Q- tz N:i Copigmentation and its Impact on the Stabilisation of Red V/ine Pigments Thesis submitted for the degree of Doctor of Philosophy By Stephanie Green Lambert B.Sc. (Honours) Department of Horticulture, Viticulture and Oenology Facuþ of Agricultural Science and Natural Resource Management THE UNIVERSITY OF ADELAIDE AUSTBALIA October 2002 TABLE OF CONTENTS vil DECLARATION vill AGKNOWLEDGMENTS IX ABSTRAGT review 1 CHAPTER 1 Literature 1 1.1 Copigmentation grapes and wines 1 1.2 The phenolic constituents of Vitis vinífelø 1 I.2.1 GraPe anthocYanins in red wines J 1.2.2 Non-pigmented phenolic constituents present wine 4 1.2.3 Coñcentration of phenolic compounds in red and polymerisation 6 1.3 Constituents involved in copigmentation 6 1.3.1 Introduction 6 1.3.2 AnthocYanins 8 1.3.3 CoPigments 9 1.4 Copigmentation 9 1.4.I Introduction 10 1.4.2 Intermolecular copigmentation 11 1.4.3 Self-association t2 1.4.4 Intramolecular copigmentation l4 1.5 Factors influencing copigmentation l4 1.5.1 Introduction 15 1.5.2 Impact of anthocyanin structure I6 1.5.3 Impact of copigment structure 17 |.5.4Influenceofconcentrationofanthocyaninrelativetocopigment 18 1.5.5 Influence of the solvent medium on copigmentation pigment formation t9 1.6 Role of copigmentation in polymeric t9 1.6.1 Introduction 19 1.6.2 Intermediate interactions leading to polymerisation wine 20 1.6.3 Mechanisms of polymer formation in red l1 20 1.6.3. 1 Interflavan bond formation 2l 1.6.4 Polymeric pigments and red wine colour stability 'Work 22 1.7 presented in this thesis 23 CHAPTER 2 Gopigmentation 23 2.1 Introduction 23 2.t.1 Intermolecular copigmentation 25 2.1.2 Types of intermolecular interactions 2.1.2.1Van der Waals interactions 25 2.1.2.2 Hydrophobic interactions 26 26 2.1.2.3 Electrostatic interactions 2.1.2.4 Charge transfer complexes 27 2.1.2.5 Energetics associated with æ-n interaction 28 2.1.2.6 Geometdcal requirements of æ-æ interactions -a set of rules 29 2.1.2.7 Anthocyanin copigmentation chemistry 31 32 2.1.3 Use of ultraviolelvisible absorption to detect copigmentation 35 2.2 Materials and methods 2.2.1 Extraction, isolation and quantification of malvidin-3-monoglucoside 35 and malvidin-3 -(p-coumaryl) glucoside 2.2.LL Determination of anthocyanin purity by HPLC 35 36 2.2.2 Extraction, isolation and quantification of grape tannins 2.2.2.1Analysis of the seed tannin extract by Thin Layer chromatography 37 2.2.2.2 Purity of the seed tannin extract assessed by HPLC 37 38 2.2.2.3 Acid catalysis of the seed tannin in the presence of phloroglucinol 2.2.2.3'lAnalysisofthereactionproductsproduced from acid catalYsis bY HPLC 38 39 2.2.3 Synthesis of procyanidin dimer 83 4l 2.2.3.1Mass spectrometric analysis of the procyanidin material 42 2.2.3.2 HPLC of procyanidin products 43 2.2.4 Other compounds used to investigate intermolecular copigmentation 43 2.2.5 PhYsical methods 43 2.2.5.1General 43 2.2,5.2 Solutions used for the spectroscopic analysis 45 2.2.5.3 Measure of copigmentation 46 2.3 Results and discussion 111 2.3.1 Intermolecular copigmentation between malvidin-3-monoglucoside and vanous copigments 46 2.3.L1 Determination of the thermodynamic parameters for intermolecular copigmentation 54 2.3.1.2 Comparìson of abilities of copigrnents to form complexes with malvidin-3-monoglucoside 61 2.3.1.3 Influence of anthocyanin concentration on intermolecular copigmentation 63 2.3.2 Self-association of malvidin-3-monoglucoside 64 2.3.3 Comparison between the self-association and intermolecular copigmentation of malvidin-3-monoglucoside 67 2.3.4 Intermolecular copigmentation of malvidin-3-(p-coumaryl)glucoside 68 2.3.5 Self-association of malvidin-3-(p-coumaryl)glucoside 68 2.3.6 The use of anthocyanin analogues to investigate the phenomenon of copigmentation 69 2.3.7 Relevance of red copigmentation on red wine colour 72 2.4 Conclusion 74 CHAPTER 3 Factors influencing copigmentation 76 3.1 Introduction 76 3.1.1 Influence of pH on intermolecular copigmentation 76 3.l.2 Influence of sulphur dioxide on intermolecular copigmentation 77 3.1.3 Influence of ethanol on intermolecular copigmentation 78 3.1.4 Influence of acetaldehyde on intermolecular copigmentation 78 3.L5 Influence of potassium bitartrate on intermolecular copigmentation 78 3.2 Materials and methods 80 3.2.1 Preparation of solutions 80 3.2.1J Influence of pH on intermolecular copigmentation 80 3.2.L2 Influence of sulphur dioxide on intermolecular copigmentation 80 3.2.1.3 Influence of ethanol on intermolecular copigmentation 81 3.2.1.4 Influence of acetaldehyde on intermolecular copigmentation 81 3.2.L .5 Influence of potassium bitartrate on intermolecular copigmentation 8l 3.2.2 Physical methods 82 3.2.2.1 Investigating copigmentation with Ultraviolet/visible spectroscopy 82 