PROTEIN STABILISATION OF

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 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 :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 , 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 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 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. Nitrogen content of musts and wines.

Nitrogen (mg/L) Musts Wines Fraction Min. Max. Ave. Min. Max. Ave.

Ammonia 0 146 44 0 143 13 Amine 15 182 75 8 348 73

Amide 1 40 4 Polypeptide 38 132 81 2s~ 273 148 Protein 28 97 51 L 125 42 Hexosamine 18 29 23 8 29 14 Nucleic 23 Vitamin 0.16 Total 98 1130 390 70 781 350

Amerine and Joslyn (1970).

TABLE 2. Nitrogen compositon of Bordeaux wines.

Nitrogen (mg/L) White Wines Red Wines Fraction Min. Max. Ave. Min. Max. Ave.

Ammoniac al N 0 13 5 1 71 18 Amine N 10 67 34 41 138 81 Amide N 1.1 7.3 2.6 1. 5 5.6 3.5 Protein N 76 363 180 139 638 312 Polypeptide N 62 294 143 94 515 227 Total N 77 377 185 143 666 330

Ribereau-Gayon, Peynaud and Sudraud (1972). 4. make any study of the factors that influence the level of individual nitro­ genous components in wine and the role that these components play in wine processing difficult.

2.1.1 Total nitrogen

In the study of the nitrogenous components of must and wine, total nitrogen is most often determined, as it is easily obtained by the Kjeldahl procedure. Total nitrogen values are given in most studies that examine the effect of various viticultural and processing conditions on wine composition; for example Castino and Ubigili (1977) examined the effect of pressing methods on white wine quality and found little analytical difference between grapes that were pressed whole and grapes that were crushed prior to pressing; Ough and Anelli (1979) examined the effect of crop level on the amino acid and protein content of the must of grapes and found that total nitrogen as determined by Kjeldahl, decreased with increasing crop level, the values for total nitrogen is of most value as an indicator of gross compositional changes. The importance of the various nitrogen containing compounds make it difficult to relate total nitrogen to any particular parameter of wine quality. The variation in total nitrogen levels of must and wine that is found in the literature (Table 3) can be explained by varietal, viticultural and

TABLE 3. Total nitrogen concentration (mg/L) reported for musts and wines.

Total nitrogen (mg/L) Musts Wines Country Range Average Range Average

Brazil 57 - 437 203 France 77 - 952 351 Germany 102 - 980 523 Italy 46 - 201 102 U.S.A. 542 - 2385 985 78 - 700 296 Others 98 - 1130 390 70 - 781 350

Amerine and Ough (1980).

rePional differences inherent in such work. Some values include: 582 to 2163 mg/1 (Bustos 1975); 310 to 1093 mg/L (Spirov 1975); and 26.1 to 76., mg/L for V. Lubruscana varieties (Kluba, Mattick and Hackler 1978). The 5 Kjeldahl method is used almost exclusively for the estimation of total nitrogen, although the procedure actually utilised is often a modification of the standard method. Factors that influence the level and type of nitrogenous compounds in must and wine will be discussed 1n Section 2.2 1n conjunction with those that effect the level of protein.

2.1.2. Amino acids

The amino acids of must and wine, both free and proteinaceous, have been extensively studied, more so than in other fruits because of the importance of amino acids as yeast nutrients during fermentation (Burroughs 1970). During champagnisation it has been found that amino acids are actively incorporated by yeast during cell metabolism and are involved in the biosynthesis of proteins and other amino acids (Kirtadze, Kalichava and Jokharidze 1976). Ferenczy (1966) has described the importance of amino acids in the development of aroma and bouquet of the finished wine, especially the aromatic compounds associated with yeast metabolism. Studies of amino acids in the brewing industry indicate that amino acids are involved in the formation of higher alcohols (Schulthess and Ettlinger 1978). Moreover, Ferenczy (1966) suggested that free amino acids might be involved in stabilising some wine constituents. It has also been shown that melanoidins are formed by reduction of amino acids during the heat treatment of red dessert wines (Fyrtsov 1977). The major amino acids found in must, wine and wine protein are alanine, arginine, aspartic acid, glutamic acid, cysteine, glycolic acid, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, Amino acids have been found to make up 60 to 90% of the total nitrogen in musts (Kliewer 1968), with the amino acids proline, arginine and glutamic acid occuring in the greatest quantity. Unlike other amino acids proline is not readily assimable by yeast during fermentation (Burroughs 1970). The large volume of literature describing amino acids 1n musts and wines can be attributed to the importance of these compounds to wine quality and may also be a result of the ready availability of automatic amino acid analysers. As a consequence, it appears that a majority of' the studies list only the amino acids isolated and how they change as a result of some treatment or process with little discussion as to the importance of the results to the winemaker. For example, Anelli (1977) 6

in her study of must proteins, although recognising the lack of information about actual protein levels present in a protein stable wine and that the majority of studies on nitrogenous compoundes have been restricted to amino acids, listed only the amino acids and protein fractions isolated from the musts; little discussion was presented of varietal, regional or maturity effects which are important when considering the role of amino acids and other nitrogenous compounds in winemaking. Similarly Kluba, Mattick and Hackler (1978) when examining the free and total amino acid content of some Vitis lubruscana varieties reported the changes in the amino acid composition that occured during maturation but advanced no suggestions as to the significance of the findings, Another example is Ough and Anelli (1978) who studied the effect of crop level on the protein and protein amino acid composition of Zinfandel must. They found that the amino acid make up of the proteins was not affected qualitatively or quantitatively by crop level; no suggestions, however, were made as to the consequence of the results and little information was given that might assist a wine­ maker improve the quality of his wine or help him come to a greater understanding as to what contributes to the quality of wine.

2.1.3. Nucleotidic materials

Little attention has been focussed on the role of nucleotidic material in wine quality, as the major part of research into nitrogenous compounds has centred on amino acids and proteins; the difficulty in separating and quantitating the nucleotides and other remaining nitrogenous compounds has limited the number of investigations. Nucleotides are now believed to be more important to wine quality than at first thought. Nucleotides and nucleosides have been shown to constitute a significant proportion o~ the UV absorbing material of wine which was previously attributed solely to phenolic substances (Somers and Ziemelis 1972). Castino and Ubigili (1977) in a study of the effect of pressing methods on wine quality found that high quality wines were characterised by high amounts of purines, pyrimi­ dines, nucleosides and nucleotides.

2. 1.4. Must and wine proteins

Proteins in wine largely originate from the grapes from which the wine was made, and the levels would vary according to the stage of grape maturity and method of vinification. The contribution of yeast to the soluble protein level of wine will be discussed in Section 2.2.2. 7

The proteins of wine are considered to be of a heterogeneous nature with a globular structure and complexed as glycoproteins (Rib~reau-Gayon et al. 1972, Lyubarevich et al. 1975). The molecular mass of wine proteins has been reported within a large range, 10 000 to 50 000 daltons (Somers and Ziemelis 1973), 17 500 to 95 000 (Tarantola 1971), 18 000 to 23 000 (Bayly and Berg 1967), 11 000 to 28 000 (Yokotsuka et al. 1977); Rib~reau-Gayon et al. (1972) considered that the molecular mass of wine protein was greater that 10 000 daltons. Vidal-Barraquer (1979), in a review of relevant literature, reported that must proteins have molecular masses between 10 000 and 200 000 daltons and that the protein fraction of the skin to be of the order of 450 000 and 571 000 daltons. These high molecular weight protein compounds of the skin would probably be complexes of protein, carbohydrates and lipids. The large variation in reported molecular masses for must and wine protein can probably be attributed to the different varieties examined, the processing conditions and analytical techniques. A wide range of isoelectric points has been reported for the soluble protein of wine. The isoelectric point of wine proteins has been found to vary from 2.6 to 7.8 (Lyubarevich et al. 1975), 3.0 to 5.0 (Molnar 1975), 3.3 to 4.0 (Rib~reau-Gayon et al. 1972), and 3.3 to 3.7 (Moretti and Berg 1965). Such variation in isoelectric points for wine protein probably reflects the genetic differences between varieties, as different varieties are claimed to have distinctive protein patterns (Koch and Sajak 1959) (See Section 2.2.1). The isoelectric points of wine proteins are generally within the range of normal wine pH, which is important when considering the flocculation of wine protein. Quantitation of soluble protein in must and wine also suffers from a large variance (100 fold) in reported values: Bayly and Berg (1967) found 20 to 260 mg/Lin must and 30 to 275 mg/Lin wine by the colori­ metric method of Diemar and Maier, after protein separation by dialysis; Rib~reau-Gayon et al. (1972), by means of the Kjeldahl method, found protein levels in wine ranging from 476 to 2270 mg/L; Spirov (1975) found 175 to 525 mg/Lin wine with higher levels, 381 to 837 mg/Lin must, and Anelli (1977) using gel-filtration and Kjeldahl procedures reported 19 to 91 mg/Lin the musts of 14 white varieties. In contrast Yokotsuka et al. (1977) from the summation of the amino acids quantitated by analysis of a protein concentrate obtained by dialysis, TCA precipi­ tation and lyophylisation reported the low protein levels of 1.5 to 9.0 mg/L for must and 0.1 to 0.4 mg/L for wine. These large differences in 8 the protein content of must and wine have been variously attributed to analytical, varietal and processing factors (factors that can affect protein levels of juice and wine will be discussed in Section 2.2). The protein levels of other fruits have been investigated by many workers, who generally report protein level as a percentage of the fresh weight of the whole furit. For example, Hansen (1970) expressed protein levels as a percentage of fresh weight of several fruits by multiplying the total nitrogen value obtained by the Kjeldahl method by the factor 6.25. Values presented were pineapple, 0.4%; apple, 0.2%; apricot, 1.0%; grapefruit, o.5%; orange, 1.0%; pear, 0.7%; strawberry, 0.7%; banana, 1.1%; cherry, 1.2%; grape, 0.6%; and tomato,1.1%;. Specific enzymes are the subject of most studies with fruit protein. Interest in fruit protein, and particularly specific enzymes, is due to importance in plant biochemistry ~hereas in wine, it is the protein 1n the juice and consequently in the wine that is of importance. The presence ofµhenolic substances in fruits makes the isolation of protein from fruit tissues difficult, which presents problems when studying specific enzymes.

2.1.4.1 Stability of w1ne protein

The formation of amorphous-type clouds in w1ne has been generally attributed to the precipitation of unstable protein. Recent studies, however, have shown that there are many other materials which may be involved with instability. Proteins exist in a colloidal state in wine and account for 7-12% of the total colloidal content in wine (Ferenczy 1966). Protein might also be expected to be in the soluble state in wine. The balance of the wine colloids would be made up of materials, such aspolysaccharide~ phenolic substances, lipids, anthocyanins, and pectins (Usseglio-Tomasset and Distefano 1977). The role of the other colloidal materials in amor­ phous cloud formation in wine, previously attributed to protein aloneJ has recently increased in importance. Kazumove and Torosyan (1975) analysed the sediments formed in white wine and found tham to be a complex of proteins, phenolic substances,polysaccharides, trace elements and acids. The involvement of polysaccharides in the formation of deposits in white wine has been supported by the studies of Datunashvilli (1975) and Nilov et al. (1975). The importance of lipids in wine cloud formation has been suggested by Mekhouzla (1979) who found that all wines ~usceptible 9 to cloud formation contained high levels of lipids, and that treatments, such as gelatin, bentonite and PVPP fining, reduced the total lipid content and thus enhanced the wines stability. Proteins were found to enhance lipid solubility allowing the formation of lipoprotein complexes, which have been shown to be unstable in the conditions that prevail in wine (Mekhouzla et al. 1976). The involvement of lipids with protein in nature is well known, especially the lipoprotein complexes of cell walls and membranes, and therefore the involvement of lipids with phenolic substances, polysaccha~idesand proteins in amorphous cloud formation is not unexpected. The majority of studies, however, have concentrated on soluble protein and its behaviour in amorphous-type cloud formation. The factors that are considered to be involved in formation of protein instabilities include protein isoelectric point, wine pH, phenolic content, metallic ion content and wine storage conditions. The isoelectric points of wine proteins are generally within the range of normal wine pH, which helps explain why slight disturbances, such as elevated temperatures, exposure to light and pH change due to acid decomposition, can lead to protein precipitation. Protein precipitation in wine involves more than a simple relation between temperature and pH and isoelectric point. The level of phenolic substances, ionic status of the wine and the colloidal materials must also be considered. Hydrogen bonding and quinone reactions and more recently hydrophobic bonding (Oh et al. 1980) are thought to be the main type of reactions involved in protein-tannin complex formation. A proposed mechanism for the protein-tannin reaction has been suggested from studies involving the problems associated with extracting active enzymes from plant tissue (Loomis and Bataille 1965, Van Sumere et al. 1973). These proposals have been summarised for the protein-tannin reactions in wine by Ferenczy (1966) and Cordonnier (1966). Some proteins, being of a hydrophilic nature, bind up water molecules forming protein hydrates. The protein hydrate probably has the following structure: O-H-0-H t C-NH- The tannins, through the phenolic hydroxyl groups, remove the water of Rolvation; such dehydration decreases the solubility of the protein which then precipitates when sufficient hydrophilic groups are occupied. 10

Precipitation may be reinforced by formation of covalent bonds through the oxidation of the phenols of the tannins to quinones and subsequent polymeric condensation. Protein-tannin complexing can result in the positively charged protein sol changing to a negatively charged dehy­ drated sol, which may precipitate by the action of small quantities of metallic salts; this is especially so with wines high in copper (Ferenczy 1966), Concern has been expressed about tannin exudates from corks contributing to protein precipitation; Pes (1976), however, claims that tannin from this source is not involved in protein stability problems.

2.2. Factors affecting levels of protein in must and wine

The range of levels for protein and other nitrogen-containing compounds reported in must and wine can be attributed to genetic, viticultural, vinification and analytical factors.

