MICROBIOLOGICAL AND CHEMICAL CHANGES DURING THE AND AGEING OF SPARKLING

A thesis

submitted to The University of New South Wales as fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

by

BRYAN TODD

Bachelor of Science with Honours (Class 1)

(UNSW, Australia)

Department of Food Science and Technology

The University of New South Wales Sydney, NSW, Australia

March 1995 j UNIVERSITY OF N.S.VV.

< 13 nr; 1995 j L 1 E R A R I E S ii

DECLARATION

The candidate, Bryan Todd, hereby declares that this thesis is his own work and that, to the best of his knowledge and belief, it contains no material previously published or written by another person nor material which has been accepted for the award of any degree or diploma of a university or other institute of higher learning, except where due acknowledgment is made in the text of the thesis.

Bryan Todd iii

ACKNOWLEDGMENTS

A most sincere thanks must first go to my immediate supervisor, Associate Professor G.H. Fleet (Department of Food Science and Technology, University of New South Wales) for providing invaluable encouragement, guidance and, most importantly, inspiration, to me during this project and production of this thesis. I would also like to thank my co-supervisor, Dr. P.A.

Henschke, (Australian Research Institute) for his keen insights and advice in key aspects of the project. It was a privilege to have had the opportunity to work with such experienced and professional scientists.

To the staff of the Department of Food Science and Technology, University of New South Wales, many thanks for all the assistance during the project. In particular, I would like to specially mention Mr. Camillo Taraborelli and Mrs.

Yvonne El-Ghetany for their help in the supply, operation and maintenance of the equipment that I used in my analyses. Similarly, a grateful thanks to Dr. Ron Haines (School of Chemistry, University of New South Wales) for his technical advice with regards to various analytical procedures.

To my fellow aspiring PhD students, Roostita Balia, Jian Zhao and Charoen

Charoenchai, I would like to say that it was a privilege to be able to work with such fine scientists from around the globe. Many thanks for all the advice and help in the project and, just as importantly, for being good friends.

I would also like to express my gratitude to the Australian Government for granting me an APRA (Australian Postgraduate Research Award) scholarship during my studies which allowed me the means to devote myself full-time to my research. iv

Finally, I would like to extend my most heart-felt thanks to my parents and my sister and her family for their support during my studies. It was a great help to know that they were behind me and supportive at all times, no matter what the reason. It is to my family, therefore, that I dedicate this thesis. V

TABLE OF CONTENTS

Page

DECLARATION jj

ACKNOWLEDGMENTS iii

TABLE OF CONTENTS v

ABSTRACT xi

1. INTRODUCTION 1

2. LITERATURE REVIEW 6

2.1 Production 6

2.11 Classification and global production 6 2.12 Production of sparkling wines by secondary fermentation 7

2.121 Sparkling wine fermented in the bottle 8 2.122 Sparkling wine produced in tanks 12 2.13 Novel methods used in the production of

sparkling wine 16 2.2 18

2.21 General description 18 2.22 Basic mechanism 19

2.23 Biochemical changes 21

2.231 and proteolytic activity 21 2.232 and p-glucanase activity 24

2.233 Other wall degrading 25

2.234 Nucleic acids and the activity of nucleases 26 vi

2.235 and lipolytic activity 27 2.236 Cytological changes 29 2.237 Industrial significance 31 2.3 Growth and Survival of Yeast During Sparkling Wine Fermentation by 'methode champeniose' 33

2.31 Factors affecting the growth of yeast during secondary fermentation 36

2.4 Chemical Changes During Sparkling Wine Fermentation 42

2.41 Changes during secondary fermentation 42

2.411 Consumption of sugar and production of and 43 2.412 Changes in concentration of amino nitrogen 43

2.413 Evolution of and organic acids 44

2.414 Production of volatile compounds 44 2.42 Chemical Changes During Autolysis 45 2.421 Occurrence of autolysis 45 2.422 Factors affecting autolysis 46 2.423 The action of enzymes during yeast autolysis in sparkling wine and the

products of their activity 48 2.43 Influence of Ageing on Yeast Upon

Sparkling Wine Quality 56 2.431 Effect upon sparkling wine aroma 56

2.432 Effect upon sparkling wine flavour 57

2.433 Effect upon the formation and stability of

bubbles (bead) 58 2.5 Effect of Carbon Dioxide upon Yeast Growth During Fermentation of Sparkling Wine 59 vii

2.51 The effect of carbon dioxide on the metabolism of 60 2.52 Inhibition of yeast by carbon dioxide 66 2.521 Physiological effects 66 2.522 Effect of carbon dioxide on the metabolic activities of yeast during fermentation 68 2.523 Effect of carbon dioxide on the growth and

metabolism of yeast during the secondary

fermentation of sparkling wines 71

2.6 Killer in the Wine Industry 72 2.61 Screening procedure for isolation of 73 2.62 Classification of killer yeasts 74 2.63 Molecular biology of killer activity 75

2.64 Mode of action of killer toxin 79 2.65 Occurrence of killer yeasts in the wine industry 80 2.66 Factors affecting the activity if killer toxin during 82 2.67 Impact of killer yeast in wine 84

3.0 DEVELOPMENT OF METHODS 3.1 Measurement of Release by Yeasts in Sparkling Wine 86 3.11 Introduction 86

3.12 Materials and Methods 89

3.13 Results and Discussion 90

3.2 Methods for the Determination of Nucleic Acids Released During Fermentation and Ageing of Sparkling Wines 94 3.21 Introduction 94 3.22 Materials and Methods 99 viii

3.23 Results and Discussion 107 3.3 Isolation and Analysis of Lipids in Sparkling Wines 120 3.31 Introduction 120 3.32 Materials and Methods 122 3.33 Results and Discussion 127

3.4 Method to Distinguish between the Populations of Killer

and Sensitive Strains of cerevisiae in Mixed Culture 135 3.41 Introduction 135

3.42 Materials and Methods 136

3.43 Results and Discussion 138

3.5 Development of a Chemically Defined Wine (CDW) 144

3.51 Introduction 144 3.52 Formulation of a chemically defined wine base (CDW) 144 3.53 Materials and Methods 150 3.54 Evaluation of the chemically defined wine base (CDW) 151

4.0 CHEMICAL CHANGES DURING THE FERMENTATION AND AGEING OF TWO COMMERCIAL AND ONE MODEL

SPARKLING WINE 153 4.1 Introduction 153

4.2 Materials and Methods 154

4.21 Base wines 154 4.211 Commercial wines 154

4.212 Model sparkling wine produced from a fermented chemically defined 155 ix

4.22 Preparation of yeast strains for inoculation into wine base (tirage) 157 4.23 Bottling, secondary fermentation and ageing 158 4.24 Analysis of sparkling wines during fermentation and ageing 158

4.241 Viable counts 158 4.242 Glycerol, fructose and 158

4.243 Ethanol 159

4.244 Headspace pressure 159

4.245 Organic acids 160

4.246 Soluble protein 161

4.247 Amino acids 161 4.248 Total nucleic acids 163 4.249 Lipids 163 4.250 Volatiles 163 4.3 Results 166 4.31 Commercial sparkling wines 166 4.32 Model sparkling wine 183 4.4 Discussion 196

5.0 INFLUENCE OF CARBON DIOXIDE PRESSURE ON THE GROWTH AND METABOLISM OF YEAST DURING

FERMENTATION AND AGEING OF SPARKLING WINES 215

5.1 Introduction 215

5.2 Material and Methods 216

5.21 Chemically defined wine (CDW) 216 5.22 Preparation of yeast starter culture for tirage 218

5.23 Analysis of CDW during the secondary fermentation 218

5.3 Results 221 X

5.4 Discussion 235

6.0 PROMOTION OF AUTOLYSIS THROUGH THE INTERACTION OF KILLER AND KILLER-SENSITIVE STRAINS OF 244 6.1 Introduction 244

6.2 Materials and Methods 245 6.21 Yeast cultures 245

6.22 Initial screening for killer and sensitive strains 245

6.23 Confirmation of killer and sensitive characteristics 246

6.24 Differentiation between killer and sensitive

strains in mixed culture 246

6.25 Strain interaction in the presence of killer activity 247 6.26 Strain interaction in the absence of killer activity 248 6.27 Analytical methods 249 6.3 Results 250 6.4 Discussion 261

7.0 CONCLUSIONS 265

8.0 BIBLIOGRAPHY 271 xi

Abstract

Utilising commercial samples and model systems, this thesis reports the microbiological and chemical changes that occurred during the secondary fermentation and ageing of sparkling wines, the effect of carbon dioxide pressure on these changes, and the potential use of killer yeasts to accelerate these changes.

The secondary fermentation was characterised by exponential growth of

Saccharomyces cerevisiae from 1 x 10® viable cells/mL to maximum populations of about 6 x 106 cells/mL over 8-10 days, a stationary phase of 15-20 days, and a death phase of 65 - 80 days after which no viable cells were detected. The main chemical changes during the secondary fermentation were : utilisation of sugars (24 g/L), amino acids (4 - 8.7 mg/L) and malic acid (0.2 - 2.5 g/L); production of ethanol (1.5% w/v), CO2 (540 kPa), glycerol (0.2 - 0.3 g/L), major wine volatiles (35 mg/L), proteins (2 - 3 mg/L) and release of nucleic acids (10 mg/L) and neutral lipids (60 pg/L). The main chemical changes during ageing (greater than 120 days) were : increase in concentration of amino acids (4.7 - 7.1 mg/L), proteins (1.5 - 3.5 mg/L), and nucleic acids (5 mg/L), and breakdown of neutral lipids (32 pg/L) and iso-amyl acetate (1.5 mg/L). The concentration of organic acids did not change during ageing.

Fermentations were conducted in chemically defined wines to give final CO2 pressures of 0 to 650 kPa in the bottle. Increasing pressure caused increased rates of consumption, ethanol production and yeast mortality following the fermentation. Other effects included depressed production of glycerol, 2-phenylethanol, reduced RNA content of yeast, and xii

decreased uptake of Group B amino acids (particularly valine, leucine, and iso-leucine).

Fermentations conducted with a combination of killer and killer-sensitive strains of S. cerevisiae gave death of the sensitive strain and greater releases of protein compared with non-mixed fermentations. The data suggested potential benefits of using such interactions to accelerate autolysis in sparkling wine processes.

Methods for the measurement of protein, lipids and nucleic acids in wines, and differentiation of killer and killer-sensitive strains of S. cerevisiae in mixed culture were also evaluated. 1

1.0 Introduction

Sparkling wine production is a relatively simple process characterised by the secondary fermentation of a base wine in a closed system whether it be a bottle or tank. This secondary fermentation encourages the absorption of carbon dioxide into the wine that is perceived as bubbles when the pressure is released - hence the term 'sparkling'.

