o (v/v) ethanol, or with no addition, then incubated at various temperatures for 20 min. The hydrolysis was stopped by the addition of 5Vo (w/v) trichloroaceric acid (3mL) and samples placed on ice (10 min) before centrifuging at 3,0009 (20 min) to remove precipitated protein. The absorbance of the supernatant was measured at 280nm in a 10mm path length cell. Two blanks were run; with the test enzyme omitted from the assay and with the test enzyme added after the trictrloroacetic acid addition. 55
3.2.4 ASsaY oF pEprrDAsE acrrvrry wrrH Hrnn Pownan AzuRE (HPA)
The procedure of Little et aI. (1979) was followed: a 2VoIIA suspension in water (lml-) and enzyme solution (lO0melL or 100Omg/L in 4pM Na2EDTA) (1mL) was added to juice or wine (8mL), and incubaæd at various temperatures. Samples (500pL) were withdrawn at intervals, treated with 207o (w/v) trichloroacetic acid (500pL), then placed on ice (10 min) before centrifuging at 10,0009 (5 min) to remove unhydrolysed, precipitated protein. The absorbance of the supernatant was measured at 595nm in a 10mm path length cell. Blanls were run with the test enzyme omitted from the assay.
Juice and wine samples for the peptidase activity assays were bentonite fined as described in Section 3.2.2.2, adjusted to pH 3.0, and heated ar 90oC for 5 min to inactivate residual endogenous grape peptidases (cordonnier & Dugal 1968, Feuillat et al. LgBo).
3.2.5 PnpPARATIoN oF PEPTIDASE INHIBIToRS
Pepstatin (lmg) was dissolved in methanol (500pL) then diluted in 6vo (w/v) potassium hydrogen tafrate buffer (pH 3.0) (saturated tarrate buffer, 7mL) to give a stock solution of 4mM. p-Chloromercuriphenolsulfonic acid (pCMPS) (1.66mg) was dissolved in sarurated tartrate buffer (pH 3.0) (1mL) ro give a stock solution of 4mM. Phenylmethylsulfonylfluoride (PMSF) was dissolved in isopropylalcohol (lml-) then diluted in saturated tartrate buffer ftrH 3.0) (5mL) to give a stock solution of 4mM. 1,10- phenanthroline (oP, 3.6mg) was dissolved in isopropylalcohol (lml-) then diluted in saturated tartrate buffer (pH 3.0) (5mL) to give a stock solution of 4mM. Iodoacetic acid (9.3mg) was dissolved in water (10mL) to give a stock solution of 5mM. N-Tosyl-L- phenylalanine chloromethyl ketone (TPCK,0.7mg) was dissolved in methanol (10mL) to give a stock solution of 0.2mM. N-cr-p-Tosyl-L-lysine chloromethyl ketone (TLCK, 3.15mg) was dissolved in water (10mL) to give a srock solution of 0.086mM. 56
3.3 RESULTS
3.3.1 PeprrDASE acrrvrry oF THE coMMERCTaL ENZyME pREpARATroNs IN BUFFER
The three proteolytic enzymes were all active on casein in citrate buffer at pH 3.0 (Figure 3.I). Boerozym and Rohapect VR Super had approximately the same level of activity per gram of preparation, whereas VínozymP showed almost three times more activity per gram than the other two enz)mle preparations.
3.3.1.1 Effect of temperature
To determine the temperature optimum of the enzymes, their activity on casein was assessed between 5 and 65oC (Figure 3.2). All the enzymes had low activity below 20oC. Figure 3.2 also shows thatVinozym P exhibited maximal activity in the range 45-55oC while for Boerozym and Rohapect VR Super this was in the range 50-60oC. An incubation temperature of 50oC was chosen for further studies because this temperature gave close to the maximal response for all enzyme preparations.
To determine the thermal stability of the enzymes under the conditions of the wine heat stability test, peptidase activity was assessed at 80oC for 6 hours (Figure 3.3). Boerozym appealed to be the most thermostable enzyme because its activity on HPA increased during the first 40 minutes. All other enzymes showed increases in activity during the first 20 minutes only, and furttrer incubation lead to rapid decreases. No activity was shown by any of these enzymes after 2 hours.
3.3.1.2 Effect of glucose, fructose and ethanol
Three major juice and wine constituents were assessed for their effect on peptidase activity. Juices generally contain equal amounts of glucose and fructose. These are the major sugars and total from 15 to257o (w/v) (Amerine et a|.1980). The effect of 0 to 257o (w/v) glucose is shown in Figure 3.4. All the enzymes were inhibited by glucose to approximately the same extent. The activity of the three enzymes was reduced by 20Vo at L2.57o (w/v) glucose. Fructose was not so inhibitory. The effects on peptidase activity of up to 207o (w/v) fructose are shown in Figure 3.5. Fructose also had a similar effect on all the enzymes but only reduced their activity by 10 to20Vo at I2.57o (w/v) fructose. 57
0.6
0.5
0.4
Peptidase activity 0.3
0.2
0.1
0 Vinozym P Boerozym Rohapect VR super
Figure 3.1 Peptidase activity of the commercial preparations Peptidase activity on casein at 37oC of the three commercial peptidase preparations at 50mg/L was determined from the absorption at 280nm due to the aromatic amino acids released by enzymatic activity and is expressed as AA4g/20 min. Details of the assay are given in Section 3.2.3.
59
1 000
800
.=
a a P ¡ I C) 600 a (ú a a ¡ l<- o Vinozym P U' (d 400 p Boerozym o- o fL Rohapect VR Super
200 ._- Pepsin
\ Bromelain -ìF 0 0 1 2 3 4 5 6
Time (hours)
Figure 3.3 The effect of incubation time at 80oC on peptidase activity Peptidase activity in wine on HPA of the five enzymes was assessed as described in Section 3.2.4 and is expressed as rate of hydrolysis under the assay conditions (AAsqs /min x 10ó). 60
s 100 I o o = BO o o (ú g (I) 60 (n o ct p 40 o- ùo 20 o Vinozym P
0 0 5 10 15 20 25
Glucose (% wlvl
100 s a BO .=
C) a (ú 60 a o a 0 0 5 10 15 20 25 Glucose (% wlv) 100 80 E .à 60 (ú() 40 (¡) an (tt 820 o- tr Rohapect VR Super rLo(l) 0 5 10 15 20 25 Glucose (% wlv) Figure 3.4 Effect of glucose on peptidase activity The peptidase activity on casein at 50oC of the three commercial peptidase preparations was determined from the absorption at 280nm due to the aromatic amino acids released by enzymatic activity. Details of the assay are given in Section 3.2.3. 61 100 I 80 o .= 8 o () (ú 60 (l) at p(ú 40 o- (l) 20 o Vinozym P fL 0 0 5 10 15 20 Fructose (% wlv) 100 s o 80 a = a o 60 al (l) (n (tt 40 E o- (l) 20 È a Boerozym 0 0 5 10 15 20 Fructose (% wlvl 100 tr * 80 .= o(\t 60 (¡) at p(ú 40 o- o 20 È tr RohapectVR Super 0 0 5 10 15 20 Fructose (% wlv) Figure 3.5 Effect of fructose on peptidase activity The peptidase activity on casein at 50oC of the three commercial peptidase preparations was determined from the absorption at 280nm due to the aromatic amino acids released by enzymatic activity. Details of the assay are given in Section 3.2.3. 62 The effect of 0 to 15Vo (v/v) etha¡rol on the peptidase activity is shown in Figure 3.6. These values were chosen because table wines generally contain up to líVo (v/v) ethanol (Amerine et al. l98O). Vinozym P exhibited the best resistance ro erhanol and retaine d,90%o activity atS%o (v/v) ethanol. At ethanol concentrations lower than this value, there appeared to be an activation of the enzyme, with approximately l70vo activity shown at 4 to 6Vo (v/v) ethanol. At 757o (v/v) ethanol the peptidase activity of Vinozym P had decreased to 40Vo. Boerozym and Rohnpect VR Super were more effected by ethanol and each responded in a simila¡ manner. Ethanol to 6Vo (v/v) had Iittle detrimental effect on the peptidase activity of Boerozym and Rohapect VR Super (Figure 3.6), but at higher ethanol concentrations, activity progressively decreased to reach ?IVo and 107o , respectively, at LSVo (v/v) ethanol. 3.3.2 PcpTIDASE AcTIvITY oN HPA IN JUIcE AND wINE The stability of the chromogenic substrate, É[PA, under the assay conditions was assessed by measuring peptidase activity in pH 3.0 ta¡rate buffer and in juice. These results are shown in Figure 3.7, and demonstrate that HPA was stable at pH 3.0 in tartrate buffer but was degraded in juice. However, this degradation could be prevented by pasteurizing the juice before use in the peptidase assays. This suggested that the juice contained endogenous peptidase activity. Further studies with exogenous peptidases were conducted in pasteurised juice and wine. The f,rve proteolytic enzymes were all active on the chromogenic proæin substrate GIPA) in Muscat Gordo Blanco (Gordo) juice and wine. The levels of activities observed at 18oC and at 50oC are shown in Figure 3.8. It was found that the commercial enzyme preparations showed maximal activity at 50oC on HPA ( as shown on casein in model solutions, see Section 3.3.1.1), but it is evident from Figure 3.8 that all five enzymes were still active at 18oC and even at an enzyme concentration as low as l0mgll-, a proportion of the chromogenic substrate was degraded in the juice medium. In wine, the activity of all the five enzymes on HPA was reduced compared to that observed in juice (Figure 3.8). Boerozym PV and Pepsin still retained more than907o of the activity they exhibiæd in juice on HPA at 50oC. Vinozym P also showed good activity in wine withTOVo of the acrivity exhibited in juice. Rohapect VR Super and Bromelain were more effected showing reductions in activity to líVo and207o respectively. 63 120 o 100 àe ¡ a I .= 80 C) o o Vinozym P (ú tr . Boerozym 60 tr o o tr Rohapect VR Super U' o (ú p 40 tr o- (t) È I o 20 024 6 I 10 12 14 16 Ethanol (% vlv) Figure 3.6 The effect of ethanol concentration on peptidase activity The peptidase acúvity on casein at 50oC of the three commercial peptidase preparations was determined from the absorption at 280nm due to the aromatic amino acids released by enzymatic activity. Details of the assay are given in Section 3.2.3. 64 o.4 o 0.3 .= a .F (J (ú -+buff sr a +untreated juice (¡) o.2 a (t, juice p(ú -tspasteurised o- (¡) fL 0.1 0.0 0 5 10 't5 20 25 30 Time (hours) Figure 3.7 Endogenous peptidase activity of grape juice The peptidase activity at 50oC determined by the absorption at 595nm of chromogenic peptide fragments liberated from HPA, and expressed as 4595, of saturated tartrate buffer (pH 3.0); untreated Gordo juice (pH 3.0); and pasteurised Gordo juice (pH 3.0). Details of the assay are described in Section3.2.4 65 500 ø n 400 300 .à '= o - 200 100 0 Boerozym VR Super Vinozym P Bromelain Pepsin I MUST 18 deg. C 10 mg/l @l MUST 1s deg. C 100 mgl ø MUST sO deg. C 10 mg/l ø MUST s0 deg. C 100 mgl tr WINE so deg. C 1oo mg/l Figure 3.8 Influence of temperature and enzyme concentration on the activity of peptidases in juice and wine Peptidase activity at 18oC and 50oC was assessed using HPA as described in the Experimental Section 3.2.4. Activity is expressed as the rate of hydrolysis under the assay conditions (AAsqs/min x 106). 66 3.3.3 PppTIDASE AcTIvITY oN GRAPE PRoTEINS 3.3.3.1 In buffer A high l\4r fraction isolated from juice (i.e. containing grape proteins and polysaccharides) was treated with the frve peptidases in pH 3.0 taftrate buffer and the result followed over 7 days at 25oC by changes in the HPLC gel permeation profîle. The data for Bromelain is shown in Figure 3.9. The action of the five enzymes altered the HPLC elution profiles in different ways prcsumably as a result of differing specificity and mode of action of each peptidase. The most generally discernible changes included a decrease in area of the N4¡ 23,000 peak with a concomitant increase in the Mr 10,000 peak, together with a loss of resolution of both these peaks. The latær phenomenon appears to be due to the formation of proteins of intermediate lvft. The peptidase enzymes themselves were not of suff,rciently high concentration to enable their detection. It is significant, however, that none of the enzymes had caused total proteolysis of grape proteins in buffer solution after 7 days. 3.3.3.2 In juice The effects of peptidase treatrnent on grape juice proteins in juice are shown in Table 3.2. After 7 days treatment at 15oC none of the enzymes produced any significant reductions in protein concentration as assessed by peak area on HPLC. After 7 days at25oC however, Vínozym P had reduced total protein to 69Vo of the original concentration. Both the M, 23,000 and 10,000 protein peaks were effected. The reduction in peak area was paralleled by a decrease in the observed haze as indicated by the Bentotest. Boerozym was the only other enzyme to show a reduction in protein concentration at 25oC, but this was not to the same extent as Vinozym P; and the effect of Boerozym was to reduce the peak area of the Mr 23,000 peak only. It must be noted that proteolysis was not complete, even in the VinozymP treated juice, after 7 days at2soc. 67 23 000 Mr 10 000 Mr \ 0 1 E lda ¡r) ô¡ (\¡ õ 2da S c .9 CL 3d o .n .cl 7d 20 30 Elution volume (ml) Figure 3.9 Effect of Bromelain treatment on isolated grape protein Bromelain in 4pM Na2EDTA was added to a grape high molecular weight fraction in 6Vo (flv) potassium hydrogen tartrate buffer (saturated tartrate buffer, pH 3.0), to give a final enzyme concentrationof.25mglL, and the mixture was incubated at 25oC. Samples (20FL) were taken at timed intervals for IIPLC analysis (Section 2.2.3). The grape high molecular weight fraction, containing protein, was isolated from Gordo juice by gel permeation chromatography (Somers &. Ziemelis 1973a). This fraction was diluted further with saturated tartrate buffer to give a solution with a protein concentration approximately half that of natural juice (as assessed from the peak area on HPLC, see Section 2.2.3). 68 Table3.2 Effect of peptidase treatment for 7 days on Gordo juicea proteins b Temp enzyme proteinc hazed (oc) (Vo inralcontent) 15 Vinozym P 99 ++ 15 Boerozym 97 ++ 15 RohapectVR Super 96 ++ 15 Bromelain 100 ++ 15 Pepsin 95 ++ 25 Vinozym P 69 + 25 Boerozym 89 ++ 25 RohapectVR Super 96 ++ 25 Bromelain 100 ++ 25 Pepsin 100 ++ o The Gordo juice used for these experiments was adjusted to pH 3.5. b The specified enzymes in 4UM Na2EDTA were added to give a f,rnal enzyme concentration of 33mg,/L, then the mixture was incubated at either l5oC or 25oC. t Prot"in concentration, analysed by gel permeation HPLC (Section 2,2.3),is expressed as a percentage of that originally present. d Jui"" heæ søbility was assessed by the Bentotest according to the directions provided by the supplier and is expressed by the degree of haze produced + =light, {+=heavy 69 3.3.3.3 In wine The HPLC elution profile of the proteins of Gordo wine was altered by peptidase treatment, as observed in model solutions (Section 3.3.3.1) and juice (Section 3.3.3.2). The effect of Virøzym P treannent is shown as a typical example in Figure 3.10. The most obvious change was a decrease in peak size of the lr4, 23,000 peak. Total protein was also reduced but the control wine showed a similar decrease in protein concentration (Figure 3.11). In addition, the chromatographic profile of the control wine after 12 weeks incubation @igure 3.12) was similar to that observed for the enzyme-treated wine after the same time period (Figure 3.10). The reduced concentration of protein and modihed FIPLC elution profiles of the enzyme treated wines were not accompanied by an observable decrease in haze potential (data not shown). Although the bentonite requirement to stabilize the wine was reduced by approximately 3OVo this improvement was observed for both the treated and control wines (Table 3.3). 70 f0üþ Mr 23üþ Mr vo 120fiþ Mr Wks.{t o 1 cE rO ô.t ô¡ (õ c '-o I o- o U) -o 12 o lo Elution volume (ml) Figure 3.10 Effect of Vínozym P treatment for twelve weeks on wine protein Gordo wine (Penfolds Wines, Nuriootpa, pH 3.5) was treated with Vinozym P in 40pm Na2EDTA to give a final enzyme concentr¿tion of 50mg , and the mixture was incubated at 15oC. Samples were taken at timed intervals for HPLC analysis (Section 2.2.3). HPLC chromatograms for samples taken at 0 to 12 weeks ate shown, with the apparent Mt of protein peaks indicated" 7T 40 o) 30 E Pc +¡o c 20 o o .g o Po L 10 fL --r control --=o- Vinozym P 0 0 4 I 12 16 Time (weeks) Figure 3.11 Change in wine protein content with Vínozym P treatment Gordo wine was treated as described in Figure 3.10 for VinozymP treated wine and Figure 3.12 for control wine. Protein content of the wines was estimated by the Bradford assay (Secúon 2.2.4). 72 10 000 Mr 23000 Mr I üo 120000 Mr Wks.{t I o l 4 I cE tr) C! C\ (õ c '-o 8 o_ o !U' 12 0 + r0 30 Elution volume (mt) Figure 3.12 Effect of incubation of wine protein at lSoC Gordo wine (as in Figure 3.10) was treated with 40pm Na2EDTA then the mixture was incubated at 15oC. Samples were taken at intervals for HPLC analysis (Section 2.2.3). FIPLC chromatograms for samples taken at 0 to 12 weeks are shown, with the apparent lVt, of protein peaks indicated- 73 Table 3.3 Concentration of bentonite needed to heat stabilize wine after periods of peptidase treatment Bentonite requirement to give heat stability (glL)" Treatment time Control wineó VirnzymP treated (weeks) winec (50rnglL) 0 1.3 t.3 2 1.3 t.2 4 1.1 1.0 8 1.0 0.8 t2 0.9 0.8 4 Bentonite f,rning was performed as described in Section 3.2.2.2.Heat søbility was assessed by the Heat Test (Section 3.2.2.L). å The *ir," was Eeated as described in Figure 3.12 " Th" *in" was treated as described in Figure 3.10 74 3.3.4 INHrBrrroN oF pEprrDAsE Acrrvrry The effect of various compounds which block the active sites of the peptidases was determined. Table 3.4 shows the degree of inhibition of the proteolysis of HPA caused by each compound after it had been preincubated with the enzyme in buffer. Under such conditions, it was possible to totally inhibit Bromelain with lmM iodoacetic acid ancl strongly inhibit Pepsin with 50pM pepstatin. The other peptidase preparations were more difficult to inhibit. VinozymP was not totally inhibited by any of the inhibitors tested but it was partially inhibited by PMSF andpCMPS. Boerozym was also not rorally inhibited by any of the inhibitors but was partially inhibited by pCMPS, iodoacetic acid, PMSF and pepstatin and activated by oP. Rohapect VR Super was parrially inhibited by PMSF, pCMPS, pepstatin and 5mM iodoacetic acid- 'When the inhibitors were preincubaæd with the enzymes in wine and then the action of the peptidases on HPA was tested in wine, the same problem of incomplete inhibition was observed (Table 3.5). Bromelain and Pepsin were totally inhibited by lmM iodoacetic acid and 50pM pepstatin, respectively. The other peptidase preparations were again d.ifficult to inhibit. Vinozym P was not inhibited by PMSF or TLCK, although slight inhibition was afforded by TPCK. Boerozym also was not fully inhibited by any of the inhibitors, although its activity was reduced to 50Vo by 5mM iodoacetic acid. However higher concentrations of this rcagent appeared to activate the enzyme . Boerozymwas also partially inhibited by TPCK. Results with Rohapect VR Super were more encouraging because it was possible to inhibit 8O7o of the activity with high concentrations of iodoacetic acid (20mM), and 50 Vo of the activity with 40pM TPCK. Since it was not possible to totally inhibit all of the enzymes, further work with the inhibitors was not pursued. 