3.2.2.2 Measurement of condensation products by HPLC 82 3.3 Results and discussion 83 IV 83 3.3.1 Influence of pH on intermolecular copigmentation 83 3.3'1'1 General trends 85 3'3.l.2Malvidin-3.monoglucosideandcopigmentsatpH<1 copigments at pH 7'80 87 3.3.1.3 Malvidin-3-monoglucoside and 3.3'|,4lnfluenceofpHonthestabilityofmalvidin-3-monoglucosideand 89 (-)-ePicatechin copigmentation 90 3.3.2 Influence of sulphur dioxide on intermolecular copigmentation 92 3.3.3 Influence of ethanol on intermolecular copigmentation 95 3.3.4 Influence of acetaldehyde on intermolecular copigmentation 96 3.3.5 Influence of potassium bitartrate on intermolecular 100 3.4 Conclusion by potentiometric titrations GHAPTER 4 Studies of copigmentation 101 and NMR 101 4.1 Introduction to determination of the ionisaüon 4.1.1 The use of Potentiomekic titrations 101 constants and the copigmentation of malvidin-3-monoglucoside t02 4.|'2Investigatingcopigmentationbynuclearmagneticresonance(NMR) 103 4.2 Materials and methods by potentiometric tihation 103 4.2.1 Ionisation constants of malvidin-3-monoglucoside 104 4.2.2 Investigation of copigmentationUyrttNMR 105 4.3 Results and Discussion 105 4.3.1 Ionisation constants of malvidin-3-monoglucoside by lH nuclear magnetic 4.3.2 Investigation of intermolecular copigmentation r07 resonance Í2 4.4 Conclusion modelling studies 113 CHAPTER 5 Molecular 113 5.1 Introduction calculations 113 background to geometry optimisation 5.2 Theoretical lr4 5.2.1 Electronic structure models studies 115 of computational chemistry to copigmentation 5.3 Application tl7 5.4 Results and discussion overlap (MNDO) analysis t17 5.4.1 Justification of Modihed Neglect of Differential copigments lzr 5.4.2 Molecular modelling of anthocyanins and V 5.4'3Relationshipsbetweenthecalculatedgeometryoptimisedstructuresof 130 copigmentsandtheirabilitiestoformcopigmentcomplexeswithanthocyanins 130 5.4.4 Intermolecular copigmentation complexes 132 5.5 Conclusion measures in a set of red w¡nes 133 CHAPTER 6 Use of cop¡gmentation 133 6.1 Introduction 133 6'1.1UV/visiblespectroscopicdeterminationcopigmentationinredwines 13s 6.2 Materials and methods 135 6.2.1 General 135 6'2.2U]traviolet/visiblespectroscopyevaluationofasetofredwines 6'2.2.|somersandEvansspectralmeasulestodetermineredwine t36 colour indices t36 6.2'2'2 The copigmentation formulas of Levengood 137 red wines by HPLC 6.2.3 Quantitative analysis of the r37 6.2.4 Statistical Methods 138 6.3 Results and discussion 138 6.3.1 Groupingofthewinesintodifferentcolourcharacteristiccategones contributing to total colour and the 6.3.2 Relationship between the components t39 measure of total colour expression of red wine r39 6.3.2.1The Shiraz red wines t45 6.3.2.2 The Pinot Noir wines anthocyanin, copigment 6,3.3 Relationship between the measures of monomeric complexandpolymericpigmentatthecompletionoffermentationandtotal 148 colour expression after 12 months ageing 148 6.3.3-l The Shiraz wines 150 6.3.3'2 The Pinot Noir wines berries and the wines 6.3.4 Relationship between the skin colour of Shiraz 151 processed from the grapes spectroscopy red wine colour 6.3.5 Relationship between HPLC and uV/visible 152 measufes 6.3.6 Comparisonoftotalcolourasdeterminedbythecopigmentationformulas t57 and the Somers and Evans measures 159 6.3.7 Improving the copigmentation formulas 6.3.STherelationshipbetweencopigmentationinredwinesandtheircolour 160 expression using the CIELAB scale vl 6.3 .9 Comparison between the colour measufes of the red wines after one months heating and 12 months ageing 161 6.4 Conclusions 163 CHAPTER 7 Summary and conclus¡ons 165 7.1 Summary 165 APPENDICES 169 REFERENCES 172 vll Declaration This work contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to this copy of my thesis, when deposited in the University of Adelaide Library, being available for loan and photocopying. SIGNED DATE .l 7 Stephanie G Lambert vnl Acknowledgments I would not have been able to complete my PhD programme without the support of many people. Thanks are due to : . Assoc Prof Graham Jones and Dr Patrick Iland of Adelaide University who kept me enthused with their encouraging and enlightening supervision. o All those involved with the Tannin Project from Adelaide University and the Australian V/ine Research Institute. o Dr Mark Buntine and Mr Phil Clements of the Chemistry Department of Adelaide University. From the former I received generous access to computational programs and advice in the molecular modelling component of the thesis, whist the latter performed the NMR analyses. Dr Kevin Wainwright of Flinders University for allowing me to use the potentiometric titration equipment. Mr Yogi Hayasaki of the Australian Wine Research Institute for performing the mass spectrometry. 'Wines 'Wines .