Sparkling wine originated in the region of France and its conception is romantically attributed to the monk, Dorn Perignon, who was the cellarer of a Benedictine Abbey in Hautvillers from 1668-1715. The for producing sparkling wine persists until the present and is termed 'methode champenoise'. It consists of the secondary fermentation of a base wine in bottles followed by a lengthy storage time which may vary from 6 months in Australia to 2-4 years or longer in France for premium quality product (Amerine and Joslyn, 1970). Today, the secondary fermentation is performed by strains of Saccharomyces cerevisiae which are able to tolerate the relatively hostile conditions of this environment of high A/ov^ temperature, ethanol concentration,Jow pH and the presence of SO2 which is added to the base wine following the primary fermentation. The subsequent ageing period on lees is considered necessary for improvements in quality which has been attributed, in part, to yeast autolysis. This process is mediated by various endogenous hydrolytic enzymes which are activated following death of the yeast cell (Arnold, 1981b). Improvements in wine flavour, bouquet and sparkling character have been attributed to the products of autolysis (Kelly- Treadwell, 1988; Charpentier and Feuillat, 1993).

Despite the controlled conditions of sparkling wine production, there are fundamental aspects of the secondary fermentation and ageing period which 2 require further study. Basic to the process are the kinetics of yeast growth, survival, death and autolysis, the biochemical events of which will impact upon the chemical composition of the wine, and time course of which will influence the economics of the process. While there is little doubt about the growth of yeasts during the secondary fermentation, precise information about the kinetics of this growth and subsequent survival and death has not been published. According to Arnold (1981b), the autolytic reactions in yeast do not commence until after the cells have died. Since autolysis is considered to be an important event in sparkling wine production, more accurate knowledge about its initiation seems justified.

An important influence on yeast viability is the presence of carbon dioxide which is produced during the secondary fermentation. Carbon dioxide pressures up to 500-600 kPa develop during the secondary fermentation (Markides, 1986). Under these conditions, the concentration of carbon dioxide dissolved in the wine may reach approximately 12 g/L at 15°C at the end of the secondary fermentation (Amerine et aL, 1980). At these concentrations, carbon dioxide impacts upon yeast growth and metabolism. It is already known from fermentation that carbon dioxide influences the production of flavour volatiles such as higher alcohols, fatty acids, and vicinal diketones and the uptake of their precursors such as amino acids

(Slaughter, 1989). It is possible that carbon dioxide will demonstrate a similar spectrum of effects during the secondary fermentation of sparkling wines which could impact upon the chemical composition and flavour of the final product.

As mentioned already, yeast autolysis during the ageing period is believed to improve the quality of the sparkling wine. The process, however, occurs over an extended period of time. This time factor introduces a significant cost to 3 the winemaker which is passed onto the consumer. Research which may lead to accelerated autolysis would provide a considerable economic advantage to the winemaker and consumer. One approach would be to exploit the killing effect of K2 killer strains of Saccharomyces cerevisiae upon sensitive strains of the same species. A possible strategy would be to encourage the growth of the killer strain for the purpose of conducting the secondary fermentation while at the same time ensuring the complete death of the sensitive strain. This would provide a pool of dead yeast cells which may begin autolysis immediately rather than after a lengthy latent period.

Most of the research conducted on yeast autolysis during sparkling wine production has focussed upon the release of nitrogen compounds such as amino acids and proteins because of the relatively high concentration of these constituents in yeast (Charpentier and Feuillat, 1993). Other cellular components, however, are degraded and released during yeast autolysis. These components include yeast lipids and nucleic acids which have not been studied in detail with respect to their behaviour during sparkling wine fermentation and autolysis. Pioneering studies on the changes in lipids and nucleic acids during sparkling wine fermentation and ageing have been reported by Troton et aL (1989a,b) and Leroy Mai- (1990). While preliminary in their concept and contribution, these studies did indicate significant changes in these compounds and the need for further study. Lipids and their breakdown components have an important role in food flavour and mouthfeel (Forss, 1969) and have been reported to influence the flavour of beer (Kirsop,

1977; Masschelein, 1986). Similarly, the autolytic breakdown products of yeast nucleic acids have an important flavour enhancing effect (Peppier, 1982; Masschelein, 1986; Benaiges et aL, 1990). 4

Sparkling wine in itself is an extremely complex chemical matrix. Its chemical composition is determined by the grape constituents, biochemical changes during the primary alcoholic fermentation, further changes during any and, finally, changes that occur during the secondary fermentation and ageing. Such complexity contributes to the difficulty in accurately measuring the changes in various chemical components during ageing. This is reflected in the limited published data on various chemical components of wine such as lipids and nucleic acids and the need to validate their reliability. One avenue that may be pursued to overcome these limitations is to develop a model system to study the specific changes caused by yeast during the secondary fermentation and ageing. This system would reflect the various influences that commercial sparkling wines have on yeast growth, and yet allow the measurement of chemical components that are normally difficult to assay reliably.

The primary objectives of this study are :

(i) to examine the kinetics of yeast growth and death during the secondary fermentation and subsequent on lees;

(ii) to measure changes in the concentrations of sugars, ethanol, glycerol, organic acids, protein, amino acids, nucleic acids, lipids and volatiles that occur during the secondary fermentation and subsequent wine storage;

(iii) to determine the effects of carbon dioxide produced during the secondary fermentation on growth and biochemical activities of the yeast and,

(iv) to investigate the possibility of using strains of killer yeast to accelerate the process of yeast autolysis. 5

As a secondary objective, it became necessary to validate or develop a range of analytical methods for measuring various chemical changes which occur during storage of sparkling wines. To facilitate interpretation of data from these analytical studies, a model wine produced from a fermented chemically defined grape juice was used to supplement the findings observed in commercial sparkling wines. 6

2.0 Literature Review

2.1 Sparkling wine production

Champagne or sparkling wine is a wine which contains a visible excess of carbon dioxide resulting in the characteristic bubbly nature of the product

(Amerine and Joslyn, 1970). The discovery of 'champagnisation1 of wine is attributed to Dom Perignon who was the cellarer in the Benedictine Abbey of

Hautvillers in France from 1668-1715 (Amerine and Joslyn, 1970). He is reputed to be one of the earliest users of Spanish to close wine bottles - an important factor in the formation of a tight seal to ensure carbon dioxide dissolution in the wine. It is suggested that the accidental discovery of sparkling wines occurred when wines were bottled too soon due to a premature winter; residual sugar then refermented in the warmer temperatures of spring to produce the sparkling character. Chappaz (1951) provides a description of the early history and development of sparkling wines in France.

2.11 Classification and global production

The method by which carbon dioxide is placed or generated in the product allows differentiation of the various types of sparkling wine (Amerine et aL, 1980). Classification of the types of sparkling wine based on this premise is given in Table 2.1, where four categories are differentiated.

Sparkling wine of type IV is by far the most dominant production method in the world today (Amerine et aL, 1980). It is produced in many countries with

France, Germany, Russia, Italy and USA being the most significant producers with an output in excess of 1000 million litres (Mann, 1986). In Australia in 7

1990-91, production of sparkling wines was approximately 32.8 million litres representing 11.16% of total wine sales (Jordan, 1994).

Table 2.1 Classification of sparkling wine based upon the source of its carbon dioxide content3.

Type Source of carbon dioxide (CO?) Origin I CO? introduced through carbonation Low cost product

II CO? produced by fermentation of A variety of Alsatian, residual sugar from the primary German, Loire and Italian fermentation wines.

III CO2 produced by a malolactic Vino Verde wines of fermentation Northern Portugal

IV CO2 produced from fermentation of Originally France, but now sugar added after the primary made in all parts of the fermentation world. a Adapted from Amerine et aj. (1980)

Notably, the production of sparkling wine compared with total wine production in Australia is proportionally greater than in any other major wine producing country (Mann, 1986). With the recent popularity of Australian wines around the world, sparkling wine therefore makes a significant contribution to the success of the local wine industry.

2.12 Production of sparkling wines by secondary fermentation

Approximately 90% of the sparkling wine produced in Australia is made using the process of secondary fermentation. The remaining sparkling wines are produced by the process of carbonation and represents the lower priced end of the market (Jordan, 1994). 8

There are two basic procedures for the production of sparkling wines by secondary fermentation - the first is when the fermentation is conducted in the bottle (methode champenoise or transfer systems) and the second is where the fermentation is conducted in tanks (Charmat or continuous systems).

2.121 Sparkling wine fermented in the bottle

The first step in the production of sparkling wines, whether bottle or tank fermented, is preparation of the wine base. A winemaker exercises control over many aspects in the production of the wine base including grape variety, degree of ripening, must handling, primary fermentation and base wine storage. These options have been reviewed by Fletcher (1986), Randall

(1986), Spagnolli (1989), Rankine (1989) and Jordan (1994) and are summarised in Table 2.2.

Considering the scope for variation depending on the desired style of the final product, Amerine et aj. (1980) gave a general description of the ideal wine base : it should have total acidity of at least 0.7 g/100 mL, pH of 3.3 or less, volatile acidity of less than 0.04 g/100 mL, content of 10.0-11.5%, low contents of aldehyde and SO2, and have a 'fresh and impeccably clean flavour'. In reality, it is very difficult to produce a single wine with all the preferred characteristics, so base wines are usually made from a blend of wines to obtain the desired flavour and aroma (Amerine et aL, 1980; Jordan, 1994). 9

Table 2.2 Important stages involved in the production of a wine base for conversion to sparkling wine of mid-to-premium quality3.

Production option Alternatives Grape variety and ripeness , or Meunier (or their various combinations)

Region Climatic and soil considerations

Harvesting Hand or machine (phenolic characters)

Must handling Whole bunch or crush, drain and press (oxidative, phenolic characters)

Skin contact Free run or with limited skin contact

Juice settling Cold settling or treatment

Juice colour Colour removal by adsorption onto carbon

Primary fermentation Yeast strain, temperature, clarification and addition of SO2

Malolactic fermentation Do not induce or induce natural or inoculated fermentation

Base wine storage Stainless steel, oak or glass.

3 Adapted from Randall (1986)

Once a satisfactory blend of wines is achieved, the wine base is prepared for the secondary fermentation by addition of sugar as sucrose or invert sugar.

The amount of sugar added depends on the amount of residual sugar in the wine after the primary fermentation and the final pressure desired in the bottle. Approximately 4 g/L of sugar is required to produce 1 atmosphere of

CO2 pressure at 10°C, so concentrations of up to 24 g/L may be added to achieve 6 atmospheres (about 600 kPa) of headspace pressure (Markides, 10 and can contain 1.0-1.5% of citric acid to assist the inversion of the sucrose to glucose and fructose (Amerine et aL, 1980).