2.2.1 Genetic and viticultural factors

The influence of grape variety on protein levels of juice and wine is unclear. Amerine (1953), from general observations in industry, indicated that certain varieties self clarify more readily than others, with wines made from the more pulpy varieties the slowest to clarify. Such an observation alludes to the colloidal status of wines and suggests that the clarification rate of individual varieties is reflected by the nature of the materials (protein, polysaccharldes, polyphenols and lipids) that make up wine colloids and also to a variation in pectic enzyme activity. Koch and Sajak (1959), by means of electrophoretic techniques, separated wine protein into several fractions and claimed that there was a specific protein band pattern for each variety examined. Moretti and Berg (1965), however, found as much variation in electrophoretic band patterns within a variety as between varieties. A compromise was reached when Bayly and Berg (1967) suggested that significant varietal differences existed between protein band patterns of both juices and wines, but that total protein content varies as widely within varieties as between varieties. Varietal differences in protein patterns have also been described by Nanitashvili, Samadashvili and Shilakadze (1975) and Molnar (1975). Wolfe (1976), using starch gel electrophoresis, 11 examined the isozyme banding patterns for over sixty varieties and found that leucine aminopeptidase, indophenol oxidase, acid phosphatase and catechol oxidase were the most usefu] for identifying varieties; this agrees with previoHs work of Drawert and Muller (1973) who also found that four proteinaceous components separated by electrophoresis from different varieties were common amongst all varieties. Differences in protein types between varieties is to be expected, as the protein patterns would be an expression of the genetic code of the particular variety. Tarantola (1971) recalling unpublished Californian results explains these conflicting results concerning the nature of protein patterns of different grape varieties as a conflict between the genetic expression by the vine and environmental influences. Varietal and environmental effects on protein patterns and levels in other fruit have also been recognised. Hansen (1970) cites literature reporting variation in protein content and composition, determined by K,jeldahl and electrophoretic techniques, between different species of cherry, currant, grape, raspberry and apple and that regional effects have been observed with the protein levels of different cultivars of apples in Australia. Viticultural factors affecting grape composition include soil type, region, climate and operations~ such as pruning, fertilisation, insect and disease control. These factors together make up the vine environment which has a significant influence on grape maturation and composition. The multi-faceted nature of the environmental influences and their interaction precludes study of the effect of any one factor. Therefore, as the environment has a profound influence on the maturation of the grape, it would be more constructive to-.study the effect of grape maturation on protein levels and patterns rather than the effect of any one environmental factor. The protein level in maturing and ripening grape berries has not been well documented; generally, work on nitrogen-containing compounds during berry development and ripening has been restricted to specific enzymes or amino acids. It is generally accepted that the stage of maturation affects the level of protein in the juice (Moretti and Berg 1965, Cordonnier 1966, Ferenczy 1966, Bayly and Berg 1967, Tarantola 1971). Koch and Sajak (1959), however, claimed that protein, estimated by the biuret method, forms only during ripening and is not detectable 1n the juice of immature berries. Such a claim is incorrect and probably reflects the insensitivity of their method; protein as enzymes 12 would be present, even at low levels, at all stages of grape development. The role of protein in the development and ripening of many fruits other than grapes has been studied extensively. In many fruits, protein, estimated by Kjeldahl, generally increases with ripening although Hobson (1974) found that the total protein of tomato did not change during ripening. Studies with apple, pear, tomato, and avocado indicate that if protein synthesis 1s inhibited, ripening does not occur (Dilley 1970). The study of protein in ripening fruits has often involved enzyme activity rather than total protein content; thus many protein studies have involved enzyme reaction methods (Sacher 1965, Hawker 1968, Drawert 1974, Hartmann 1974, Hobson 1974, Skakoun and Daussant 1974, Young, Salminen and Sornsrivichai 1974, Latche et al. 1974). Protein activity has been shown to increase during ripening and has been attributed to changes in cell membrane permeability (Sacher 1965, Hansen 1970). Rhodes (1970), however, considers that these permeability changes and consequent increases in protein activity play a secondary role to actual protein synthesis as the major cause of fruit ripening. Molnar (1975) considered that the protein content of grapes was independent of maturity. The demonstrated importance of protein synthesis in the ripening of other fruits suggests the contrary and that protein levels of grapes would be expected to show signs of change during maturation. Vos and Gray (1979) found an inverse relation between protein nitrogen and titratable acidity in grapes and suggest that it implies_an increase in protein content during fruit maturation. A further obervation that supports the importance of protein during ripening of grapes is that protein levels are lower in non-maturing years (when high acidities and immature grapes are expected) than in maturing years (Koch and Sajak 1959). Ripening can be described as a period of metabolic reorganisation founded on a change in protein synthesis in which new enzymes are synthesised to catalyse the ripening process. Studies of soluble protein during grape maturation have been restricted to the activity of specific enzymes with little quantitative data available. Hawker (1968) examined the changes in activity of malate dehydrogenase, phosphopyruvate carboxylase and pyruvate decar­ boxylase during ripening of Sultana berries and found that, in general no large consistent increase occurred in the activity of these enzymes during the period in which malic acid concentration decreased. But there was an unexplained increase in the activity of these enzymes in the sixth week after verai son, which could not be related to changes 13

1n berry metabolism. Datunashvilli (1974), however, found, that the enzyme activities of must varied considerably depending on variety, maturity and growing conditions. The free and total amino acids have taken precedence in the study of nitrogenous compounds during the ripening of grapes. Total and free amino acids have generally been shown to increase during ripening (Lafon-Lafourcade and Guimberteau 1962, Nassar and Kliewer 1966, Pandey et al. 1974). Kliewer (1968) described a two-to fivefold increase in total amino acid content during ripening of grapes, and Kluba, Mattick and Hackler (1978) found that both the free and total amino acid content of Vitis labrusca varieties increased during ripening. Separation and estimation of the free and total amino acids of grapes during ripening indicated that arginine increased 100-fold, while other amino acids variously increased or showed little or no quantitative change (Marcy, Carroll and Young 1981). The acceptance of the importance of amino acids to wine quality could indicate that a study of the use of amino acid content as an aid to deciding optimum grape maturity could be beneficial. The role of other viticultural factors is more difficult to discern because of the effect climate, soil type, etc. has on the vine environ­ ment. Koch and Sayak (1959) reported no effect of fertilisation on protein levels, but Moretti and Berg (1965) found that protein content was dependent (among other factors) on fertiliser levels. Ough and Anelli (1978) examined the effect of crop level of Zinfandel grapes on protein and amino acid content of the juice and found that amino acid composition did not vary with crop level but that protein levels of the juice increased with increasing crop level the difference in protein levels were not significant at 62-95 mg/L, as Ough and Anelli (1978) separated protein by gel filtration and then employed the Kjeldahl method to determine protein nitrogen. The sum total of viticultural effects on protein levels of grapes and grape juice can best be examined by studying the protein levels at various stages of maturity which to date has not been done satisfactorily.

2.2.2 Vinification factors

The processing of grapes to wine can affect the level of soluble protein in wine; harvesting, crushing, clarification, fermentation, fining, stabilisation, filtration and bottle maturation are operations 14 that may contribute to the variation of reported levels of soluble protein in juice and wine. The general effect of processing conditions has been noted by Cordonnier (1966), Tarantola (1971) and Nanitashvili et al. (1975). The advent of machine harvesters has lead to higher levels of phenolics and nitrogenous compounds in juice and wine due to greater skin contact with juice as a result of berry damage incurred during harvest (Burtov, Razuvaev and Mindadze 1977). Crushing ensures intimate contact between juice, skins and other grape solids and as a consequence the nitrogen content and one would expect protein content of the juice increases the longer juice is in contact with skins and solids (Ough, Berg and Amerine 1969); furthermore one would expect free run juice (low solids) to have a lower protein content than press juice (high solids). Press juice would be expected to have higher protein levels as it would contain a larger proportion of grape cell structural material, such as cell walls and cell membranes which are in part made up of protein. The current trend 1n white wine making 1s to reduce the solids level of juice to at least 1% solids - some winemakers specify filter bright juice - before the commencement of fermentation. Thus several methods, such as cold settling, addition of pectic enzyme and proteo­ lytic enzyme preparations, centrifugation, filtration, fining, or combinations of thesP trPat~ents, have bePn d·ve1 oped to clarify juice to the required solids level. There is, however, no data available on the influence of clarification treatments on soluble protein levels of juice, although one could surmise that due to the nature of drained juice-colloidal particles that contain protein in a juice solutjon-the method of clarification could considerably influence the soluble protein content of clarified juice. Observations by winemakers have certainly confjrmed such a suggestion. Pectic enzyme prPparations can modify the co~position of must, but nothing 1s known of the effect of these preparations on the level of nitrogenous compour.ds or specifically soluble protein. Ough and Crowell (1979) examined the effect of clarification method on the composition and quality of white wine and found that pectic enzymes were not involved in the anal)~ical and sensory changes which occured during fermentation. These changes were prjmarilv dependent on temperature and skin contact time. Protein or nitrogen levels, however, 15 were not estimated in this study, and thus no information appeared as to the effect of these enzyme additions on soluble protein levels. Recent Russian work suggests that protein levels of grape juice can be reduced with proteolytic enzyme preparations and that a wine can be produced that is stable with respect to protein (Datunashvili 1974, 1975, Nanitashvili and Samadashvili 1974, Tikonova and Platsynda 1976), although Adler-Nissen (1976) questioned the success of proteolytic activity in juice and wine; he found that native globular proteins generally resist proteolysis, because the compact tertiary structure of the protein protects most of the peptide bonds from enzymatic attack. Centrifugation of beer has been reported to remove protein and to produce a stable product with respect to protein (Ihle 1975). It is considered that centrifugation would remove insoluble proteins bound up in the skin and grape solids, and thus if the must is centrifuged immediately after crushing the protein solubilisation that could occur while the skins and grape solids remain in contact with the juice might be prevented. Centrifugation may also disrupt or remove colloids associated with proteins, physically removing the protein or facilitating its flocculation. Filtration, in a similar way to centrifugation, may also influence the soluble protein levels of grape juice. Fermentation is a complex biological process that dramatically changes the composition of grape juice. It is generally accepted that nitrogen levels decrease during fermentation; the major cause for such a reduction is the absolute requirement for assimilable nitrogen, ammonium salts and .aminoacids by yeast for cell growth (Tarantola 1966). Kluba et al. (1978) reported a general decrease in the levels of amino acids of V. lubruscana juices during fermentation except for that of proline which was apparently not utilised by yeast during fermentation: similar results have been reported for V. vinifera varieties (Kozub, Balanuga and Furfure 1977). The constancy of the proline level before and after fermentation could also be due to active acretion and secretion by yeast (Tarantola 1966). As with other fermentation by-products, the production of amino acids by yeast is probably dependent on the grape juice, i.e. the medium, and the physiological state of the yeast cells (Pavlenko, Pekur and Bur'yan 1976). There is some evidence to suggest that protein levels decline during fermentation, possibily because increased alcohol content, decreased sugar content, changing redox potential and acidity lead to denaturation, complexing and hydrolysis of proteins. For example, 16

Ferenczy (1966) and Spirov (1975) found that protein levels during fermentation decreased from 15% to as much as 75% of the level in the juice. Different conditions of fermentation could account for the large variation in the reported reduction of soluble protein during fermentation; fermentation variables include temperature, yeast strain and juice composition. High fermentation temperature can lead to a greater loss of nitrogen than at low temperatures (Ough and Amerine 1965), probably because of increased yeast activity; yeast strains would be expected to have different nitrogen assimilation patterns; the initial nitrogen level of juice and its availability for yeast metabolism, together with any nitrogen added to the juice by the winemaker and the presence of any yeast inhibitors in the juice, could alter the pattern of nitrogen utilisation during fermentation. The contribution by yeast to the level of soluble protein in wine has not been clarified. Moretti and Berg (1965) suggested that yeast protein could be involved in protein clouding of wine; subsequently Ferenczy (1966) claimed that yeast can add 20 to 30 mg/L protein to wine, but Bayly and Berg (1967) demonstrated that yeast probably contributes peptides to wine but that they are not involved with wine instability. In addition Pavlenko and Kuridze (1977) found that yeast adds protein to wine bµtonly at low concentrations and furthermore this protein was not responsible for protein clouding of wine. Somers and Ziemelis (1973), however, could find no evidence for the presenceof yeast protein in new wine. The secretion of extracellular proteolytic enzymes is considered the source of yeast protein in wine {Pavlenko et al. 1976). Vos and Gray (1979) considered that this protease secretion was in response to low levels of assimila­ ble nitrogen present in the juice, and that proteolytic activity is associated with the formation of H2s from the breakdown of sulphur containing amino acids in the proteins. On completion of the primary fermentation, the level of soluble protein in the new wine can be affected by the length of time the wine spends in contact with the yeast lees and whether bacterial growth occurs. Yeast autolysis can increase soluble protein levels (Ferenczy 1966). The growth of bacteria, either during or other bacterial spoilag~ is favoured by extended contact of the wine with yeast lees and such activity might be expected to increase protein levels through secretion of protein by the bacteria. A decrease in protein levels with bacterial activity would not be expected as Feuillat 17

(1977) found that protein does not support the growth of lactic acid bacteria. Lactic acid bacteria may also be responsible for the reappe­ arance of ammoniacal nitrogen in wine (Cordonnier 1966). Normal cellar operations, such as fining,pH and acid adjustments, filtration, cold stabilisation, and storage, can also influence the soluble protein level of wine. Since the isoelectric point of wine protein is generally regarded to lie within the normal pH range of wine, any pH or acidity adjustment could result in protein destabili­ sation. Filtration, especially through charged filter media, could absorb some proteins or destabilise wine colloids resulting in further destabilisation of protein. Ion-exchange resins may also absorb protein either by the ion-exchange mechanism or by active adsorption onto the resin. As wine is a complex mixture of many compounds it would be expected that many reactions may take place, albeit slowly in a wine during storage. Apparently protein hydrolysis is one of these reactions as Nanitashvili and Shilakadze (1974) found that protein levels decrease during storage. Ermachkova (1975) also reported that protein levels decrease during storage of semi-sweet .

2.2.3 Effect of analytical methods on reported protein levels

The methods used for protein estimation probably contribute more to the variation in reported soluble protein levels in juice and wine than any other factor. Total nitrogen can be reliably determined by the standard Kjeldahl method, provided all organic nitrogen is decomposed into ammonia (Kichkovski and Mekhouzla 1967). The reliabi­ lity of the Kjeldahl method in practice, however, is possibly over­ -emphasised as the technique required to achieve meaningful results is not easy to acquire. The total nitrogen content cannot be used to obtain the protein content as there are many other nitrogen containing substances that would contribute to an unrealistic figure for protein (Bayly and Berg 1967). Thus for accurate estimation of protein or specific nitrogen-containing compounds they must first be separated from wine. Protein has generally been separated from wine with a reagent that specifically precipitates protein. The inherent complexity of wine contributes to the unreliability of precipitation methods. Wine

components, such aspolysaccharides 1 pectins, lipids, phenolic substances and metal ions, can alter the solubility of protein, reduce the sensiti­ vity of protein towards the reagent or may interfer with the reagent and partake in the flocculating reaction (Cordonnier 1966, Ferenczy 18

1966, Somers and Ziemelis 1973). Koch and Sa,jak (1959), Greenberg and Shipe (1979) and Peterson (1979) found that when separating protein with various reagents, such as ethanol, trichloroacetic acid, ammonium sulphate and phosphomolybdic acid, the amount of protein precipitated varied with the precipitant and with the type of protein; these procedures often precipitated non-protein nitrogenous compounds as well. The wide range of precipitants utilised for protein separation would appear to contribute to the variation in reported levels of wine protein. The modification of the protein estimation procedures by investigators to their own design may also enhance the inconsistency of reported protein levels; Koch and Sa,jak (1959) modified Voits procedure, Moretti and Berg (1965) modified the procedure of Diemar and Maier and Bayly and Berg (1967) used a further modification of Diemar and Maier's method when estimating protein in wine. Chemical methods of protein separation use either Kjeldahl or a colorimetric method for quantitation of the protein content of the precipitate. Modifications by researchers of the colorimetric methods of Diemar and Maier (1962) Lowry et al. (1951) and Gornal et al. ( 1949) and further inconsistencies. The waveleng·th at which the developed colours of the biuret and Folin-Ciocalteu methods are measured vary between investigators. Somers and Ziemelis (1973) reported that different proteins give different colours with the Folin-Ciocalteu reagent. The inconsistencies and inadequacies of these protein estimation methods explain the lack of meaningful investigations into total soluble protein of grapes and wine. The accurate estimation of soluble protein levels in must and wine, there­ fore, requires that the protein fraction be separated from any inter­ fering substances and quantitated by a method specific for proteins.

