PROTEIN STABILISATION OF WINE Presented as a Thesis for the Degree of Master of Science (Food Technology) of The University of New South Wales by Paul Joseph Tyson Submitted: June 1982 DECLARATION The candidate Paul Joseph Tyson, hereby declares that none of the work presented in this thesis has been submitted to any other University or Institutio~Jo\:ee. ACKNOWLEDGEMENTS There are many people to whom my sincere gratitude and appreciation is owed without whose help this study could not have been completed. I particularly would like to thank Dr. T.H.Lee, associate professor, School of Food Technology, University of New South Wales, for his willing assistance and advice during the course of this study and for his valuable suggestions and editing during the preparation of this thesis. I am indebted to Waters Associates for the loan of the HPLC system, the use of their laboratory facilities and to their staff for their willing assistance. I particularly want to thank Mr Roy Day, and Mr Brian Walker. I would like to thank Mcwilliams Wines Pty Ltd, for allowing me the time, opportunity and the use of the Yenda Research Laboratory, for this project. I particularly want to thank Mr Doug Mcwilliam for his assistance at Yenda. I owe a great deal of thanks to my parents for their support and encouragement during this project. I would also like to thank Mrs G.Dias and Mrs C.Petroulakis for their expert typing of this thesis and Mr.David Duckworth for his exce1lent draughting of the figures. - ii - SUMMARY Soluble protein is generally considered to be responsible for the formation of amorphous type clouds and deposits in wine. Clarity and stability of wine is best obtained by fining before bottling to remove suspended particles, some of the colloidal materials and other possible unstable substances. There is a large volume of literature on wine protein, however, the protein level at which white wine :is stable and how ripening and processing conditions affect wine proteins is still unknown. The recent literature describes electrophoretic analysis of wine protein but generally does no more than list the number of electrophoretically distinct proteins and the amino acid content of the must (juice), wine and/or protein. The few studies that have examined the affect of variety and ripening on the nitrogenous components of juice and wine have been restricted to free amino acids or specific enzymes. The values quoted for the protein content of juices and wines vary considerably and probably reflect varietal, maturity, processing and analytical differences. The lack of a rapid, reliable and quantitative procedure for determination of protein in grape juice and wine 1s probably the main reason for the variation of reported results and to the paucity of information on the relationship between protein levels and wine stability. A rapid method has been developed for the estimation of total soluble protein (protein) in must and wine by high performance liquid chromatography (HPLC). The protein levels during ripening of grapes from eight white varieties grown in the Griffith district of NSW have been examined with the method along with the changes in protein level that occur during vinification. Protein levels in the ripening grape increase substantially between a sugar level of a0 and 11° Be, with for example, a five-fo1d increase to 840 mg/L for Traminer at 11° Be. Protein level of juice increases by unto ~0% during cold settling and ~ay increase, decrease or remain unchanged during fermentation. Bentonite rapidly removes protein from wine until a protein level of between 20 to ~0 mg/Lis reached - which is common for all white wines examined - when the rate of protein removal decreases significantly and to such an e::~e~~ that some protein remains even at high levels of bentonite addition. The protein level of wine where the rate of removal falls significantly coincides with stability as indicated by - iii - most commonly used protein stability tests. When wines are bottlectat a orotein level above that required for stability, the protein level falls bv sometimes as much as ~0% during the four months after bottling without any sign of protein instability. The question is raised of whether better wines can be made by reducing the amount of bentonite used to stabilise a wine, and/or by a search for a more sui tab-le stability test. - lV - CONTENTS Acknowledgements Summary 1. INTRODUCTION 1 2. LITERATURE REVIEW 3 2.1 Nitrogenous components of juice and wine 3 2.1.1 Total nitrogen 4 2.1.2 Amino acids 5 2.1.3 Nucleotidic materials o 2.1.4 Must and wine proteins 6 2.1.4.1 Stability of wine proteins 8 2.2 Factors affecting levels of protein in must and wine 10 2.2.1 Genetic and viticultural factors 10 2.2.2 Vinification factors 13 2. 2. 