Other nutrients added to support the growth of the yeast include a nitrogen source and (Vine, 1981). Nitrogen is usually added as diammonium (DAP) to a concentration of 50-200 mg/L (Rankine, 1989) to avoid problems such as stuck fermentations and H2S production which can be associated with a limiting nitrogen supply. Vitamins, which play an important role in biosynthetic pathways (Suomalainen and Oura, 1971), are usually added as a complex mixture and commercial preparations such as 'cerevit'

(Lallemond) are available for this purpose.

A suitable strain of the yeast Saccharomyces cerevisiae is then inoculated

(levurage) into the base wine at levels of 2-3% (Amerine and Joslyn, 1970) to achieve an initial viable population of 1-3 x 10^ cfu/mL. The yeast may be added as a rehydrated dry yeast culture or propagated over several days to acclimatise it to the conditions of the wine base (Markides, 1986). It is common for flocculating agents such as isinglass (5-60 mg/L) or bentonite (20-150 mg/L) to be added to encourage clumping of yeast cells and facilitate their removal at a later stage (Rankine, 1989).

Following addition of the yeast, the wine base is stirred to obtain an even distribution of the starter culture before addition to the bottles. Filling into bottles was traditionally performed manually but is now most often an automated process (Farkas, 1988). The bottles are specially made to withstand the high pressures of CO2 evolved during the secondary fermentation and should not contain bubbles or scratches as these may become weak points giving rise to breakages and loss of product (Farkas,

1988). After filling, the bottle is closed by crown caps or champagne corks. If 11 corked, an clamp (agraffe) is added to ensure the cork stays in place (Amerine et aL, 1980). Bottles are then stored horizontally for the duration of the secondary fermentation.

Fermentation is usually conducted at temperatures of 12°-15°C in France and 15°C in Australia (Rankine, 1989) and has been reported to last as long as 6 months (Amerine and Joslyn, 1970), although many factors such as temperature, yeast culture and wine base composition will affect the length of the fermentation. These factors and their effect on the fermentation will be discussed in a later section. Following secondary fermentation, the bottles are stored on lees for an extended period of time - 6 to 18 months or for 2-4 years in France for premium quality sparkling wine (Amerine and Joslyn, 1970).

At the completion of ageing on lees, the bottles are then prepared for removal of the yeast. In the 'methode champenoise' process, the bottles are shaken and restacked with the neck downwards to facilitate settling of the yeast sediment toward the neck of the bottle. The bottles are then periodically twisted to loosen the yeast from the walls of the bottle, and replaced neck downwards. With time, the bottles are repositioned at a more acute angle until the yeast forms a plug in the neck of the bottle. This process is termed 'riddling' or 'remuage' (Amerine et aL, 1980) and was traditionally performed manually. More recently, mechanically operated containers (such as the

'gyro-palette') holding multiple cases (50-400 dozen bottles) of sparkling wine perform this procedure automatically (Jordan, 1994).

The yeast is removed from the bottle neck by a process termed disgorgement. The yeast plug is first frozen by dipping the neck of the bottle into a chloride solution at -24°C and then the cork or crown cap 12 removed, allowing the internal pressure to force the yeast plug out. The loss of wine volume by this process is replaced by addition of the dosage containing base wine and sugar. The amount of sugar added depends on the style of sparkling wine desired. 'Sec' or dry sparkling wine, for example, will be sugared to a level of 20-40 g/L (Morlaes, 1985). The dosage frequently contains other added substances such as SO2 and citric and ascorbic acids, the amounts of which vary according to the (Rankine, 1989).

Disgorgement and dosage in the modern winery are now automated processes. Following these operations, the bottles are corked, an agraffe applied and are washed and labelled for sale.

A modification of the methode champenoise procedure (termed transversage) is to transfer the bottle contents to a pressurised tank following disgorgement for addition of the dosage and to perform post-tirage blending (Jordan, 1994).

In sparkling wine prepared by the transfer method, the processes of riddling and disgorgement are replaced by a filtration system which is maintained under positive CO2 pressure (Amerine and Joslyn, 1970; Amerine et a[., 1980). Following storage on lees, the bottle contents are chilled and emptied into a pressurised tank; the sparkling wine is then drawn off, filtered and returned to clean washed bottles. The dosage is added to the wine in the pressurised tank before it is returned to the clean bottles. After refilling, the bottles are processed as in the methode champenoise system.

2.122 Sparkling wine produced in tanks

In tank (or Charmat) fermented sparkling wines, the fermentation is carried out in a pressurised tank to which the wine base, nutrients and yeast are added. Following fermentation, the wine is chilled and clarified by centrifugation before addition to a second tank containing the dosage. The 13 wine is then filtered to remove the yeast and bottled for sale. Carbon dioxide in solution is maintained by performing all manipulations under CO2 pressure

(Rankine, 1989).

Tank fermented sparkling wines offer several economic advantages over bottle-fermented sparkling wines. These advantages include decreased labour and capital costs and increased control over the fermentation (Amerine et aL, 1980). For example, clarification and uniform addition of the dosage is simplified by use of large tanks and developing pressure may be easily controlled through use of pressure safety valves.

However, tank fermented sparkling wines are generally considered to be of inferior quality when compared with sparkling wines made by the methode champenoise. It has been suggested that this is due to the lower concentration of yeast autolytic products in tank fermented sparkling wines

(Amerine and Joslyn, 1970; Farkas, 1988). The large volumes of wine involved in the fermentation (30-50 hi) encourage thick layers of yeast to form at the bottom of the tank. To avoid formation of H2S through yeast decomposition, the yeast are removed from the product at the end of the secondary fermentation, thereby preventing any ageing on lees (Amerine et aL, 1980).

Tank fermented sparkling wines have now been adapted to a continuous process (Farkas, 1988; Kunkee and Bisson, 1993). Base wine is kept in a

reservoir which is supplied to the fermentation vessel at a steady rate. After fermentation, the product is placed in a sedimentation tank and the yeast

(and other precipitates such as tartrates) allowed to sediment out. The clarified sparkling wine is then directed to a reception vessel prior to bottling. 14

A summary of the procedures used for the production of sparkling wine in the industry is given in Figure 2.1 (Jordan, 1994). u_ 0 irment in bottle Ferment in pressure tank Carbonate and bottle O M T3 T3 To >k CO C CO 0 3 CD CO 3_ —

CD _0 .E .E CO _0 b V. o o 3 CO cd o 3 0 CO CO o o 3 0 3 o CO CD CD O 0 "O X) ■a -o 0 CO C 0 c a. 0 CO o o CO 0 CO 0 o 0 o 0 0 k Q. 0 c O 0 CD CO 0 — ' O To 0 O 0 0 0 o 0 c 0 Q. 3 T "D 0 0 0 0 CD 0 0 C 0 C 3 3 3 i 0 CD .E c O O 0 o 0 3 0 CD 3 ■a c 0 0 o o 0 Q. 0 0 3 J 5 0 ithode Transversage Transfer Charmat (bulk) Carbonai £ o 15 il 16

2.13 Novel methods used in the production of sparkling wine

Sparkling wine made by the traditional 'methode champenoise1 procedure is an expensive process that incurs significant costs associated with various labour- intensive steps, and requirements for a large amount of storage space for ageing the product. Not surprisingly, manufacturers have investigated several alternative technologies designed to decrease production costs involved with the traditional method. In the main, these options have dealt with reducing costs associated with the riddling process and shortening the ageing period required for encouraging yeast autolysis.

2.131 Methods for reducing costs associated with riddling

Several technologies have been developed for the reduction of riddling costs, including encapsulation of the yeasts in alginate beads (Bidan et aL, 1978; Fumi et aL, 1987; Fumi et aL, 1988; Divies, 1989; Pisanelli et aL, 1989; Zamorani et aj, 1989; Godia et aL, 1991), containment of the yeast in a membrane-bound plug attached to the bottle cap (Lemonnier and Duteurtre,

1989; Jallerat, 1990), and selection of highly flocculant yeast strains with an enhanced ability to settle out of the wine following the secondary fermentation

(Markides, 1986; Thorton and Bunker, 1989).

In the first procedure, the yeast inoculum is embedded in beads of calcium alginate before addition to the bottle. In theory, the yeast is expected to conduct the secondary fermentation as normal and (following a suitable ageing

period) the bottle is riddled by the simple process of inverting the bottle so that the beads form a plug in the neck. In practice, however, several technical difficulties have been encountered. These include problems associated with achieving equal distribution of beads during bottling, release of yeast cells from the alginate beads into the wine, and impaired release of autolytic components 17

during ageing (Kunkee and Bisson, 1993). It would appear, therefore, that the process requires further development before becoming commercially viable.

Yeast cells have also been contained in a membrane-bound plug termed the 'Millispark' (produced by Waters-Millipore) as reported by Lemonnier and Duteurtre (1989). The plug is designed to allow transfer of nutrients from the wine to the yeast cells, and the release of fermentation and autolytic by­

products back into the wine. As yet, only initial trials have been conducted

which indicate that there is no difference between this system and the

traditional method; however, long term trials have yet to be completed and reported (Lemonnier and Duteurtre, 1989). However, Divies (1993) notes that the secondary fermentation is retarded in bottles utilising the 'Millispark'. The author attributes this phenomenon to depressed exchange of nutrients due to insufficient membrane surface area and local supersaturation of dissolved

carbon dioxide.

The selection of strong flocculative ability is a genetically modifiable trait that is desirable in sparkling wine yeasts because it can substantially reduce the amount of riddling required prior to disgorging (Thornton, 1986; Thornton, 1989;

Thornton and Bunker, 1989). Flocculative ability has been introduced to winemaking strains of S. cerevisiae using spore-vegetative cell mating with any undesirable oenological properties being eliminated by selective backbreeding (Thornton, 1985).

2.132 Acceleration of autolysis by the addition of yeast autolysate

Charpentier and Feuillat (1986, 1993) reported the possibility of accelerating the autolytic process by addition of yeast autolysate during bottling. The influence of yeast strain and method of preparing yeast autolysate (effect of pH, ethanol, temperature and physiological condition of the yeast) was assessed by 18 the evolution of nitrogen and compounds in the wine, the formation of foam, and sensory analysis. The authors concluded that the addition of yeast autolysates successfully accelerated the process of yeast autolysis, and claimed that wine produced by this method was of superior quality to sparkling wine of the same age produced by the traditional method. Studies are continuing to identify the polysaccharide component(s) responsible for improving the foaming character of the wine.

2.2 Yeast Autolysis

2.21 General description

Yeast autolysis is a term that describes the cellular breakdown of yeast after death and is mediated by the action of endogenous hydrolytic enzymes. These enzymes are normally kept separate from the rest of the cell by compartmentalisation, but this organisation is disrupted as the cell dies. This change results in the enzymes and their substrates coming into contact, and subsequent release of these reactants and their products into the external environment.