2.3 Separation methods for soluble protein

Separation of protein from wine, other than by chemical means, can be achieved by dialysis, ultrafiltration, permeation chromatography and electrophoretic techniques. Dialysis with cellulose membranes has been used extensively to separate wine proteins from wine by removing the small molecules that can interfere with normal chemical separation and quantitation methods; Rib~reau-Gayon et al. (1972) considered that one of the characteristics of wine protein was that it was not dialysable through cellulose membranes. In general,dialysis is carried out against a trisglycine buffer, the pH of the buffer varies between reports and a 19 period of several days is required before separation is complete (Moretti and Berg 1965, Bayly and Berg 1967, Yokotsuka et al.1977). Dialysis methods are slow and involved and not suitable for rapid separation of protein from wine. The quantitation of the protein in the concentrate could still suffer from interference from other high molecular weight material retained by the dialysis membranes. Dialysis is a normal preliminary step before electrophoretic examination of the concentrate. Ultrafiltration could also separate proteins from wine by spliting the wine into several streams of differing molecular size with membrane filters of varying molecular weight limits. Ultrafiltration has not been used as an analytical tool in wine protein studies. Ultrafiltration has found some applications in the actual vinification processes of concentration, sterilisation and stabilisation (Section 2.4.2). Liquid permeation chromatography is well suited for the separation of soluble proteins from other soluble wine components. Permeation chromatography separates a mixture of molecules according to molecular size. When a mixture of molecules is passed through a column of porous gel granules, the molecules generally appear in the effluent in order of decreasing molecular size. Fractionation is believed to occur when diffusion of the molecules into the gel pores is restricted but not prevented, because of their size and because they pass through the column at rates that are related inversely to the fluid volume accessible to them within the column (Andrews 1964). Gel permeation chromatography withpolysaccharide-type gels such as Sephadex (Pharmacia) has been a common technique for protein investiga­ tion in recent years. Somers and Ziemelis (1973) developed a method for the direct determination of wine protein on Sephadex G-25 fine, with dilute acetic acid as the mobile phase; the benefits claimed were· rapid separation (the protein fraction eluted first along with polymers such aspolysaccharides), the facility for automated quanti~ation and limited pretreatment of the wine. Anelli (1977) and Ough and Anelli (1978) studied the proteins and their amino acid composition of grape juice with a similar system. Gel filtration was the method chosen by Ochi and Nakanishi (±975) to examine the nitrogenous components of various media utilised in alcoholic fermentation of several substances. Ferenczy (1966), Bayly and Berg (1967), Kichkovski and Mekhouzla (1967), Nanitashvili and Samadashvili (1974) and Aivazov, Gorinova and Tsarkov (1975) have all used gel filtration in their studies of wine protein. 20

The gel riltration systems, however, have not been able to fractionate the protein of wine into individual components (Bayly and Berg 1967). Gel permeation chromatography with polysaccharide-type columns has other disadvantages; the gel columns can be easily disrupted by excessive pressure, are subject to microbial degradation and the length of column required to give good protein separation is such that separation time can be of the order of one hour. Protein separation can only be obtained with columns of sufficiently high exclusion limit, i.e. the molecular weight above which the molecules pass straight through the column and are eluted with the void volume with consequent loss of protein resolution. Gel filtration, however, can provide a valuable tood 1n wine protein studies. Recent developments in high-performance liquid chromatography (HPLC) afford the opportunity of separating protein from wine many times faster than with conventional chromatography. Liquid/ liquid chromatography, both normal and reverse phase, ion-exchange and gel permeation chroma­ tography, are available in the high-performance mode. Protein studies with HPLC systems have generally been restricted to mixtures of proteins of biological and clinical importance. All modes of liquid chromato­ graphy have been employed to separate mixtures of proteins; reverse phase has been utilised by Rivier (1978) with trialkylammonium phosphate buffers as the mobile phase, by Hancock et al. (1979) who studied the effect of various cationic reagents on the separation of peptides, and by Monch and Dehnen (1977) who separated a mixture of proteins with a gradient elution with a phosphate buffer and isopropanol. Anion exchange and steric exclusion chromatography has been used to separate enzymes of clinical importance (Chang, Gooding and Regnier 1976). Furthermore Thrall and Spelsberg (1979) have reported the separation of proteins and nucleotides by permeation chromatography with sodium acetate buffers. A difficulty with the HPLC separation of proteins is the tendency of the proteins to be absorbed onto the silica based packing material of the columns. Thrall and Spelsberg (1979) make mention of this problem but relied on the covalently bonded phase of the column packing and adjustments of the ionic strength of the mobile phase to minimise the absorption effects. It has been found that denaturing solvents, such as dilute solutions of sodium dodecylsulphate (SDS), urea and guanidine hydro­ chloride (GyHCl), prevented the absorption of protein on agarose 21 columns (Fish 1977) and on controlled pore glass columns (Frenkel and Blagrove 1975). These surfactants (ion pairs) and denaturing agents are also widely used in electrophoretic studies of proteins. Monch and Dehnen (t977) included 2-methoxy ethanol in the mobile phase (as it behaves like a surface active agent) to separate protein on a reverse phase support. Rivier (1977) found that recovery of protein from reverse phase systems was dramatically increased with the inclusion of a detergent (ion pair), such as SDS, in the mobile phase. Thus it appears that with appropriate column conditions, a mixture of proteins can be separated by most commonly available HPLC columns. But the separation of protein from a complex beverage system such as wine has not been reported as far as can be ascertained. A rapid separation of protein from wine would offer the advantage of studying protein behaviour in detail during the short time of vintage. HPLC methods have successfully separated and quantitated organic acids, sugars,polysaccharides>Phenolic substanc0 and anthocyanins from wine (Rapp and Ziegler 1976, Wulf and Nagel 1976, Ong and Nagel 1978, Subden, Brown and Noble ~978). The quantitation of a protein isolate from wine has generally been determined by Kjeldahl or colorimetric method and these have certain disadvantages (Section 2.2.3). The problems associated with accurate protein quantitation mean that investigators have preferred qualitative electrophoretic techniques to examine the separated must and wine protein (Nanitashvili and Shilakadze 1974, Nanitashvili et al. t975, Nanitashvili and Samadashvili 1975, Lyubarevich 1975, Anelli 1977). Electrophoretic studies have shown that wine protein is of a heterogeneous nature, the number of protein fractions isolated from wine varying between reports. Electrophoresis relies on the differen­ tiation of the protein fractions by means of their characteristic movement in an electric field. Since proteins are charged molecules under most conditions, the rate of movement in an electric field depends on the charge intensity on the molecule. Gel electrophoresis uses the electric field as a driving force for a separation of proteins on the basis of size. Moretti and Berg (1965) separated wine protein into two fractions with low voltage electrophoresis and into four to five fractions with high voltage electrophoresis. More recent work has resolved the protein of wine into 17- 28 distinct components (Kichkovski et al. 1 975), but Kazumov and Torosyan (1975) found only seven components in protein isolated from white wine deposits, possibly 22

indicating that only certain fractions are involved in protein instabi­ lity. Somers and Ziemelis (1973) concur, as they considered there were two protein fractions, with one complexed with flavonoid material and responsible for protein instability. Bayly and Berg (1967) also consi­ dered that there were basically two protein fractions in wine and that only one was responsible for protein instability, although they had isolated 16 different components of wine protein by electrophoresis. Nanitashvilli and Shilakadze (1974) also separated protein into 13 to 16 fractions by electrophoresis and characterised these into two types; they found 8 - 10 acidic fractions and 5 - 6 alkaline fractions. Studies of the protein fractions of beer haze have shown that the'acidic fractions have a greater tendency to cause haze formation than the more basic protein fractions (Savage, Thompson and Anderson 1975); a similar role for the acidic fractions in wine would be expected as the acidic fractions have a low isoelectric point, close to the normal pH of wine. Electrophoresis has proven to be a valuable tool for the investigation of wine proteins especially for the characterisation of grape varieties. Wolfe (1976) continuing the work of Drawert and Muller (1973) found that four isozymes could be used to distinguish different grape varieties. Variation in methodology, lack of an accurate means of quantitation of soluble protein and slow speed of operation has prevented greater success with this technique. Proteins have a uv absorption maximum at 280 nm, which provides a convenient method for the specific detection and quantitation of protein. The continual monitoring of the absorbance of the effluent from a liquid chromatographic column at 280 nm can produce a uv absorbance chromatogram of the wine introduced onto the column, and if a protein peak can be iden­ tified it can be quantitated by comparison with a standard protein, gene­ rally bovine serum albumen. During liquid permeation chromatography high molecular weight constituents are eluted first-in wine these are proteins, polysaccharides and pectins. As polysaccharides and pectins are trans­ parent to uv light, monitoring the column eluate at 280 nm detects only the proteins which would be expected to appear as the first peak of the chromatograph as they are the highest molecular weight uv absorbing material in wine (Somers and Ziemelis 1972). Thus continuous uv absor­ ption provides a specific and accurate method for protein detection and quantitation easily adapted to automatic analysis. Other means of detection and quantitation of protein from a liquid chromatographic separation include polarography (Kichkovski and Mekhouzla 1967) and enzyme detectors. 23

2.4 Fining theory

Fining can be defined as the addition to wine of a clarifying agent which adsorbs the suspended material and settles to the bottom of the vessel leaving the supernatant wine clear (Rankine and Emerson 1963). The purpose of fining is to (a) effect a rapid clearing of the wine, (b) clear wines which will not clear naturally, (c) remove unstable substances prior to bottling and (d) effect sensory changes to the wine. When assessing the clarity of wine three states are generally recognised (a) brilliant - wine free of all suspended matter, (B) clear - a slight haze may be detected and (c) cloudy - a definite cloud or deposit is detected (Amerine 1953). The need for fining has increased over the last 20 years as wines are being bottled much earlier than has been done in the past. Early bottling means that a wine has little time in which to self-clarify, and a winemaker must be able to guarantee that the bottled product is stable. This guarantee is provided in part by fining. When fermentation is complete a new wine is always cloudy. Wine clarification can be achieved by addition of a fining agent or by natural self-clarification. When wines were cellared for long periods before bottling, self clarification could be achieved with wine being decanted or racked off the deposit several times during this period - fining agents were used only where a wine would not self-clarify. Natural clarification allows the wine to come to equilibrium over a long period of time. Some wineries in Europe and other countries still follow this course of action in the production of high quality red wines. The removal of unstable protein is the most common reason for fining. Bentonite fining is the method generally preferred in industry for protein removal. Other methods available for protein stabilisation of wine include pasteurisation, treatment with proteolytic enzyme and ultrafiltration. Heat treatment of wine is a common method for removing unstable protein. Heating for two min at 75-1oo0 c induces precipitation of the heat labile protein of wine (Holden 1955, Koch and Sajak 1959, Moretti and Berg 1965, Ferenczy 1966). Heat treatment may itself result in the formation of a heat haze (Berg 1953, Joslyn 1953); further disadvantages include the detrimental effect of heat treatment on wine quality (Holden 1955) and it may not always guarantee the production of a protein stable wine. Ultrafiltration offers another method for achieving a protein stable wine. By separating wine into fractions of defined molecular weight with 24 fine membranes, the fraction containing the protein can be eliminated, thus rendering the wine stable with respect to protein. Wysocki (1977) and Wucherpfenning (1978) reported the successful removal of protein from wine by ultrafiltration methods; complete protein stability was achieved with membranes having a molecular mass cut off of 10 000 daltons. A problem that could occur with ultrafiltration for protein stabilisation is the removal, with the protein fraction, of other high molecular weight components that are important to the organoleptic quality of wine, i.e the polymers that are considered to contribute to the body and mouthfeel of a wine. Proteolytic enzyme applications have not been confined to operations with must and fermenting juices (Section 2.2.2). The protein stabilisa­ tion of wine by proteases has been reported by Datunashvili (1975), Tikhonova and Platsynda (1976) and Gaina and Sirota (1979). The success of proteolytic enzymes is surprising as the enzymic preparations have to act on the protein under conditions generally not favourable for enzyme action, i.e. high alcohol and low pH. Proteolytic enzymes are prohibited in Australian winemaking operations.

2.4.1. Bentonite fining

The most common fining agent for wine clarification is the clay bentonite. Bentonite is a highly colloidal, plastic clay, with the correct nomenclature of montmorillonite. Bentonites are hydrated aluminium silicate clays with a general formula of [(Al,Fe) 1 _67 Mg0 _33] si4o10 (0H) 2 (Na,Ca 0.33). The clay structure (Fig.1) consists of small plate-like particles, 10R thick by 50ooR across, and when dispersed in water these particles separate and give negatively charged particles that form a homogeneous colloidal suspension. 25

Figure 1. Simplified dimensional structure of bentonite (Anon. 1970)

o.._ ...... o.._._...o I Silicon I I o Oxygen 0 V 0 ,1/ ' -OH ■ /1, ■ Aluminium 0 V 0 I I o_,,•,o--•--o o.._.--o ...... _....o I I 0 V 0 ,1/ /I'• 0 V 0 I I o.,,,,.• ...... o--•---o

Such a colloidal system has a surface area of 750 m2/g dry bentonite (Anon. 1970). The proteins of wine are generally positively charged at wine pH and, therefore, are absorbed onto the negatively charged bentonite within 1-2 min of its addition to wine (Ferenczy 1966, Kichkovski and Mekhouzla 1967) in an ion exchange-type reaction with the sodium and calcium ions that are bound to the bentonite. Sodium bentonite is the most commonly used form of the clay as it absorbs more protein and floculates more readily than calcium or hydrogen bentonites (Rankine 1962). The proteins are probably denatured when they are absorbed by the bentonite (Rankine 1962). Bentonite also adsorbs protein by formation of hydrogen bonds. Particles of matter are not only adsorbed onto the surface of the bentonite but are probably also absorbed within its lattice-like structure. Bentonite carrys an overall negative charge when dispersed in 26 water. The negative charge is spread over the surface of the platelets, but there is a positive charge around the edges of the particles which means that bentonite will adsorb other material from wine, bringing about alterations to wine composition (Somers and Ziemelis 1973). Ough, Berg and Amerine (1959) observed that bentonite fining also reduced colour, total nitrogen, tartrate and potassium contents, but not pH, total acidity or total phenolic substances; however, Taran, Bychenko and Sereda (1977) found that phenolic substances are absorbed to some extent by bentonite during clarification. This does not appear correct as phenolic substances are generally assumed to have a net negative charge that would be expected to disperse the bentonite rather than react with it and precipitate. Bentonite fining may also help reduce the level of microorganisms in a wine (Nilov, Rabinovich and Zudova 1975). Whether bentonite preferentially absorbs a certain fraction of the wine protein or absorbs all fractions equally is not clear. Koch and Sa,jak (1959) considered that bentonite removed the several protein fractions at the same rate and that quantitative differences between the protein fractions after fining meant that the smaller fraction was removed first; Moretti and Berg (1967), however, found that bentonite removed proportionally more of one fraction than of the other three fractions that had been separated by electorphoresis. Somers and Ziemelis (1973) agreed with Koch and Sa,jak as they found that bentonite was non-selective between the two wine protein fractions: that they isolated by gel chromatography. It is generally accepted that the amount of protein removed by bentonite is propor­ tional to the amount of bentonite added, and it is assumed that this relationship varies between wines. It is also assumed that if sufficient bentonite is added, it can remove all the protein from wine, however, recent work suggests that this may not be true. For example, Vos and Gray (1979) found that a small amount of protein remained in grape ,juice even after excessive bentonite additions and Somers and Ziemelis (1973) found that there were large differences in the amount of protein removed from wine fined at the same level of bentonite addition. Such data highlight the fact that the removal of protein from wine by bentonite and its effect on composition is dependent on the bentonite type, the composition of the wine and fining conditions; they also help explain why fining procedures have remained an empirical operation. 27

How well a wine fines with bentonite depends on several factors, such as (a) acidity and pH, (b) phenolic substances, (c) alcohol level, (d) colloidal status, (e) ionic status of the wine, (f) temperature and (g) bentonite type. The acidity and pH are important as they determine the charge intensity on the proteins of wine. A low pH means a greater positive charge on the protein and hence greater reactivity with the negatively charged bentonite; as pH is lowered the isoelectric point of many proteins is approached which can lead to the removal (by precipitation) of the proteins independently. Moretti and Berg (1965) found that if the pH of a wine was lowered then protein levels declined and if lowered to less than pH 3.0 sufficient protein was precipitated to make the wine stable. No comment was made as to the effect of the pH reduction on wine quality. Rankine (1962) quotes literature sources stating that four times as much bentonite is required at pH 3.6 as at pH 3.0 to give a protein stable wine, however, this would surely depend on the particular wine being fined and the type of bentonite used. Kichkovski and Mekhouzla (1967) also found that reduction of protein levels by bentonite was more efficient at a pH value less than 3.0 thanat 4.0. Tannins lower the efficiency of protein removal from wine by the formation of a negatively charged complex with protein. Such a complex, however, may lead to floculation of the protein (Section 2.1.4.1). The presence of negatively charged colloids, such as phenolic substances tannin~protein complexes, polysaccharidesJ pectins and lipids, reduces fining efficiency by preventing access to the protein by the bentonite or by dispersion of the bentonite through neutralisation of the positive charges on the edges of the bentonite platelets (Rankine and Emerson 1963). Similarly ion­ -exchanged wines with a high concentration of monovalent cations and a low concentration of divalent cations have been shown to fine poorly because of dispersion of the bentonite (Rankine and Emerson 1963). The fining efficiency of bentonite also depends on the source and batch of the bentonite; Taran and Burlak (1967) found that fining efficiency in wine was dependent on the mineralogical composition, degree of dispersion of the particles and the specific surface makeup of the bentonite. 28 2.4.2. Fining practice