75 Table 3.4 Effect of inhibitors on peptidase activity in buffer Inhibitor concentration ActivityToa Bromå Peps. Vino. Boer. Roha pCMPSc 1mM r4 80 74 60 d pepstatin 5OpM 2l 99 89 80 PMSF 1mM 66 85 60 oP 1mM 93 t17 100 iodoacetic acid 1mM 0 83 80 5mM 75 49 o Th" (10mg/L,4QrL) were preincubated with the inhibiton (50UL) in sau¡raæd taÍrate buffer þH "nry*es 3.0, 1l0pl) at room temperature for 30 min, fhen at 50oC for 30 min, with the exception of iodoacetic acid which was incubated at 50oC for 60 min. Enzyme activity was then determined by adding 4Vo WA suspension (lml) and saturated tartrat€ buffer (2.8mL) then incubating the mixture at 50oC. Samples (500Uf) were taken as described in Section 3.2.4. Tfuee blanks were run for each inhibitor: with the enzyme omitted, the inhibitor omitted, and both omiced; the last blank containing only the solvent used to dissolve the inhibitor, e.g.7Vo methanol in the case of pepsøtin. å abbreviations used: Brom., Bromelain; Peps., Pepsi ni Vino.,Vinozym Pi Boer., Boerozym; Roha., Rohapect VR Super c abbreviations used: pCMPS, p-Chloromercuriphenolsulfonic acid; PMSF, phenylmethylsulfonylfluoride; oP, l,l0-phenanthroline d -,notdetermmed 76 Table 3.5 Effect of inhibitors on peptidase activity in winea Inhibitor concentration A.ctivityVob Bromc Peps. Vino. Boer. Roha pepstaûn 5OpM e 0 PMSFd 1mM 100 iodoacetic acid 1mM 0 2rnÀd ìo 5mM 50 88 1OmM 80 75 20mM 110 20 TPCK 23tl'jvlr 9T 86 78 40[tM 81 82 53 TLCK 17pM 100 93 9L ø The Chenin Blanc wine used for ttrese experiments con[ained 11.57o alcohol, and had pH 3.1. b_ " Enzyme activity was determined after preincubation with the specihed inhibitors by adding 47o IÐA (500ttl) and wine (3.3mL) and then incubating the mixtu¡e at 50oC. Samples were taken as described in Table 3.4. The preincubation mixes were identical to those in Table 3.4 except san¡raæd târtrate buffer was replaced with wine. c abbreviations used: Brom., Bromelain; Peps., Pepsin; Vino.,Vinozym P;Boer.,Boerozym;Roha., RohapectVR Super d abbreviations used: PMSF, phenylmethylsulfonylfluoride; TPCK, N-Tosyl-L-phenylalanine chloromethyl ketone; TLCK, N-a-p-Tosyl-L-lysine chloromethyl ketone ¿ -,not determined 7l 3.4 DISCUSSION The three commercial enzyme prepamtions chosen for this study were active in model solutions buffered to juice and wine pH, and one preparation, Vinozym P, showed approximately three times the activity of the other two. It was not established if Virwzym P contained a more active enzyme, and/or it contained a greater amount of active enzyme per gram of preparation. The optimum temperature for maximum peptidase activity was similar for all three commercial preparations, with greatest activity around 50oC and little peptidase activity below 20oC. These results are analogous to those reported by Ngaba-Mbiakop (1981) who found that the optimum temperature of an experimental peptidase from Röhm (i.e. the peptidase now present n Rohapect VR Super) was 50oC. Low activity observed below 20oC may limit the effectiveness of the enzymes in white wine making where processing temperatures rarely exceed 20oC. The enzymes were also active at 80oC, although enzyme activity did not persist for time periods longer than 2 hours. However, this short lived activity may cause serious problems if the heat test (80oC, 6 hours, Section 3.2.2.1) is used to assess the effectiveness of enzyme treatment of juices and wines. A juice or wine treated with enzyme at 2æC and then subjected to the heat test may have more protein degraded during the heat test than at 20oC. The resultant juice or wine may then appear to be stable because of the protein degradation caused by the heat stability testing rather than the treatment at 20oC. A potential way to avoid such an artefact would be to inhibit the enzymes during the heat test. Preliminary studies with active site inhibitors were undertaken in an attempt to find inhibitors that were effective in juice and wine for each enzyme preparation. Unfornrnately, it was not possible to completely inhibit the three commercial enzyme preparations. There are two possible reasons for this: firstly, the crude commercial preparations may contain a mixed population of enzymes such that with any given inhibitor only a proportion of the active peptidases in each preparation was affected. Secondly, some of the active site inhibitors used a¡e specific for particular classes of enzymes and these enzymes generally have pH optima far removed from wine pH. For example, PMSF is an active site inhibitor of serine dependent peptidases which have pH optima near neutrality. oP is an active site inhibitor of metal dependent peptidases which have alkaline pH optima (Storey & Wagner 1986). The inhibitory compounds are designed to bind strongly at the pH optimum of the enzyme but at juice and wine pH the compounds may not bind as eff,rciently, and therefore 78 not be effective inhibitors. Despite these problems, the present study gave an indication of the classes of peptidases present in the various preparations. The most effective active site inhibitors of Bromelain in model solutions and in wine, were iodoacetic acid and pCMPS. Both these inhibitors are specific for cysteine dependent peptidases (Storey & Wagner 1986, Phillips & W'allace 1989). Such results are consistent with the literature which classifies Bromelain as a cysteine dependent peptidase (Whitaker 1972). Pepsin was successfully inhibited by pepstatin in model solutions and in wine. Pepstatin is a powerful inhibitor of most aspartic acid peptidases, the class in which pepsin belongs (Fruton 1976). Virnzym P was only partially inhibited by inhibitors of the following types of peptidases in model solutions (Storey & Wagner 1986); PMSF (inhibits serine dependent peptidases), marginally inhibited by pCMPS (cysteine), and not signihcantly inhibited by pepstatin (aspartic acid) or oP (metal). This suggests that Vinozym P may be a mixture of peptidases with the predominant forrl being serine dependent. Flowever, in wine, it was not possible to even partially inhibit Vínozym P with PMSF or additional irreversible serine active site inhibitors (chloromethyl ketones: TPCK and TLCK)(Storey & Wagner 1986). Boerozym, which is alleged to be a mixture of two cysteine dependent peptidases, papain and chymopapain (see Table 3.1), was also not successfully inhibited by atty inhibitors tested- The most effective inhibitors in model solutions were compounds which specifically inhibited cysteine dependent peptidases, pCMPS and iodoacetic acid, but these compounds did not effect total inhibition. Pepstatin (aspartic acid) and PMSF (serine) were also partially inhibitory which suggests that Boerozym was a mixture of peptidases, predominated by cysteine dependent enzymes, but containing some aspartic acid and serine dependent peptidases. The metal dependent peptidase inhibitor, oP, activated Boerozym , which is consistent with the preparation containing predominantly cysteine dependent enzymes, because such enzymes are inhibited by metals which oP would sequester (Storey & V/agner 1986). In wine, it was possible to inhibit 5O7o of the peptidase activity with iodoacetic acid, however, higher concentrations appeared to reverse the inhibition. The two irreversible serine dependent inhibitors, TPCK and TLCK, were also partially inhibitory, confirming that ttre preparation was a mixture of cysteine and serine dependent peptidases. Rohapect VR Super was partially inhibited by afl the inhibitors tested in model solutions, except oP, which had no effect. The greatest inhibition was produced by iodoacetic acid, which suggests that Rohapect VR Super is a mixture of cysteine, serine and aspartic acid 79 dependent peptidases, with the majority being cysteine dependent. The results obtained in wine support the hypothesis that Rohapect VR Super contains predominantly cysteine dependent peptidases. This is because it was possible to inhibit 80Vo of the peptidase activity in wine with high concentrations of iodoacetic acid, and 50Vo with the ireversible serine dependent inhibitor, TPCK. Although the peptidases were active in model solutions buffered to juice and wine pH, this result did not ensure that the enzymes would be active in juice and wine. For the enzymes to be effective in juice they must be able to tolerate the high sugar content. Juices generally contain equal amounts of glucose and fructose and they total from 15 to 25Vo (w/v) (Amerine et al. L980). The activity of all the commercial enzymes was reduced by approximately 2OVo in model solutions containing l2.5Vo glucose, and model solutions containing t2.57o fructose caused a reduction of activity of approximately L57o. One could therefore predict a reduction in activity in grape juice to approximately 60 to 80Vo of that shown in model solutions due to the effect of fructose and glucose. The inhibitory effect of the sugars could be avoided by treating wine rather than juice, provided that the enzymes can tolerate ethanol. From the results obtained by assaying peptidase activity in model solutions containing ethanol, it seems that one could expect almost a total reduction in activity in wine for Boerozym and Rohapect VR Super, and a reduction to approximately 407o of the activity shown in model solutions for Virnzym P, due to the effect of ethanol alone. However, these values are only approximations and to obtain a true picture of the effect of juice and wine constituents on peptidase activity, the enzymes must be assayed in juice and wine. This required the development of a peptidase assay which could be used in juice and wine. The standard assay used for peptidase activity in model solutions correlates casein breakdown with an increase in the amino acids; tryptophan, tyrosine and phenylalanine, which absorb at 280nm. Such an assay can not be used in juice and wine due to the high concenmtion of endogenous amino acids and other interfering substances in grape juice and wine. Therefore some other measure, which can distinguish between released peptides and amino acids and those in the background, must be employed to monitor proteolysis in juice and wine. The use of a chromogenic subsnarc offered one solution to this problem. These substrates are proteins labelled with covalently bound dye molecules which on proteolysis give light- absorbing peptide fragments. The latter could be assayed spectrophotometically in solution 80 at wavelengths gteater than 400nm. Because the background absorbance of white juice and wine was low above 400nm, the activity of a peptidase towards a chromogenic substrate could be readily determined in these media. Hide powder az'$e (HPA) was used in this study. It is prepared by covalently linking Remazobrilliant blue dye to collagen (hide powder). This substrate has previously been used in oenology but its chromogenic properties have not been fully utilized. In such studies, HPA was used to measrue peptidase activity in model solutions but not in juice or wine and it was shown that a peptidase preparation in buffer medium was not seriously inhibited at juice and wine pH or by sulphur dioxide or ethanol (Ngaba-Mbiakop 1981, Heatherbell et a|.1984). The results given here with the non-chromogenic protein substrate, casein, confirm these findings, and in addition, show that neither fructose or glucose are particularly inhibitory to the activity of the commercial enzyme preparations in model solutions. The use of the chromogenic substrate HPA, has taken these results one step further and demonsüated that the peptidases all showed proteolytic activity in grape juice and wine and that this activity increased with temperature. All the enzymes showed some reduction of activity in wine, as was predicted from the casein study, although Boerozym only showed a reduction of LÙVo. For Vinozym P the casein study predicted a reduction in activity in wine to 40Vo of that shown in model solutions, or 50Vo of that shown in juice, but its activity on HPA was only reduced to707o of that shown in juice. The results for Rohapect VR Super were more consistent with those predicted by the casein study; the enzyme preparation showed an almost total reduction in activity in both media. The reduction in activity caused by ethanol may be due to changes in conformation of the enzymes affecting their active sites and thus reducing their efficiency. In addition, ethanol may affect the conformation of the substrate and change sites recognized by the enzymes. The differences in the degree of reduction was presumably a function of differing specificities, active sites and modes of catalysis between the enzymes and differences between the substrates and their behaviour in wine solutions. In practice, partial inactivation in wine may not necessarily be a serious constraint since opportunities for prolonged treatment are greater in wines, e.g. during storage and conservation, than in juices during the pre-fermentative stages of wine making. The use of the chromogenic substrate also revealed the presence of endogenous protein- degrading activity in grape juice. HPA was degraded in untreated juice. The degradation 81 could be prevented by pasteurisation suggesting that this peptidase activity was due to an enzyme. Endogenous grape peptidases have been previously described (Cordonnier & Dugal 1968, Pallavicini & Dal Belin Peruffo 1977,Feuillat et al. 1980, Feuillat & Ferrari 1982). However, at present, it is not known if any of the endogenous peptidases would have activity on grape proteins in wine even though this study has shown that endogenous peptidases have activity for the chromogenic substrate ItPA. The activity of the five commercial enzymes on the substrate of interest, grape and wine protein, was studied in model solutions and in juice and wine. It was clear from HPLC studies that grape and wine proteins were modified by the action of the exogenous peptidases in all these media, but under the conditions examined, the 10,000 to 23,000 M¡ protein fractions were only partially hydrolysed to material with M¡ less than 10,000. In wine, the reduced concentration of protein and modified Mr of the fractions were not accompanied by an observable decrease in haze potential and although the bentonite requirement to stabilize the wine was reduced by approximately 30Vo, this improvement was observed for both the treated and control wines (Table 3.3). Similar results have been reported in the literature (Lagace & Bisson 1990). The effect of two peptidases from Yarrowia lipolytica andTorulopsis magnolia at3}oC on Chenin Blanc and Chardonnay wine proteins were examined by protein separation on a IIPLC anion exchange column. The profiles showed evidence of some degradation of wine and peptidase proteins, together with an increase in proteins of lower isoelectric point, but not complete hydrolysis of all protein. However, profiles of the two relevant controls (wine without enzyme held at 30oC, and enzyme held at 30oC) were not published. Hence it is not possible to determine if the effects observed were due to added enzyme or endogenous grape peptidases. In this project, treatment of juice and wine with peptidases did not appear to be a viable alternative for haze control and it was uncertain as to whether ttre small effect observed was due to the added peptidases or those naturally present. The treated juices and wines contained proteins with different characteristics from those initially present (as seen by the HPLC profiles), but the proteins could still be precipitated and form hazes. 