The process of yeast autolysis has been the subject of considerable study because of its significance as a source of industrially useful extracts. Such

research has focused on the release and activity of various endogenous

enzymes, the products of autolysis, factors affecting its kinetics, and

biochemical modelling of the process. Early studies on autolysis were reviewed by Joslyn (1955), Joslyn and Vosti (1955) and Farrer (1956). More recent

reviews include those of Hough and Maddox (1970), Arnold (1981b), Babayan and Bezrukov (1985), Halasz and Lasztity (1991) and Charpentier and Feuillat 19

(1993). The review of Charpentier and Feuillat (1993) is focussed on yeast autolysis as it occurs during wine fermentations.

2.22 Basic mechanism

In a summary of existing literature, Charpentier and Feuillat (1993) have

proposed a five step process for the biochemical basis of autolysis. These

steps are :

(i) breakdown of cell membrane systems and internal compartmentalisation allowing the liberation of hydrolytic enzymes;

(ii) inactivation of specific inhibitors of these enzymes by the action of

proteases;

(iii) enzymatic degradation of intracellular macromolecules leading to a build-up of the breakdown products within the cell;

(iv) partial degradation of the cell wall, giving an increase in its porosity which

allows the release of degradation products into the external environment; and

(v) continued degradation of substances in the extracellular environment.

According to early studies by Vosti and Joslyn (1954 a,b) and conclusions of

Arnold (1981b), the environmental conditions that initiate onset of yeast autolysis also cause cell death, but should not be so severe that they inactivate key enzymes involved in the autolytic process. Babayan and Bezrukov (1985)

have reviewed the factors which initiate yeast autolysis and list them under the headings of physical, chemical and biological inducers of autolysis. These factors are summarised in Table 2.3. 20

Table 2.3 Summary of factors reported to induce the onset of yeast autolysis (Babayan and Bezrukov, 1985)

Classification Description Comments Physical Temperature Elevated temperatures (45°-60°C)a Freeze/thaw processing

Osmotic Increased NaCI concentration8 pressure

Irradiation UV or X-raysb

Mechanical Sonication and release of autolytic enzymes8 disruption

Chemical pH Most effective near neutralityb

Ion composition Metal ion cations (Co, Mg, Ca)b

Membranotropic Detergents, organic compounds, proteins8 compounds

Antibiotics Causes imbalance between synthetases and depolymerasesb

Biological Starvation Partial or complete removal of nutrientsb

Enzymes Degradation of cellular membranes8

Aeration Removal or addition of to aerobic or anaerobically cultured cells, respectively13 a Autolytic response is primarily due to direct effects upon internal membrane structures. b Autolysis occurs in response to the inability of the yeast cell to maintain internal membranes as a result of impaired biochemical processing.

The time course of autolysis in yeast cells varies according to the conditions initiating the process. Vosti and Joslyn (1954a) illustrated the effect of temperature and pH upon the autolytic release of nitrogenous compounds from baker's yeast. Autolysis for 24 hours at 45°C resulted in the release of 41% of total nitrogen while at 34°C for the same time period only 3.2% of total nitrogen was released. The optimal pH for autolysis shown by nitrogen release was found to be near neutrality. Similarly, Hough and Maddox (1970) described the 21

extensive autolysis of Saccharomyces carlsbergensis over 14 hours at 45°C and pH 6.5 in distilled water. In sparkling wine, however, autolysis of champagne strains of S. cerevisiae is believed not to begin until the product has been aged on the yeast lees for at least 9-12 months (Feuillat and Charpentier, 1982). Charpentier and Feuillat (1993) specifically note that the low pH of wine (3.0-3.5) and its storage at low temperature (generally less than

15°C) are two important factors which decrease the rate of yeast autolysis in

sparkling wine environments. Yeast autolysis in conditions such as that found

in sparkling wine will be discussed separately in a later section.

2.23 Biochemical changes

The process of autolysis is mediated by endogenous hydrolytic enzymes resulting in the release of intracellular material into the external environment (Arnold, 1981b). The most widely studied cellular components that are degraded and liberated are proteins and protein derivatives. Other components include polysaccharides, lipids (including fatty acids), nucleic acids, esters, alcohols and (Vosti and Joslyn, 1954 a,b; Farrer, 1956; Hough and Maddox, 1970; Thorne, 1966; Arnold, 1981b; Babayan and Bezrukov, 1985;

Charpentier and Feuillat, 1993)

2.231 Proteins and proteolytic activity

Breakdown and release of proteins as a result of proteolytic activity have been

more thoroughly studied than any other aspect of yeast autolysis. This is probably related to the high protein content of yeast (approximately 50% by dry

weight) and the ease with which protein is solubilised during autolysis. The

activity of these proteases results in the release of proteins, peptides and amino acids (Babayan and Bezrukov, 1985). Proteins released into the 22 external medium may be further broken down to peptides and amino acids (Vosti and Joslyn, 1954a).

As indicated already, early studies of protein degradation during yeast autolysis suggested that environmental conditions influence the kinetics of nitrogen release. Vosti and Joslyn (1954a) showed that temperature and pH determined the time course of nitrogen release as well as the composition of the autolysate in S. cerevisiae. Increasing the pH of the buffer from 3.0 to 7.0, for example, increased the nitrogen content of the autolysate from 40% to 65%. The effect of temperature was shown by conducting autolysis at 34°C and 45°C for 24 hours; incubation at 34°C resulted in the release of 3.2% of total cellular nitrogen, while at 45°C this amount increased to 41%. Temperature also influenced the form in which nitrogen was released. When autolysis was conducted at 35°C for 72 hours, about 4% of the nitrogen released was acid- soluble and 2.5% was amino nitrogen. Increasing the temperature to 45°C changed the composition of the released nitrogen to 70% and 25% for acid- soluble and amino nitrogen, respectively. A similar result was obtained by Babayan et aL (1981) who demonstrated that increasing temperature from 40° to 60°C progressively increased the amino nitrogen content of the autolysate.

The release of proteins, peptides and amino acids during autolysis is the result of the activity of proteases and peptidases (Hough and Maddox, 1970; Kelly-

Treadwell, 1988). To date, more than 40 different proteinases have been identified in yeasts (S. cerevisiae) and their properties have been reviewed by Achstetter and Wolf (1985) and Hrisch et a\. (1989). However, only a small proportion of these enzymes (the vacuolar proteinases A, B and C) have been implicated in the process of autolysis (Table 2.4). 23

Table 2.4 Vacuole proteases reported to be involved in the process of yeast autolysis (Achstetter and Wolf, 1985).

Name Class of protease Products of activity pH optima3

Protease A Carboxylic Peptides of molecular 2-3 endoprotease weight 1-10 kDa

Protease B Endopeptidase Peptides of varying size 7

Protease C Exopeptidase Peptides and amino acids 5-6 a As determined by the breakdown of denatured haemoglobin

The activity of these autolytic proteases are controlled by the presence of specific inhibitors (|A, |B ancj |C) This control mechanism has been discussed by Lenney (1975), Saheki and Holzer (1975) and Behalova and Beran (1979) who considered the inter-related activity of the autolytic proteases and their inhibitors. It appears that protein degradation during autolysis is initiated by small amounts of the active form of protease B which hydrolyses the inhibitor of protease A (|A). Protease A in turn activates proteases B and C by hydrolysing their respective inhibitors (lB and |C).

Albara et aL (1970) also showed that protease C is initially synthesized in an inactive pro-enzyme form. Subsequently, this fact was reported for proteases A and B (Mechler et aL, 1982). The role of these pro-enzyme forms is not certain.

It is thought that the precursors function to keep the hydrolytic enzymes inactive during transport to the site of activity or, more simply, act as a recognition sequence for guidance to the appropriate cell compartment (Mechler et aL, 1982).

It is apparent that protein-proteinase interactions in yeast during autolysis is very complex. Many enzymes, activators and inhibitors are involved, and 24 precisely how they operate under various conditions during autolysis remains unclear.

2.232 Polysaccharides and B-glucanase activity

The cell walls of S. cerevisiae consist primarily of glucans (30-60%) and mannoproteins (25-50%) (Fleet, 1991). Glucans, consisting of glucose residues connected by (1->3)-3-linkages are mainly responsible for the rigid structure of the wall. Enzymes of the (1->3)-0-glucanase type are involved in the process of wall turnover during the normal consequence of yeast growth and it has been suggested that they play a role in yeast autolysis (Fleet and Phaff, 1974; Hien and Fleet, 1983; Fleet, 1991). Isolated cell walls of yeast exhibit autolytic degradation through the action of endogenous (1->3)-0- glucanases. Although walls are not completely broken down during this form of autolysis, some of their (1-^3)-f3-glucans are degraded because (1 —>3)-3- oligosaccharides are major products of the reaction (Hien and Fleet, 1983).

Although (1-»3)-3-glucanases hydrolyse (1—>3)-3-glucans within the wall to generate oligosaccharide products, they also cause the release of peptido- polysaccharide compounds such as mannoproteins (Sanz et aL, 1985;

Charpentier et aL, 1986; Fleet, 1991). Charpentier and Feuillat (1993) claim that the majority of this material originates from the periplasmic zone and is covalently linked to glucans. It is thought that these compounds are released when the glucan material to which they are attached is hydrolysed by autolytic (1->3)-6-glucanase activity.

Yeast autolysis does not result in complete cell wall dissolution (Vosti and

Joslyn, 1954a; Babayan et al., 1981) although the cell does shrink in size and appears wrinkled due to the loss of intracellular material (Takeo et aL, 1989). Charpentier et aj. (1986) demonstrated that the yeast cell wall does undergo 25 partial during autolysis which has the effect of increasing cell wall porosity. This is believed to facilitate the diffusion of autolytic products to the external environment. These authors showed that the cell wall decreased in thickness in strains of S. cerevisiae and S. bayanus by 48% and 63%, respectively, during autolysis in buffer at pH 3.0 and 10% ethanol (%v/v). The decrease in wall thickness was matched by a decrease in polysaccharide content by 18% and 41% for S. cerevisiae and S. bayanus, respectively.

2.233 Other wall degrading enzymes

As noted already, the cell wall contains significant amounts of mannan polysaccharide. However, the literature is inconclusive about the existence of mannanase activity in cell walls. Maddox and Hough (1971) reported the action of mannanases in the autolysis of walls of brewers yeast and determined that its activity increased with the disappearance of mannan in cells stored at 5°C over 10 days. In contrast, other authors have failed to detect mannanase activity in cell walls (Fleet, 1991). It has been suggested that misleading results may have been obtained due to problems associated with the mannanase assay (Fleet, 1991).