The practices of fining and of stability testing are largely empirical. Bentonite fining requirements for a wine are generally determined by examining the performance of small samples (100 mL) of a wine with varying amounts of added bentonite and evaluating the 'potential' stability of the treated wine with one of the commonly used 'stability' tests. These methods are rather arbitrary and not based on a sound knowledge of protein reactions during vinification, the mechanisms of protein removal by bentonite or of the relationship between 'potential' stability indicators and 'actual' stability of the product. Little is known about the relationship between the stability testing of 100 mL cylinders of wine and the fining efficiency and potential instability of the same wine in a 100 000 litre tank. Bentonite is more efficiently added to wine as a 5 - 10% slurry either in water or wine to ensure complete separation of the colloidal plates (Holden 1955, Rankine 1962). The separation (swelling) of bentonite is enhanced if the bentonite is slurried in hot water and allowed to stand for several days before use. Fining is an exercise in thorough mixing, as incomplete mixing will lead to incomplete fining. Gelatin or polyacrylamide are often added to wine in conjunction with bentonite to facilitate its flocculation (Rankine and Emerson 1963, Ferenczy 1966). When a wine is incorrectly fined certain problems can arise: overfining, i.e. excess bentonite is added to what is required for stability, can change the composition of the wine and hence its sensory properties and can mean a costly loss of wine in the lees and wasted bentonite; if a wine is underfined the stability of a wine can not be guaranteed and there may also be problems with filtration of the product. Considerable attention has been focussed on the addition of bentonite before and during fermentation. Such addition has been practised mainly in France and South Africa where it is claimed that bentonite addition during fermentation gives a more even fermenta­ tion rate because of the mixing effect and dispersion of yeast (Somers and Ziemelis 1973). It is also claimed that wine is more readily clarified and stabilised when fined during fermentation than if fining is left until after fermentation (Milisanilyevic 1969). There has been many counter claims as to the success of this technique, with some workers claiming that there was little advantage in fining 29 during fermentation and a more efficient fining would be expected at the end of fermentation where there is less protein, less protec- tive colloidal material and conditions conducive to fining. (Ough, Berg and Amerine 1969, Somers and Ziemelis 1973). Prolonged contact of fermenting juice with bentonite may bring about competition between the protein and other nitrogen compounds for adsorption sites on the bentonite. Bentonite fining of the juice prior to fermentation may also result in high loss of juice in the lees formed (Ough and Amerine 1965). The higher concentration of colloidal and particulate material of juice, which can interfere with bentonite, suggests that fining of juice would not be as efficient as fining the finished wine. Original interest in the problems of fining during frementation occured during the late 1960s and early 1970s, however, in recent years there has been renewed studies into the effect of bentonite additions during fermentation with the increasing demand for highly clarified or low solids juices for fermentation. Some difficulties have been experienced with the low solids juices, viz. slow fermentation rates and 'stuck' fermentations. The addition of inert solids, such as talc and bentonite, has alleviated some of the problems associated with clear juices. Groat and Ough (1978) showed that the addition of bentonite to juice with less than 0.1% solids improved fermentation rate and enabled the wine to ferment out to dryness, however, no mention was made as to the effect of the added bentonite on the protein stability of the finished wine. The benefits of bentonite fining following fermentation of clear juices would be similar to those mentioned above, as fermentation of clear juices would also be expected to reduce protein levels, particularly as the removal of solids from a juice would also remove protective polysaccharide colloids and thus render the protein more susceptible to precipitation during fermentation. Clear juices may also lack an adequate supply of easily assimilable nitrogen, forcing the yeast to breakdown proteins for their growth, which may lead to other problems such as H2S production (Vos and Gray 1979). At present it appears that fining at the completion of fermentation is more efficient than fining before or during fermentation with respect to protein stabilisation.

2.4.3. Protein stability testing

Wine stability may be defined as the attainment of that state or condition in which a wine for some definite period will not exhibit 30 undersirable physical or organoleptic changes (Berg 1953). Determination of when a wine has attained that state is probably the most difficult aspect of protein stabilisation yet to be resol­ ved. The assessment of a wine's potential instability by industry currently involves either heat tests or some chemical precipitation method. Heat tests assume that subjecting a wine to high tempera­ tures for some short period is equivalent to long term storage at ambient temperatures. The conditions of the heat tests vary with different studies and through out industry. For example Rankine (1962) suggested that holding the wine at 80°C for 30 minutes and then cooling in running water was a sufficient indicator of potential protein instability; 100°c for 30 minutes and then overnight at room temperature was employed by Somers and Ziemelis (1973); Moretti and Berg (1965), Bayly and Berg (1967) and Ough et al. (1969) suggested a heat test of 48 hours at 5o0 c followed by 48 hours at o0 c; a short heat test involved 2 minutes at 100°c at natural pH and at pH 2.95 but was discarded as it was found to be too eratic (Moretti and Berg 1965). Pocock and Rankine (1973) discussed heat testing for protein stability and recommended 80°c for 6 hours, temperatures below 10°c for period of up to 24 hours were disregarded as being insufficient to produce maximum haze. The notable feature of the different studies on wine protein and stabilisation is the variation that occurs between reported methods and results, in this case the conditions for heat testing. The high temperatures are acceptable in theory, however, do high temperature tests indicate stability at ambient temperatures over long term storage or do they merely indicate the potential stability of a wine if it is stored at these elevated temperatures? High temperature testing may indicate the temperatures at which the various wine protPin fractions will precipitate at these high temperatures and that long term storage at lower temperatures will not result in protein precipitation. The perfect stability test, therefore, is to store the bottled wine at room termperature for the expected storage term of the product; this, however, is not a practical operation. Somers and Ziemleis (1973) touch on this subject when they mention the notion of 'practical stability'; do the heat tests indicate 'practical stability' or maximum possible instability?. Other methods of testing wine stability entail a chemical precipitation of the unstable protein. The two most common chemical stability tests are the proprietary Bentotest and the TCA test. The 31 relevance to the 'practical stability' of wine of the results of chemical stability tests must also be questioned. The effect of interfering compounds on the test results must be considered, as the test reagents would be expected to suffer from the same problems that hinder protein separation by chemical precipitation reagents. It has been generally found in industry that Bentotest is a very sensitive test for residual protein (Somers and Ziemelis 1973), which may suggest reaction with other materials beside protein. As long ago as 1953 it was suggested that more data be gathered on the relationship between protein and clarity and stability (Amerine 1953), and yet Anelli as recently as 1977 acknowledged that there was no information as to what levels of protein actually constituted stability. Studies during the mid 1960s endeavoured to examine the relationship between protein and protein stability, and in several reports total protein was not considered to be a good indicator of protein stability (Moretti and Berg 1965), Ough and Amerine 1965, Bayly and Berg 1967): whether this finding was based on initial protein levels or protein levels at stability was not indicated,Moretti and Berg (1965) isolated four protein fractions with electrophoretic techniques. One fraction was found to be more heat labile than the others and the ratio of the sum of the three less heat labile fractions to the heat labile fraction was claimed to correlate with stability, however the range given, less than 4.2, unstable - greater than 6.6, stable, leaves a large area where the stability is poorly defined. The ratio also allows the possibility of stabilising wine by adding protein. Ough and Amerine (1965) found several wines that were indicated to be protein unstable although they contained no protein; such an observation can be taken to support the argument that protein can not be used as a stability indicator or that the 'stability' tests do not indicate stability. Pocock and Rankine (1973) determined protein levels before and after high fining levels and examined the stability of the wines by several tests, however, there was no attempt to examine the effect of different levels of bentonite on protein content and there was no comment as to any relationship between protein level and stability which should be important considerations in any protein-stability studies. Danilatos (1978) also failed in this area by not determining the protein levels at different fining levels, concentrating on stability tests and initial protein levels only. These studies would have benefited 32 from a reliable, fast protein quantitation method in order to produce accurate protein levels at various levels of bentonite addition and at 'stability'. 33 3 MATERIALS AND METHODS

3.1 Grapes

Traminer, , Rhine , and Semillon grapes were obtained from the of McWilliarn's Wines Pty. Ltd. at Hanwood in the Riverina district of NSW, French from the vineyards of the NSW Department of Agriculture, Viticultural Research Station at Griffith in the Riverina district of NSW, Muscat Gordo Blanco (Gordo) from vineyards of an independent grower in the same district and from McWilliarn's Wines vineyards at Robinvale in Victoria.

3.1.1 Grape sampling procedures

Grapes were harvested from the beginning of one row of each variety, stripping the vines of bunches until 30 kg (two picking buckets) were obtained. At the next harvest date picking continued along the same row from where it had stopped previously, until another 30 kg has been obtained. This procedure was repeated until five or six lots had been taken from vines of each variety at increasing maturity.

3.2 Juices

The Semillon, and Gordo juices required for the clarification trials were commercial juices obtained from McWilliam's wines Yenda winery. The juice samples were drawn immediately after the juice was drained from McWilliam's tower-type drainers and prior to any juice clarification. The Gordo juice employed for studies on fortification, ion-exchange, ammonium sulphate precipitation of protein and ultrafiltration of wine were obtained in a similar manner.

3.3 Wines

Wines employed in stability studies involving bentonite fining, ion-exchange, ultrafiltration and heat treatment were commercial wines drawn at completion of fermentation from McWilliarn's Wines Yenda and Beelbangera wineries in the Riverina district of NSW. 34

3.4 Winemaking procedure

3.4.1 Standard method

Approximately 30 kg of grapes, sampled in the manner described 1n Section 3.1, were crushed with a small, custom-made crusher­ -destemmer of the beater type; so 2 was added immediately after crushing to a level of approximately 90 mg/L. The juice was left in contact with the skins for one hour, after which the juice was lightly pressed by means of a small, custom-made, hydraulic basket press. The expressed juice was run into an open stainless steel can of approximately 13 L capacity and a sample taken for analysis. The can was then placed in a cool room at 10°C· the top of the can ' was covered with a thick glass plate. After 24 hours the juice was racked off its settlings into a clean, stainless steel can and inoculated with yeast Saccharomyces cerevisiae strain 729 ( Research Intitute); the can was transfered to a cool room at 13°c. The sugar level in the juice was monitored throughout fermen­ tation. At the completion of fermentation, as indicated by the Clinitest tablet method, the wine was racked off the gross lees, bentonite added at a level of 1.0 g/L and the can placed in a cool room at 20 C for two days after which the wine was racked off the bentonited lees into clean glass jars, stoppered, and left in the 2°c room for two weeks or until recognisable sedimentation had ceased. The wine was then racked into another glass jar with a further addition of 25mg/L so2 and transfered to the 13°C room until bottling.

3.4.2 Clarification

The samples of Gordo, Trebbiano and Semillon juices, obtained as described in Section 3.2, were each split into six 4.5 L glass jars. The clarification treatments examined were: (1) cold settling at 10°c for approximately 20 hours; (2) bentonite addition of 1.0 g/L with several hours at room temperature and then overnight (approximately 16 hours) at 10°c; (3) enzyme clarification with three pectic enzyme preparations, (a) Ultrazyme (Ciba-Geigy), (b) Klerzyme (Pfizer), (c) Clariphase (Pfizer), at a level of 11 mg/L juice and two proteases, (a) Protease B (Pfizer) and (b) Protease M-6 (Pfizer) both at a level of 11 mg/L juice except for the Gordo juice where 22 mg/L was added. The enzyme-treated juices were maintained at 35 room temperature for several hours before being placed in the 10°c room overnight. The lees volume and degree of juice clarity, esti­ mated visually, was noted at the end of the clarification period. The juice was racked off its settlings and inoculated with a suspension of Sacch. cerevisiae 729 yeast and fermented to dryness at 13°C. Protein levels were determined in the juice before and after clarification and after fermentation. Upon the completion of fermentation all samples were trial fined with bentonite; the protein level and the protein stability of the wines were measured at each level of bentonite addition.

3.4.3 Non-standard winemaking procedures

The production of the fortified sweet white and dry white wines from Gordo juice entailed clarification by cold settling and with a pectic enzyme preparation (Ultrazyme, 11 mg/L); the clarified juice was split into two 101 lots, inoculated with Sacch. cerevisiae 729 and fermented at 13°C. When the sugar level had been reduced to 7.2°Be, one sample was fortified to 18.1% v/v alcohol with rectified grape spirit, reducing the density of the wine to 3.8°Be; the other sample was fermented out to dryness, as indicated by the commercial Clinitest tablet method. At the completion of fermentation both samples were racked off gross lees and 25 mg/L so2 added. The wines were held for two days at 2°c, racked again and cold stabilised for two weeks at 2°c, after which time they were racked once more and transferred to the 13°C room to await bottling.

3.4.4 Bottling procedures

The clarified wine was racked from the 4.51 glass jars, with an addition of 25 mg/L so 2 , into 10 L pressurised stainless steel cans. The wine was filtered through a hot water sterilised Ekwio 15 cm round plate and frame pad filter containing eight D9 pads, followed by a 0.45 um Sartorius membrane filter in a Sartorius 13 cm round, single sheet, membrane housing which had been steri­ lised in an autoclave. Bottles were steam sterilised before filling and sealed with presterilised corks (Jones, Steains & Waller (NSW) Pty. Ltd, Thornleigh, NSW). 36

3.5 Fining

A 5% slurry of bentonite was prepared by mixing the dry bentonite (Wyoming Green Bond, granular) with water in a blender until a homogeneous suspension was obtained. The suspension was stored for two days before addition to wine. Fining trials consisted of dividing each wine sample into 100 ml or 10 ml aliquots and adding the bentonite at levels of 0.0, 0.25, 0.5, 0.76, 1.00 g/L or higher until it was considered that the bentonite addition had reduced the protein to negligible levels. The samples were thoroughly mixed and the bentonite allowed to completely settle. The settled wine was decanted off the sediment and filtered through a 0.45 pm Sartorius membrane.

3.6 Protein stability tests

The stability of the fined and filtered wines was determined by two tests commonly used by wineries: (a) the proprietary Bento­ test solution was used according to instructions, i.e. 1 mL of Bentotest solution was added to l0mL of each sample of fined wine, mixed and the appearance of a cloud or haze was taken to indicate instability; (b) the heat and tannin.test - tannic acid (50 mg) was added to 100 mL of the fined and filtered wines which were heated for 16 hours at 5o 0 c and then cooled to room temperature. Instability was indicated by the appearance of a haze or cloud in the wine.

3. 7 Analytical methods.

3.7.1 Protein estimation

The protein of .iuices and wines was separated from the other constituents by high-pPrformance liquid permeation chromatography (HPLC) and quantitatively estimated by continuous monitoring of the absorbance of the column effluent at 280 nm. Crystaline bovine serum albumin (Calbioch~m electrophoreticallypure) was the protein standard for column calibration. The HPLC system consisted of a Waters Associates model 6000A solvent delivery system, a model U6K injector and a model 440 dual channel absorbance detector with a 280 nm filter. Absorbance of the column effluent was recorded with a dual pen 0mniscribe recorder at two attenuation levels, 0.02 and 0.2 AUFS. Chart speed was set at 1.0 cm/min. 37

Waters Associates E-linear µBondagel and E-300 µBondagel columns were used in series. The mobile phase for the system was 0.01 M ammonium acetate plus 0.01 M dodecyl hydrogen sulphate (Mench) at a pH of 6.7. The flow rate of the mobile phase was maintained at 2.0mL/min. Injections of 10 pL were made with a Hamilton 25 pL syringe. Juice and wine samples were filtered through a 0.45 µm membrane filter before injection into the HPLC. It was necessary to centrifuge ,juice samples for 10 minutes at 3400 rpm before membrane filtration.

