Heat Unstable Wine Proteins and Their Interactions with Wine Polysaccharides
Total Page：16
File Type：pdf, Size：1020Kb
WÂll tr ,rr.$fTTtlS 2$. ;t- ay LIB&'$.KT tct tr.{il 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