Other enzymes thought to be involved in cell wall degradation include lecithinase C (Arnold, 1981b) and periplasmic chitinase (Elango et a}., 1982). The latter enzyme is associated with the budding process because of its peak activity during exponential growth (Fleet, 1991). However, the role of these enzymes during autolysis is not clear. 26

2.234 Nucleic acids and the activity of nucleases

Nuclease activity has been demonstrated during autolysis of S. cerevisiae and is responsible for the breakdown of nucleic acids (Vosti and Joslyn, 1954a; Hough and Maddox, 1970; Trevelyan, 1977, 1978a).

The concentration of DNA in S. cerevisiae varies from values reported as low as 0.07% (Polakis and Bartley, 1966) to 0.5% (Eddy, 1958) on a dry weight basis. The fate of DNA during autolysis is not clear as conflicting reports have appeared in the literature. Initial reports suggested that DNA is extensively degraded during autolysis. Hough and Maddox (1970) induced autolysis of S. carlsbergensis at pH 6.5 and 45°C over 12 hours and determined that approximately 80% of the DNA was excreted from the cell. Similarly, Suomalainen (1975) found that DNA was gradually lost from baker’s yeast during storage from 7 days and continued up to 20 days until almost all of the cellular DNA was lost from the cell. In contrast, Trevelyan (1978b) reported that DNA was not lost from S. cerevisae during heat-induced autolysis, even when 42% of dry matter and 50% of protein was solubilised from the cells. In this study, however, autolysis was conducted over three hours only which may have been too short to observe any loss of DNA at later stages of autolysis.

Alternatively, the author acknowledged that the heat-treatment used to induce autolysis may have inactivated any DNA'ase enzymes or other autolytic enzymes modifying the porosity of the cell wall.

The RNA content of yeast cells is generally 20-50 times greater than DNA and may range from 3.60-11.20% by dry weight basis (Mounolou, 1971). In contrast to DNA, RNA is very susceptible to RNA'ase hydrolysis during autolysis. The rapid loss of RNA from S. cerevisiae during autolysis has been noted by many authors (Hough and Maddox, 1970; Trevelyan, 1976, 1977, 1978a; Babayan et aL, 1981). Hough and Maddox (1970) showed that 27

autolysis of brewer's yeast at 45°C in distilled water solubilised 70% of yeast RNA within 12 hours. Accelerated loss of RNA from S. cerevisiae has been induced by first disrupting internal membrane structures through the use of organic solvents (such as ethanol) and dehydration/rehydration of the cells (Trevelyan, 1977) or through the use of heat shock (Trevelyan, 1978 a,b).

Typically, reductions of RNA from the yeast cell were observed to be greater

than 90%.

Babayan et aL (1981) also studied the effects of temperature (40°-75°C) and membranotropic chemicals such as ethanol and (hydrophilic additives) and lecithin and lauric acid (hydrophobic additives) upon nuclease activity during the autolysis of baker's yeast. Optimal temperature for nuclease activity was found to be 70°C, although addition of the membranotropic agents tended to lower this temperature. Significantly, ethanol (3%) was found to be the most effective agent for nuclease activation at 60°C.

Ohta et aL (1971) characterised the changes in RNA composition during heat- induced autolysis of utilus. Initially, 90% of the nucleic acid material

within the cell was native, polymeric RNA. Within 30 minutes of application of the heat-shock, 40% of the polymeric RNA was degraded to 3'-mononucleotides, but remained within the cell. After 2 hours, only 12% of the RNA remained in polymeric form with the remaining RNA being degraded to 3'-mononucleotides and excreted outside the cell.

2.235 Lipids and lipolytic activity

The composition of S. cerevisiae varies between 3.5-20% depending upon the conditions of growth of the organism (Ratledge and Evans, 1989). Yeast

lipids may be considered to belong to two major groups within the cell - neutral 28 lipids forming the bulk of the energy storage capacity of the cell and , which form an integral part of cell membranes.

Early researchers suggested that degradation of phospholipids in cell membranes are reactions which are likely to occur during autolysis because this would increase the permeability of the cell and therefore loss of intracellular components (Joslyn, 1955; Farrer, 1956). Nurminen and Suomalainen (1970) demonstrated the presence of lipase and phospholipase activity in isolated cell envelope fractions of baker's yeast. The majority of the lipases and phospholipases were situated in the plasma membrane and it was proposed that these enzymes were responsible for altering the structure of the plasma membrane for transport purposes. This phenomenon has been recorded by many authors, particularly when autolysis is initiated by organic solvents such as toluene and acetone (Letters, 1968), ethyl acetate (Ishida-lchimasa, 1978;

Arnold, 1981a) and (Watanabe et aL, 1983). Autolytic degradation of phospholipids during the storage of compressed baker's yeast has also been reported (Takakuwa and Watanabe, 1981).

Phospholipase activity in yeast has been attributed to phospholipase A (van den Bosch et aL, 1967), phospholipase B (Watanabe et aL, 1983) and phospholipase C (Harris and Trevelyan, 1963). Major products of degradation are free fatty acids via the action of phospholipases A1 and A2

(Letters, 1968; Ishida-lchimasa, 1978; Takakuwa and Watanabe, 1981;

Watanabe et aL, 1983). However, the conditions of autolysis influence the products of lipid degradation during autolysis. For example, the autolytic products described above were observed at acidic pH - when autolysis was repeated at pH 8.0, diglycerides and not free fatty acids were the main products which corresponded to the activity of phospholipase C (Watanabe et aL, 1983).

Clearly, the conditions of autolysis determine the activities of the various 29

lipolytic enzyme systems, which in turn will determine the products of the reaction.

The changes occurring in neutral lipid composition during yeast autolysis are not well defined. Piton et aL (1988) studied the changes in polar and neutral lipid classes in yeast cells ageing in sparkling wine over 19 years. Following

the secondary fermentation at 6 weeks, the polar lipids (free fatty acids and

phospholipids) decreased in concentration as did the neutral lipids,

monoglycerides and diglycerides. Triglycerides, however, were observed to

accumulate in vesicles. The authors suggested that phospholipids were being converted to triglycerides with diglycerides as the intermediate. However, more direct studies observing the accumulation of degradation products of neutral lipids in the autolysate are required before conclusions may be reached.

2.236 Cytological changes

Microscopic observations have shown that yeast autolysis does not cause rupturing of the cell wall and that autolysing cells still retain their basic cell shape (Farrer, 1956; Babayan et a]-, 1981). Arnold (1981b) suggests that autolysis results in the formation of pores in the cell wall through which

intracellular material passes.

Babayan et aL (1981) determined that intracellular membranes are degraded during the initial stages of autolysis. Continued autolysis resulted in a loss of turgor and organelle structure and a decrease in cell diameter by 1.5 fold. The cell wall was observed to thicken and remain intact throughout all stages of autolysis. Contrasting results were found by Charpentier et aL (1986) during ultrastructural studies of autolysing cells of S. cerevisiae and S. bayanus previously propagated in wine and a synthetic medium. It was shown that the cell wall thickness of yeasts grown in wine medium did not change during 30

autolysis, although wall structure was observed to slacken. For yeasts grown in synthetic medium, cell wall thickness decreased by about 50% during autolysis. These changes were attributed to a loss of amino acids and glucans as a consequence of autolytic enzyme activity. The results indicated that the method used to propagate the cells influenced the subsequent autolytic events. Enzymes associated with the cell wall such as proteases (Kusunose et aL,

1980) and glucanases (Hien and Fleet, 1983) were proposed as being

responsible for the observed changes to the cell wall. The structural slackening

in cell walls of yeast grown in wine media was related to a lower content of

mannoproteins which are considered to protect the glucans of the cell wall from f3-glucanase activity (Zlotnik et aL, 1984).

Changes to the yeast plasma membrane during autolysis are not well defined. It is generally assumed that, upon cell death, the plasma membrane undergoes structural modifications which allow the passage of intracellular material (Arnold, 1981b). The most significant structural change observed with electron microscopy is that the plasma membrane becomes detached from the cell wall during autolysis (Avakyants, 1982; Piton et. aL, 1988).

Nurminen and Suomalainen (1970) demonstrated the presence of lipolytic and

phospholipolytic activity in yeast plasma membranes. These enzymes were thought to cause changes to the plasma membrane structure for the purpose of

solute transportation. These enzymes can be activated by compounds such as ethanol (Letters, 1968), so they may be triggered during autolysis in foods where ethanol is present (as in wine). It is conceivable, therefore, that the

plasma membrane does participate in autolysis by allowing the passage of materials as suggested by Arnold (1981b). 31

2.237 Industrial significance of yeast autolysis

Yeast autolysates are noted for their strong desirable flavour and content of nutrients such as proteins, amino acids and vitamins (Lyall, 1962; Peppier, 1979; Dziezak, 1987a; Reed and Nagodawithana, 1991). They are widely used in the food industry as flavouring agents and for nutritional fortification (Reed and Peppier, 1973; Peppier, 1979; Reed, 1982; Dziezak, 1987b; Reed and

Nagodawithana, 1991; Schoenberg, 1993; Nagodawithana, 1993). Examples

of food products where yeast autolysates or extracts are used for these

purposes include various meat products (sausages, pie fillings, meat pastes,

seasonings, fish cakes), in meat curing brines, soups (canned, dried and stock cubes), canned vegetables (peas), sauces and savoury spreads.

Further processing of yeast autolysates can yield specific protein or polysaccharide fractions (derived from the cell wall) which may be used as ingredients in foods to modify texture and viscosity (Arnold, 1981b; Kinsella, 1986).

Schmidt (1987), Reed and Nagodawithana (1991) and Longo et a]. (1992) describe some novel uses of yeast autolysates. One product is a bioemulsifier called 'Liposan' which is an extract from Candida lipolytica claimed to have

useful emulsification and stabilisation properties. Another potential use for

yeast extracts is as an antioxidant due to their content of sulphydryl amino

acids and proteins. Suggested applications were for prevention of rancidity in processed animal fats and for the protection of natural colourings such as

0-carotene. Certain yeasts such as species of Rhodotorula, Cryptococcus,

Sporobolomyces and Phaffia may be used as a source of pigments. Extracts of the pink yeast, Rhodotorula rubra, included in the diet of farmed salmon gave the flesh the pink colour found naturally in wild salmon (Reed and Nagodawithana, 1991). 32

Autolysis of yeasts is used to produce commercially important enzymes such as lactase (B-galactosidase), invertase (Peppier, 1982) and melibiase (Reed and Nagodawithana, 1991) for breakdown of sugars. Reed and Nagodawithana (1991) also describe the use of highly purified yeast alcohol dehydrogenase in biochemical studies as a biocatalyst.