3.7.2 Analysis of ,juices and wines

Methods of analysis for sulphur dioxide, titratable acidity, pH, alcohol, total phenolics, potassium and organic acids are listed in detail in Appendix 1. 38 4. RESULTS AND DISCUSSION 4.1 Development of a rapid protein estimation system

Protein in must and wine has been estimated by K,jeldahl, colorimetric and spectrophotometric procedures, but the accuracy of these procedures can be reduced by interfering substances, such as non-protein nitrogenous compounds, by variable colour development among proteins and by variation in reported maximum absorption wavelenghts of the developed colour. Some of these problems may be avoided by separating the protein from the wine by precipitation, dialysis, ultrafiltration and gel filtration. Often other materials that interfere with protein estimation are also precipitated with the protein. The separation of protein from wine by the three physical methods, although allowing accurate measurement of protein, are too slow for estimating protein levels in the large number of samples of juice and wine that are available during the short period of vintage. For example, protein has been separated from wine by gel filtration, with the protein in the column effluent estimated either by absorption at 280 nm (Somers & Ziemelis 1972, Ochi & Nakanishi 1975) or by polarography (Kichkovski & Mikhouzla 1967). Recent developments in high-performance liquid chromatography (HPLC) indicate that it may be possible to separate protein from wine many times faster than with conventional liquid chromatography. Thrall and Snelsherg (1979) separated and recovered the proteins from a mixture of nroteins on most commonly available HPLC -type columns, however, the separation by HPLC of protein from a complex beverage such as wine has not been reported.

4 .1. 1 µBondagel size separation of protein from juice and wine

The success of gel filtration column chromatography in separating protein mixtures and protein from wine (:Somers & Ziemelis 1973) suggested that size separation offered the best means for separating protein from wine with HPLC. The molecular mass of wine protein is considered to be in the range 10 000 to 100 000 daltons (Section 2.1.4). The µBondagel columns from Waters Associates are gel permeation columns packed with a controlled porosity silica with a permanently bonded ether phase creating a hydrophilic surface. The columns are available with various molecular exclusion limits and separation ranges (Table 4). 39

Figure 2. lN spectrum of unfined 1978 Semillon wine(-) and of bovine serum albumin(----).

2·0

1·6

II C "CD .a.. ~ 0·8 .a

0 20~0~-~-~2~50~-~-~30~0~-~-~3~50~----- Wavelength (nm)

Table 4. µBondagel column specifications

Nominal molecular Nomina] molecular Designation Mass exclusion Mass senaration (daltons) range (na1tons)

E-125 50 000 2 000 - 50 000

E-300 100 000 3 000 -100 000 E-500 500 000 5 000 -500 000 E-1000 2 000 000 50 000 -2 000 000 F.-Linear 2 000 000 2 000 -2 000 000

Waters Associates information sheet F68 (1976).

The E-300 µBondagel column, with a molecular mass separation range of 3000-100 000 daltons, appeared to be the most suitable column ~or seoarating protein from wine, however, none of the mobile phases listed in Table 5, eluted BSA from the several pBondagel columns examined. 40 These results are at variance with those of Thrall and Spelsberg (1979) who were able to separate a mixture of proteins with 100% recovery on pBondagel columns with 0.01 M sodium acetate as the mobile phase. Protein recovery was reduced at higher salt concentrations in the mobile phase, which was attributed to hydrophobic absorptive retention on the column. The difference between the present results and those of Thrall and Spelsberg (1979) cannot be explained, but it is probably related to the history of treatments of the columns.

Table 5. Mobile phases and pBondagel columns examined

Mobile phase Column systems

0.1 M Sodium acetate E-300 0.1 M Sodium acetate/2% ,E-125 acetic acid tE-Linear 2% Acetic acid E-1000 0.01% Phosphoric acid E-300 + E-Linear 0.1 M Ammonium acetate E-300 + E-1000

Before injection, the BSA was dissolvedin the mobile phase under test at a concentration several orders higher than that expected for protein 1n wine. Single µBondagel columns provided little separation of the UV absorbing material found in wine (Fig.3a}, however, separation of this material was achieved to varying degrees with two columns in series (Figures 3&4). The failure of these mobile phases to elute protein from pBondagel columns indicated that none of the observed peaks resolved by the two column system could be classified as protein. The mobile phases that gave the best separation of the UV absorbing material were 0.01% phosphoric acid, 0.1 M ammonium acetate and 0.1 M sodium acetate/2% acetic acid. 41

Figure.3. Elution profiles of 10 pL unfined 1978 Semillon wine from a single µBondagel column (a) and from two pBondagel columns in series (b,c). P indicates the protein peak.

a b C

p \

0 2 4 ~ 5 7 9 1 3 5 Time (min)

Conditions of chromatography: a - E-300 µBondagel column, mobile phase 2% acetic acid, AUFS 0.05, flow rate 2mL/min; b - E-linear and E-300 pBondagel columns in series, mobile phase 0.1 M sodium acetate, AUFS 0.1, flow rate 1 mL/min; c - E-linear and E-300 pBondagel columns in series, mobile phase 0.01 M ammonium acetate+ 0.01 M sodium dodecylsulphate, flow rate 1.5 mL/min. 42

Figure. 4. E]ution profile of 10 pL unfined 1978 Semillon wine. Chromatography conditons: E-linear and E-300 pBondagel columns in series, mobile phase 0.1 sodium acetate, wave length 254 nm, AUFS 0.05, flow rate 1.0 mL/min.

0 2 4 Time (min) 43

4 .1. 2 Reverse phase separation

The inability to elute protein from pBondagel columns led to an examination of the Waters Associates reverse phase µBondapak system. Mobile phases of methanol/ water mixtures of low and high pH gave inconsistent results on pBondapak-C18 , -palkyphenyl and -pCN columns; when BSA was eluted from these columns the peak was either not symmetrical or not a single peak even though the BSA was electrophoretically pure. Furthermore the absorption of BSA within the HPLC system varied with the solvent in which the BSA was disolved. The solvent probably changed the conformation and polarity of the BSA, as well as the affinity effects of residual silanols in the stationary phase (Hancok et al.1979). Methanol/ water mixtures separated the UV absorbing material of wine poorly on all reverse phase columns: in contrast 0.01% phosphoric acid gave good separation of these components with the µBondapak-c 18 column, however, BSA was not eluted from this column with this mobile phase.

4.1.3 Effect of addition of sodium dodecylsulphate to the mobile phase

SOS, a widely used reagent in electrophoretic and chromatographic work with proteins, acts as a denaturing and complexing agent. Protein denatured in the presence of SOS assumes the shape of a random coil and its dimensions are a unique function of molecular weight (Frenkel & Blagrove 1975). Many proteins bind identical amounts of SOS at monomer equilibrium concentrations of SOS above 0.5 mM (Peterson 1971). BSA was eluted with good recovery from all reverse phase columns with the addition of sodium dodecylsulphate (SOS) to the mobile phase. Excellent recovery of BSA was obtained from pBondapak-palkyphenyl and -µCN columns with a 0.01 M sodium acetate mobile phase containing 1% SDS. Rivier (1978) similarly found that the presence of SOS in the mobile phase greatly increased the recovery of protein from a reverse phase HPLC system. The success of the mobile phase containing SOS in eluting BSA from the reverse phase columns indictes that the protein- SOS complex was not absorbed within the HPLC system. This is probably due to either the lack of reaction between the SOS-protein complex and the column packing material or the negation of any electrostatic effect of the charge on the protein. Fernstrom and Moberg (1977) noted that at neutral pH the total charge of the protein-SOS complex is almost entirely dependent on the charge of the SOS molecules. 44

Figure 5. Elution profile of 10 µL unfined 1978 Semillon on pBondapak c 18 column. Conditions of chromatrography: pBondapak-c18 column, mobile phase 10% acetonitrile + 0.01 M SDS (pH 2.5), AUFS 0.02, flow rate 2.0 mL/min.

•

. 0 4 8 12 Time{min) 45

The reverse phase columns un~er certain conditions gave good separation of the UV absorbing materials of wine (Fig.5), however, hecause separation is based on polarity it was difficult to relate quantitiatively the BSA standard and its retention time with that of must and wine protein. Thus pBondagel columns were reconsidered, since separation is based on molecular size and the molecular weight of BSA was within the range of reported values of wine protein and of similar globular structure. Separation and quantitation of protein rrom wine was achieved with an E-linear and a E-300 µBondagel column in series, with 0.01 M ammonium acetate containing 0.01 M SDS at pH 6.8 as the mobile phase (Fig.6). Total soluble protein in must and wine could be determined in 10min. BSA standards gave a linear response up to 1.0 g/L. The E-300 column alone resolved finished wine samples in less time than with the two columns in series and with slightly greater sensitivity, however, the two columns in series were chosen as it was considered that the pBondagel E-linear column would enhance protein separation and also help protect the E-300 from inadvertent clogging by high molecular weight material that might precipitate from grape juice. 4.1.4 Protein separation system In mid-1979 Waters Associates released a Protein Separation System (PSS) for the separation of relatively low molecular weight proteins. The column was a gel permeation column, but the column packing material had been improved to reduce or eliminate protein absorption onto the column material that occurred in the pBondagel columns. The PSS was evaluated under a variety of conditions including high and low pB mobile phases, with and without SDS; at high pH (0.01 M ammonium acetate) BSA was eluted linear1v up to 400 mg/L but at low pH (0.5% acetic acid,pH 3.1 BSA was not eluted. When SDS was added to either of the mobile phases, BSA was eluted, and the response at 280 nm was considerably enhanced. The effect of SDS addition on wine protein separation and estimation is illustrated in (Figure 7) for an unfined Semillon wine. The low pH mobile phase containing SDS altered the order of elution of the other UV absorbing materials of wine (Fig.8), resulting in the failure to separate the wine protein; further more,the time for completion of a chromatogram was increased with a mobile phase of low pB. The low pH of the mobile phase changes the charge and configuration of moiecule, altering its molecular size in solution and changing its reactions with materials in the HPLC system. The nominal pore size of the protein column is 125 nm with an exclusion limit of 80 000 daltons. It has been observed (Fig.7), that the protein from wine and must was consistently split into two peaks. 46

Figure. 6. Elution profile of 10 µL unfined 1978 Semillon. Conditions of chromatography; E-Linear pBondagel and E-300 pBondagel columns in series, mobile phase 0.01 M ammonium acetate+ 0.01 M SOS, AUFS 0.05, flow rate 1.5 mL/min. P protein peak.

0 4 8 Time (min) 47

Figure.7. Elution profiles of 5 µL unfined 1979 Semillon. Conditions of chromatography; Protein Separation System column, mobile phase 0.01 M ammonium acetate, AUFS 0.05, flow rate 1.5 mL/min:b - mobile phase contained 0.01 M sodium dodecylsulphate, c- wine was ultrafiltered through a membrane of 10 000 daltons cut-off before chromatography. P protein peaks.

8 b C

p \

1 ib 2k-...... __..,!I---'-' --.lg.--'-' -.ig,----L-' --.!,fb.------' 4 e Time (min)

4 .1. 5 Detection and quanti tation of soluble· protein The separated protein was detected by monitoring the absorbance of the column effluent at280nm (Somers & Ziemelis 1973, Ochi & Nakanishi 1975). The UV spectrum of unfined Semillon wine and bovine serum albumin (BSA) (Fig.2) indicate that 280 nm, the chosen wavelength of the Waters UV detector in the HPLC system, is suitable for detection and estimation of soluble protein in wine. BSA (Calbiochem electrophoretically pure) was chosen as the calibration protein because it has a molecular mass of 69 000 daltons, an isoelectric point of 5.1 (Righetti & Carvaggio 1975) and a globular structure that is similar to that reported for wine protein (Tarantola 1971, Somers & Ziemelis 1972). 48

Figure.8. Elution profile of 5 pL unfined Semillon. Conditions of chromatography; PSS column, mobile phase 0.5% acetic acid + 0.01 M SOS, flow rate 1.0 mL/min, AUFS 0.05.

0 4 8 12 Time (min)

4. 1.6 Confirmation of protein peak as total soluble wine and must protein

Much circumstantial evidence indicates that the first two pea~s from the PSS represent the total soluble protein in must and wine: BSA and the protein peaks appear at similar elution volumes; protein is the largest molecular weight, UV absorbing material in wine and as such would be expected to elute first in gel permeation chromatography; only the protein peak in wine is reduced with any significance by bentonite fining; and ultrafiltration of wine through a membrane of 10 000 dalton cut-oFF (Amicon PA2) removed only the protein fraction (Yig.7). Wine protein was also precipitated with ammonium sulphate, but when the precioitate, diso1ved in !1% acetic acid, was chromatograohed it was ohvious that manv UV 49

Figure.9. Elution profile of (NH4)2 S04 precipitate (60-80% cut) from wine after disolution m 5% acetic acid. Conditions of chromatography: in column PSS, mobile phase 0.01 M ammonium acetate+ 0.01 M SDS, flow rate 1.5 mL/min, AUFS 0.005, injection volume 5 pL.

0 4 8 12 Time(min) absorbing material other than protein was precipitated by protein precipi­ tating agents. The non-selective nature of ammonium sulphate, precipitation of UV absorbing components of wine precluded (NH4) 2so4 from being a suitable agent to confirm that the peak under consideration represented total soluble protein in wine, and further indicated that (NH4 )2s04 is not suitable as a protein precipitation agent forquantitative~olorimetric protein analysis. 50

4.2. Total soluble protein in white wine grapes

Thoukis (1974) in a review of the chemistry of wine stabilisation states 'Unfortunately, in spite of the published literature on wine proteins, we do not know the actual protein levels at which table or dessert wines are stable. The changes during production and processing of wines are stjll not known with sufficient accuracy to predict their behaviour'. Such a criticism can also be levelled at the knowledge available on how grape variety and maturity affects the soluble protein level in juice. The development of a rapid method for quantitation of soluble protein in juice by HPLC (Section 4.1) made it possible to examine in some detail the effect of variety and maturity on juice protein levels during the short period of vintage. These studies during the 1979 vintage in the Riverina district of NSW were an extension of studies of the 1976 and 1978 vintages, (Tyson 1976, Tyson unpublished results) in which the soluble protein content of juices and wines was determined by the gel filtration technique of Somers and Ziemelis (1972). Winemakers have often observed that juices and wines of grape varieties vary considerably in the ease with which they clarify and thus stabilise. Indeed, as long ago as 19~3, Amerine noted that juices and wines of several varieties clarified at different rates and that,iuice protein content varied considerably between varieties. Moreover winema~ers consider that maturity and seasonal factors affect the ease of clarification of wine, 1.e. even for a particular variety, maturity and climate can affect the soluble protein level of the juice and of the corresponding wine. Although the effects of maturity and variety will be discussed separately, it is emphasised that the protein level in grape juice is a result o~ the inter­ action between environmental inf]uences and genetic expression by the particular variety. 4.2.1 Effect of variety on juice protein level

Measurements of the total soluble protein by ~PLC in nine grape varieties indicated that there is a significant varietal effect on juice protein level. Table 6 lists the maximum soluble protein level recorded for the nine varieties examined, with data also for the 1980 vintage for Gordo, and for Traminer for the Rooty Hill area of NSW. The level recorded for Rhine Rielsing and Chardonnay may not be the maximum as these two varieties were harvested before the protein Jevel had oeaked (Section 4.2.2). 51

The variety Traminer, grown 1n two districts of NSW, had consistently the highest soluble protein content at about 870 mg/L - Luis and Lee (1980 unpub. results) recorded similar maximum levels in Gordo grown in the Riverina district of NSW. Rkatsiteli and Colombard were low protein varieties achieving maximum soluble protein levels slightly greater than 100 mg/L.

Table 6. Maximum protein levels in grape varieties grown at Griffith and Rooty Hill, NSW and at Robinvale, Victoria.