82 Chapter 4 IDENTIFICATION OF UNSTABLE PROTEIN 4.1 INTRODUCTION AND LITERATURE REVIE\ry There is a need to formulate alternative and novel techniques to remove unstable haze- forming proteins from wines because the currently used technique of removal by absorption onto bentonite is non specific and can impair the quality of wine. Paramount to the success of such a goal is the need to establish the unstable proteins which ate responsible for haze, and to characterize them. In early work on wine instability it was observed that unstable wines tended to have higher levels of the lowest pI band of four major protein fractions separated by disc electophoresis (Moretti & Berg 1965). Other work suggested that protein haze was mostly caused by protein fractions of low pI and M¡ (Mesrob et a\.1983). Recently it was demonstrated that wine protein fractions of low lvL (13,000 and 20,000 to 30,000), low pI (4.1 to 5.8) and containing glycoproteins were the most important since their removal by bentonite conferred heat stability to the treated wines (Hsu & Heatherbell 1987b). However bentonite is not a selective absorbent and fining with this material tends to deplete the treated wines of all protein fractions. An ideal strategy to target the particular proteins involved in heat instability is to demonstrate haze prevention on removal of specif,rc proteins and haze induction on back- addition of that material. This approach was adopted by Bayly & Berg (1967) who used anion exchange chromatography to sepafate wine proteins into four fractions, added these fractions back to a bentonite-fined and protein-stripped wine, and showed that the fraction which gave the greatest fesponse to heat treaünent had the lowest pI. This observation is in agreemenr with the findings of Hsu & Heatherbell (1987b) discussed above. The back- addition technique was also apptied recently to fractions isolated by chromatofocussing (Paetzold et aI 1990) who showed that three glycoprotein fractions with pI of 5.4,6.7 and groater than 9, and with IvIr in the range 20,000 to 30,000, were capable of producing haze. However, the haze potential of these fractions was assessed by heating them in unbuffered, aqueous solution, rather than in wine. This protocol may give misleading results because the haze potential of proteins is likely to change as a function of pH and to depend on other 83 components present in the wine. Indeed, the results from this study (Paetzold et al \990) conflict with data in the literature which demonstrated that wine proteins of low pI were responsible for protein haze (Bayly & Berg 1967, Hsu & Heatherbelt 1987b). The mechanism of haze fomtation in grape juice and wine is still poorly understood. Koch & Sajak (1959) were among the first to suggest an interaction between wine polyphenolic compounds and protein and reported the presence of 57o tannin in isolated wine hazes. Other workers also asserted that hazn,in wine was a tannin-protein complex (Danilatos & Sotiropoulous 1968, Cordonnier l97O).It has been suggested that about 507o of wine protein was bound to a much smaller quantity of flavanoid material and this interacúon made the proteins prone to precipitation (Somers & Ziemelis 1973a). A thorough study by Yokotsuka et al. (1983) showed that isolated protein fractions in model solutions at wine pH interacted with grape tannin fractions to produce significant turbidity. It is likely that this interaction occurs in wines and is responsible for haze however, this hypothesis has not yet been tested. Phenolic oligomeric compounds have a well known ability to complex with and precipitate proteins. This phenomenon is responsible for hazes in beer (Gardener & McGuiness 1977, Asano et aI. 1982, Section 1.3.1) and is involved in the mouth sensation of astringency (Asquith et al. 1987), the impaired nutritional quality of some cereals and the inactivation of digestive enzymes (Pierpont 1935). It has been exploited by plant biochemists who use proteins such as bovine serum albumin, and proline derivatives such as polyvinylpyrrolidone, to scavenge phenolic constituents during plant extractions (Loomis L955, Throneberry 1961, V/einbach & Garbus 1966, Hobson 1970). Polyphenolic compounds are effective as complexing agents because they have many potential binding sites and this, in addition to their molecula¡ size and conformational flexibility, enables them to form stable cross-linked structures with proteins. Phenolics bind to proteins principally by hydrophobic interactions with hydrogen bonding also involved at neutral or higher pH (Haslam & Lilley 1985). Oh and colleagues studied the interaction of grape polyphenolic compounds with model proteins and. showed that, at juice and wine pH, complex formation was the result of hydrophobic bonding between the polyphenolics and the proteins (oh et al. 1980,oh et al.1985, oh & Hoff 19g7). Haslam & Lilley (1985) proposed that polyphenolic compounds bind to protein creating a surface layer which is more hydrophobic than the protein itself, causing aggïegation and further interaction with other polyphenolics. The association of phenolics with proteins is 84 monomefs can bind to strengthened as the phenolic size increases. Although phenolic responsible protein, they lack the crosslinking ability of the polyphenolics, hence afe not protein are also for protein precipitation (Haslam & Lilley 1935). The properties of the have high impoftant. In general, proline-rich and conformationally flexible proteins Ovttrry et al' affinities for phenolics (Flagerman & Butler 1980, Hagerman & Butler 1981, for such 19gT) and it has been confirmed that grape polyphenolics have similar affinity phenolic proteins (oh et aI. 1980). This property is exploited in wine making where proteins compounds are fomoved by precipitation with proline- and hydroxyproline-rich 1987)' Grape such as gelatin, isinglas and casein (Seddon & Eschenbruch 1985, Zoecklein 1979' proteins also have high levels of these amino acids (Anelll t977, Ough & Anelli for many years' Villettaz et at. t982) and complexes with polyphenols have been suspected of haze formation' This chapter characterizes wine haze and discusses possible mechanisms in terms of Wine proteins were fractionated by a variety of techniques and chatactenzed purifred and shown to their M and amino acid composition. Two major wine proteins were be important conEibutors to wine haze. 85 4.2 EXPERIMENTAL The methods relating to individual experiments are given in the relevant Figure or Table legend. General experimental details are given in this section. 4,2.1 Mt rERraLS Ammonium sulphate (Univar grade) was purchased from Ajax Chemicals (NSW, Australia). Acrylamide, N'N'-bismethylone-acrylamide, ammonium persulphate, N,N,N',N'- tetramethylethylene-diamine (TEMED), B ioRad molecular weight standards and BioRad Silver Stain kit were from BioRad Laboratories Pty Ltd (NSW, Austalia). The Boehringer Mannheim Glycan Detection Kit containing: 5-bromo-4-chloro-3-indolyl- phosphate, DlG-succinyl-Ê-amidocaproic acid hydrazide, nitroblue tetrazolium chloride, polyclonal sheep anti-DIG Fab fragments conjugated with alkaline phosphatase and Ponceau S, was from Boehringer Mannheim Australia Pty. Ltd. (NSW, Australia) Bovine serum albumin (BSA), Coomassie Brilliant Blue G and ovalbumin were obtained from Sigma Chemical Company (MO, USA). Bromophenol blue and glycerol were from May & Baker Australia Pty. Ltd. (VIC, Australia). Filter paper (No 1) was from Whaunan (W & R Balson Ltd., England). 2-Mercaptoethanol (2-ME) and sodium dodecyl sulphate (SDS) were from BDH Ltd., England. Nitrocellulose paper (BA 85) was purchased from Schleicher & Schüll. SEP-PAK C-18 cartridges were purchased from'Waters Associates, (MA, USA). Tris(hydroxymethyl)-aminomethane (Tris) was from Merck Pty., (VIC, Australia). 'Water used was purified by a MilliQ reagent water system (Millipore Pty. Ltd., NSW, Ausralia). All other reagents were of the highest purity available. Muscat Gordo Blanco (Gordo, syn. Muscat of Alexandria) wine was sourced from the 1988 and 1989 vintages (Lindemans Wines, Karadoc) and stored at -20oC. 86 4.2.2 GBNERAL METHoDs 4.2.2.L Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (sDs PAGE) Discontinuous SDS PAGE was performed according to the method of Laemmli (1970). The gels consisted of a separating (lower) gel and a stacking (upper) gel. The stacking gel acted to concentrate large (up to 40¡tL) sample volumes, resulting in better band resolution than was possible using the same volumes on a gel without a stacking gel. Molecules were then separated in the separating gel. 4.2.2.1.L Gel polymerization The separating gel monomer solution was prepiled by combining water (3.35mL), 1.5M Tris-HCl (pH 8.8) (2.5mL), 1.07o (wlv) SDS (100pL), acrylamide/bis stock solution (4.0mL) and TEMED (5pL), to give a I2Vo separating gel. The acrylamide/bis stock solution consisted of acrylamide (87.69) and NN'-bismethylene-acrylamide (2.4g) in water (300mL). The separating gel monomer solution was degassed under vacuum for 15 min and then polymeiznd by the addition of freshly prepared I07o annonium persulphate (50FL). The solution was mixed gently and then cast into the glass plate sandwich using a BioRad gel casting stand, to a height of 5cm. The gels were overlaid with isobutanol (approximately 2mL) and allowed to polymerize for at least 90 min. The isobutanol was removed and the gel rinsed with water and then dried using filter paper before the stacking gel was cast on top. The stacking gel monomer solution \¡/as prepared by combining water (6.lrnl-),0.5M Tris- HCI (pH 6.8) (2.5mL),lOVo (Vv) SDS (100pL), acrylamide/bis stock solution (1.3mL) and TEMED (10[L), to give a 4Vo stacking gel. The stacking gel monomer solution was degassed under vacuum for 15 min, then freshly prepared lÙVo annonium persulphate (50ttl-) was added, mixed gently and the polymerizing solution cast on top of the separating gel. A 10 well comb was placed in the top of the stacking gel and the gel polymerized for at least 90 min before the comb was removed and the wells rinsed with water. The gel dimensions were 80 x 60 x lmm. 87 4.2.2.L.2 Sample preparation Samples were diluted at least 5 fold in sample buffer, boiled for 4 min, and then loaded into the wells. Electrophoresis sample buffer was prepared by combining water (425¡tL),0.5M Tris-HCl (pH 6.8) (1251.t"L), glycerol (100pL), l1Vo (w/v) SDS (200pL), 2-ME (100pL) and0.O5%o (w/v) bromophenol blue (50pL). 4.2.2.1.3 Electrophoretic conditions A BioRad Mini-Protean II unit (BioRad Laboratories Pty Ltd) was used to run the gels. They were electrophoresed at a constant voltage setting of 200V until the bromophenol tracker dye was 5mm from the bottom of the gel (usually 40 min). 4.2.2.L.4 Staining After electrophoresis, the separated proteins were fixed and stained by incubating the gel in fixative (methanoVacetic acid/water 4:1:5) containing Coomassie Britliant Blue R-250 (O.IEI) for 30 min and then destained with either fxative or water. Alternatively, gels were fixed after electrophoresis by incubation in fixative for 30 min and then stained for protein with the BioRad Silver Stain kit (BioRad Laboratories Pty Ltd) based on the procedure of Merril et aI. (1981), and according to the instructions provided with the kit. Staining to detect carbohydrate material was accomplished with the periodic acid-Schiff (PAS) stain procedure of Allen et aI. (1976). After fixing, gels were incubated in 0.27o aqueous periodic acid at 4oC for 45 min, in Schiffs reagent at 4oC for 45 min and then destained in L07o acetic acid. Schiffs reagent was prepared by dissolving basic fuchsin (1g) in boiling ',vater (20OmL), stirring for 5 min and then cooling to 50oC. lM HCI (20mL) was added and the mixture cooled to 25oC. Sodium metabisulphite (1g) was added and the solution left in the dark overnight. Activated charcoal (2g) added and the mixture was shaken vigorously for 1 min then filtered. Following PAS staining, some gels were then stained with Coomassie Brilliant Blue as described above. BioRad molecular weight standards (BioRad Laboratories Pty Ltd) were used as M¡ markers. The N4¡ values of unknown samples were calculaæd from the regression equation of log lvl¡ vs. mobility. 88 4.2.2.1.5 Glycoconjugate detection with enzyme immunoassay The Boehringer Mannheim Glycan Detection Kit was used to detect glycoconjugates in fractions which had been separated by SDS PAGE and then transfened to nitrocellulose via the'Western blotting procedure. 4.2.2.1.5.1 Digoxigenin (DIG) labetting The sample was diluted at least 1 in 1 with 0.1M sodium acetate (pH 5.5) to give a2Oþ solution containing between 0.1 to 10¡rg protein. Sodium periodate (6.6791L,10pL) was added and the solution incubated in the dark at room temperature for 20 min. Excess periodate was destroyed by adding sodium metabisulphite (159/L, 10FJ-) and incubating at room temperatue for a further 5 min. The samples were labelled with DIG by adding DIG- succinyl-e-amidocaproic acid hydrazide (15pL of solution in dimethylformamide as supplied by Boehringer Mannheim) and incubating at room temperature for 60 min. 4.2.2.t.5.2 SDS PAGE DIG sample buffer was prepared by dissolving SDS (0.8g) in 0.1M Tris-HCl (pH 6.8) (2.5mL) and adding glycerol (4.0mL), z-l'/ÍE (2.0mL), 0.057o (w/v) bromophenol blue (500É) and water (1.0tiL). For SDS PAGE, the entire sample was diluted into DIG sample buffer (15[L), boiled for 4 min and then loaded into the wells and run as described in Section 4.2.2.1.3. 4.2.2.1.5.3 Transfer to nitrocellulose paper After SDS PAGE, the gel was equilibrated in transfer buffer for 30 min. Transfer buffer consisted of 25mM Tris, 192mM glycine and207o methanol. Fibre pads (supplied with BioRad Mini-Trans-Blot cell), nitrocellulose paper and filter paper were also soaked in transfer buffer for 30 min. The gel sandwich was constructed by placing a pre-soaked f,rbre pad on one panel of the gel holder cassette. A piece of saturated filter paper was placed on top of the fibre pad and further saturated with transfer buffer (3mL). The equilibrated gel was placed on top of the paper and aligned in the centre of the cassette. The surface of the gel was flooded with transfer buffer (3mL), and the pre-wetted nitrocellulose was lowered onto the gel surface and any bubbles removed by rolling a test tube over the top of the membrane. The surface of the membrane was flooded with transfer buffer (3mL) and then the sandwich completed by placing a piece of saturated filter paper on top of the membrane 89 and placing a saturated fibre pad on top of the filter paper. The cassette was closed and placed in the buffer tank so that the gel was on the cathode side and the nirocellulose paper was on the anode side. The tank was filled with buffer and blotting was achieved at constant voltage of 100V for 60 min. The heat generated during the transfer was absorbed by Biolce (as supplied by BioRad). After transfer, the proteins were visualized on the membrane by staining with Ponceau S. The membrane was incubaæd in}.2%o (w/v) Ponceau S in3Vo (w/v) trichloroaceric acid for 5 min and rinsed in water until the bands were visible. The standard proteins and other bands were marked with a pencil dot because the Ponceau S staining disappeared during the following steps. 4.2.2.1.5.4 Glycoprotein detection All the following steps were performed with ttre membrane on a shaking table. The filter was incubated in blocking solution (20mL) for 30 min to cover any sites on the membrane that did not contain transferred protein. Blocking solution was prepiled by dissolving skim milk powder (0.5g) in Tris buffered saline (pH 7.5) (TBS 7.5) (0.05M Tris-HCl, 0.15M NaCl; 50mL), and heating at 60oC for t hour. The filter was washed 3 times for 10 min each with TBS (pH 6.5) (TBS 6.5) (0.05M Tris-HCl, 0.15M NaCl; 50mL), to remove excess blocking protein. The filær was then incubated in DIG antibody conjugate solution for 60 min. This solution was prepared by diluting antiserum (polyclonal sheep anti-DIG Fab fragments, conjugated with alkaline phosphatase F50U/mLl, 10¡rl) into TBS 6.5 (10mL). The filter was washed 3 times for 10 min each with TBS 6.5 to remove excess antibody, and then stained by immersing the filær in staining solution without shaking. The staining solution consisted of 5-bromo-4-chloro-3-indolyl-phosphate (50g/L in dimethlyformamide, 37.5¡tL), nitroblue tetrazolium chloride (77g/L in 70 Vo (vlv) dimethlyformamide, 50pL) in 0.1M Tris-HCl buffer (pH 9.5) containing 0.05M MgCt2 and 0.lM NaCl (10mL). The reaction of these compounds with the alkaline phosphatase bound to the glycoconjugates via the anti-DIG antibody was stopped by rinsing the filter several times in water. 90 4.2.2.2 Infrared analysis Samples, after drying in a vacuum desiccator at room temperature, were analyzed as a KCI disc against a KCI standa¡d between 4000 and 400cm-1, on a Perkin-Elmer Infra¡ed Spectrophotometer 9 8 3. 4.2.2.3 Ultrafiltration For sample volumes less than 10mL, the procedure in Section 2.2.2 using centricon-lO's was followed. Ultraf,rltration of volumes greater than 10mL was performed in a 50mL capacity stirred cell (Amicon Corporation, Danvers, MA, YM-10 membrane, nominal cut off of Mr 10,000) at 5oC under nirogen pressure of 350KPa. The filtrate was collected under a nitrogen blanket to minimise oxidation. The retentate was exchanged with the buffer concerned (50mL) or desalted by reconstituting with water (50mL) and reconcentrated by ultrafiltration. 4.2.2.4 Micromethod for haze potential To determine the haze potential of different proteins, aqueous solutions of protein (0-50pL, made up to 50pL with water) were added to ultrafiltered Gordo wine (1050UL) obtained by ultrafiltration (Section 4.2.2.3). After sparging with nitrogen, samples were sealed. and heated for 6 hours at 80oC and24 hours at 4oC following the procedure of Pocock and Rankine (1973). The samples were then transferred to lml- disposable UV grade microcuvettes (10mm path length) and the haze measured by the absorbance at 540nm. Values were corrected by subtraction of A54g for a control (no protein added before heat testing). Data were subjected to linear regression analysis. Protein concentration of standards was determined spectrophotometrically using published extinction coefficients (Gill & von Hippel 1989) while protein concentration of grape and wine prorein fractions was deærmined from the sum of amino acids after acid hydrolysis in hermetically sealed, microcapillary tubes (Section 2.2.5). 91 4.2.3 ISOLATIoN oF wINE H^zE 1988 Gordo wine was centrifuged (19,0009, 60 min, 4oC) and filtered (0.45pm membrane) to remove sediment. Haze was induced by heating filtered wine (6 hours, 80oC) under N2. After cooling, the wine appeared unchanged in colour but contained a fluffy sediment. This wine was centrifuged as above, the pellet washed twice with water, then transferred to a¡ound bottom flask with water and freeze-dried. The residual haze after lyopholizing was dried to constant weight in a vacuum desiccator at room temperature (yield:98m9,/L). The haze material was a brown solid. 