The significance of yeast autolysis during the production of fermented

beverages has been appreciated for some time. In the production of beer,

autolysis of yeast cells during the lagering stage is considered to be an

important part of flavour maturation (Masschelein and Van de Meerssche, 1976; Masschelein, 1986). Autolytic products of yeasts believed to contribute to the development of beer flavour during the maturation stage include amino acids, peptides (Masschelein, 1986), free fatty acids (Van de Meerssche et aL, 1979, Chen et a!., 1980) and nucleotides (Qureshi et aL, 1979; Masschelein, 1986), although it has been suggested that the contribution of the flavour nucleotides 5'-guanylic acid (5'-GMP) and 5'-inosinic acid (5'-IMP) are of secondary importance (Masschelein et aL, 1973).

In wine fermentation, yeast autolysis may have several effects. Although noted for its subtle effect on flavour and aroma development (Fleet and Heard, 1993), the autolysate may act as nutrients and encourage the growth of spoilage or

malolactic bacteria (Wibowo et aL, 1985). In 'methode champenoise1 style

sparkling wine production, ageing on yeast lees for at least 6-12 months is practiced so that the sparkling wine is enriched with the autolytic components of yeast. Improvements in wine flavour, aroma, mouthfeel and foam stability have been attributed to autolysis of the champagne yeasts. More detailed aspects of yeast autolysis and its effect on sparkling wine production are described in a later section. 33

2.3 Growth and survival of yeast during sparkling wine fermentation by 'methode champenoise'

The main yeasts used for the secondary fermentation are strains of Saccharomyces cerevisiae. Unlike the primary fermentation of grape juice, where many naturally occurring Saccharomyces and non-Saccharomyces yeasts can take part (Heard and Fleet, 1993), the secondary fermentation of

sparkling wine is mostly conducted by a single inoculated strain of

S. cerevisiae. This occurs because many filter-sterilise the wine base

before tirage to prevent undesirable microbial growth (such as malolactic bacteria) which may complicate subsequent riddling operations (Markides, 1986). The wine base at the end of primary fermentation is also an extremely hostile environment (high alcohol content, low pH) which will prevent the growth

of microbial contaminants. Furthermore, the wine base is subject to various

procedures which are also hostile to microorganisms and include clarification by centrifugation, cold stabilisation and the addition of sulphur dioxide to a free level of 15-20 mg/L (Rankine, 1989).

Strains of S. cerevisiae for the secondary fermentation are selected on the following criteria (Kunkee and Amerine, 1970; Zambonelli et ah, 1982; Bidan et

aL, 1986; Markides, 1986) :

(i) Ability to ferment to dryness under conditions of low temperature (10°-15°C),

8-12% (v/v) ethanol, low pH (sometimes below 3.0), low nutrient status and

CO2 pressures up to 600 kPa;

(ii) Minimal production of undesirable fermentation by-products such as H2S and volatile acids;

(iii) Tolerance of free SO2 concentrations up to 20 mg/L; 34

(iv) Flocculation and sedimentary properties following the fermentation so as to facilitate the riddling process;

(v) Produce desirable esters and aldehydes during fermentation, and

(vi) Produce a favourable autolytic character during ageing on lees.

Markides (1986) reports that yeasts are usually inoculated to a cell density of

1-3x10^ viable yeast/mL of wine base. The yeast population then increases to

concentrations of 1-3x107 cells/mL, undergoing a maximum of about four

generations.

However, despite the controlled conditions encountered in sparkling wine fermentation, little quantitative information has been published on the kinetics of yeast growth and death and this represents a significant gap in fundamental knowledge. A limited amount of information concerning yeast growth may be gained from the work of Cahill et aL (1980) who examined the effect of yeast adaptation to carbon dioxide pressure on fermentation rates during tirage at

15°C. These authors grew yeast starter cultures in wine base under 0.0, 0.3

and 0.6 atmospheres of CO2 pressure to observe whether increased rates of fermentation could be obtained for use in continuous sparkling wine production. Tirage was conducted in bottles of base wine containing 1.65 % (w/v) of

glucose for the development of 4 atmospheres of pressure at the end of fermentation. Lag phases of 0, 3.5 and 7 days were observed for yeast cultivated under 0.0, 0.3 and 0.6 atmospheres of CO2, respectively. However, yeast cultivated under 0.3 atmospheres of pressure conducted the secondary fermentation at an increased rate, achieving sugar exhaustion in about 18 days compared with 23 and 30 days for yeast cultured under 0.0 and 0.6 atmospheres of pressure, respectively. The maximum rate of carbon dioxide production for the yeast cultivated under 0.3 atmospheres of pressure was 35

0.228 atmospheres/day compared with 0.144 and 0.166 atmospheres/day for yeast cultivated under 0.0 and 0.6 atmospheres CO2 pressure, respectively. The authors concluded that growth of the starter culture in low concentrations of carbon dioxide successfully adapted the yeast to the extreme conditions of the secondary fermentation. However, no data concerning the survival of the yeast following the secondary fermentation were presented.

Monk and Storer (1986) examined the kinetics of yeast growth in tirage in response to different methods of starter culture preparation (aerobic and anaerobic growth in wine media and aerobic growth in grape juice media) and inoculation level. Total and viable cell numbers were determined using fluorescence microscopy after staining with acridine orange. It should be noted, however, that all experiments were conducted in flasks saturated with

CO2 at atmospheric pressure. In contrast, fermentations in 'methode champenoise1 are usually conducted under increasing pressures and concentrations of dissolved carbon dioxide (Rankine, 1989). As explained elsewhere (Section 2.5), this increasing pressure and carbon dioxide concentration will influence both yeast growth rate (Kunkee and Ough, 1966; Cahill et af., 1980) and metabolism (Dixon and Kell, 1989). Monk and Storer

(1986) found that the specific growth rate of yeast prepared in grape juice medium (aerobically) was unaffected by inoculum size, whereas increases in specific growth rate with larger inocula were observed for yeast propagated in wine media both aerobically and anaerobically. During linear growth, only yeast grown anaerobically in wine media showed increased sugar catabolism and linear growth with increasing inoculum levels. Yeast mortality was only discussed during the linear growth phase and was not investigated following the secondary fermentation. 36

Bidan et aj. (1986) described the general trends of yeast growth and death during sparkling wine fermentation. The viable population of the yeast increased during the first 30 days of fermentation and then appeared to decline to undetectable levels by 80 days. However, the data were presented graphically without giving details about the conditions of fermentation and other important parameters such as storage temperature and yeast strain. Additionally, the kinetics of yeast death were not rigorously examined with data provided for only 50 days and 80 days of fermentation. At 80 days, the yeast population was merely indicated as being less than 10^ cells/mL.

In summary, therefore, the question of yeast growth during secondary fermentation appears not to be studied in detail, or has been studied under conditions different to those encountered in commercial situations. Additionally, there appears to be no information on the kinetics of yeast viability or death following the end of secondary fermentation. This gap in knowledge leaves unanswered questions about links between death of the yeast cells and subsequent autolytic events. Feuillat and Charpentier (1982) indicated that the initiation or onset of autolysis (indicated by release of amino acids) was a delayed process occurring after 6-12 months ageing on yeast lees. It is likely that this delay is linked to the survival kinetics of the yeast following secondary fermentation and, therefore, is an area which requires investigation.

2.31 Factors affecting growth of yeast during secondary fermentation

Table 2.5 gives some variables which may affect the growth of yeasts during sparkling wine fermentation. 37

Table 2.5 Factors affecting yeast growth during the secondary fermentation of sparkling wines.

Type of factor Description Intrinsic pH Ethanol concentration Development of CO2 pressure Metabolic products of microorganisms from the primary fermentation Chemical composition and nutrient status of wine baseP inc’lodincj c»ojgeT\a4'ion

Extrinsic Temperature of fermentation and storage

Processing Propagation conditions of yeast Chemical additions (SO2, DAP, vitamins, bentonite)

Wine pH (2.9-3.3) plays an important role in the survival of S. cerevisiae in sparkling wine production by increasing the toxic effects of SO2 (Markides,

1986). The effect of SO2 on wine microorganisms has been reviewed by

Romano and Suzzi (1993). Although the majority of SO2 in wines exists in 'bound' form through combination with various wine components such as sugars (Somers and Westcombe, 1987) and carbonyl compounds (Burroughs,

1975), a small amount exists in the antimicrobial form of molecular SO2 (Ingram, 1948). Sulphur dioxide in this conformation passes into the cell by simple diffusion (Stratford and , 1989) and disassociates to form (in the main) the HSO3' 'on which exerts a multitude of antimicrobial effects as summarised by Romano and Suzzi (1993). Wine pH plays a role in the efficacy of SO2 by influencing the formation of its dissociated forms in solution - decreasing wine pH increases the amount of molecular SO2 and therefore the toxicity of added sulphur dioxide. Bidan et aL (1986) demonstrated the effect of

SO2 on yeast viability during tirage; increasing the free SO2 content from 11-31 38 mg/L gave a tenfold increase in the lag phase of the secondary fermentation and free SO2 levels above 18 mg/L resulted in stuck fermentations.

Ethanol produced during primary fermentation is present in the wine base at concentrations of 9-11% (v/v) at tirage (Rankine, 1989). Ethanol is an antagonistic substance, for which the effects on yeast have been reviewed by van Uden (1989) and Jones (1989). A summary of these effects is given in

Table 2.6.

It is generally considered that a major site of activity for the inhibitory effects of ethanol is the cell membrane (Jones, 1989; van Uden, 1989). It is, therefore, not suprising that ethanol enhances the sensitivity of yeast to sulphur dioxide (Amerine et aL, 1980), the efficacy of which is dependant upon passage from the extracellular environment to inside the cell.

The cell membrane is also the major site of activity for the inhibitory influence of carbon dioxide upon yeasts. During the secondary fermentation, CO2 pressures may reach 5 to 6 atmospheres or 500 to 600 kPa (Markides, 1986) which results in about 15 g/L of dissolved CO2 in the wine when stored at 10°C

(Amerine et aL, 1980). The effects of CO2 on yeast metabolism are reviewed in a later section (Section 2.5). The inhibitory effects of carbon dioxide are aggravated by the presence of ethanol, although ethanol tends to decrease the solubility of CO2 in aqueous systems (Jones and Greenfield, 1982).

In addition to the effects of ethanol, other metabolic products from the primary fermentation may also affect yeast metabolism. Larue and Lafon-Lafourcade

(1989) reviewed this concept and reported that medium chain fatty acids (octanoic, decanoic and dodecanoic acids) and their ethyl esters may be responsible for some instances of 'stuck' fermentations where a wine 39 fermentation is halted prematurely before exhaustion of sugars. Removal of these compounds by charcoal or yeast ghosts (Lafon-Lafourcade et aL, 1984) was shown to stimulate growth of yeast in a synthetic medium which contained the toxic metabolites at the concentrations encountered in wines.