Protein (mg/L BSAE') Variety 1979 1980

Rkatsitelli* 110 Colombard 130 Rhine Riesling 280 Chardonnay 380 Sauvignon blanc 480 Gordo 480 878+ Semillon 540 Traminer 850 Traminer'' 874+ 875+ Sultana 239+

*Grown at Robinvale, Victoria +Data form •mpublished results of Luis and Lee "Grown at Rooty Hill, NSW 'BSAE BSA equivalents.

Such data supports the suggestion put forward by many workers that there are large varietal differences in juice soluble protein levels (Koch & Sajak 1959, Tarantola 1971, Molnar 1975, Nanitashvili, Samadashvili and Shilakadze 1975). However, more data is required for these varieties grown under more than one climatic condition and for several vintages before a clear picture will emerge of the effect of variety, maturity and climate on juice soluble protein levels. Table 6 indicates that varieties, with distinctive varietal aroma and bouquet such as Traminer and Gordo, generally have a higher protein level than the less intensely flavoured varieties, such as Colombard and Rkatsitelli. 52

T~e maximum protein levels for the nine varieties examined 1n this study ranged from 110 to 878 mg/L (Table 6) and are of a similar order to those reported in other studies of proteins in grape juice and wine (Section 2.1.4). Some levels quoted include 20 to 260 mg/Lin juice and 30 to 275 mg/Lin wine as determined by dialysis separation and Diemar and Maier colorimetric quantitation (Bayly & Berg 1967) and 175 to 525 mg/Lin wine and 381 to 837 mg/Lin juice (Spirov 1975). The studies generally do not mention the number of or the varieties studied or at what stage of maturity the results were recorded; the drawbacks of the quantitation methods employed can further emphasise the variation. The UV absorbance profiles for each of the eight varieties examined are shown in Figures 10 and 11. The profiles were recorded for each variety for juice expressed from berries harvested at approximately the same maturity, as measured by sugar density, i.e. 10 - 10.5° Be. The profiles appear to be characteristic of individual varieties, which would suggest that such a chromatographic system could be used to obtain a 'finger print' specific for the juice of each variety. Somers and Ziemelis (1972) observed similar specific chromatographic profiles at 280 nm for varietal wines subjected to Sephadex gel filtration chromatography. The accurate comparison of the UV profile of different varieties requires that the grapes be at about the same maturity level. There are several maturity indexes available, such as sugar density, as measured by 0 Be, sugar/acid ratio, and malate/tartrate ratio, but none of these are considered more suitable than the others as an absolute indicator of maturity. Thus the choice of maturity index may influence the variation in the chromatographic profiles observed for different varieties. Figures 10 and 11, show that differences do exist in the UV profiles of different varieties, however, the profile changes during ripening (Fig. 15) which probably rules out the use of such a chromatographic system for varietal classification. Regional effects, not examined 1n this study, would add further variables that would also limit the use of UV profiles for varietal classification. However the profiles are distinctive enough to warrant further investigation.

4.2.2 Effect of grape maturity on juice protein level The interaction of variety and maturation conditions, 1.e. the vine environment, determines the protein level of grape juice; it has been suggested that maturation conditions play the more important role ( Tarantola 1971). The processes involved in the ripening of fruit have been the sub.iect !'i3

Figure. 10. Elution profile of (a) Rkatsitelli juice harvest date 20/3/79, (b) Rhine Riesling juice harvest date 16/2/79, (c) Sauvignon blanc ,juice harvest date 9/2/79, ( d) Chardonnay .iuice harvest date 9/2/79. Conditions of chromatography; column µBondage} E-Linear and pBondagel E-300, mobile phase 0.01 M ammonium acetate and 0.01 M SDS, flow rate 2.0 mL/min, injection volume 10 µL. a b

Eu:> C 0 CX) NV.., co .-. NN I 0,- >< u,O LL ~ C d ..._,,< G) CJ coC -e (0 0 .0"' < V

0 0 3 6 0 3 6 Time ( min) 54

Figure. 11. Elution profiles of (a) Traminer harvest date 26/2/79, (b) Semillon juice harvest date 14/2/79 (c) French Colombard juice harvest date 7/3/79 (d) Gordo juice harvest date 5/3/79. Conditions of chromatography; column pBondagel E-Linear and pBondagel E-300, mobile phase 0.01 M ammonium acetate and 0.01 M SDS, flow rate 2.0 mL/min, injection volume 10 pL.

a b E C 0 C04 N .., (Q .-.2 N 0,- >

of much detailed research. Both catabolic and anabolic reactions occur, with a concurrent increase in enzyme activity. Whether the increase in enzyme activity 1s due to increased protein synthesis, enzyme activation, disap­ pearance of enzyme inhibitory substances or analytical differences 1s an unresolved question. Dilley (1970), however, cites results for many rruits which indicate that if protein synthesis is inhibited, ripening is prevented. 55

The total soluble protein level in the juices of the eight varieties examined in this study showed a rapid and large increase followed by a decline as the grapes approached winemaking maturity (Figs 12-14). Figure 12 shows the protein levels in ripening Traminer, Semillon and Chardonnay grapes from the Griffith district of NSW. The protein level greatly increases during the latter stages of ripening and reaches a maximum at 9.5°Be for Traminer and at 10.8°Be for Semillon after which it declined. The increase in protein level during this period when must density increases by 3°Be can be as high as five fold. The protein level in Chardonnay and also in Rhine Riesling was still rising at 11°Be when the plots were mistakenly harvested. Similar patterns of protein accumulation can be seen forFrer-chColombard, Rkatsitelli, Sauvignon blanc and Gordo (Figs 13 & 14 ). The increase in protein level observed in this study for grapes supports the contention that ripening of fruit involves protein synthesis. It should be noted, however, that the vineyard sampling technique may have contributed to the observed fluctuations in the protein levels, despite the care taken to obtain representative samples; the consistent patterns of protein accumulation observed in the varieties studied appear to indicate that the sampling techniques were successful. Hawker (1969) determined the activity, rather than protein content, of the enzymes malate dehydrogenase, phosphopyruvate carboxylase and pyruvate decarboxylase during ripening of grapes and did not find any large increase in activity in these enzymes, however, a close examination of Hawker's results does show an increase in activity of the three enzymes in the eleventh week after flowering, which could not be explained in terms of changes in berry metabolism and was attributed to higher than average temperatures in the vineyards at that time. The increase in enzyme activity found by Hawker could correspond to the large increase in protein level observed in this study as both increases in content and activity occur at approximately the same stage of maturity. Vos and Gray (1979) suggested that protein levels increase during grape maturation, following studies that indicated an inverse relation between protein nitrogen and titratable acidity during grape ripening. Such an observation supports the finding of the direct relation between protein content and sugar accumulation shown in Figures 14 - 16. 56

Figure. 12. Protein levels of juice during grape ripening

800

640

-. w ~480 m ...J...... C) E ._.320 C ·-..,Cl) • 0... a. 160

0

6 8 10 12 Must density ( 0 Be )

• Semi l lon 6. Traminer O Chardonnay Figure.13. Protein levels of juice during grape ripening

480

...... J.... ~20 w <:( en m --160 C ·-4) ~ 0 ~ 0. 0

6 8 10 12 Must density ( 0 Be)

0 Gordo A Rhine Riesling 0 Sauvignon blanc Figure. 14. Protein levels of juice during grape ripening

'w'

• Rkatsitelli 6 French Colombard 59

Figure 15. F.lution profiles of Chardonnay ,juice Harvest dates during rioening (a) 6.2.79, (b) 9.2.79, (c) 14.2.79, Conditions of chromatography; (d) 19.2.79. column pBondagel E-Linear E-300, mobile phase and µBondagel 0.01 M ammonium acetate and 0.01 rate M SDS, flow 2.0mL/min, injection volume 10 pL.

a b

6

4 N- I 0.,.. >< 2 en LL ::J -

Cl) CJ caC 4 .0... 0 en .0 2

0

0 2 4 6 0 2 .4 6 Time (min) 60

Table 7. Analytical data for varietal white juices

Harvest 0 se Titratable Malate Tartrate p..,otein Acidity pH K ( g/L) ( g/L) (m g/L) Date Variety ( g/L (mg/L) Tartaric)

Traminer 31.1.79 8.5 9.6 3.43 6.2.79 8.5 8.0 3.46 2080 7.4 220 7.2.79 8.6 7.2 3.58 400 9.2.79 9.2 7.7 3.72 2800 4.8 7.0 450 14.2.79 9.5 5.85 3.80 2600 4.7 6.0 850 19.2.79 9.5 5.6 3.91 2760 3.1 6.7 700 26.2.79 10.2 5.4 3.95 3000 2.8 7.0 650 5.3.79 10.2 5.4 3.9 3120 2.5 5.5 620 Chardonnay 31.1. 79 9.0 12.9 3.25 6.2.79 9.8 10.86 3.33 2200 5.5 7.25 130 9.2.79 10.3 9.15 3.'56 2760 '5.1 7.0 170

14.2.79 10. fi 7.5 3. fi 1 2400 11.0 'i.8 270

19.2.79 10.R 6.7, 3.79 2720 3.4 6. fj :mo Sauvignon 31. 1. 79 9.0 13.8 2.95 blanc 6.2.79 10. 7 9.4 3.17 HiOO 3.5 7.2'5 1'5, 9.2.79 10.4 9.10 3.36 2120 4.3 7.6 230 16.2.79 11.0 6.0 3.32 1720 2.6 5.9 280 22.2.79 11.0 5. 2 '5 3.56 1920 2.0 6.7 340

28.2.79 11.0 '5. 0 3.62 2000 1.8 fj • 6 480 7.3.79 12.0 4.4'5 3.78 2080 1.2 '5 . 7 300 15.3.79 14.0 4.35 3.85 2440 1.9 '5. 9 390 Rhine 31.1.79 8.0 17.2 2.85 Riesling 6.2.79 9.2 14.8 2.90 1fi80 5 . '5 9.0 '50 16.2.79 10. '5 7.3 3.13 1'560 3.5 6.2 180 22.2.79 11.0 7.1 3.39 2000 3.3 6.8 280

Cont/over ...... 11 l

Table 7. Analytj cal data for varietal white ,bices

Jfarvest OBe Titratable Malate Tl:lrtrate Protejn Acidity pH K (g/L) ( g/L) (f'tg /L) Variety Date (g/L Tartaric) (mg/L)

Semillon 31.1.79 7.5 8.9 3.15

n.2.79 8.5 1. i; 3.23 1440 2.5 6.5 230 14.2.79 10.3 6.3 3.45 1920 2.7 6.6 ,oo 19.2.79 9.8 5.85 3.49 1840 1.8 6.45 400 26.2.79 10.8 5.4 3.5n 1920 1.4 c; • 7 540 5.3.79 10.7 5.0 3.62 1920 1.4 5.7 365 French 28.2.79 10.0 7.2 3.28 1720 2.6 7.0 110 Colo'!lbard 7.3.79 10.0 5.9 3.37 1520 2.3 5.8 130 15.3.79 10. 5 5.25 3.34 1360 1.9 4.7 115 R'katsiteli 28.2.79 9.0 6.9 3.13 1160 2.2 IL!'i 40 20.3.79 10.0 5.7 3.21 1240 1.2 7.0 110 ,.4.79 11.0 !'i. 1 3.34 1200 1.2 5.4 120 Gordo 7.2.79 7.5 8.3 3.21. 280 13.2.79 8. !'i 6.75 3.27 290 20.2.79 8.7 6.0 3.29 23.2.79 9.2 5. 90 3.38 4R0 27.2.79 9.6 !'i. 1 3.39 3/i0 5.3.79 10. 5 4.8 3.49 210 9.3.79 10.5 4.5 3.59 2/i0 23.3.79 13.0 4.4 3.8 3n0 62

Analytical data for the juices (Table 7) show the typical compositional changes that occur during grape ripening, i.e. decreasing total acidity, increasing potassium level, pH and must density ( 0 Be). The compositional data do not provide any additional information that might further explain the obvious relationship between protein and grape maturity found in the trials presented here. The changes in grape composition that occur during grape ripening are illustrated by the chromatographic profiles for the UV absorbing materials of juice from ripening Chardonnay grapes at Reveral maturity levels (Fig.15). The changes observed in the chromatographic profiles during ripening probably mean that 'finger printing' a .iuice or wine according to variety by means of such a profile is not possible. The UV profile of ,juice is perhaps a unique 'finger print'of that particular ,juice and not generally of the variety or region. The ident­ ification of the individual UV absorbing components of ,juices and wines separated by HPLC could also warrant careful investigation, especially the influence of these compounds on wine quality and how they are affected by variety, maturity and processing conditions.

4.3 Protein levels of must and wine during vinification

Wine proteins mostly originate from the grape and occur at various levels which depend not only on grape variety, maturity, year of vintage, vine environment but also on juice clarification conditions, fermentation conditions, stabilising methods, bottle aging and analytical methods. Vinification operations can reduce soluble protein levels by physical removal, provide conditions that result in protein flocculation, precipitation or hydrolysis, or processing may allow conditions suitable for protein solubilisation or conditions that prevent natural defaecation of the protein.

4.3.1 Effect of clarification method on total soluble protein of juice and wine

Fermentation of clarified or low solids ,juices has allowed the production of the light, delicate and fruity white wines that are presently in demand by the consumer. Clarification by enzymes, bentonite and cold settling has been examined for the effect on protein levels of juice and the corresponding wine. The protein levels before and after clarification and at the end of fermentation are presented in Table 8. 63

Table 8. Protein levels of ,Juice and resultant wine before and after various clarification treatments

Protein (mg/L) BSAE Semillon Trebbiano Gordo juice wine juice wine juice wine

Befor~ clorification 190 25 435 Cold settling 280 200 75 70 450 430 Bentonite(lg/L) 110 25 100 25 450 160 Enzyme 1* 290 190 85 70 500 400 2 240 220 70 90 525 450 3 300 210 90 130 510 435 4 285 210 85 110 510 410

*Enzyme 1 Protease B (Pfizer) llmg/L juice (Gordo 22 mg/L) 2 Protease M-6(Pfizer) 11mg/L juice (Gordo 22 mg/L) 3 Clari phase (Pfizer) 11mg/L juice 4 Ultrazyme (Ciba-Geigyhlmg/L juice An important observation from Table 8 is the increase in soluble protein in the juice after clarification in all trials except for Semillon clarified with bentonite. The increase in soluble protein during clarification could be a result of the dissolution of proteins from juice solids. Proteins can be complexed with polysaccharides, polyphenols and lipids in juice solids and so reactions that break up these complexes during clarification would contribute to the observed increases in soluble proteins. The solubilisation of protein from grape solids after crushing suggests that a rapid removal of juice solids from the must by centrifugation or filtration, could lead to the production of a protein stable wine. Such a technique would also prove valuable in the study of the susceptibility of wine to protein cloud formation. The effect of rapid clarification on sensory properties and style of wine produced would also need examination. The increase in protein levels of the Trebbiano and Gordo juices and the marginal reduction in protein content of the Semillon juice after bentonite treatment was not expected. The bentonite may have been dispersen by negatively charged colloids which would prevent its acting on the soluble protein. 64

These results are not in accord with the work of Vos and Gray (1979) who found that bentonite fining of must could effectively reduce protein concentration. Ough, Berg and Amerine (1969) also reported that bentonite removed protein from must, but was more effective in removing protein after fermentation. It is interesting to note that the juices clarified with bentonite had substantially lower protein levels after fermentation than those clarified by the other treatments. In this case the bentonite may have removed some protective colloids in the juice, thus sensitising the protein to the conditions present during fermentation, or the fermentation conditions may have changed the colloidal status of the fermenting juice allowing any dispersed bentonite remaining in the juice to act on the proteins. At this stage bentonite fining of juice is not favoured due to the large amount of lees generated. Proteolytic enzymes have been claimed to reduce protein levels of juice and wine to a level where the protein stability of the product can be guaranteed (Nanitashvili & Samadashvili 1974, Datunashvili 1975, Gaina et al. 1976, Tikhonova and Platsynda 1976). The protease preparations (Table 8) did not reduce the soluble protein levels in the juices examined. Adler-Nissen (1977) suggests that native globular proteins are resistant to the action of proteases which may explain the failure of the proteases examined to reduce the soluble protein levels of the juices. The concentration of the proteases added to the juices was based on that suggested by the suppliers for the pectic enzyme preparations. Double the concentration of the proteolytic enzymes was added to the Gordo juice because of the expected high protein levels and the previous lack of activity of the proteases in the Trebhiano and Semillon juices. The higher proteolytic enzyme concentration did not reduce the soluble protein content of the Gordo juice. Failure to emo1oy optimum conditions for the emzymes could explain the observed lack of pro­ teolytic activity as recommended enzyme concentrations and incubation times and temperatures were not supolied with the product. The pectic enzymes did not reduce the level of soluble protein in the juices; the observed increase in protein levels during clarification with pectic enzymes was similar to that of the untreated .iuice. The site of action of the particular enzymes present in these commercial pectic enzyme preparations is thus independent of the reactions in the juice which bring about the protein solubilisation observed in the trials. Work during the 1980 vintage (Luis and Lee, unpublished results) has conrirmed that protein 65

content of the juice held at temperatures between 10 and 20° C increases during cold settling, and the presence of either pectic or proteolytic enzymes had little effect on the increase in protein content of the clarified juice. It was shown that the protein content of the untreated juice increased by about 34% after 12 hours clarification compared to 40% for juice treated with pectic enzymes. The rate of increase in protein content of the protease treated juice was much slower than that of the untreated or pectic enzyme treated juice, but reached a protein content similar to that of the latter juices after 36 hours of clarification.