4.2.4 TS0LATION oF WINE PROTEIN 4.2.4.1 Gel permeation HPLC The methodology was identical to that given in Section 2.2.1 except that sample volume was increased to 200pL, detector setting to 2.0 AUFS and integrator attenuation to 16mV. The sample was desalted and concentrated 10 - 40 fold by ultrafiltration through a Centricon-10 microseparation device (Section 2.2.2) before injection. This sample is referred to as Gordo wine protein concentrate. Peaks were collected directly into fresh Centricon-10 devices and concentrated by ultrafiltration (Section 4.2.2.3). Protein was assessed qualitatively by SDS PAGE (see Section 4.2.2.1) and infrared spectroscopy (see Section 4.2.2.2> and quantitatively by the Bradford protein assay detailed in Section 2.2.4. Samples were stored frozen (-20oC) until used. 4.2.4.2 Ammonium sulphate fractionation All procedures were performed at 4oC unless otherwise stated. (NII¿)zSO+ was added to a 1989 Gordo wine þH 3.8) to 60Vo saturation (0.39glml).The precipitate (fraction A) was collected by centrifugation (19,0009, 2 hours), suspended in 0.1M Tris-HCl (pH 6.8) at OoC, and desalted into 67o w/v ta¡trate (pH 3.2) by ultrafiltration (Section 4.2.2.3). The wine supernatant was subjected to further (NFI+)zSO4 addition and the proteins precipitating at 657o saturation (0.43g/nL, fraction X) and 70Vo saturation (0.47g|mL, fraction B) were collected and treated as above. TheT}Vo (NlI+)zSO+ saturated supernatant (fraction C) was dialyzed against four changes of Mitli-Q water (2L), then concentrated by ultrafiltration. One half of this material was further fractionated by six passes through an 92 ultrafiltration membrane with nominal cut off of M¡ 30,000 (YM-30 membrane). The retentate is referred to as D. The pooled filtrates (fraction E) were concentrated by ulrafiltration (Section 4.2.2.3). All fractions were stored at-20oC. 4.2.4.3 Anion exchange chromatography The (NHa)zSO¿ precipitate (70Vo saturation of 1989 Gordo wine) was collected by centrifugation (19,0009,2 hours), suspended in 0.lM Tris-HCl (pH 6.8) ar 0oC, then desalted into water by ultrafiltration (Section 4.2.2.3). The solution was filtered through a SEP-PAK C-18 cartridge to remove phenolic contaminants. The SEP-PAK had previously been activated with methanol (zmL) and washed with warer (5mL). The final stages of the purification were undertaken on a Pharmacia FPLC system at room temperature. The sample was diluted 10 fold in 20mM Tris-HCl (pH S.0) and centrifuged (10,0009, 5min) before loading on a Mono-Q (HR 5/5) column which had been equilibrated with the above buffer. The NaCl concentration was increased from 0 to 500mM over a period of 30 min. Peaks containing the lr,fr 24,000 and 32,000 proteins (as assessed by SDS PAGE, Section 4.2.2.1) were collected and rechromatographed as necessary to obtain both proteins in a purifïed state. Purity was assessed by SDS PAGE (Section 4.2.2.1). Amino acid composition and protein quantification were determined by the procedure in Section 2.2.5, and SDS PAGE was performed as described in Section 4.2.2.1 93 4.3 RESULTS 4.3.1 CSIRACTERIzATIoN oF wINE Id.AzE 4.3.1.1 Characterization of protein SDS PAGE of the grape proteins from a Gordo wine (Figure .l,lane 2) showed that the major constituents were proteins of M, 32,000, 26,000 and 24,000. (The nature of the regression equation used to estimate the lvl, of unknown protein bands results in the assignment of lvf. 32,000 to the fust major band in this lane even though it is not at a position that is clearly higher than the standa¡d protein with known M¡ 31,000). These proteins were also present in a heat-induced precipitate, oÍ haze, isolated from the wine (Figure 4.1, lane 1). 4.3.I.2 Infrared spectroscopy An infrared absorption spectrum obtained (Figure 4.2), indicated that the haze sample tested was predominantly protein. Amide I and II peaks at 1651 and 1510cm-1' respectively, were app¿ìrent and there was little contribution from carbohydrate material judged by the weak absorptions of peaks in the C-O stretching region of 1000 - 1200cm-1 (Bellamy 1975). 4.3.1.3 Solubility properties The amount of haze that dissolved in various solvents is shown in Figure 4.3. All of the haze dissolved in 0.lM NaOH, which is consistent with the haze being proteinaceous. No measurable amount of the haze dissolved in methanol which is an excellent solvent for phenolic material. 207o of. the haze dissolved in 8M urea, a reagent that breaks hydrogen bonds. The components of the SDS PAGE sample buffer were also investigated for their haze solubilizing ability after it was noted that all the haze dissolved in the complete SDS PAGE sample buffer. None of the haze dissolved in the Tris buffer without SDS or 2-l'/ß^ l5%o of the haze dissolved when SDS was included in the buffer, andZOVo dissolved when 2ME was included with the Tris buffer. This gave a total of 357o, whereas buffer containing both of these reagents completely dissolved the haze. 94 ,I 2SKDa 97.4 66.2 42.7 a- 31.0 rI 21.5 14.4 - Figure 4.1 Etectrophoretic separation of the proteins in Gordo wine and haze protein SDS pAGE of Gordo wine Qane 1) and heat-induced haze (lane 2). S = standard mixtrue. Relative molecular mass (Mr x tü3) of standa¡ds are given on the right side of the gel. The gels were run and stained as described in Section 4.2.2.1. Haze was isolated as described in Section 4.2.3. 95 100 BO 60 2931 1230 1059 8 c .9 U' .1t, 3281 E Amide II 240 1510 Lct F Amide I 1651 20 0 4000 3000 2000 1600 1 200 800 400 Wavenumber (cm-1¡ Figure.4.2 Infrared spectrum of Gordo wine haze Ahazn fraction isolated as described in Section 4.2.3 was analyzed in Section 4.2,2.2. 96 100 olto d 80 i s 60 s o 40 I v 20 e d 0 l.laOH urea methanol T ris Tris + Tris + SDS PAGE SDS 2T/E buffer Figure 4.3 Solubility of wine haze The amount of wine haze dissolved in various solvents was determined as follows. flazn (950-1500pg) isolated by the procedure in Section 4.2.3 was placed in preweighed Eppendorf microcentrifuge tubes and either 0.1M NaOH, 8M urea or methanol were added to give an approximaæ f,rnal concentration of lmg/ml-. After 16 hours at room temperature, the samples were centrifuged (10,0009, 5 min), and the pellets resuspended and washed 3 times with water (lml) andrecentrifuged. The final pellet was dried to constant weightin a vacuum desiccator at room temperature. The weight of the Eppendorfs after drying was compared to the initial weight to determine the amount of material that had dissolved in the solvent of interest. The SDS PAGE sample buffer and its components were also investigated for their haze solubilizing ability. The complete sample buffer was composed of water (425ttL),0.5M Tris-HCl (pH 6.8) (125UL), glycerol (100pL), lOTo (wlv) SDS (200pL), 2ME (100UL) and0.l%o (w/v) bromophenol blue (50ttl-). The "Tris + SDS" buffer had 2ME replaced with water (100FL).The "Tris + 2ME" buffer had the SDS replaced with water (200FL). The "Tris" buffer had both the 2ME and the SDS replaced with water (300É). Known amounts of the above haze were placed in preweighed Eppendorf microcentrifuge tubes and to oach, one of the three buffers was added to give a final concentration of lmg/ml-. After boiling for 4 min, the samples were centrifuged (10,0009, 5 min), and the pellets washed once with the appropriate buffer, then 2 times with water (lml-), before drying to constant weight in a vacuum desiccator at room temperature. 97 4.3.2 WTNE FRACTIONS SEPARATED BY GEL PERMEATIoN 4.3.2.L Characterization of protein Two fractions were selected from a preparative I{PLC separation of the grape proteins from a Gordo wine (Figure 4.4). SDS PAGE analysis of the first fraction permitted the observation of faint bands at Mr 64,000, 37,000 and 24,000 (Figure 4.5, lane 3). These proteins were possibly associated in the native state, so that they co-eluted on gel permeation HPLC at an apparent Mr of 120,000 (Figure 4.4). The second fraction isolated was the more abundant protein of the wine and comprised the partially resolved pair of peaks on gel permeation HPLC with apparent Mr of 23,000 and 10,000 (Figurc 4.4). SDS PAGE analysis of this material revealed at least 10 protein bands with Mrranging from 36,000 to 10,000 (Figure 4.5, lane 2). The major constituents had M. of 32,000, 26,000 and 24,000. 4.3.2.2 Infrared spectroscopy Infrared spectra were obtained for the starting material (ultrafiltration retentate) and the two fractions isolated by gel permeation chromatography. These are shown in figures 4.6,43 and 4.8, respectively. The infrared absorption spectrum of the starting material (Figure 4.6) showed a major peak at 1M4cm-1 which is in the C-O stretching region of 1000 - 1200cm-1, but the amide I and tr peaks were not apparent (a:rride I peak near 1650cr¡-1, amide II peak in the range 1570 - 1510cm-1, Bellamy t975). This suggests that the starting material was predominantly carbohydrate in nature. The infrared absorption spectrum of the lst fraction @igure 4.7) also showed a major peak at IO52 in the C-O str,etching region of 1000 - 1200cm-1 in addition to the amide I and II peaks at L652 and 1543cm-1. This result suggests that the fraction contains both polysaccharide and proteinaceous material. 98 I t I T 10 000 Mr I 1 23 000 Mr cE ro C\l c\l (ú c o '<= o_ o¡_ U) _o Fraction 120 000 Mr 1st F ractio n 10 20 30 Elution volume (mL) Figure 4.4 Preparative HPLC separation of proteins in Gordo wine Gordo wine protein concentrate (approximately 40 fotd, ultrafiltration retentate) was separated by HPLC and the peaks marked lst Fraction and 2nd Fraction were collected. The estimated Mt of these peaks is shown. See Section 4.2.4.1 for conditions of chromatography and sample preparation. 99 KDa S 123 97 .4 66.2 I lr- 42.7 at 31.0 .t 21 .5 14.4 (l- Figure 4.5 Electrophoretic separation of proteins in Gordo wine and Gordo wine fractions (lane Fraction (lane SDS pAGE of Gordo wine proteins (lane 1), 2nd Fraction 2) and lst mass (Mr x t0-3) of standards is given 3). S = standard protein mixture. Relative molecular lst Fraction and on the left side of the ge|. The gels were run as described in Figure 4'l' Figure 4'4' 2nd Fraction welp obtained by preparative HPLC as shown in rNsTfiulË UpRARY 01 100 80 8uo c 1404 'ano .9. II E (t, 1543 c 2933 ,s ¿o Amide I 1652 1 052 20 3345 0 4000 3000 2000 1600 1 200 800 Wavenumber (cm-l¡ Figure 4.7 Infrared spectrum of lst Fraction 1st Fraction, isolated by gel perrneation chromatography (Figure 4.4) was analyzed as described in Figure 4.2. t02 The infrared absorption spectrum obtained for the 2nd Fraction (Figure 4.8) showed amide I and II peaks at 1655 and 1543cm-1 respectively, indicating that the fraction tested was predominantly protein, but with some contribution from ca¡bohydrate material judged by the weak absorption of peaks in the C-O stretching region of 1000 - 1200cm-1. This profile was similar to that shown by a haze sample (Figure 4.2). 4.3.2.3 Micromethod for haze potential The development of a micromethod for measuring haze was essential to enable quantitative comparisons of haze potential between different proteins. The commonly used haze assessment by eye was subjective and not appropriate for small scale research. In Figure 4.9,the results obtained when the micromethod (Section 4.2.2.4) was applied to the standard proteins, BSA and ovalbumin are illustrated. The induced heathaze of BSA can be compared on the same basis to that of ovalbumin because concentration of both proteins has been determined from their published extinction coeff,rcients. BSA gave more haze than ovalbumin per unit mass and hence has a higher haze potential in wine. 4.3.2,4 Haze potential When the two fractions shown in Figure 4.4were added back to an ultrafiltered wine, and their heat induced haze poæntial tested, they each exhibited very different responses to heat (Figure 4.10). Protein concentration for these experiments was estimated by the Bradford procedure which was later shown to grossly underestimate wine protein concentration (Section 2.3.3.2). Nevertheless the data show that the 2nd fraction, i.e. that containing protein of Mr 36,000 to 10,000, gave a linear response to heat as did the model proteins shown in Figure 4.9. In contrast to the linear response of concentration to heat shown by all proteins discussed 'When so far, the behaviour of the lst fraction was unexpected- increasing concentrations of this material were added back to a protein-free wine and heated (Figure 4.10), the haze produced by this fraction was maximal at about sttg/nL of added protein and further additions elicited no further turbidity. 80 64 Ð4sG' c o '-u, 1411 .9, ç-2 32 2934 (ú 1 050. Amide II 16 r543 3297 Amide I 1655 0' 400 4000 3SOO gOO0 2SOO 2OOO 18OO 1600 1400 12OO 1OOO 800 600 Wavenumber (cm-1¡ Figure 4.8 Infrared spectrum of 2nd Fraction 2nd Fraction, isolated by gel permeation chromatography (Figure 4.4) was analyzed as described in Figure 4.2. (,O to4 0.400 a BSAC") 0.300 O ovalbumin ('-') cE o st rf) 0.200 (l) N -GI 0.100 0.000 0 20 40 60 80 100 120 140 Protein concentrat¡on (mg/L) Figure 4.9. Influence of the concentration of BSA and ovalbumin on heat-induced haze in wine. BSA and ovalbumin were added to protein-free wine and incubated as detailed in Section 4.2.2.4. (***) significant linea¡ correlation at 0.0017o level. 0.800 E 1st frac{ion 2nd fraction ('*') 0.600 I E c, os rog 0.400 (l) N ct I 0.200 0.000 0 10 20 30 40 50 Protein concentration (mg/L) Figure 4.10 Influence of protein concentration on the heat induced haze of wine protein fractions in the wine 1st Fraction and 2nd Fraction were obtained by preparative HPLC as described in Figure 4.4. These fractions were added to Gordo wine ultrafiltrate and haze was induced and measured as described in Section 4.2.2.4. Protein concentration was measured by the Bradford assay (Section 2.2.4). (***) signifrcant linear correlation at 0.0017o level. 105 4.3.3 Wrxn FRAcTroNs sEpaRATED By AMMoNTuM suLpHATE PRECIPITATION 4.3.3.1 Characterization of protein The fractionation of 1989 Gordo wine proteins by (NII¿)zSO4 precipitation is shown in Figure 4.11. The major protein component of fraction A [precipitated by 607o satwation of (NH¿)zSO¿] had a Mr of 32,000. In fraction B [precipitated by 65 to707o saturation of (NIIa)2SOa] the major component was a protein of Mr 24,0ffi. Material precipitating between 60 and 65Vo saturation (fraction X) was of composition intermediate between that of A and B, (it contained both the N4r 32,000 and 24,000 proteins in equal proportions), and as such was not sufficiently distinct to warrant further study. The protein composition of the fraction that remained soluble at 70Vo saturation of (NII+)zSO+ (C) differed from that of fractions A and B in having two major proteins of M¡ 26,000 and 24,000, a minor protein band at l\,fr 63,000 (Figure 4.11) in addition ro several apparently higher M¡ bands which stained strongly with the PAS reagent (PAs-srained electropherograms not shown). This positive PAS response suggested a polysaccharide component and these macromolecules a¡e discussed in Chapter 5. The proteins of fraction C were separated from polysaccha¡ide material by ultrafiltration through a membrane with a nominal cut off of Mr 30,000. This brought about an enrichment of the lvlr 26,000 and 24,000 proteins in the fîltrate E (Figure 4.11). The retentate (fraction D) showed a decreased level of these proteins but retained the PAS- positive polysaccharide material (not shown) in addition to the Mr 63,000 protein band @igure 4.11). 106 KDa StdA X B C D E 97.4 66.2 42.7 31.0 21 .5 14.4 Figure 4.11. SDS PAGE of (NH4)2SO4 fractions of wine. Gordo wine protein was fractionated by (NHa)zSO¿ precipitation. Fraction A was the material precipitated with 607o saturation of (NHf2SO4 and is show in lane A. The other lanes are as follows: X - material precipitated between 60 and 65Vo saturation of (NHa)2SOa; B - material precipitated benveen 65 and TOVo saturation of (NHa)2SOa; C - material soluble at7ÙVo saturation of (NlIa)2SOa; D - component of C retained by an ultafilter with M, 30,000 cut off; and E - component of C not retained by an ultrafilter with 30,000 cut off. Relative molecular mass (l\4. x t0-3) of protein standards (Std) are given on the left side of the gel. r07 4.3.3.2 Evidence for glycoproteins In addition to the sugar specifîc PAS stain discussed above, a sensitive enzyme-linked immunoassay was used to test for the presence of glycoproteins in the fractions. Before SDS PAGE separation of the fractions and transfer to nitrocellulose, the adjacent hydroxyl groups in sugars of any glycoconjugates present were oxidised to aldehyde groups by mifd periodate treatment and the steroid hapten, digoxigenin (DIG) covalently attached to these aldehydes via a hydrazide group. DlG-tabelled glycoconjugates were subsequently detected on nitrocellulose paper in an enzyme immunoassay using an antibody alkaline phosphatase conjugate. This protocol is schematically described in Figure 4.12. DlG-conjugation did not noticeably effect the SDS PAGE mobility of the fractions or the standard proteins (Figure 4.13) and the high M. components of fractions C and D were now visible with the silver stain (lanes 7 and 8 respectively). V/hen untreated, rhese components could only be seen after PAS staining. DIG labelling also appeared to increase the sensitivity of the silver stain for all proteins and not just the high M, glycoconjugates. Both the untreated and treated standard proteins were loaded at the same concentration but the treated standa¡ds stained much stronger than the untreated standards. After transfer onto nitrocellulose, the proteins were detected with the protein stain Ponceau S and marked with a pencil dot @onceau S staining disappeared in the subsequent staining steps). The DlG-conjugated compounds were immunologically bound to an enzyme-linked antibody. The bands to which this complex bound were then revealed by the enzyme reaction, resulting in a dark colouration (Figure 4.14). The glycoprotein standard, transferrin (lane 1), gave a strong positive result. The untreated protein standards (lane 2) did not stain, however, all the treated standa¡ds (lane 3) showed some response to the immunoassay. Carbonic anhydrase (band at Mr 31,000, lane 3) gave a strong positive response, simila¡ to transferrin (lane 1). BSA (band at Mr 66,200, tane 3) and phosphorylase b (band at Mr 97,4D,lane 3) responded very faintly as did ovalbumin (band at Mr 42,700,Iane 3). The response shown by ovalbumin was expected because ovalbumin contains 47o sugar and is a glycoprotein (Dubray &Bezard 1982). Carbonic anhydrase, phosphorylase b and BSA are not recognized as glycoproteins, but other workers have noted that phosphorylase b and carbonic anhydrase stained with a different carbohydrate stain, and they suggested that this result indicated that the proteins were indeed glycosylated (Dubray & Bezard 1982). The instructions provided with the Glycan 108 glycoconjugate l. tn ox¡dat¡on with periodate l-r solution labeling with D I G-succinyl-e-am ido- caproic acid hydrazide Ða sDS polyacrylamide gel electrophoresis and transfer onto nitrocellulose (DlG> + AP on the f ilter + substrate for the reaction with AP (X-phosphate, NBT) o sugar I oxidized sugar DIG-succinyl-e-amidocaproic acid hydrazide Figure 4.12 Principle of glycoconjugate detection Adjacent hydroxyl groups in sugan of glycoconjugates a¡e oxidised to aldehyde groups by mild periodate treatment. The spacer linked steriod hapten digoxigenin (DIG) is then covalently attached to these aldehydes via a hydrazide group. Digoxigenin labeled glycoconjugates are subsequently detected in an enzyme immunoassay using an antibody alkaline phosphatase conjugate ( KDa 123456789 97.4 66.2 42.7 + 31.0 + 21.s + 14.4 + Figure 4.13 SDS PAGE of DIG - conjugated (NH+)zSO+ fractions of wine. Gordo wine protein was fractionated by (Nþ)zSO+ precipitation as described in Figure 4.11. The fractions, the protein standards and the glycoprotein standard, transferrin, were conjugated with DIG as described in Section 4.2.2.L.5.1 before analysis by SDS PAGE (Section 4.2.2.1.5.2.) and staining with the silver stain (Section 4"2.2.1.4). DlG-conjugated tansferrin is shown in lane 1. The untreated standards are shown in lane 2. The DlG-conjugated standards are shown in lane 3. Their relative molecular mass (lr4 x 1g-3¡ are indicated on the left side of ttre gel. DlG-conjugated (NH¿)zSO¿ fraction A is shown in lane 4. DlG-conjugated (Nþ)zSO¿ fraction X is shown in lane 5. DlG-conjugated (Nþ)zSO¿ fraction C is shown in lane 7. DlG-conjugated (NH¿)zSO+ fraction D is shown in lane 8. DlG-conjugated (Nþ)2SOa fraction E is shown in lane 9. 