Table 2.6 Reported effects of ethanol on the growth, survival and metabolic activities of Saccharomyces cerevisiae.

Aspect of Reported effect Reference metabolism Cell yield Decreased by 50% and 94% when exposed Jones and Greenfield to 70 and 150 g/L ethanol, respectively (1982)

Cell viability Death of 50% of population at Chohji et aL (1981) concentrations of 90-100 g/L. Lethal effects increase with increasing temperature and in actively growing cultures.

Cell growth Incubation with 100 g/L ethanol at 25°C Herman et aL (1980); inhibits replication. Older cells more Sawada and Chohji sensitive than smaller daughter cells. (1978)

Sugar Depressed uptake of glucose by inhibition of Thomas and Rose (1979); uptake permease enzyme Leao and van Uden (1985)

Nitrogen Depressed uptake of NH4+ and amino Leao and van Uden uptake acids. More sensitive than sugar transport (1983); systems. This would explain why growth is Leao and van Uden more sensitive than alcoholic fermentation to (1984); the effects of ethanol. van Uden (1989)

Formation Increases the occurrence of respiratory Kojima et aL (1979) of mutants by mutation of mitochondrial DNA respiratory mutants 40

Being itself a product of intensive alcoholic fermentation, the wine base as a substrate for yeast growth is relatively poor. In accordance with this, many winemakers provide nitrogen and supplements to support the secondary fermentation (Markides, 1986). It is usual practise to add 50-200 mg/L of (Rankine, 1989) which will stimulate yeast growth and prevent an incomplete fermentation (Vos et aL, 1978; Monk, 1982). Another important oenological consideration is the repressing effect that ions have on the production of hydrogen sulphide, a major wine

quality defect (Vos and Gray, 1979; Monk, 1982; Henschke and Jiranek, 1993).

Addition of vitamins is also practised because of their importance in many metabolic reactions (Suomalainen and Oura, 1971), although under certain

circumstances vitamin supplements may have adverse effects such as increasing acetic acid production (Eglinton and Henschke, 1993). Monk (1982) found that vitamin supplementation of grape juice had no effect on yeast growth, although it would be anticipated that the vitamin status of wine base is poorer than grape juice.

The temperature of fermentation plays an important role in fermentation by the yeast. In general, an increase in fermentation rate will be observed as temperature increases. However, several other factors must be considered. Firstly, increases in temperature increase the sensitivity of the yeast to ethanol (van Uden, 1989). The other aspect of concern is the effect that temperature

has on autolysis of the yeast. Good quality sparkling wines are obtained by

storage at low temperature (10°-15°C) which is thought to support a slow autolytic reaction and favourable secondary chemical reactions (Kelly- Treadwell, 1988). Accelerated autolysis at higher temperatures may result in modified reactions and yeast-like flavours and aromas (Molnar et al., 1980a). The final point is a practical one in that storage at higher temperatures 41 increases the headspace pressure of CO2 in the bottle and thus increases the risk of breakages.

Preparation of the yeast for inoculation into the wine base is a significant factor in growth of the yeast during the secondary fermentation. The influence of yeast preparation on subsequent growth during the secondary fermentation has been investigated by Valade et aL (1984) and Tzvetanov et aL (1989) and reviewed by Markides (1986) and is summarised in Table 2.7.

Table 2.7 Influence of yeast preparation upon growth of the yeast during the secondary fermentation.

Aspect of culture Description Reference preparation Temperature Progressive adaptation to low Markides (1986); temperature to minimise temperature Juroszek et aL shock and increase ethanol tolerance (1987)

Aeration during Increased content in Thomas et aL growth phospholipid component of cell (1978); membrane to increase ethanol Larue and Lafon- tolerance Lafourcade (1989)

Adaptation to Gradual exposure to wine base for Markides (1986) wine base adaptation to antagonistic components components such as SO? and ethanol.

The aim of the winemaker is to maximise the viability of the starter culture upon addition to the wine base. The impact of starter culture preparation upon the survival of the yeast following the secondary fermentation is an area which has not been studied. This point is of interest because autolysis in sparkling wines is a lengthy process requiring many months of storage on lees and depends upon the decrease in viability of the yeast. Recent work by Mauricio et aL (1991) indicated that aeration of yeast and supplementation with 42

increased the survival time of S. cerevisae in completed wine fermentations. It is possible that such practises by winemakers, in an effort to increase ethanol tolerance, are contributing to the lengthy ageing component currently required in sparkling wine production.

2.4 Chemical changes during sparkling wine production

The chemical changes that occur during sparkling wine production will be considered in two phases :

(i) the changes that occur during the relatively short but distinct phase of secondary fermentation, and

(ii) the subsequent phase of ageing and yeast autolysis.

2.41 Changes during secondary fermentation

The secondary fermentation in the bottle occurs under relatively hostile conditions of high ethanol and sulphur dioxide, low pH and temperature, and increasing concentration of carbon dioxide. However, the effects of these parameters on aspects of yeast metabolism such as secondary by-product formation during fermentation have not been studied in detail. Instead, most

research has been directed towards understanding the chemical changes that occur during the period of ageing and yeast autolysis (Section 2.42). To an extent this is understandable as the secondary fermentation is a relatively short phase where, in comparison with the primary fermentation, only a small concentration of sugars are metabolised. However, considering the marked effect that parameters such as carbon dioxide pressure have on volatile 43 production in the industry (Section 2.5), this absence of knowledge represents a significant gap in wine research.

2.411 Consumption of sugar and production of ethanol and carbon dioxide

The conversion of sugars into carbon dioxide and ethanol represents the major biochemical activity of yeasts during the secondary fermentation. The production of carbon dioxide from sugar follows a first order kinetic reaction (Merzhanian and Kozenko, 1972) with approximately 100 kPa (1 atmosphere) of pressure being produced for every 4 g/L of sugar (monosaccharide) that is fermented

(Bidan et aL, 1986). Approximately 1 g/L of sugar remains as residual sugar at the end of the secondary fermentation (Amerine et aL, 1980). Thus, sparkling wine bases that typically contain between 22 to 24 g/L of glucose will develop about 525 to 575 kPa of headspace pressure by the end of the secondary fermentation. The time required for conversion of sugars to carbon dioxide and ethanol will vary according to factors discussed in the previous section. Bidan et aL (1986), for example, showed that secondary fermentations conducted at 11° ,15° and 20°C took approximately 55, 30 and 15 days to complete, respectively.

Ethanol concentration in these wines increased from 11.3% to 12.8%, representing an increase of 1.5%. A similar result was obtained by Suarez et aL (1979) in a wine base containing equivalent concentrations of sugar, with approximately 1.7% additional ethanol being produced by the end of the secondary fermentation.

2.412 Changes in concentration of amino nitrogen

Amino nitrogen in the form of or amino acids represents the major source of nitrogen consumed by yeast during the secondary fermentation.

Initially, the concentration of amino nitrogen decreases until about 25 days when 44 approximately 17 mg N/L of ammonia nitrogen has been consumed (Bidan et aL, 1986). This is followed by a small, passive efflux of nitrogen between 25-40 days which is considered to be a physiological response to exhaustion of sugars (Charpentier et aL, 1986). Bidan et aL (1986) reported an efflux or release of approximately 9 mg N/L during this phase. Following these stages, the concentration of amino nitrogen does not change significantly until autolysis begins after several months of ageing (Kelly-Treadwell, 1988).

2.413 Evolution of glycerol and organic acids

Changes in concentration of glycerol and organic acids during the secondary fermentation have been studied by Silva et aL (1987) and Postel and Ziegler (1991a). Silva et aL (1987) found that the concentration of glycerol increased from 5.75 g/L to 6.13 g/L, a rise of 0.38 g/L. In contrast, Postel and Ziegler (1991a) did not find any significant increase in glycerol concentration from 6.7 g/L during the secondary fermentation.

Silva et aL (1987) found that the concentrations of tartaric and lactic (L+ and D-) did not vary significantly during secondary fermentation. However, the malic acid concentration was observed to decrease from 0.52 g/L to 0.45 g/L, whereas the concentrations of and a-ketoglutarate increased by 3 mg/L and

8 mg/L, respectively. As with glycerol, Postel and Ziegler (1991a) did not observe any changes in the concentration of organic acids (acetic, pyruvic, malic, tartaric, citric, and lactic acids).

2.414 Production of volatile compounds

Postel and Ziegler (1991b) studied the changes in concentration of and twelve higher alcohols, two , seven carbonyl and nineteen esters during secondary fermentation of sparkling wines prepared in bottles, tanks and 45 following transfer processes. In two separate trials, total volatiles were observed to increase by between 60 to 100 mg/L during the secondary fermentation. The majority of the changes observed was due to increases in ethyl lactate, and to a lesser extent, diethyl succinate, ethyl acetate and . No difference in formation of aroma compounds were observed between the various methods of sparkling wine production.

2.42 Chemical changes during autolysis

Yeast autolysis during sparkling wine storage is a subtle process which occurs over several years (Feuillat and Charpentier, 1982). It is considered essential in the production of a superior quality product and is the reason why sparkling wine is aged on yeast lees for extended periods of time. The process of yeast autolysis during sparkling wine ageing and its importance to product quality have been reviewed by Bidan et a]. (1986), Kelly-Treadwell (1988) and Charpentier and Feuillat (1993).

2.421 Occurrence of autolysis

Yeast autolysis during ageing does not begin until all available nutrients in the sparkling wine have been utilised and the cell dies from starvation. Charpentier et aj. (1986) specifically cite glucose exhaustion for induction of autolytic proteolysis of yeasts grown in wine. However, autolysis in sparkling wine occurs only after lengthy periods of ageing. According to Suarez et aL (1979), autolysis does not begin (as evidenced by amino acid release into the wine) until 12 months of ageing. Similarly, Feuillat and Charpentier (1982) suggested that autolysis of sparkling wine yeasts is not initiated until approximately 6 months after the secondary fermentation. These authors used release of amino acids as a marker of autolysis to study the course of this process over 4 years. After passive excretion of amino acids at the conclusion of the fermentation, the 46 concentration of amino acids remained relatively stable until the onset of autolysis where they increased again. The greatest enrichment occurred between 6-12 months ageing where the amino acid concentration in the wine was 24.5% higher than the base wine. This was correlated with an increase in intracellular protease activity of the yeast deposit and, from these observations, the authors concluded that autolysis was occurring. Leroy et ai- (1990) showed that intracellular protease activity in the yeast deposit following the secondary fermentation actually increased for a period of 6 years. Again, this was correlated with a decrease in intracellular protein content and increase in amino acid and total nitrogen contents of the wine. However, differences in the extent of autolysis were observed between yeast strains and methods of starter culture propagation, suggesting it would be difficult to predict how long autolysis will actually continue during sparkling wine ageing. The factors affecting the occurrence and extent of yeast autolysis in sparkling wine are discussed in the next section.