4.3.2 Changes in soluble protein levels during fermentation Fermentation is a complex biological phenomenum,during which dramatic chemical and biochemical changes occur; it could be reasonably expected that protein would also be involved or be affected by these changes. For example the increase in alcohol content would be expected to decrease the solubility of the protein, but on the other hand could also facilitate the formation of stable colloids that was not possible at lower alcohol concentrations. Protein levels of the fementing ,juices examined in these trials were estimated each day from inoculation to the end of the fermentation. The fluctuating protein levels during fermentation (Figures 16 - 22) reflect the changing conditions of a fermentation. There is no steady decrease in protein from the start to the conclusion of the fermentation, but fluctuation from day to day. There is no common pattern in the fluctuations of protein content even for one variety. Patterns that might be discerned are the rapid and sometimes severe fluctuations in protein levels that occur immediately after inoculation. These fluctuations did not occur in every fermentation but are apparent in a majority of fermentations and are probably due to the intense yeast metabolic activity which occurs at the initiation of fermentation. Another pattern of protein level variation that was observed in several fermentations was a rapid increase in protein level near the completion of fermentation; this peak in protein content is possibly related to the physiological activity of the yeast at the final stages of fermantation. It has generally been considered that the protein content of musts declines between 1~ and 75% during fermentation (Ferenczy 1966, Amerine and Joslyn 1970, Somers and Ziemelis 1973, Spirov 1975). Such a decrease in protein content has not been confirmed in this study during the fermentation of musts from six varieties at several levels of maturity. Protein content in the new wine varied from that in the must by as much as a 33% decrease to a 40% increase The effect of fining .iuice with bentonite prior to fermentation on protein levels of the final wine can be seen from Table 8. The juice 6fi

Figure lfi. Protein levels of Semillon .iuices during fermentation. Harvest dates. 0 14.2.79, £26.2.79 ■ 5.3.79 •

...... w 520 < ·~ ■ enco • /\ ..J ■ ..... /\ ■ C) .-----1 ./ .._.,E 360 ·-C ..,G) ■ 0 ~ CL ·~ ■ 200 12 8 4 0 Must density ( 0 Be)

was racked off gross bentonite lees with little effect on protein level. Dispersed bentonite remaining 1n the juice after could account for the reduction in protein at the end of fermentation when conditions for bentonite action are more ruitable. The practice of fermenting with hentonite is a common occt•1'rCi,Ce in West Germany, France and South Africa, however,

information as to its effect on fermentation rate1 clarification, protein stability and wine quality is contradictory, with no definite agreement as to whether bentonite is best added to the fermentation or the finished wine (Groat and Ough 1978). Previous experience in commercial wineries(McWilliam's Wines Pty Ltd, personal communication) has indicated that there is little 67

Figure. 17. Protein levels of Traminer juice during fermentation. Harvest dates O 9.2.79, ■ 14.2.79, 6 26.2.79.

880------

■

720 ...... UJ ~

C ■ ·-...C1) ...0 C. 400

12 8 4 0 Must density ( 0 Be)

difference in the total amount of bentonite required to produce a protein stabl~ wine whether added after fermentation or during fermentation, in many jnstances it was found that a wine fermented on bentonite required additional bentonHe at the end of fermentation to stabilise the wine with respect to protein and consequently with a further loss of wine in the lees. The effect of bentonite added during fermentation varied with variety and vintage. Ough et al. (19fi9) also found that bentonite addition to a finished wine was more effectjve than adding it to the juice prior to fermentatjon. The fluctuations in protein levels dui'ing fermentatjon observed in this work and the variation Figure.18. Protein levels of Chardonnay ,iuices during fermentation Harvest dates.9.2.79, 0 14.2.79, ~ 19.2.79 .

...... w ~320 al -I 'C) ...... E ·=160 • Cl) ----■-· +I 0 ~ 0.

0 L--~---L-----L------L---L---.J.-.---'-----' 12 8 4 0 Must density ( 0 ee)

in the final protein level in the wine with respect to the level in the initial ,iuice could explain the contradictory results reported by various workers on the effect of bentonite added before and during fermentation. 69

Figure. 19. Protein levels of Rhine Rielsing juices during fermentation ~arvest dates O 16.2.79, .& 22.2.79.

- ~320 en m ..J C) 'E - 160 C ·-..Q) 0 ~ 0.

12 8 4 0 Must density ( 0 ee) 70

Figure 20. Protein levels of Rkatsitelli ,iuices during fermentation Harvest dates 0 28.2.79, 6 20.3.79.

~40 "'cc ..J a,' ...... E ·= 80 · ..,Q) 0 ~ 0. 10 6 2 Must d ens1·t y ( 0 ae·) 71

Figure 21. Protein levels of Sauvignon blanc juices during fermentation Harvest dates • 9.2.79, 6 22.2.79, 0 28.2.79., O 7.3.79 .

.-.440 w c:r: Cl) al ...J 'C) .....,E280 C ·-Cl) ~ 0... a. 120 ..,___L------1------l--__.______., _____.__ _ __.______. 12 8 4 0 Must density ( 0 Be) 72

Figure 22. Protein levels of French Colombard juices during fermentation Harvest dates D 28.2.79, .& 7.3.79, () 15.3.79.

.--.. 320 w 160 ..._.E C ·-..,Q) ...0 C. 0 12 8 4 0 Must density ( 0 ee) 73

4.3.3 Protein stabilisation of white wine

The procedures used by the wine industry for the determination of the correct amount of bentonite required to produce a protein stable wine are imprecise, and there is little information as to how protein levels are affected by bentonite addition or by various bentonites. There is no precise instrumental Or chemical method for the accurate assesment of the correct level of bentonite that will render a wine protein stable during the period between bottling and consumption. At present the level of bentonite to be added to a wine is determined by the application of one of several arbitrary tests to the wine after trial fining. These tests include the proprietary Bentotest, the TCA test, the heat and tannin test and the heat test (Section 2.4.3 or 2.3.6). The protein level at which a wine is stable is not known(Thoukis 1974). The lack of a rapid, reliable method for the quantitation of protein in must and wine has probably contributed to the scarcity of data for protein behaviour during fining. The development of a reliable and rapid HPLC method for quantitation of soluble protein has allowed a large volume of data on protein levels to be gathered in a short time. The protein level of a wine was followed during fining, by fining the new wine wit½ a range of bentonite levels then determining the protein level of the wine after settling of the bentonite. These fining trials were carried out for all the wines made in the maturation study and for many commercial wines from McWilliam's Wines Yenda winery. Protein levels estimated during fining show that protein is rapidly removed from wine by increasing levels of added bentonite (Figures 23-32). For most of the wines there are slight differences between the rates of protein removal. The rate of protein removal for higher pH wines such as Chardonnay and Traminer, is slower than that for the wines of lower pH, such as Rhine Riesling and Rkatsiteli; the varieties with the higher protein levels before fining also tended to have slower fining rates, the exception being Chardonnay. It appears that low pH wines are more easily fined by bentonite, but it must also be noted that wines of low pH generally had low protein levels, whether this is because of the low pH or a varietal effect, as demonstrated by Chardonnay, is difficult to determine. Previous work 74

(Tyson, 1976) indicated that the rate of protein removal is significantly higher for wines of low pH; this was based in part on Chardonnay grown both in the Hunter Valley of N.S.W. and the Riverina District of N.S.W. Both wines had similar protein levels but different pH, although the low pH wine from the Hunter Valley had a significantly higher protein removal rate; it should be noted that a regional effect, such as higher polysac­ charides and lipids in warm areas, could also affect the fining efficiency. A feature of all the fining trials (Figures 23-32) is the rapid decrease in protein removal rate when the protein level of the wine is in the range of 20 - 50 mg/L, even though the initial protein level of the wines ranged from 40 - 650 mg/L. This pattern of protein removal from wine by bentonite has been consistently seen for all wines from the major white varieties grown in the Griffith and Hunter Valley areas of NSW for three vintages (Tyson 1976,Tyson unpublished results). Such a relationship can be seen in the published data of Rankine and Emerson (1973) and Danilatos (1978) but has not been specifically commented on. The protein level of wine at which the rate of protein removal by bentonite declines so quickly also corresponds to that level at which the Bentotest and the heat and tannin test indicate stability. A small fraction of wine protein appears not to be affected by bentonite or is removed at a slower rate than that of the heat sensitive protein. These two protein fractions could be the same as those referred to by Somers and Ziemelis (1973) who believed that wine protein consisted of two fractions and that one fraction of protein was complexed with a small amount of flavonoid material and was responsible for protein instability, however, they found no difference between the rate of removal of the two fractions by bentonite. One further feature of the data presented in Figures 23-32 is that some protein, approximately 10 mg/L, is detectable even after large additions of bentonite. The position of the remaining protein on the HPLC chromatogram indicates that it has a slightly higher molecular weight than that of the wine protein that is easily removed by bentonite. Later develop­ mPnts of the HPLC method appear to indicate that this peak may have been an 7 !5

Figure. 23. Protein levels of Rkatsiteli wines during bentonite fining. • Harvest date 28.2.79, O Harvest date 20.3.79, 6 harvest date !5.4.79.

~00 w < ~ ...I '~00

C ·-Q) 0 £t 0 0 0.8 1.6 Bentonite level ( g1L)

artifact, perhaps a reaction between polypeptides and the SDS in the mobile phase (Tyson et al. 1981). However, the discovery that a certain amount of protein remains in wine even after addition of high levels of bentonite is supported by Vos and Gray (1979) who found a similar relation­

ship for protein removal from grape ,juice1 that is 1the protein level, determined by micro-K,jeldahl, was reduced to a constant low level for all ,juices - al though no values were given- by high levels of bentoni te and independent of the initial protein level in the juice. Fining of juices in this study did not exhibit this pattern; it was found that bentonite addition to grape ,iuice did not effectively reduce the protein content. These conflicting 76

Figure 24. Protein levels of Chardonnay wines after bentonite fining 6 harvest date 9.2.79, e harvest date 14.2.79, D harvest date 19.2.79.

300

...... ~ 200 en al ..J d\ ...... E 100 C ·-...,G) 0.... 0. 0 0 0.8 1.6 2.4 3.2 Bentonite level { g/l)

results could be explained by differences in the colloidal content of the .iuices used by Vos and Gray and those studied in this investigation. The disadvantages of the K,jeldahl method have been described (Section 2.2.3) and may have contributed to misleading results in the work of Vos and Gray (1979); the fine tuning of the HPLC method and electrophoretic techniques should see this problem resolved. Bayly and Berg (1967) fractionated wine protein by disc electro­ phoresis and considered that one of the several fractions observed was responsible for protein instability, but that total protein content was not a suitable indicator of fining requirements or of the potential stability of a w1ne. 77

Figure. 25. Protein levels of Rhine Rielsing wines after bentonite fining.

■ Harvest date 16.2.79, 0 harvest date 22.2.79.

~200 w ~en al ..J c,,100 ._.E C ·-....,(1) 0 ~ 0 0 0.8 Bentonite level ( 91L)

Moretti and Berg (1965) also suggested that the protein content could not be used as a measure of the protein stability of a wine and consequently proposed a formula based on the ratios of four protein fractions separated by electrophoresis. Values of less than 4.2 indicated unstable wine, and those greater than 6.6 indicated stable wine, however, a significant range of values from 4.2 to 6.6 remains where the protein stability of a wine is ill-defined. In contrast to the claims of Berg and his students data from fining trials of this study indicate that the 78

Figure. 26. Protein levels in Semillon wines after bentonite fining Harvest dates ~14.2.79, 019.2.79, 0 26.2.79, • 5.3.79.

450 •

300 ...... w <( Cl) al ..J ' ...... ,r 150 C ·-..,G) ~

0 0 0.8 1.6 Bentonite level { g/L)

protein level of wine is an excellent indicator of wines stability. It appears that protein stability, as defined by the described tests, occurs when the soluble protein is reduced by bentonite to level in the range of 20 to 50 mg/L (Table 9); as previously noted this is the range where the protein removal rate by bentonite shows a rapid decrease. Protein content can not as yet indicate what fining level is required to reduce the protein to within the stability range. 79

Figure. 27. Protein levels of Traminer wines after bentonite fining Harvest dates~ 9.2.79,. 14.2.79,D19.2.79,Q 5.3.79.

600

...... ·w 450

150 • ~ • 0 0 1.2 2.4 Bentonite level (9/L) 80

Figure. 28. Protein levels in Sauvignon Blanc wines after bentonite fining Harvest dates. 9.2.79, 0 16.2.79, /:).. 28.2.79.

300

200 ......

UJ I c:r: en al _. 100 CJ) 'E """' C ·-..,Q) 0 00 a.~

0 0 0.8 1.6 Bentonite level { Q/L)

The words stability as defined by the tests described must be emphasised1 as bottle maturation experiments suggest that these stability tests are quite severe and probably do not reflect the level to which protein should be reduced so that a wine remains bright between bottling and consumption. 81

Figure 29. Protein levels in French Colombard wines after bentonite fining Harvest dates O 28.2.79, 0 7.3.79, A 15.3.79.

C 50 ·-..,Q) 0 a.. 0 a. I 0 0.8 Benton ite level (g/L)

Moretti and Berg (1965) also found that the normal stability test procedures gave variable nephelos values for wines of similar protein levels and suggested that these data further support the contention that protein levels of wine could not be used as an indicator of stability. These variable nephelos values on the contrary could highlight the unsuit­ ability of the common testing methods for determining the potential of wine to become protein unstable. A wine from this study was shown (Table 13) to have no protein, as determined by HPLC after an ion-exchange treatment, and yet the Bentotest method indicated that the wine was unstable. Such an unexpected result was from one trial only, but poses the questions as to what 82

Figure. 30. Protein levels 1n commercial Trebbiano wines after bentonite fining A Beelbangera vat RF8, 0 Yenda vat 78, Q Yenda vat 620.

...... 200 w <( Cl) m ...J en 100 ._.E C ·-..,Q) ...0 C. 0 0 0.8 1.6 2A Bentonite level (g/L) wine components, apart from wine protein, react with the reagents in the Bentotest solution and how do these affect the accuracy of protein stability determinations. The Bentotest and the heat and tannin test were used to evaluate the stability of the wines in this study; contrary to most opinion in industry, the Bentotest did not prove to be more sensitive than the heat and tannin test, and with most wines the level of bentonite addition at which each test indicated stability represented a difference of less than 0.2~ g/L bentonite. The trichloroacetic acid test and heat test have been shown to be insensitive and variable and were consequently discarded (Tyson 1976). 83

Figure. 31. Protein levels in commercial Semillons from the Yenda winery after bentoni te fining • vat 603, D vat 78, ~ vat 622, 0 vat 204.