110 KDa123456789 97.4 -> 66.2 -> 42.7 è 31.0 > 21.5 + Figure 4.14 Western blot of DIG - conjugated proteins transferred to nitrocellulose The wine fractions and standards described in Figure 4.13 were stained for sugars with the immunoassay described in Section 4.2.2.1.5.4. The dots indicate the position of bands which stained for protein with Ponceau S (Section 4.2.2"I.5.3). DlG-conjugated Eansfenin is shown in lane 1. The untreated standards a¡e shown in lane 2. The DlG-conjugaæd standards a¡e shown in lane 3. Their relative molecular mass (Mr x 10-3) are indicated on the left side of the gel. DlG-conjugated (NIIa)2SO+ fraction A is shown in lane 4" DlG-conjugated NH¿)zSO¿ fraction X is shown in lane 5. DlG-conjugated (NH¿)zSO¿ fraction B is shown in lane 6. DlG-conjugated (NII+)zSO+ fraction C is shown in lane 7. DlG-conjugated (NlI¿)zSO¿ fraction D is shown in lane 8. DlG-conjugated (NlI¿)zSO¿ fraction E is shown in lane 9. 111 Kit state that sugars are sometimes bound to BSA in commercial preparations, which would explain the response seen here (band ar Mr 66,2}O,lane 3) with BSA in the protein standa¡d mixture (Boehringer 1989). Nevertheless, the faint response seen with these "non-glycosylated" standard proteins demonstrated that there may be some non sugar specific staining with this assay, and faint staining could not be taken as evidence of glycoconjugation. The results Of DlG-conjugation of fractions A, X, B-E are shown in lanes 4-9 (Figure 4.14). Fraction A showed a strong band at Mr 34,000 (Figure 4.I4,Iane4). This band was not visible by the protein stain Ponceau S, but its location indicated that it is the fi¡st faint brown band seen on silver staining (Figure 4.13, lane 4). This same band gave a stronger response in fraction X (Figure 4.I4,lane 5), which also contains more of the thin brown band at 14 34,000 seen with silver staining @igure 4.13, lane 5). Other bands in these two fractions and fraction B (Figure 4.l4,lane 6) either stained faintly, as d.id the non- glycosylated standards, or did not stain at all, despite a strong reaction with the protein stain Ponceau S. There was also a reaction in the high M. region of the filter in fractions A, X and B, indicating an incomplete separation of the (NH4)2SO4 precipitated proteins from the heavily glycosylated material which was soluble in (NH4)ZSO¿ (Fraction C, Figure 4.14, lane 7). For fraction C and D (Figure 4.14, lanes 7 and.8), there was a stong positive reaction in the high Mr region of the filter as seen previously with the PAS stain, confirming that the high lvlr components were glycosylated. The other low lvl, proteins that stained with Ponceau S, in these two fractions and fraction E (Figure 4.l4,Iane 9), did not stain with the immunoassay. 4.3.3.3 Amino acid composition The amino acid composition of fractions A, B and E were determined by HpLC analysis of acid-hydrolysed protein samples as described in Section2.2.5.In order to minimize losses of labile amino acids, hydrolysis was ca:ried out in hermetically sealed microcapillary tubes (Lui & Boykins 1989) with 2ME included to improve the recovery of uyptophan (Gruen & Nicholls L972, Ng er al. 1987). Asparagine and glutamine are converted to aspartic acid and glutamic acid respectively, on hydrolysis. Results for the asparagine plus aspartic acid and glutamine plus glutamic acid are thus reported as ASX and GLX, rcspectively. proline was not detected with the derivatization technique employed (IÆkhart et aL l9B2). IT2 The amino acid composition of the (NH¿)ZSO4 fractions is shown in Figure 4.15. Taken in total, these were consistent with reports on amino acid composition of proteins from grapes, juices and wines of more than 15 varieties (Koch & Sajak Lg5g, Pavlenko & Datunashvili 1969, Anelli 1977, Yokotsuka ¿r al.1977,Ough & Anelti lgTg,yillettaz et al.1982, Nakanishi et al.1989). All fractions were rich in the amino acids ASX and GLX. Since it is known that grape and wine proteins are acidic (Radola & Richter lgT2,Anelli 1977, Yokotsuka et al. 1977, Hsu & Heatherbell 1987a, Correa et al. 1988, Moio & Addeo 1989) one can assume that the ASX and GLX fractions were dominated by aspartic and glutamic acids, respectively. The aliphatic amino acids, glycine, alanine, serine and threonine, were also abundant in the fractions. 4.3.3.4 Protein quantification Initially, protein concentration in the (NHa)2SO4 fractions was determined by the Bradford assay (Section 2.2.4), but, as discussed in Chapter 2, this assay considerably underestimated the true values (Section 2.3.3.2). Such inaccuracy was unacceptable when comparison between different proteins, based on absolute concentration, was needed. For all further studies, protein was quantified from the sum of masses of amino acids released after acid hydrolysis of the protein. 4.3.3.5 lIaze potential The data recorded in Figure 4.16 were obtained when the micro-method was applied to the (NH¿)zSO4 fractions A to E. It can be seen that fractions B and E gave the greatesthaze per unit mass of protein. It may be significant that these two fractions share the lrrlr 24,0t0 protein band as a major component (Figure 4.11). Fraction A, the least soluble, gave a heat-haze response approximating half that of B or E. This observation suggests that the lv1¡ 32,000 major protein component of A may not be as important to haze formation as the major proteins of B or E, because, at the same concentration, the M¡ 32,000 protein only gave half the haze of the latrer two fractions (see Figure 4.16). In comparison, fractions C and D gave little turbidity, indicating that the components they share are not contributing greatly to haze formation. Their concentration versus induced haze curves were not fitted by a linear regression model (see Figure 4.16). Instead, the data points for these two may reach a plateau, as previously observed with the 1st fraction from gel permeation (Section 4.3.2), which has an SDS PAGE profile similar to rhat of fraction D. These carbohydrate-rich fractions a¡e discussed in chapter 5. 113 l- Aliphatic--1 Aromatic ¡Basicl Acidic 20 15 10 A 5 0 20 15 10 B 5 0 20 c 15 -E€o'õ >8 10 E *Eo () 5 0 q à ã 3 _s ffi Ë ËËÊ s-q? ãð =h AfúINOACID Figure 4.15 The Vo molar composition of amino acids detected in (NH¿)zSO4 fractions of wine Amino acid composition of the fractions obtained by (NHa)2SO4 precipitation (Figure 4.11) was determined as described in Section 2.2.5, and is expressed as Vo molar composition. tr4 0.300 o Ê c 0.200 -to lr) o o N (ú 0.100 :E o o A 0.000 0 25 50 75 100 125 150 Protein concentration (mg/L) rA (***) trB (***) oc (ns) AD (ns) oE (***) Figure 4.16. Influence of concentration of (NHa)zSO+ wine protein fractions on heat- induced haze in wine. Fractions A to E isolated as described in Figure 4.11, were back-added to protein-free wine and incubated as detailed in Section 4.2.2.4. (***)significant linea¡ correlation at 0.0017o level (ns) no signihcant linea¡ correlation at 0.052o level. 115 4.3.4 PunTnTnn wINE PRoTEINS ISoLATED BY ANIoN ExCHANGE CHROMATOGRAPHY 4.3.4.L Chromatographic separation The separation of wine proteins on an anion exchange column at pH 8.0 is shown in Figure 4.17. A small amount of material was not bound to the exchange resin but most of the material eluted as a group of partially resolved peaks. SDS PAGE analysis of 2mL fractions collected tlroughout the ch¡omatogram indicated that the first peak contained a lylr 24,OW protein, the middle section contained a mixture of a I\4. 24,000,26,000 and 32,000 protein, and a lvfr 32,000 protein eluted last. A preparative run was performed and the two peaks of interest (N4 24,000 and 32,000) were collected. Their composition was assessed by SDS PAGE and the electropherogram is shown in Figure 4.18. 4.3.4.2 Amino acid composition Figure 4.19 shows the amino acid compositions of the À4 24,000 and 32,000 proteins as well as of the starting material before and after treatment to remove phenolic compounds. The latter was achieved by passage through a C-18 SEP-PAK. The untreated starting material is thus coded "G707o pellet" and the treated "C-18 treated. sample". C-18 treatment was necessary to preserve the life of the FPLC column, because phenolic compound.s bind irreversibly to these high resolution columns (Jervis & pierpont 19g9). As expected, the amino acid compositions of the 0-70Vo pellet and the C-l8 treated sample were almost identical, indicating that the protein composition was unaltered by passage through the reverse phase adsorbent. Both samples contained large proportions of the acidic amino acid, ASX, and were also rich in the aliphatic amino acids, glycine, alanine, serine and threonine, and the aromatic amino acids phenylalanine and tyrosine. The amino acid composition of the two purified proteins were distinct from each other. The main difference was in their serine and threonine content, the ìvÍr 32,000 had. more serine and the lvfr 24,000 more threonine. There were also differences in aromatic and basic amino acids. The lt4 24,000 protein had more phenylalanine, and more of all the basic amino acids. 116 2.O r-- 1.00 1.5 o.75 I o @ c (\t .9 S 1.0 (ú ) 0.50 c c q) () Þ c I o o- O õ z(ú 0.5 0.25 0.0 0.00 0 B 15 24 32 Elution volume (mL) Figure 4.17 Separation of wine proteins by anion exchange chromatography The wine protein fraction precipitated bV NII¿)zSOa (0-707o saturation) was separated on a FPLC-Mono Q column at pH 8.0 with a 0 to 0.5M salt gradient (Section 4.2.4.3).The hatched regions were collected" lt7 12 97 "4 66.2 < 42.7 + 31"0 < 21"5 <14"4 Figure 4.18 Electrophoretic analysis of FPlC-purified proteins Purified proteins were obtained by preparative anion exchange chromatography as shown in Fignre 4.17 and described in Section 4.2.4.3. SDS PAGE of the fraction collected in the hatched region L: i: i¡ is shown in lane I and of the fraction collected in the haæhed region Íl/////tÀ is shown in lane 2. The positions of the standard proteins, with their relative molecula¡ mass (lvt, x t0-3) are given on the right side of the gel. The gels were run as described in Section 4.2.2.I. 118 0-70% pellet c o 20.00 T"m p 15.00 mo os 10.00 ti at 5.00 ri 0.00 o GLY ALA VAL ILE TEU SER THR PFE TYR TRP LYS ARG HIS ASX GIX [/ET n C-18 treated sample c o o/o lfl 20.00 P 15.00 mo OS 10.00 li at 5.00 ri 0.00 o GLY ALA VAL ILE I.EU SER THR PI€ TYR TRP LYS ARG HIS ASX GTX [/ET n 32K protein c o 20.00 !"m p 15.00 mo os 10.00 t¡ at 5.00 ri o 0.00 n GLY ALA VAL ILE Lzu SER THR PI+ TYR TRP LYS ARG HIS ASX GU( h/ET 24K protein c o 20.00 1"m p 15.00 mo os 10.00 ti at 5.00 ri o 0.00 SËR ARG GIX n GLY ALA VAL ILE Lzu THR Pl+ TYR TRP LYS HIS ASX I/Et Figure 4.19 The molar 7o composition of amino acids detected in (NH¿)zS o ¿ precipitated protein, FPLC starting material, and FPl,c-purifïed proteins The amino acid composition of the fractions described in 4.2.4.3 was detennined as described in Section 2.2.5, and is exprcssed as 7o molar composition. 119 The protein concentration and the yield of the purification a¡e shown in Table 4.1. These results demonstrate that the C-18 pretreatrnent to remove phenolic compounds depleted the total protein concentration by 607o.However, the protein composition of the sample afær C-18 treatment was not altered as evidenced by the amino acid composition @igure 4.19) and the electrophoretic profile (data not shown). These results suggest that the C-18 removed an equal proportion of all the proteins present, possibly as a result of their associaúon with polyphenolic compounds. The two purified proteins totalled 74Vo of the starting material, or 40Vo of that loaded onto the column (i.e.4O7o of C-l8 treated sample). 4.3.4.3 Haze potential The haze potential of the two purified proteins was established by the micromethod (Section 4.2.2.4), and the induced heat haze versus protein concentration graphs are shown in Figure 4.2O.'the l\& 24,000 protein caused more haze, at the same concentration, than the lvI, 32,000 protein. t20 Table 4.1 Protein concentration and yield from anion exchange chromatography * Fraction mg/L wine 7o yield 0-707o pellet 99.49 100 C-18 treaæd sample 40.21 40 l\4 24,000 protein 7.93 8 lvlr 32,000 protein 6.36 6 Mr24,000 + 32,000 t4.29 L4 * proæin concentration is expressed as mg protein per lite of wine from which all fractions were obtained. I2l 0.400 a O 0.300 cE o rf)s 0.200 a O Mr 32,000 protein o N O Mr 24,000 protein (ú I 0.100 0.000 0 20 40 60 80 100 Protein concentration (pg/ml) Figure 4.20 Influence of concentration of FPlC-purified proteins on heat-induced haze in wine. The M, 24,000 protein (hatched region [::::::1, Figure 4.17) and M, 32,000 protein (hatched region futzttttf,, Figure 4.17) were back-added to protein-free wine and incubated as detailed in Section 4.2.2.4. (***) significant linear correlation at 0.0017o level L22 4.4 DISCUSSION V/ine haze was shown to be predominantly protein without major quantities of carbohydrate or phenolic material. The formation of haze and. is insotubitity appeared to be the result of hydrophobic interactions amongst wine proteins combined with the formation of disulfide bridges. In view of the necessity of both z-M{E and SDS for complete solubilisation, and their synergistic action, it seemed that the disulfide bridges were both intra- and intermolecular. One can surmise that 2-ME could only effect complete solubilisation if SDS was present to expose intramolecular disulphide bonds. All the major wine proteins were present in haze, as observed by Hsu & Heatherbell (1987b). Thus haze doesn't result from precipitation of a single grape protein fraction. Nevertheless, some proteins may be more heat-unstable than others and to investigate this it was necessary to separate, isolate and assess each fraction for haze potentiat. This in turn required development of a micromethod for measuring heat instability because the commonly used method of haze assessment by eye was not objective and was inappropriate for small scale research. The micromethod for haze potential developed here used spectrophotometry to determine both the presence and extent of haze formation. Haze from proteins back added to and heated in wine ultrafiltrate was correlated with the absorbance of a lml. sample at 540nm. In a clea¡ sample, absorbance was negligible, but as the solution became cloudy, haze particles scattered the light and transmittance was diminished. Ultrafiltered wine was chosen as a medium to mimic the environment under which protein precipitation naturally occurs. Because ultrafiltration removes proteins and polysaccharides with M, $eater than the filter cutoff size (10,000), the ultrafiltered wine was essentially coltoid free and stable to heat. This allowed an analysis of the heat potential of specific proteins which were back- added. Not all wines can be heat stabilized,by ultrafiltration because some wines have low Mt proteins which can pass through the membrane (Hsu et al. 1987, Flores et al. L990). Such wines would therefore be unsuitable for the micromethod due to the background.level of unstable protein. The typical heat response of proteins in wine was shown by the model proteins BSA and ovalbumin, the latter a glycoprotein. As their concentration increased, more haze was produced. BSA also gave more haze per mass of protein than ovalbumin demonstrating that proteins have a specific, distinct response to heat. t23 The haze potential of the fractions isolated in the first step of the purification scheme, gel penneation chromatography, were very different to each other. In contrast to the model proteins, the lst fraction gave a low and non-linear response to heat. This fraction comprised high lt4 components and carbohydrate material and a possible explanation for its behaviour was that some of these components may have acted as protective colloids, preventing haze. The phenomenon of haze protection from such macromolecules is discussed in Chapter 5. The 2nd fraction which contained the majority