3 Effect of analytica1 methods on reported protein levels 17 2. 3 Separation methods for soluble protein 18 2 .4 Fining theory 23 2.4.1 Bentonite fining 24 2.4.2 Fining practice 28 2.4.3 Protein stability testing 29 3. MATERIALS AND METHODS 3.1 Grapes 33 3.1.1 Grapes sampling procedures 33 3.2 Juices 33 3.3 Wines 33 3.4 Winemaking procedure 34 3.4.1 standard method 34 3.4.2 Clarification 34 3.4.3 Non-standard winemaking procedures 35 3.4.4 Bott]ing procedures 35 3.5 Fining 36 3.6 Protein stability tests 36 3.7 Analytical methods 36 3.7.1 Protein estimation 36 3.7.2 Analysis of musts and wines 37 - V - 4. RESULTS AND DISCUSSION 4.1 Development of raped protein estimation system 38 4.1.1 pBondagel size separation 38 4.1.2 Reverse phase separation 43 4.1.3 Effect of addition of sodium dodecylsulphate to the 43 mobile phase 4.1.4 Protein separation system 45 4.1.5 Detection and quantitation of protein 47 4.1.6 Confirmation of protein peak as total soluble wine 48 and must protein 4.2 Total soluble protein in white wine grapes 50 4.2.1 Effect of variety on juice protein level 50 4.2.2 Effect of grape maturity on juice protein level 52 4.3 Protein levels of must and wine during vinification 62 4.3.1 Effect of clarification method on total soluble protein 62 of must and wine. 4.3.2 Changes in soluble protein levels during fermentation 65 4.3.3 Protein stabilisation of white wine 73 4.3.3.1 Stabilisation of Muscat Gordo Blanco: a special problem 85 APPENDIX I 93 BIBLIOGRAPHY 94 I. INTRODUCTION The formation of clouds and deposits in wine is a common instability problem confronting oneologists. Clouds and deposits may be classified into three tY.)es; microbial, crystalline and amorphous. Amorphous-t:voe clouds pose special problems of identification and correction, and in white wines are generally attributed to the presence of unstable proteins. Other wine components, such as polysaccharides phenolic substances and lipids have also been found to be involved in amorphous cloud formatjon. These unstable polymeric compounds are generally removed from wine by the addition of fining agents such as bentonite, gelatin and polyvinylpoly pyrrolidone. Fining agents when added to wine generally absorb or react with the unstable material and then flocculated, leaving the wine clear and if correctly fined, stable with respect to the formation of amorphous clouds. Bentonite is probably the most common fining agent added to wine to remove unstable protein. The accurate assessment of the correct amount of bentonite required to obtain a protein stable wine is at the centre of the problem, as the methods available for determining the stability of a wine with respect to protein are of an empirical nature and not based on a detailed knowledge of the behaviour of proteins during grape maturation, vinification, stabilisation and bottle maturation. Overfinin~, i.e. adding more fining agent than is required for stability, costs money in excess bentonite and in loss of wine in lees, and can alter wine compo­ sition thus resulting in sensory changes. Underfining, i.e. addin~ less fining agent than is required for stability, means that stability can not be guaranteed and may also lead to filtration problems. This project will examine high-performance liquid chromatopraohy as a new reliable method for estimation of soluble protein in must and wine that is more rapid and specific than conventional methods, such as K.jeldahl, colorimetric and gel-column methods currently in use. Such a technique will allow a quantitative examination of protein behaviour in several commercially important white wine varieties durinP­ grape ripening, must preparation, fermentation, fining, stability testing and bottle maturation. By monitoring the protein levels during grape ripening and through out vinification, it is considered that an imoroven understanding will be obtained of the role of protein in amorphous clourl formation and that an appropriate method for estimating correct fining procedures will ensue. Aspects of wine and juice comnosition, such as 2. pH, cation content, organic acids and phenolic substances, will also be monitored during vinification, and their role along with protein in determining wine stability will be examined. 3. 2. LITERATURE REVIEW 2.1 Nitrogenous components of must and wine. The nitrogenous constituents of must and wine, of which protein is one, are important components as they are involved directly in reactions that contribute to wine quality. Nitrogen-containing substances in wine and grape .juice include inorganic ammonium salts, amino acids, amides, amines, organic bases, nucleic acid derivatives, peptides and proteins, purines, pyrimidines and vitamins. The large number and the low and variable levels of nitrogenous compounds in must and wine (Tables 1 and 2) TABLE 1.