300

...... w <( cn200 m ..J C) 'E

C 100 ·-Q) ...0 a.~ 0 0 0.8 1.6 2A Bentonite level (g/l)

The Bentotest was less consistent than the heat and tannin test, which was most apparent when evaluating the stability of the pulpy varieties such as Muscat Gordo Blanco. How the results of the stability tests relate to the actual potential for a wine to throw a protein haze during bottle age is not known. Short time heat stability tests may only indicate the instabilities that occur at the elevated temperatures of the tests and not the long term potential instability at ambient temperatures. To support this contention,Table 10 shows data for protein levels in experimental wines four months after bottling._ 84

Figure. 32. Protein levels after bentonite fining; a comparison of dry white wines and fortified sweet white wines. Dry white wines Q Beelbangera Gordo vat RF4 D Beelbangera Gordo vat RF10; sweet fortified white wines 8, Beelbangera Gordo,. Yenda Semillon vat 603.

450

300

..J ~ ._,.E 150 C ·-..,Cl) ...0 ~

0 0 0.8 1.6 2.4 Bentonite level (g/L) All wines were fined with 1 g/L bentonite prior to bottling and thus were bottled at protein levels well above the stability range of 20 - 50 mg/L. Four months after bottling the protein level in most wines was approximately half that at bottling and more importantly none of the wines exhibited protein instability, i.e. formation of a haze. The large decrease in protein after only four months bottle age was not expected, as it was considered that any hydrolysis reactions that take place would be extremely slow. The bottled wines were stored at room temperature and although the temperature varied the wines were not subject to extremes. Observation of experimental wines made at the Yenda research laboratory of McWilliam's Wines in previous vintages confirm this finding as wine bottled without any bentonite treatment showed no sign of a cloud or deposit after two years storage under far from ideal conditions. Nanitashvili and Shilakadze (1974) and Ermachkova (1975) also found that protein levels decrease with bottle maturation. The reduction of soluble protein means that many white wines could be over fined which may have affected the sensory qualities of the wine. The decrease 1n protein level with bottle age and the lack of signs instability suggests that the stability tests employed are severe and probably are not an indicator of the real potential of a wine to form a protein haze within the expected shelf-life of the product. A detailed study is required to evaluate the effect of storage conditions, variety, area and production conditions on the reduction of protein with bottle age and the relationship between appearance of protein haze and the indicated stability by the protein stability tests procedures.

4.3.3.1 Stabilisation of Muscat Gordo Blanco; a special problem

Over 50 000 tonnes of Gordo are crushed in Australia each vintage for the production of white table wine and fortified sweet wine and as such is of considerable economic importance to the Australian wine industry. Many fortified sweet Gordo wines are difficult to stabilise, with stability tests often indicating that bentonite additions in excess of 3 g/L are required to produce a protein stable wine.

In an effort to understand the behaviour of the proteins of Gordo under processing conditions, a dry table wine and a fortified sweet wine were made from commercial Gordo juice in a manner similar to that in a winery. The protein content of both samples was monitored throughout clarification, fermentation, fortification and fining: after bottling without bentonite fining the protein content was again determined after

four months storage with further fining and fortification trials. Table 9. Protein levels of commercial wines at stability as indicate~ by Bentotest and heat and tannin tests

Wine Protein mg/L BSAE*) Initial at stability by at Stability by Bentotest Heat and Tannin

Semillon 145 15 30 Semillon 220 40 Semillon 210 40 30 Semillon 210 30 30 Semillon 190 45 45 Semillon 200 30 30 Semillon 190 40 Semillon 270 30 40 Semi llon 220 25 35 Semillon 195 5 10 Semillon 70 30 30 40 15 15 Palomino 130 40 Palomino 65 10 15 Trebbiano 70 70 40 Trebbiano 90 40 40 Trebbiano 70 30 30 Trebbiano 130 35 35 Trebbiano 110 15 40 Trebbiano 110 25 25 Trebbiano 170 25 2 5 Trebbiano 125 25 nO Gordo 430 10 10 Gordo 365 20 20 Gordo 425 25 25 Gordo 430 25 25 Gordo 450 30 30 Gordo 370 15 15 Gordo 400 7 7

* BSAE - Bovine serum albumin equivalents. 87

Table 10. Effect of bottle ~ge on ,£EOtein levels

PROTEIN (mg/L) VARIETY HARVEST OATE AT BOTTLING AFTER 4 MONTJ-JS

9.2.79 200 100 14.2.79 300 180 Traminer 19.2.79 340 170 26.2.79 245 140 5.3.79 560 270

9.2.79 85 20 Chardonnay 14.2.79 20 15 19.2.79 70 1 '5

9.2.79 20 10 16.2.79 10 10 Sauvignon 22.2.79 10 10 Blanc 28.2.79 45 20 7.3.79 90 40

Rhine 16.2.79 30 10 Riesling 22.2.79 10 20

14.2.79 50 1 'j Semi llon 19.2.79 5 15 26.2.79 10 20 5.3.79 30 20

French 28.2.79 5 15 Colombard 7.3.79 5 10 15.3.79 5 15

28.2.79 5 10 Rkatsite]i 20.3.79 "i 10 5.4.79 5 5 Table. 11. Analytical data for white wines

Harvest T.A. pl-f K Malic Tartaricc Protein Total Alcohol Date g/1 Tartaric (mg/L) (g/L) ( g/L) (mg/L) phenoJics % V/V mg/L GAE

9.2.79 6.65 3.79 1320 3.86 1.85 460 0.22 8.5 14.2.79 5 .55 3.83 1280 2.6 1.64 560 0.23 8.8 Traminer 19.2.79 4.80 3.99 1400 2.6 1. 7 550 0.25 9.25 26.2.79 *5.30 3.71 1040 2.1 1. 7 550 0.2n 9.9 5.3.79 *4.75 3.84 1120 2.0 1.8 650 0.30 10.16 * Juice was acid djusted prior to fermentation 9.2.79 6.90 3.75 1320 3.6 1. 7 170 0.33 9.88 Chardonnay 14.2.79 6.35 3.80 1320 3.0 2.0 210 0 .3!'i 10.2n 19.2.79 !'i. 70 3.97 1560 2.7 1.9 270 0.37 10.36

9.2.79 6.7, 3.38 800 3.2 2.3 210 0.24 11 . 0 16.2.79 !'i. 95 3.!H 760 2.0 2.1 200 0.20 11. 7 Sauvjgnon 22.2.79 4.70 3.57 760 1.5 1.8 260 0.21 11.7 Blanc 28.2.79 4.20 3.59 840 1.1 2.3 380 0.22 12.0 7.3.79 **4.80 3.47 680 1. l 2.5 360 0.25 13.4 **Juice was acid adjusted = 2 g/L prior to fermentation Rhine 16.2.79 6.10 3.28 760 2.5 2.7 150 0.20 11 .1 Riesling 22.2.79 5.90 3.37 800 2.1 2.4 230 0.30 11.,;

14.2.79 5.25 3.44 740 1.4 2.2 360 0.23 10.1 19.2.79 4.80 3.45 720 1.3 2.2 365 0.22 10.2 · Sem-i11on 26.2.79 4.60 3.50 740 1.1 2.3 390 0. 2,; 11.3 5.3.79 4.25 3.61 800 LO 2.2. 420 0.27 12.1

French 28.2.79 5.75 3.23 640 1.8 2.5 75 0.25 10.4 7.3.79 5.00 3.34 660 1.8 3.0 l!'iO 0.25 10.2 Colombard l!'i. 3. 79 5.10 3.38 680 1.5 2.6 130 0.24 11. 2

28.2.79 6.10 3.06 520 1.5 3.6 80 0.28 9.05 Rkatsjteli 20.3.79 !'i.30 3 .14 480 1.0 3.3 12'5 0.29 10.4 5.4.79 5 .10 3.27 ,20 0.8 2.9 95 0.30 10.9

Gordo D/W 4.85 3.99 1160 340 0.34 13.8

Gordo S/W 3.55 4. 115 1080 235 0.32 1 R. 1 Figure 33. Protein levels in unfortified dry Gordo (0) and fortified sweet Gordo (I) after bentonite fining.

Muscat Gordo Blanco

300 .-. w c( Cl) m • ..J..... 0) 200 ._.E C fortified ·-..,Cl) ~ ..0 •'Zweet (18·1%alc. 3·8°Be) 0. •, 100 ., ...... •- dry (13·8% ale.)

0·5 1 1·5 2 2·5 Bentonite (g/L) Table 12. Protein levels in bottled Gordo wines

Alcohol Protein (mg/L) style (% v/v) At bottling After 4 mo

Dry 13.8 370 265

Sweet* 18.1 245 265

*Sugar level 3.8° Be 90

Figure 34. Protein levels in unfortified dry Gordo O fortified dry Gordo (0) and fortified sweet Gordo (I)

•~ Muscat Gordo Blanco ~w <( • (/) m 200 • ..J "' "'-!ortif ied sweet 'c,, • 18·1%alc. 3·8°8e ._E '• C ~ ·-..G) dry •---.___ 0 13·8%&Ic. • a: 100

D fortified dr 18·1% ale. 0 0 0·5 1 1·5 2 Bentonite (mg/l) Figure 33 shows the rate of removal of protein from a dry Gordo wine of 13.8% alcohol and from a fortified sweet Gordo of 18.1% alcohol and 3.8°Be; both wines were made form the same juice. A high alcohol level in wine is reported to promote efficent fining (Rankine 1963), however, the ineffectiveness of bentonite in these high alcohol (18.1%) wines 1s well illustrated (Fig.33) and can probably be attributed to the high colloidal content of the sweet Gordo. Gordo is a pulpy variety, high in pectins, gums and other polysaccharides. Thes~ polysaccharides combine with wine

proteins to form protective colloids which prevent bentonite acting on the 91

protein. In fact it appears that either the higher alcohol level, the sugar content or a combination of both have somewhat stabilised the colloidal system present in the fortified sweet Gordo and made the system more resistant to the action of bentonite. This proposal is supported by the observation (Table 12) that the protein level of the fortified sweet Gordo wine does not fall in the four months after bottling, whereas that of the dry 13.8% alcohol Gordo wine decreased significantly, about 30%. To examine further the relationship between alcohol and sugar levels and protein removal rate, the two Gordo wines were subjected to bentonite fining after four months bottle age, i.e. when both wines had a protein content of 265 mg/L. A portion of the dry 13.8% alcohol Gordo was fortified to 18.1%. Figure 34 shows that apart from an initial lowering of the protein level of the dry 18.1% Gordo by the addition of the fortifying spirit, the protein removal rate was not greatly affected by alcohol level and thus it seems that the colloids of the fortified sweet Gordo are probably protecting the protein against the action of bentonite. Several treatments to either remove the protein from Gordo wines or make the protein more reactive to the bentonite have been examined. For instance a heat treatment of 6 min at 600C decreased the protein content of fortified sweet Gordo wine from 300 to 75 mg/L, however, the rate of protein removal by bentonite was not improved. Testing of the heat treated wine for protein stability gave inconsistent and unexplained results - wines with low protein levels were indicated to be stable by one test and grossly unstable by another, or again slightly unstable by both tests. Fortified sweet Gordo wine was ion-exchanged (Table 13) to lower pH and to increase the charge intensity on the protein and thus increse the reactivity of the protein with the bentonite. Again conflicting results were obtained for the stability tests on these ion-exchanged wines: in one instance the protein was completely removed by the ion-exchange operation - this was attributed to the phenolic materials absorbed onto the resin from previous use with red wines - and yet the wine was indicated to be unstable by the Bentotest. An apparent increase in protein level was observed 1n another ion-exchange trial at the begining of the ion-exchange run, this increase was attributed to the low pH 2.1 of the wine altering the apparent molecular size of some wine components which would then elute from the HPLC system 1n a different order. The conflicting results for stability testing of these Gordo wines highlight the do•1btful relation between the protein stability as predicted by the commonly used tests and actual protein stability. 92

Table 13., Gordo sweet white fining and stability trials

Control Bentonite fining rates (g/L) 0.0 0.25 0.5 0.75 1.0 1.5 2.0 Protein level (mg BSA equiv./L) 195 170 135 100 60 Bentotest = = Heat & tannin = =

After heat treatment (75°c for 2 min)

Bentoni+;e fining rates (g/L) 0.0 0.5 1.0 1.5 2.0 2.5 Protein level (mg BSA equiv./L) 30 25 25 22 20 20 Bentotest = = Heat and tannin = =

After ion exchange to pH 2.0 of heat treated wine Bentonite fining rates (g/L) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Protein level (mg BSA equiv./L) 0 0 0 0 0 0 0 Bentotest = = Heat & tannin + + + + + + +

= grossly unstable - unstable + stable

Even though the results of the trials with Muscat Gordo Blanco discussed here have shed some light on the difficulties in stabilising sweet Gordo wines, they have shown that there needs to be an extensive study into what constitutes practical protein stability Tor Gordo wines and how this state might be achieved and what tests might be utilised to predict the appropriate treatments required to obtain this state. The investigation would have to include studies as to the effect of grape maturity and various vinification operations on the levels and reactions of proteins, polysaccharides and lipids which are undoubtedly involved in 'protein' stability problems. 93

APPENDIX I Analysis of juices and wines Sulphur dioxide

Wine (20mL) and 25% H3P0 4 (10 mL) were refluxed together for 15 min with nitrogen purging. The non-condensable gases were passed through 3% hydrogen peroxide containing methyl red-methylene blue indicator. The hydrogen peroxide was titrated with 0.01M NaOH to an olive green end point. This method is based on the Monier-Williams distillation as modified by Rankine (1962). The only variation from Rankines method was the use of nitrogen sparging instead of air aspiration. Titratable acidity and pH After vacuum degassing, wine (10 mL) or juice (5 mL) was titrated with 0.1M NaoH to a pH 8.3 end point. The total acidity was calculated as g/L tartaric acid; pH was measured with a Pye Unicam pH meter. Alcohol Wine (50 mL) and distilled water (20mL) were distilled until 50 mL collected in a volumetric flas~. The refractive index of the distillate was measured at 20°c with a Zeis model 32957 refractometer, and the% v/v alcohol determined from AOAC tables. Total phenolics Wine (1 mL), diluted to about 60 mL with distilled water, was reacted with Folin-Ciocalteu reagent (5 mL) and 20% sodium carbonate (15 mL). The reaction mixture was made up to 100 mL with distilled water and the colour allowed to develop for two hours; the absorbance of the reaction mixture was read at 765 nm with a Hitachi soectrophotometer. Total phenolic content was calculated as g/L gallic acid equivalents. (Singleton & Possi 1965) with gallic acid standards. Pot assi t:m The wine or juice was diluted one to ten with distilled water and the absorption of this diluted sample was determined with a Varian Techtron atomic absorption spectrophotmeter with the following instrument settings: wavelengh, 404.4 nm; lamp current, 10 mA; and slit width, 0.5 nm. The potassium level of the diluted sample was determined from a standard curve of the absorption of standard potassium chloride solutions of 25,50,100 and 150 mg/L. Organic acids Tartaric, malic and lactic acids were separated by ion-exchange 94

chromatography and estimated by conductivity (Monk & Forrest unpublished results). Bio-Rad AG,0W x 2 cation exchange resin (200-400 mesh) was packed into a 1.3 m x 6 mm teflon tube, and developed with a mobile phase of 0.075% v/v n-butonoic acid maintained at a flow rate of 20 mL/h by a peristaltic pump. The conductivity cell consisted of two stainless steel annular electrodes 1 mm thick with a 2 mm bore and spaced 0.2 mm apart. The column was maintained at constant temperature in a water bath. Injection volume was 5uL. The organic acid peaks were recorded on an 0mniscribe chart recorder (Huston Instruments). The acios were quantitated by comparing peak heights to those of standard solutions of tartaric, malic and lactic acids.

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