of wine proteins, with lr4, rarige of 36,000 to 10,000, gave a linear response to heat as did the model proteins d.iscussed above. This result supported earlier work (Ngaba-Mbiakop 1981, Heatherbell et al.19g4, Hsu & Heatherbell 1987a, Hsu & Heatherbell 1987b, Hsu ¿r al. L987,paetzold et at. I99))which identified a fraction of M, 30,000 to 20,000 as the most important to protein instability. Ammonium sulphate precipitation was then used to isolate three fractions each dominated by the major wine proteins present in the 2nd fraction, i.e. the M. 32,000,2g,000 and 24,000 proteins. These proteins were charactenzed.and their haze potential assessed before further purification begatt. All the fractions were rich in the acid.ic amino acids and contained low concentrations of the basic amino acids. This result is consistent with all reports in the literature (Koch & Sajak 7959' Pavlenko & Datunashvili 1969, Anelli 1977, Yokotsuka et al. 1977,Ough & Anelli 1979, VillettaT et 1982, al. Nakanishi et at. 7989)excepr that of pae tzo?d. etøl. (1990) who found that juice proteins contained between 8Vo and,76Vo of thebasic amino acid, arginine, including a fraction with surprisingly low pI (nominatly less than 4) which contuned32zo arginine and only ITvo of total acidic amino acids. The low concentration of basic amino acids in the proteins examined in this present study may also account for the underestimation of protein concentration by the Brad.ford assay, as discussed in Chapter 2. The concentration of the amino acids serine and threonine was also high suggesting that glycoconjugated proteins may be prosent in wine. This is because the hydroxyl groups of these amino acids have a functional role in o-glycosid.ic linkages of glycoproteins @eeley 1985)' The higher M. components of wine (i.e. greater than Mr50,000) were indeed glycosylated giving a strong positive response to the sugar specific pAS stain and to a sensitive immunoassay for glycoconjugates. The lower I\4, proteins however, did not react with the PAS stain and only a minor protein with lr4, 34,000 was shown to be a glycoprotein with the more sensitive immunoassay. Hsu & Heatherbell (l9g7a) utilizing a 124 reagent based on the lectin, Concanavalin A, detected th¡ee glycoprotein fractions of lv1, 13000, 25000 a¡rd 28000 in Gewürztraminer wine. These proteins were not detected in this present study, perhaps because the concentration of sugar in these proteins was below the detection limit of the stains used or they were not present in the wine under study. The haze potential of the (NlI¿)zSO4 fractions was assessed by the micro-method. The fractions containing the lr4 24.900 protein as a major component, gave the greatest hazeper unit mass of protein. The fraction containing the M, 32,000 protein, gave aheat-haze response approximating half that of those containing the M, 24,000 protein. This observation suggested that the l\4 32,000 protein may not be as impo,rtant to haze formation as the IvIr 24'000 protein. Purification of these two proteins to homogeneity demonsgated that although the Mr 32,000 protein contributed significantly to wine haze, the Mr 24,000 protein fraction was indeed more important, because it gave twice the haze of the M¡ 32,000 protein at the same concentration. Identification of the lvlr 24,000 protein as the most important to heat haze has been independently supported by a recent rcport which suggested that juice fractions containing a 21,000 IVI¡ protein were important to hazn (Paetzold. et aI. 1990).The photograph of the gel presented by Paetzold et al. (1990) showed sufficient simitarity to the gel shown in Figure 4.11 to suggest that the protein designated as Mr 21,000 may be the same as the Mr 24,000 species studied here. This study has shown that wine proteins respond differently to heat and although all the proteins are present in haze, some give more haze on a weight basis than others. The contribution of these proteins to wine instability is therefore greater and their removal from the wine more critical. r25 Chapter 5 HAZE PROTECTIVE POLYSACCHARIDES 5.1 INTRODUCTION AND LITERATURE REVIEW The role of complex carbohydrates in haze formation of various fruit juice beverages has been well established. In apple juice and wine it is thought that pectins form associations with phenolics, proteins and cations which in turn prevents their precipitation (Fleatherbell I97 6). Lemon juice can develop a cloudiness due to a complex of protein and pectin, where 50Vo of the pectin is entrapped in a protein matrix (Klavons & Bennett 1985, Klavons & Bennett 1987). In both cases pectins act as stabilizing-protective colloids which hold insoluble particles in suspension and prevent their aggregation and precipitation. Gum arabic, a polymer of galactose with side chains of rhamnose and glucuronic acid, is a typical protective colloid (Ribéreau-Gayon et al. 1977). Ir is exuded. from the bark of African acacias and when added to wine it either prevents the flocculation of suspended particles or the formation of deposits and cloudiness (Peynaud 1984). It has been suggested as a preventative measure against iron and copper haze (Ribéreau-Gayon et al. 1977). Protective colloids also exist naturally in wine and hinder clarification (Ribéreau- Gayon et al.1977). It has been proposed that pectinase enzymes improve the efficiency of fining agents by hydrolysing the pectins acting as protecúve colloids. Their elimination then allows contact between the components to be removed and the fining agents (Flores et al. 1e88). Usseglio-Tomasset (1978) speculated that the phenomena of wine protein haze was a result of molecular associations between proteins, polysaccharides and tannins. More recently, small amounts of polysaccharides were found associated with protein sediments of ultrafiltered juices (Flores et al.1988). Interaction of polysaccharides with other molecules has been well documented. The cyclic polysaccharide: cyclodextrin, interacts with the milk protein, ß-casein, and inhibits its aggregation. It has been established that cyclodextrin forms inclusion complexes with fatty acids, amino acids and some flavour compounds of suitable size, shape and polarity and it was thought that cyclodextrin also formed an inclusion complex with ß-casein, such that the hydrophobic amino acid groups we e included in its hydrophobic cavity (Lee & r26 Fennema 1991). Two methylated polysaccharides from Mycobacterium smegmatis also formed helices. It was suggested that they regulate fatty acid metabolism by binding long- chain acyl coenzyme A derivatives in the nonpolar cavity of the coiled polysaccharide chain (Hindsgaul & Ballou 1984). Yeast cell wall mannoprotein is likewise thought to form inclusion complexes with volatile compounds (Voilley et a\.1990). It also has excellent emulsifying properties, solubilizing a range of oils, hydrocarbons and organic solvents (Cameron et aL.7988). In this study, the interactions of wine polysaccharides with wine proteins has been investigated and shown to effect protein haze formation, resulting in an altered degree of visible haze. 127 5.2 EXPERIMENTAL The methods relating to individual experiments are given in the relevant Figure or Table legend. General experimental details are given in this section. 5.2.1 M¡,TERIALS Concanavalin A Sepharose and disposable columns (50 x 1.5 mm) to contain the gel were purchased from Pharmacia Australia Pty. Ltd. (NSW, Australia). The BPl fused silica capillary column (20m x 0.33mm I.D., 0.25pm film thickness) was purchased from SGE International Pty. Ltd. (VIC, Australia). Citric acid, methyl-ø-D-mannoside, myo-inositol, p-nitrophenyl-ß-D-galactopyranoside and piperazine-HCl were obtained from Sigma Chemical Company (MO, USA). Resorcinol (Univar grade) was purchased from Ajax Chemicals (NSW, Australia). Trisil reagent and trifluoroacetic acid were purchased from Pierce (IL, USA). Water used was purified by a Milli-Q reagent water system (Millipore Pty. Ltd., NSW, Ausffalia). All other chemicals were of the highest purity available. Muscat Gordo Blanco (Gordo) wine made from grapes grown in the Riverland, SA, was sourced from the 1989 and 1990 vintages (Lindemans Wines, Karadoc) and 1991 vintage (Penfolds'Wines, Nuriootpa). The wine was stored at -20oC. 5.2.2 GpNERAL METHoDS 5.2.2.1 Polysaccharide quantification 5.2.2.1.1 Total neutral sugars The total neuffal carbohydrato content of samples was estimated by the resorcinol assay (Monsigny et al. 1988), as follows: 69ll. resorcinol (200pL) and 757o sulphuric acid (lml-) were added to the sample (200pL). The sulphuric acid was propared by adding 987o (v/v) sulphuric acid (100mL) to water (24ÍnL). The samples were incubated at 90oC for 30 min and left on ice for 30 min, before their A43g ¡6 wÍts determined. p-Nitrophenyl-ß-D- galactopyranoside was used as the standard for calibration. The concentration of the 128 standard was determined from the A3gg ¡¡¡ using the published extinction coefficient of 10,800 (Monsigny et al.1988). All assays were made in triplicate. 5.2.2.1.2 AcÍd hydrolysis and monomer analysis For the analysis of sugar monomers present in purif,red polysaccharides, the samples were hydrolysed in the presence of the intemal standa¡d, myo-inositol, in 2M trifluoroacetic acid at 110oC for 6 hours. After cooling, the acid was removed under a stream of dry nitrogen gas and then the hydrolysate was dried under reduced prcssure in a desiccator over separate containers of phosphorous pentoxide and sodium hydroxide pellets. Trisil reagenr (150FL) was added to the residue from the acid hydrolysate. By this treatment the components sugars, as well as the internal standard, were converted quantitatively to their trimethylsilylates. A sample (lUL) of this mixture was injected into the gas chromatography column. Gas chromatography was performed on a Varian instrument equipped with a flame ionization detector. A BPl fused silica capillary column (20m x 0.33mm I.D., 0.25pm f,rlm thickness) with helium as the carrier gas was used with a temperature program of 1 min isothennal at LãOOC,2oC/min from 120oC to 180oC, and then 10 min isothermal at 180oC. Injections were made with a split injector at 250oC (1/10 split ratio). Peaks were integrated by a LDC Milton Roy integrator. The identity of peaks was determined from the retention times of standards chromatographed under the same conditions. Under these conditions, the second peak of both the mannose and galactose anomeric pair co-eluted. The proportion of each sugar in this peak could be determined from the peak area of the fust peak because the expected peak area ratio of the anomers had previously been determined, separately, for galactose and mannose under identical sample preparation conditions. The amino sugars, galactosamine and glucosamine, also co-eluted and they are reported as "hexosamine". The relative amount of the component sugars was determined by comparing their peak ar€as. 5.2.2.2 Micromethod for haze potential To determine the effect of carbohydrate additions on the haze potential of proteins, the micromethod for haze potential (Section 4.2.2.4) was modified in the following manner. To a constant known amount of protein (5¡rL of either wine protein precipitated by 0-70Vo saturation of (NFIa)2SO4 or BSA), aqueous solutions of carbohydrate rich fractions (0- 50pL made up to 50pL with Milli-Q water) were added to ultrafiltered Gordo wine 129 nitrogen' (1050pL, obtained by the procedure in Section 4.2.2.3)' After sparging with at 4oc, as described in samples were sealed and heated for 6 hours at 80oc and24 hours Section 4.2.2.4. 5.2,2.3 Particle size measurement using a The particle size of hazes was measured by Photon Correlation Spectroscopy (4mL) were analyzed at Malvern Autosizer IIc (Malvern Instruments, England). Samples 25oC after vigorous shaking to disperse the flocculatdhaze. 5.2.3 PUUFICATION 5.2,3,1 Concanavalin A affinity chromatography l'5mm, column volume Con-A Sepharose was packed into a disposable column (70mm x (pH 5'6) approximately 7mL) and equitibrated with 50mM sodium acetate buffer (starting buffer). containing 150mM NaCl and lmM each of CaCl2, MgCl2 and MnCl2 (10,0009, 5 min) before Samples were diluted 10 fold in starting buffer and centrifuged metþ1-cr-D-mannoside loading. Elution of bound material was achieved by adding 500m\4 the eluant' When to the stafting buffer (elution buffer). Protein was detected by AZSO of until no more material used preparatively, the unretained fractions were rechromatographed repetitive runs were eluted with the elution buffer. The retained fractions from these pooled. 10 volumes of Regeneration of the column was achieved by washing with at least column 10 column volumes of 0.1M Tris-HCl buffer (pH 8.5) containing 0.5M NaCl and a further 0.1M sodium acetate buffer (pH 4.5) containing 0'5M NaCl' 5.2.3.2 Anion exchange chromatography FPLG system at foom Anion exchange chromatography was undertaken on a Pharmacia 5'5) and temperature. The sample was diluted 10 fold in 20mM piperazine-HCl buffer þH which had centrifuged (10,0009, 5 min) before loading on a Mono-Q (HR 5/5) column was increased from 0 to been equilibrated with the above buffer. The NaCl concentration the 4280 decreased to 60mM over a period of 3 min, then held at this concenüation until 60mM to 250mM ovef a the baseline. The NaCl concentration was then increased from to the baseline' period of 10 min, then held at this concentration until the A4g decreased 130 The NaCl concentration was then increased from 250mM to 1.0 M over a period of 1 min, and held at this concentration until the 446 decreased to the baseline. 5.2.3.3 Cation exchange chromatography Cation exchange chromatography was undertaken on a Pharmacia FPLC system at room temperature. The sample was diluted 10 fold in 20mM sodium citrate buffer þH 3.0) and centrifuged (10,0009, 5 min) before loading on a Mono-S (HR 5/5) column which had been equilibrated with the above buffer. The NaCl concentration was increased from 0 to 50mM over a period of 2 min and held at this concentration until the A2g¡ decreased to ttre baseline. The NaCl concentration was then increased from 50mM to 120mM over a period of 4 min and held at this concentration until the A4¡ decreased to the baseline. The NaCl concentration was further increased from 120mM to 150mM over a period of 2 min and held at this concentration until the AZSO decreased to the baseline. The NaCl concentration was subsequently increased to 1.0M over a period of I min and held at this concentration until the AZSO decreased to ttre baseline. 131 5.3 RESULTS 5.3.1 CTnBoHyDRATE-RrcH FRAcrroNs TsoLATED By aMMoNruM SULPHATE FRACTIONATION 5.3.1.1 Characterization of protein and polysaccharide by SDS PAGE In the ammonium sulphate fractionation procedure described in Section 4.2.4.2 the following carbohydrate-rich fractions were isolated. The fraction that remained soluble at 707o saturation of NH+)zSO4 (C) was rich in carbohydrate as demonstrated by the presence of several high ìvf¡ bands on SDS PAGE which stained strongly with the PAS reagent (PAS-stained electropherograms not shown). One of the PAS-positive bands appeared at the same R¡ as a band which stained faintly for protein with lvI, 63,000. The fraction also contained two major proteins of M, 26,000 and 24,000 @gure 4.1 l). The M. 26,000 and 24,000 (low Mr) proteins of fraction C were separated from polysaccharide maærial by ultrafrltration through a membrane with a nominal cut off of l\4, 30,000. This brought about an enrichment of the proteins in the filtrate E (Figure 4.lI). The retentate (fraction D) showed a decreased level of the proteins but retained the PAS- positive polysaccharide material in addition to the lvlr 63,000 protein band (Figure 4.11). 5.3.1.2 Amino acid composition The amino acid composition of carbohydrate-rich fractions C and D is shown in Figure 5.1. Both fractions contained abundant quantities of serine and threonine in addition to high levels of the acidic amino acids, ASX and GLX, and the ariphatic amino acids, glycine and alanine. 5.3.1.3 Haze potential The data recorded in Figure 5.2 were obtained when the micro-method. was applied to the three fractions which were soluble in (NFIa)zSO+. The protein-rich fraction E gave the most haze. In comparison, the carbohydrate-rich fractions, C and D, gave little turbidity indicating that the components they share were not contributing greatly to haze formation. It is signif,rcant that, although C is a mixtue of both D and E, the haze response of D was expressed by the mixture. Fraction D components appeared to have a repressing effect on the haze potontial of the proteins in E. 132 Fraction C c o 20 Y"m p 15 mo os 10 li at 5 ri o 0 n GLY ALA VAL TEU ILE SER THR FHE T\R TRP LYS ARG I{IS ASX GIJ( MET Fraction D c o 20 "/" m p 15 mo os 10 li at 5 ri o 0 n GLY ALA VAL tEU ILE SER THR FHE TYR TRP LYS ARG HIS ASX GX MET Figure 5.1 The 7o molar compòsition of amino acids detected in fractions C and D isolated from wine by (NHC)ZSO4 precipitation Amino acid composition of fractions C and D, isolated as described in Section 4.2.4.2, was deærmined as described in Section 2.2.5, and is expressed as 7o molar composition. 133 0.300 a cE 0.200 so ro a o à I 0.100 0.000 0 25 50 75 100 125 150 Protein concentration (mg/L) -È c D -*- E (***) Figure 5.2 Influence of the protein concentration of wine fractions on the heat induced haze in wine Fractions C, D and E, isolated by (NH+)ZSO4 precipitation (Section 4.2.4.2) were back- added to protein-free wine and incubated as detailed in Section 4.2.2.3. (***) significant linear conelation at 0.0017o level t34 5.3.2 IoBNTIFICATIoN oF THE HAzE-MINIMIzING FAcToR 5.3.2.1 Effect on the haze potential of wine proteins The effect of increasing concentration of the carbohydrate-rich fraction (fraction D) on the haze induced from total wine proteins is shown in Figure 5.3. The protection afforded by fraction D was extended to all wine proteins and not just those present in fraction E. This protection became greatcr, reducing the visible haziness caused by heating, as the ratio of added carbohydrate-rich fraction to wine protein increased. The relationship between the extent of haze protection and concentration of added polysaccharides was exponential, such that a residual low level of haze was still detecæd at the highest level of fraction D tested. 5.3.2.2 Haze and supernatant protein and polysaccharide composition Hazes and supernatants from the samples described in Section 5.3.2.I were analyzed,by SDS PAGE (Figure 5.4). There was no signif,rcant difference between the haze samples as estimated by band intensity after staining with the protein reagent, Coomassie Brilliant Blue. All the proteins present before heating, as well as some lower I\4, bands, were found in all hazes regardless of the level of polysaccharide addition. SDS PAGE analysis of the supernatants (Figure 5.4B, lanes 5, 6 and 7) for the samples with 0, 390 or 7g0pg polysaccharide per 100pg protein, showed no measurable protein. Some of the hazes and supernatants analyzed by SDS PAGE were stained with both the protein stain and the carbohydrate stain @igure 5.5). The haze samples gave a very poor response to the pink PAS stain, indicating that they did not contain large amounts of polysaccharide material. In comparison, the supernatants showed an increasing response to the PAS stain, evident by the increase in intensity of the pink hue in rhe high M, region of the gel, as the concentration of added polysaccharide was increased- 5.3.2.3 Haze particle size The particle size of suspended haze was measured by Photon Correlation Spectroscopy (Figure 5.6). The results showed that the size of the haze particles decreased as the polysaccharide concentration increased. The relationship between the haze particle size and polysaccharide concentraúon was exponential, with the decrease in particle size of the precipitate diminishing at high polysaccharide additions. There was an excellent correlation between visible haziness and haze parricle size (Figure 5.7). 135 0.5 Y = 0.41512' 10n(-1.1314e-3x) **) o (* 0.4 cE o rto o o¡ rr) 0.3 a o r.u o N 0.2 r o o 0.1 a 0.0 0 200 400 600 800 Fraction D (pg gal. equiv/100 pg protein/ml) Figure 5.3 The effect on wine protein haze formation of increasing concentration of carbohydrate-rich fraction D Increasing concentration (expressed as ¡rg galactose equivalents by the resorcinol assay Section 5.2.2.1-1) of Fraction D (see Figure 5.1 and Section 4.2.4.2) was back-added to wine containing 100pg/ml wine protein and incubated as detailed in Section 5.2.2.2. (***) significant linea¡ correlation at 0.00l7o level Figure 5.4 SDS PAGE of wine hazes and supernatants. Samples (lrtl-) from the previous study (Figure 5.3) were centrifuged (10,0009, 10 min)' the supernaranr removed, and the pellet washed twice with Milli-Q watel (250pL). The hazes, redissolved in SDS PAGE sample buffer, and the supernatants were analyzed by SDS PAGE as described in Secúon 4.2.2.1'. A Relative molecular mass (M¡x 1g-3¡ of protein standa¡ds (S) a¡e given on the left side of the gel. The other lanes are as follows: lane 1 - hazefrom sample containing 100pg/ml wine protein but no added fraction D; lanes 2-7 - hazes from samples containing 100pg/ml wine protein and fraction D at the following levels, 80, 150, 230, 310, 390, 460, 540pg, respectively' B Relative molecular mass (Mr x t0-3) of protein standards (S) are given on the right side of the gel The other lanes arc as follows: lanes 1-3 - hazes from samples containing 100¡rg/ml wine protein and fraction D at the following levels, 620, 7 N, 7 80pg, respectively; lane 4 -hazefrom sample containing 100pg/ml wine protein but no added fraction D, and at lOTo of the loading in lanes 1-3; lanes 5-6 - supernatants from samples containing 100pg/ml wine protein and fraction D at 390 and 780pg, respectively; lane ? - supernatanr from sample containing 100pg/ml wine protein but no added fraction D. 136 s1234567 97.4 66.2 (r¡D - 42.7 A -) 31.0 -) ¡fft *í¡È 21.5 #ËF 4{tçv - 14.4 .- n 8r.ü. ltÉ' 1234567S 97.4 .- 66.2 42.7 31.0 B - -- 21.5 - 14.4 - t37 123456789S 97.4 66.2 42.7 31.0 21 .5 14.4 Figue 5.5 SDS PAGE of haze and supernatants. Hazes and supernatants from samples with va¡ious carbohydrate additions were isolated and analysed as described in Figure 5.4, except the gels were stained \Mittr both Coomassie Brilliant blue for protein, and PAS stain for carbohydrate (pink). For details of staining procedures, see Section 4.2.2.1. Relative molecular mass (lrd¡ x t0-3) of proæin standards (S) are given on the right side of the gel. The other lanes are as follows: lane 1 -haze from sample containing 100pg/ml wine protein but no addedFraction D; lanes 2-5 - hazes from samFles containing lO0pg/ml wine protein and Fraction D at the following levels, 4, 17,35, 60pg, respectively; lanes 6-9 - supernâunts from sâmples containing 100pg/rnL wine protein and Fraction D at the following levels; 60, 35, L7,4¡tg,respectively. 138 30 Y =22.464 * 10n(-1.015'le-Sx) R^2 0.997 25 = E 1 20 o .N U' 15 -9 .9 Ë 10 (U o_ 5 i 0 0 200 400 600 800 1 000 Fraction D (pg gal. equiv/100 pg protein/mL) Figure 5.6 The effect of the concentration of carbohydrate-rich fraction D on haze particle size Increasing concentration (expressed as pg galactose equivalents by the resorcinol assay Section 5.2.2.I.1) of Fraction D was back-added. ro wine containing 100pg/ml wine protein and incubated, as described in Figure 5.3. The particle size (expressed in pm) of the resultant haze was measured by Photon Correlation Spectroscopy as described in Section 5.2.2.3. 139 0.5 0.4 T I cE ott lo 0.3 o N 0.2 Tct ¡T 0.1 y = 2.4141e-2 + 1.4182e-2x R^2 = 0.933 0.0 0 5 10 15 20 25 30 Particle size (pm) Figure 5.7 The relationship between haze particle size and visible haziness The particle size of the samples as shown in Figure 5.6 was compared to the visible haziness of the samples, measured as described in Section 5.2.2.2. 140 5.3.3 PURIFICATIoN oF THE HAZE.MINIMIZING FACToR A summary of the purifîcation of the haze protective factor is given at the end of this Section in Figure 5.16 (page 152). 5.3.3.1 Affinity chromatography Separation of fraction D by Concanavalin A (Con A) affrnity chromatography demonsrated that this technique gave further purification of the haze protective factor (data not shown), hence a preparative procedure was developed. The starting material was an ultrafrltration retentate containing the full complement of wine proteins and polysaccharides. SDS PAGE analysis of this material, and of the unretained and retained fractions from preparative Con A chromatography, are shown in Figure 5.8. The unretained fraction contained the bulk of the protein in the M, range of 36,000 to 10,000, with a faint response to the PAS stain as a smear near the gel interface. The rctained fraction contained the bulk of the PAS staining material which smeared ar the high Mr (top) of the gel, in addition to a PAS- and protein-positive band at Mr 63,000, and a PAS staining band at approximate M, 54,000, and faintly staining protein bands at Mr 40,000, 32,000 and 23,000 werc apparent when the gel was freshly srained. The concentration of neutral polysaccharides in the retained fraction, as determined by the resorcinol assay (Section 5.2.2.I.1) was 10.68g/L, which is equivalent to 137mg per L wine. The yield of neutral polysaccharides in the unretained fraction was 108mg per L wine. The neutral polysaccharide concentration of the starting material was 302mg/L thus, in total, atSLVo recovery of neutral polysaccharides was achieved from the preparative Con A ch¡omatography. 5.3.3.1.1 Effect on protein haze potential The effect of increasing concentration of the Con A retained fraction on the haze induced from wine proteins and from the model protein, BSA, is shown in Figure 5.9. The Con A retained fraction reduced the visible haziness produced by heating both of these protein fractions in wine. Haze decrease diminished at higher polysaccharide concentrations leaving a residual low level of haze. 141 s123 97.4 66.2 42.7 31.0 21 .5 14.4 Figue 5.8 SDS PAGE of fractions from preparative Concanavalin A chromatography The high N[r retentate from ultrafiltered Gordo wine (600mI., Section 4.2.2.3) was fractionated by Concanavalin A chromatography as described in Section 5.2.3.1. The protein and polysaccharide composition was assessed by SDS PAGE. Relative molecula¡ mass (M¡ x t0-3) of protoin standards (S) are given on the left side of the gel. The other lanes are as follows: lane I - Gordo wine ultrafiltration rctentate (the starting material); lane 2 - ttre r¡nretained ûr¿ction; lane 3 - the retained ùaction. Gels were run as described in Section 4.2.2.1, and stained for protein with Coomassie Brilliant blue, and for carbohydratc with the PAS stain (phk). r42 o.4 -+ wine protein E03 ---o--. BSA \fo rf) S o.2 uJ N r 0.1 0.0 0 1 00 200 300 400 500 600 Con A retained fraction 0rg gal. equivalents/ml) Figure 5.9 The effect on the haze potential of proteins in wine of the Concanavalin A retained fraction Increasing concentration (expressed as pg galactose equivalents by the resorcinol assay Section 5.2.2.1.1) of Con A retained fraction (Figure 5.8, lane 3) was back-added to wine containing sOpg/ml- wine proæin, or 100pg/ml BSA, and incubated as detailed in Section 5.2.2.2. r43 5.3.3.2 Anion exchange chromatography The Con A retained fraction was further separated by anion exchange chromatography on an FPLC Mono Q column at pH 5.5. The resulting chromatogram is shown in Figure 5.10. Most of the material loaded onto the column was retained and eluted with an increase in NaCl concentration. The first fraction eluted at 0.06M NaCl, then a strongly UV-absorbing fraction eluted at approximately 0.2M NaCl. A weakly UV-absorbing and tightly bound. fraction was eluted by an increase in NaCl concentration to 1.0M. The protein and polysaccharide composition of these peals and the fractions between them, as revealed by SDS PAGE, are shown in Figure 5.11. The material not exchanged with the Mono Q column (lane 2) showed a faint protein band at N4 52,000 when first stained. Fractions collected between the unretained and fîrst peak (lane 3) and the first and major UV-absorbing peak (lane 5) did not contain any detectable material. The frst peak (lane 4) contained much of the PAS staining components of the starting material that smeared at the high l\4 (top) of the gel, in addition to PAS staining bands at Mr 80,000 and 63,000, but no protein bands. The major UV-absorbing peak (lane 6) contained some of the PAS staining components of the starting material rhar smeared at the high I\4, (top) of the gel in addition to a PAS- and protein-positive band at Mr 63,000, a PAS staining band at lr4, 54,000, and protein bands at Mr 40,000, 32,000 and 23,000. The strongly retained material (lane 7) contained a band staining weakly for protein at Mr 63,000, which was apparent when the gel was freshly stained. 5.3.3.2.1 Effect on protein haze potential The haze protective properties of the fractions isolated by anion exchange chromatography were assessed by detennining their effect on the haze potential of BSA (Figure 5.12). Each fraction can be compared to the starting material because the results are expressed on an equivalent volume basis (r.e. the fractions were concentrated to the same volume of the starting material loaded onto the column). Neither the unretained or strongly retained fraction showed a signihcant haze protective effect. The first peak showed some haze protection but the majority of the haze protective factor appeared to be localised in the second peak. r44 0.1 25 0.1 00 I 1.00 I I I I I I I 0.075 I 0.75 I I I Strongly ¡ o Retained @ , c\l Peak I c o 0.050 I 0.50 È c I / (U 'o L I c o I ()o ù I c I o() I 0.025 ) 0.25 () z(ú 0.000 0.00 0 5 10 15 20 Elution volume (mL) Figure 5.10 Separation of Con A retained fraction by anion exchange chromatography Con A retained fraction (Figure 5.8, lane 3) was separated by anion exchange chromatography on a FPLC Mono Q column at pH 5.5 as described in Section 5.2.3.2. 145 s1234567 97.4 66.2 42.7 31.0 21 .5 14.4 Figure 5.11 SDS PAGE of anion exchange chromatography fractions The protein and polysaccharide composition of fractions isolated by anion exchange chromatography (Figure 5.10) was determined by SDS PAGE. Relative molecula¡ mass (Mr x 19-3¡ of protein sundards (S) a¡e given on the left side of the gel. The other lanes a¡e as follows: lane I - the starting material: Con A retained fraction (Figure 5.8, lane 3); lane 2 - material not exchanged with the Mono Q column; lane 3 - fractions collecæd be¡veen the r¡nretained and first peak; lane 4 - firstpeak; lane 5 - fractions collected between first and major UV-absorbing peak; lane 7 - srongly retained peak. Gels wero stained as described in Figure 5.8. L46 140 120 100 o BO N --t- start¡ng material (d :E unretained fraction òe 60 -È first peak strongly UV-absorbing peak strongly retained peak 40 20 0 0 20 40 60 80 100 Volume (¡rL) Figure 5.12 The effect on the haze potential of BSA of wine fractions isolated by anion exchange chromatography Increasing volumos of the fractions isolated by anion exchange chromatography (Figure 5.11, Section5.2.3.2) were back-added to ultrafiltered wine conraining 100pg/ml BSA and incubated as detailed in Section 5.2.2.2. The volume of the anion exchange fractions is expressed as þLlper mL wine and equivalent to the volume of the surting material (Con A retained fraction: Figure 5.8, lane 3; l0.68pg gal equivalents/pl). I4l 5.3.3.3 Cation exchange chromatography The major UV-absorbing peak from the Mono Q column (Figure 5.10) was further fractionated by cation exchange chromatography on a FPLC Mono S column at pH 3.0 (Figure 5.13). Most of the material loaded onto the column was rotained and eluted with an increase in NaCl concentration. The first peak eluted at 0.05M NaCl, then a strong UV- absorbing peak eluted at 0.15M NaCl with a third peak as a shoulder at0.26Mr NaCl. A small amount of tightly bound material was eluted by an increase in NaCl concentration to 1.0M. The protein and polysaccha¡ide composition of these peaks, as revealed by SDS pAGE, is shown in Figure 5.14. The material not retained by the cation exchange column (lane 3) contained some of the PAS staining material of the stafling material rhat smeared at the high Mr (top) of the gel and a faint PAS and protein staining band at Mr 46,000 was apparent when the gel was freshly stained. Fractions collected between the unretained peak and the first peak contained no detectable material (not shown on rhis gel). The f,rsr peak (lane 4) contained only smeared PAS staining material and no detectable protein. The second peak (lane 5) contained all the protein bands of the starting material. These proteins were also present in the third fraction (lane 6) although at ¡educed concentration. The strongly retained fraction (lane 7) contained the PAS staining band at N4 54,000 and a faint protein band at Mr 40,000. 5.3.3.3.1 Effect on protein haze potential The result of increasing concentation of the cation exchange fractions on the haze induced from BSA is shown in Figure 5.15. The effect of each fraction can be compared to the effect of the original starting material (Con A retained fraction) because the results are expressed on an equivalent volume basis. Neither the unretained, the second peak or strongly retained fraction showed ahazn protective effect. On the contrary, the strongly retained fraction increased the haze potential of added BSA. The first peak showed the majority of the haze protection, indicating that the haze protective factor was contained in this peak. Hazes and supernatants from the samples in Figure 5.15 containing the first peak and the strongly retained peak rvs¡e analyzed by SDS PAGE (data not shown). None of the 148 0.20 Second 1.00 Peak r / 0.15 0.75 I I I I E o o @ (ú ñl 0.10 S 0.50 +¿c ()o .s c g ()o e CL o z(ú 0.05 0.25 F lrst Peak Strongly Retained ____J Peaks + / Third Peak + + 0.00 0.00 0 5 10 15 20 Elution volume (mL) Figure 5.13 Separation of the major UV-absorbing fraction from anion exchange chromatography by cation exchange chromatography The major UV-absorbing fraction isolated by anion exchange chromatography (Figure 5.10) was separated by cation exchange chromatography as described in Section 5.2.3.3. 149 s1234567 97.4 66.2 42.7 31.0 21 .5 14.4 Figure 5.14 SDS PAGE of catÍon exchange chromatography fractions The protein and polysaccharide comÐosition of fractions isolated by anion exchange chromatography was determined b¡, SDS PAGE. Relative molecular mass (ltlI¡ * 19-3¡ of protein standards (S) are given on the left side of the gel. The other lanes are as follows: lane 1 - Con A retained fraction (Fig¡re 5.8, lane 3); lme 2 - the starting material (major UV-absorbing fraction from anion exchange chromatography, Figure 5. 1 0, Secti on 5.2.3.2) lane 3 - un¡etainedfraction; lane 4 - firstpealq lane 5 - secondpeak; lane 7 - srongly retained peak. Gels were stained as described in Figure 5.8. 150 160 A 140 Â 120 100 -ù star4ing material o o N :E 80 A first peak òe A E second peak A A O unretained fraction 60 A strongly retained peak A A 40 20 0 0 10 20 30 40 50 60 Volume (¡rL) Figure 5.15 The effect of cation exchange chromatography fractions on the haze potential of proteins in wine Increasing volumes of the cation exchange fractions (Figure 5.13, Section 5.2.3.3), and the original starting material (Con A retained fraction, Figure 5.8,lane 3) were back-added to wine containing 100pg,/ml BSA and incubated as detailed in Section 5.2.2.2. The volume of the cation exchange fractions is expressed as pl/per mL wine and equivalent to the volume of the starting material (Con A retained fraction: Figure 5.8, lane 3; 10.68pg gal equivalents/¡rl). 151 supernatants contained any protein nor was there a significant difference between the protein present in the different haze samples as observed previously with the unpurified. haze protective factor (Figure 5.5, Section 5.3.2.2). The hazes examined here could only be differentiated with the PAS reagent.Hazn samples containing increasing amounts of the first peak showed an increasing response to the PAS reagent, clearly indicating that the haze protective factor was associated with the precipitated protein and that its increasing concentration correlated with lower visible turbidity. 5.3.4 PnoPERTIES oF THE HAar' PRoTECTIvE FACToR A summary of the purification of the haze protective factor is given in Figure 5.16. 5.3.4.1 Composition The haze protective factor comprised polysaccharide (967o) and protein (4Vo) and, thet composition is given in Table 5.1. The carbohydrate component of the factor was dominated by mannose (787o) with some glucose (l3Vo) and traces of apiose, galactose, arabinose and hexosamine. The presence of apiose, galactose and a¡abinose may be due to the incomplete separation of the haze protective factor from other grape-derived polysaccharides. The amino acid composition of the protein component of the factor was dominated. by serine (3LVo) and threonine (L3Vo), with major contributions from glycine (9Vo), asx (9Vo) and glx (97o). 5.3.4.2 UV Spectrum The UV spectrum of the haze protective factor is shown in Figure 5.17, and its second order derivative in Figure 5.18. The UV spectrum of the haze factor with a maximum at 278nmis typical of protein. Derivative spectroscopy offers the advantage of producing sharper spectral features than conventional absorbance (i.e., zero order derivative) spectroscopy, allowing one to identify aromatic amino acids in proteins and peptides (Patladino & Cohen 1991). In the specrrum of the haze protective factor, minima were clearly observable at 281 and 255nm correspond to the primary minima of tyrosine and phenylalanine, respectively, thus conf,rming their 152 Gordo wine ultrafiltered to concentrate high Mr components (Section 4.2.2.3) Gordo w¡ne UF retentate Concanavali n A (affiníty) eluted with 50mM sodium acetate pH5.6, 150mM NaCl, plus 50mM methyl-mannoside (Section 5.2.3.1) Con A reta¡ned f raction Mono Q (anion exchange) eluted with 20mM piperazine-HCl pH5.5, at 200mM NaCl, Section 5.2.3.2 Major UV-absorbing f raction Mono S (cation exchange) eluted with 20mM sodiun citrate pH3.0, 50mM at NaCl, Section 5.2.3.3 HAZE PROTECTIVE FACTOR Figure 5.16 Purification of the haze protective factor The main purif,rcation steps are summarised with reference to the relevant Sections 153 Table 5.1 COMPOSITION OF THE IJAZE LIMITING FACTOR Vo weight Carbohydrate 96 Proæin 4 Neutral sugar compositiona Sugar (Vo molar ratio) mannose b 78 glucose b 13 apiose c 3 galactose c 3 arabinose c 2 c hexosamine 1 Amino acid compositiond A¡nino acid (Vomolar ratio) ser 31 thr L3 asx 9 glx 9 glv 9 val 6 ala 5 ile 5 tyr 5 leu 4 phe 2 lys 1 a determined as described in Secrion 5.2.2.1.2 b confirmed by coinjection c peaks eluting at the same retention times as standa¡ds d determined as described in Section 2.2.5 r54 1 278 250 260 270 280 290 300 wavelength (nm) Figure 5.17 UV spectra of the haze protective factor The UV absorption spectra from 250 to 300nm of the haze protective factor in pH 5.0 buffer (50mM sodium acetate) was measured in 10mm path length cell. The maximum is arrowed. 273 288 t/ \, 1 1 255 1 260 293 1 1 1 268 1 28L 285 ns 250 260 270 280 z9O 300 wavelength (nm) Figure 5.18 second order derivative spectrum of the haze protective factor The second order derivative spectrum from 250 to 300nm of the haze protective factor in pH 5'0 buffer (50mM sodium acetate) was measured in 10mm path length cell by a Varian spectrophotometer, at a scan rate of 200nm/s and. slit width of 2nm. Minima and. zero ordinate axis intersections a¡e arowetl. 155 presence in the haze protective factor (Table 5.1). Minima, at 275 and 285nm and the intersection of the zero ordinate axis atZT3nmare also reportedly characteristic of tyrosine, as are minima at268 and 260nm of phenylalanine (palladino & Cohen 1991). Second order derivative spectroscopy also allowed examination of the presence of tryptophan. No tryptophan was detected in acid hydrolysates of the haze protective factor, but this amino acid can be destroyed during acid hydrolysis especially when carbohydrate is present (Ng er al. 1987). Unfortunately, its identification in the haze factor by second order derivative spectroscopy was hampered by the large proportion of tyrosine present in the factor (5Vo,Tab\e 5.1). In proteins with a tyrosine:tryptophan ratio greater than 4:1, it has been shown that the primary minimum of tryptophan (290nm) is obscured by minima of tyrosine at 28lnm and 285nm. The contribution of tryptophan to the spectrum is thus reduced to a characteristic intersection of the zero ordinate axis upscale from 282nm (Palladino & Cohen 1991). Such a feature is evident in Figure 5.18 at 288nm suggesting that there may be some ûyptophan present, but its quantity is only minor compared to the amount of tyrosine present in the haze factor. 156 5.4 DISCUSSION A wine polysaccharide which has a haze-proæctive role has been isolated, characterized and its effect on protein precipitation and thus wine haze formation studied. The fi¡st step in the isolation was the (NHa)2SO4 procedure described in Chapter 4 for wine protein purification. A carbohydrate-rich fraction (C) which contained the wine polysaccharides and some wine proteins was easily obtained by this proced.ure. After ultrafiltration, the carbohydrate-rich fraction (D) was relatively free of low M' protein. It contained a number of high l\4 PAS positive bands in addition to a major PAS and protein staining band at Mr 63,000. This positive PAS response suggested a polysaccharide component and since the l\lr 63,000 band stained for both protein and carbohydrate, it is probable that the Mr 63,000 band was a glycoprotein. The carbohydrate-rich fraction contained abundant quantities of the amino acids, serine and threonine. This composition is considered significant because serine and threonine ¿ìre commonly involved in O-glycosidic linkages (Beeley 1985), consistent with the possibility that some protein was covalently linked to carbohydrate material. In the micromethod for haze potential test, both the carbohydrate-rich fractions (D and C) gave little turbidity. The response was non-concentration dependent, and the data points for these two may have reached a plateau, as previously observed with a grape protein fraction isolated by gel permeation chromatography and having a SDS PAGE profile similar to that of fraction D (Section 4.3.2). This nonlinear response was observed regardless of whether the polysaccharides were present with the wine proteins which accompanied it in the original isolate (C) or separated from these (fraction D). This is significant, because the proteins in fraction C, on their own (fraction E), were capable of producing a large amount of haze (Section 4.3.3.4). When these proteins were present with the carbohydrate-rich fraction (fraction C), the high hazing potential of these proteins was not expressed. The presence of fraction D components had a repressing effect on the high hazing ability of the proteins of E. Fraction D extended its haze protective effect to all wine proteins a¡rd not just to those in fraction E. Visible haziness was ¡educed as the ratio of added carbohydrate-rich fraction to wine protein increased. The relationship between the extent of haze protection and the concentration of the added polysaccharides was exponential, leaving a residual, persistent low level of haze at higher polysaccharide concenftations. t57 The mechanism of haze protection was not to prevent protein precipitation or effect the types of proteins precipitating but to change the manner in which haze formed. The particte size of the haze decreased as more polysaccharide was present. This had the visual effect of making the wine appear less hazy. Some of the polysaccharide material was present in the hazes but most appeared to remain soluble after heating. This may be because the polysaccharide fraction examined was not purified and contained many polysaccharides which wero not involved in the haze protective effect. Indeed, when this experiment was repeated with the purif,red haze protective factor, polysaccharide material was shown to be present only in the hazes, and its level to increase in tandem with a decrease in visible turbidity. The size of particles detected in wine haze was greater than those observed in beers, ranging from 30pm in the control samples decreasing to 5¡rm at the highest polysaccharide level æsted. Beers that appeared visually hazy contained partictes with size no greater than L2¡tm (Leedham & Carpenter 1977). Although wine particle size was decreased from 30¡tm to 5pm by polysaccharide addition, the hazes could still be detected with the naked. eye. Indeed, particles grcater than 5pm in beers are considered commercially significant because the consumer may be able ro see them (I-eedham & Carpenter lg77). In the further fractionation of this carbohydrate-rich fraction its retention on a Concanavalin A (Con A) affinity colu.mn was used to advantage. Con A is specific for mannose and glucose residues, which suggests ttrat the retained haze protective factor contained mannose and or glucose. The Con A retained fraction also contained most of the high M. carbohydrate material, including the PAS- and protein-positive band at lvlr 63,000 and other proteins at M, 40,000, 32,000 and 23,000. Passage of this fraction through an anion exchange column separated some of the high M, carbohydrate material from the haze protective factor, but the fraction retained the PAS- and protein-positive band at Mr 63,000 and other proteins at Mr 40,000, 32,000 and 23,000. However, the protein components were not responsible for the haze protective effect because they were separated. from the haze protective factor by cation exchange chromatography at pH 3.0, and precipitated normally when heated. The PAS- and protein-positive band at l\4 63,000 could be grape inveftase. This enzyme has been previously isolated from wines (Nakanishi et a\.1991), and had lvlr of 65,000 and pI of 3.9, (i.e. it would be capable of binding to an anion exchange column at pH 5.0 and cation exchange at 3.0). In addition, the enzyme was shown to be a glycoprotein, 158 containing approximately 33Vo sugar (Nakanishi & Yokotsuka 1990b), and was retained on a Con A column (Ishikawa et al.1989). The purifiedhaze protective factor did not stain with either of the protein stains but showed a diffuse smear in the stacking gel and in the high M, region of the separaring gel with the PAS stain. It was composed predominantly of carbohydrate (96Vo) with some protein (4Vo). Although the protein component of the haze protective factor was minor, the amino acid composition of the protein was significant because it was dominated by serine (3lVo). As discussed above, this amino acid can be involved in O-glycosidic tinkages (Beeley 1985), and its extremely high occurrence in the haze protective factor suggested that the protein component was covalently linked to some of the polysaccharide chains at serine residues. Threonine is also abundant in the haze factor (l3Eo) and linkages to the polysaccharide may also occur via O-glycosidic linkages to threonine residues. A similarly high proportion of these two hydroxyl bearing amino acids was reported in the protein component of yeast structural cell wall mannoprotein, although this macromolecule contained more threonine (307o) than serine (157o)(Freverr & Ba[ou 1985). The presence of the UV absorbing amino acids, tyrosine (5Vo) and,phenylalanine (27o), in the protein component of the haze protective factor explains the UV absorbance of the factor, enabling its detection at 280nm in chromatography eluants. The most intensely UV absorbing amino acid, tryptophan, was not detected in acid hydrolysates, nor was there conclusive evidence of its presence in the intact haze protective factor by UV derivative spectroscopy. This result indicates that the UV absorbance of the haze protective factor is largely due to the presence of phenylalanine and tyrosine. The carbohydrate poftion of the factor was dominated by mannose (78Vo) with some glucose (137o) and traces of apiose, galactose, arabinose, and a hexosamine. The extremely high proportion of mannose suggests that the haze protective factor is of yeast cell wall origin. The SDS PAGE results also suggest a yeast cell wall origin because purified yeast mannoprotein is reported to have smeared in SDS PAGE stacking gels and only stained with the PAS stain and not the two protein stains (Coomassie or silver)(Novick & Scheckman 1983, Frevert & Ballou 1985). The wine used for the isolation of the haze protective factor had not been produced. by a method designed to increase yeast autolysis producs (i.e. methode champenoise, or stored 159 on yeast lees), but it is likely that the fermentation process alone released some yeast products. Saulnier et al. (1991) have shown that storage on yeast lees was not nocessary for colloid release and that by the end of fermentation (i.e. approximately 8 days), yeasts had released about 150mg/L of polysaccharide material. They suggested that mannoproteins are released by yeast into wine from the yeast cell wall by hydrolytic enzymes and perhaps this is the source of the haze protective factor in the wine used in this study. 160 Chapter 6 CONCLUSIONS Purification of two of the major wine protein fractions has led. to the positive identification of proteins that are involved in wine instabitity. These proteins have different and distinct hazing potentials, the M, 24,0N protein gtuirg almost double the visible haze in wine per unit mass of the lvlr 32,000 protein. V/ine protein haze is thus a summation of the instabilities of a number of wine proteins, some of which produce more haze than others. Such proteins are therefore of greater concern to the wine industry and their removal is critical for white wine stabilization. The study of these proteins has also revealed the difficulties associated with measuring grape and wine protein concentration. In this work it was necessary to use a method which involved acid hydrolysis of purified protein and then quantification of protein content by the sum of the masses of the amino acids liberated- The advantages of using this method over the more commonly used colourimetric assays were its reliability and accuracy and lack of susceptibility to phenolic interference. However, this procedure is complex and requires sophisticated equipment and thus is not suitable for routine use by a winemaker. The development of a simple kit to measure grape juice and wine protein concentration may be an avenue for future research. The identifrcation and isolation of the N4 24,000 protein as a major contributor to heat hazing has provided a substrate and a convenient starting point for the design of novel protein removal techniques. This is because the removal of this protein would be crucial to successful protein stabilization. One alternative technique, í.e. degradation and thus removal of grape and wine protein with peptidases, does not appear to be a reality. This is not because the enzymes were inactive in juice and wine; on the contrary, they were capable of rapidly degrading other protein substrates such as casein or dye-labelled collagen (HPA) in these media. However, all the grapo proteins, including the M¡ 24,000 protein, appeared extremely resistant to enzymatic attack. Even after prolonged incubation, treated wines did not show a significant decrease in protein concentration. If one considers that these proteins have survived the adverse conditions and hostile environment of the winemaking process, perhaps it is not unreasonable to expect them to be fairly resistant to proteolytic attack. The reason for their resistance has not been elucidated although resistance to proteolysis is not unique to these grape and wine proteins and numerous examples of other resistant proteins exist in the literature. Glycosylation can sometimes confer protoction to proteins (Semino et aI. 1985, Maruyama et al. 199O, Seymour et aI. l99l) but this study only detected one wine 161 glycoprotein with Mr 34,000 and Hsu & Heatherbell (1987b) only detected 3 glycoproteins with lvlr 13000, 25000 and 28000, suggesting that glycosylated proteins may not be common in juice and wine. Alternatively, it has been suggested that wine polysaccharides protect the proteins from proteolytic attack by surrounding them and thus physically hindering the enzymes (Heatherbell et al.1984). Wine polysaccharides certainly have an important part in protein haze formation besides this hypothetical role in proteolytic resistance and the wine proæinhazeproblem can not be simply considered in terms of the wine proteins alone. The polysaccharide status of the wine also needs to be appreciated because interactions of polysaccharides with wine proteins can reduce the visible haziness of a wine. Such haze protection results from the formation of protein haze with a smaller particle size. The outcome of this work has been the identification of natural haze protection in wine. This has shifted the problem of wine protein haze into an entirely different light and raises the possibility of addressing the problem by exptoiting the phenomenon of haze protection, rather than devising new methods for removing the unstable proteins. The wine polysaccha¡ide responsible for haze protection appears to have originaæd from the yeast cell wall. It may therefore be possible to enhance the natural haze protection present in a wine by deliberately enriching that wine in yeast cell wall polysaccharides through prolonged aging on yeast lees, or by adding a purified sample of the haze factor during wine production. t62 REFERENCES p roteins and p eptide s', I aboratory techniques in biochemistry van Knippenberg, P.H., Eds.; Elsevier: BîU*,R.H.; Allel n. 9.i Spicer, S. S.; Zenr, D. 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