2.422 Factors affecting autolysis

Since autolysis is an enzyme mediated process, the factors controlling this process will be those that affect autolytic enzyme activity. The most significant factors in sparkling wine production include pH, the presence of ethanol, temperature of storage and the conditions of growth of the yeasts during starter culture preparation (Bidan et ai., 1986; Kelly-Treadwell, 1988).

The effects of wine pH (3.0-3.5) and ethanol concentration (10.0-11.5%) upon major autolytic proteases have been studied by Sugimoto (1974). It was found that at pH 3.0 and at ethanol concentrations greater than 5%, the activity of the major autolytic protease (A) was significantly inhibited. Despite this observation, however, protease A remains the most active autolytic protease at acid pH (Charpentier and Feuillat, 1993). This conclusion is based on the findings of 47

Lurton (1987) and Lurton et aj. (1989) who studied the effect of inhibitors on autolytic protease activity at acid pH. These authors showed that the majority of nitrogen release at pH 3.0 is peptide material of molecular weight 1-10 kDa representing the products of endoprotease activity of protease A. Similarly, when pepstatin was added to inhibit the activity of protease A, almost all autolytic proteolytic activity was observed to cease.

Temperature of storage has a major influence on yeast autolysis in sparkling wines. As most sparkling wines are generally aged at temperatures less than

15.6°C (Amerine et aj, 1980), it is probably this reason more than any other that the onset of autolysis is delayed for several months during ageing (Suarez et aj., 1979; Charpentier and Feuillat, 1993). Molnar et aL (1980b) showed that protease activity in autolysing yeasts is temperature-dependant. These authors used loss of C1^ from labelled cells as a measure of autolysis. It was found that in the range 4-40°C, the rate of autolysis in sparkling wine yeast was linearly related to temperature - a 10°C increase in temperature corresponded to a 6-7% increase in the rate of autolysis with maximum activity occurring at 40°C.

The composition of the wine during growth of the starter culture is another factor affecting the rate of autolysis in sparkling wine (Kelly-Treadwell, 1988). Lenney

(1975) showed that the activity of autolytic proteases A, B and C was low in yeasts grown on media with glucose as the carbon source; when the yeasts were transferred to media with no carbon source, the autolytic protease activity was correspondingly enhanced. A similar observation was made by Charpentier et aj. (1986) who suggested that glucose starvation is a key event in the activation of autolytic proteolysis during ageing of sparkling wine yeast.

Finally, it is interesting to note that carbon dioxide has been implicated in causing a transient inactivation of hydrolases associated with autolysis of fish 48 flesh (Mitsuda et aL, 1980). If this is the case, there is a good possibility that this effect will occur also in sparkling wine production as the secondary fermentation produces 500-600 kPa pressure (at 10°C) which will increase the concentration of CO2 in solution. Further research is required to confirm the influence of CO2 on autolysis in sparkling wines.

2.423 The action of enzymes during yeast autolysis in sparkling wine and the products of their activity

The most significant biochemical changes that occur during autolysis of sparkling wine yeasts is the activation of autolytic enzymes and the products of their activity. Autolysis of yeasts during sparkling wine storage results in the release of nitrogenous compounds, nucleic acids, polysaccharides, lipids and fatty acids, esters and various carbonyl compounds and has been reviewed by

Charpentier and Feuillat (1993).

2.4231 Release of nitrogenous compounds

Nitrogen release during yeast autolysis in sparkling wine has been most studied because of the high protein content of yeast cells. Cologrande and Silva (1981) classified the nitrogen compounds released during sparkling wine ageing: polypeptides and proteins of molecular weight > 5000 (2.55-4.56%); peptides of molecular weight 5000-1000 (15.65-24.94%) and amino acids and oligopeptides of molecular weight < 1000 (72.90-79.99%).

The release of individual amino acids, in particular, has been most extensively studied (Suarez et aL, 1979; Feuillat and Charpentier, 1982; Cologrande et aL, 1984; Silva et aL, 1987). In these reports, amino acid release follows the same general pattern after completion of the secondary fermentation. Initially, there is a passive release of amino acids in response to glucose starvation (Feuillat and 49

Charpentier, 1982; Charpentier et aL, 1986). This is followed by a latent phase of several months before a further stage of amino acid release occurs due to autolysis (Feuillat and Charpentier, 1982). As indicated earlier, the length of time of ageing on lees required before autolytic release of amino acids may vary from 6 months (Feuillat and Charpentier, 1982) to 12 months (Suarez et aL, 1979). Notably, the increase in amino acid concentration is relatively small, being in the range of a few mg/L (Charpentier and Feuillat, 1993). Additionally, there is considerable variation in which amino acids are reported to increase during autolysis (Table 2.8). However, this variation is understandable when the influence of yeast strain is taken into consideration. Tzvetanov and Bambalov

(1994) studied the effect of yeast strain upon changes in amino acid nitrogen content of sparkling red wines during ageing on lees. These authors examined 16 yeast strains over 36 months and found that 5 strains gave an increase in amino acid nitrogen (ranging from 0.9 to 7.2%), 8 strains a decrease in amino acid nitrogen (ranging from -1.4 to -5.4%), and 3 strains had no effect on amino acid nitrogen content in ageing sparkling red wines.

This has prompted some authors to suggest that the contribution of autolysis in the methode champenoise process is of little significance (Usseglio-Tomasset et- aL, 1983; Margheri et aL, 1984; Usseglio-Tomasset, 1985). However, as pointed out by Charpentier and Feuillat (1993), autolysis involves the transfer of other intracellular components such as nucleic acids, polysaccharides, volatiles and lipid material which may also impact upon sparkling wine quality. 50

Table 2.8 Amino acids reported to increase in sparkling wines due to yeast autolysis during ageing on lees.

Reference Amino acid Suarez et a\. Feuillat and Cologrande et Silva et ai. (1979) Charpentier af. (1984) (1987) (1982) Aspartic acid / y Serine / / Threonine / y Glutamic acid / y y Alanine / y Glycine / y y Valine / y y y Methionine y y Tryptophan y Phenylalanine y y Leucine / y y y Iso-leucine y Lysine / y Tyrosine y y Histidine y y /

Changes in the concentration of soluble protein in sparkling wine during ageing have not been extensively studied, probably due to the technical difficulties associated with such measurements in wine (Chapter 3, Section 3.1). The concentration of protein in wines have been reported to range from 1.5 mg/L

(Yokotsuka et aL, 1977) to 840 mg/L (Somers and Ziemelis, 1973). Tzvetanov and Bambalov (1994) utilised sulphate precipitation to measure protein changes during storage of red sparkling wines fermented with sixteen different yeast strains. For five strains, the aged wine contained significantly more protein than the control wine (ranging from 13.8 to 28.6%), while the opposite was true for three strains (ranging from -7.6 to -17.3%). Eight strains gave no significant difference in protein content between the control wine (disgorged 51

after the secondary fermentation and aged for the same period of time) and wine aged on lees. For sparkling wines with reduced protein contents, the authors suggested that autolytic protease A had been released into the wine where it degraded protein that had been solubilised from the yeast cell.

2.4232 Release of nucleic acids

The release of nucleic acids during ageing of sparkling wine on lees has been

studied by Leroy et aL (1990). These authors concluded that the release of

nucleic acids was greatest during the first 5 months of ageing following the

secondary fermentation, although these components continued to be released at slower rates during the next 21 months of the experiment. The total increase

in nucleic acid content of the wine was of the order of 18 to 20%, where the concentration increased from about 410 to 480 mg/L. The concentration of nucleic acids in sparkling wine of different was also found to increase with age (4-20 years) except for a plateau between 6 and 10 years. There are no reports on the specific concentration of RNA and DNA in sparkling wines.

2.4233 Release of polysaccharides

Polysaccharide compounds are released as a normal part of sparkling wine fermentation and consist, in part, of glucan (31%) and mannan (43%) components (Feuillat, 1986). Feuillat et a\. (1988) showed that the concentration of these compounds increased by 300-400% during ageing over eight months following the secondary fermentation. Charpentier et aj. (1986) investigated polysaccharide release due to autolysis after the yeast were propagated in a synthetic or wine medium. Changes to cell wall /glucose ratios of autolysing S. cerevisiae and S. bayanus indicated a loss of glucans as a result of autolytic glucanase activity. A significant decrease lin cell wall amino acids was also noted; after 14 days autolysis, the loss in 52 amino acids from the cell wall was 46% and 72% for cells propagated in the synthetic and wine media, respectively. This was thought to be due to degradation of the protein component of cell wall mannoproteins. The loss of amino acids was seen to indicate the destruction of mannoproteins that may be responsible for protecting cell wall glucans against R-glucanases (Zlotnic et ah, 1984).

Charpentier and Freyssinet (1989) isolated and characterised by gel filtration polysaccharides solubilised from isolated yeast cell walls during autolysis.

During the first 24 hours, the released polysaccharides ranged from 2-400 kDa.

Over the subsequent 14 days, the autolysate was enriched with polysaccharides predominantly 300-400 kDa in size and contained a significant amount of mannose suggesting a mannoprotein origin. Using these results, Feuillat and Charpentier (1993) suggest that cell wall autolysis occurs by the following pathway :

(i) the initial stages in the autolysis of yeast cell walls are characterised by the activity of exo- and endo-f3-(1,3)-glucanases, releasing a heterogenous mixture of polysaccharides and short chain oligosaccharides including a small percentage of mannoprotein;

(ii) sustained autolysis weakens cell wall integrity which allows further solubilisation of high molecular weight mannoproteins likely to originate from the periplasmic zone;

(iii) in the latter stages of autolysis, cell wall glucans are attacked by f3-(1,3)- glucanases remaining in the wall and by glucanases released into the autolysate; 53

(iv) exo-f3-(1,3)-glucanases released into the autolysate will cleave any glucan material linked to mannoproteins. Solubilised mannoproteins will be degraded by a-mannosidases while proteolytic enzymes will also degrade the protein component of mannoproteins.

2.4234 Release of lipids

Changes to the lipid composition of yeasts associated with sparkling wine during ageing was studied by Piton et aL (1988). A gradual conversion of polar lipids to non-polar glycerides and their subsequent accumulation by the yeasts was observed. The decrease in polar lipids was ascribed to the destruction of organelle membranes since the cytoplasmic membrane appeared to remain unchanged under electron microscopy. Lipid excretion during these chemical transformations was suggested to be the source of lipids found in ageing sparkling wine. This was confirmed by Ferrari et aL (1987) who studied the release of fatty acids in a synthetic medium during yeast autolysis. These authors found that the major fatty acids released during autolysis were saturated and were composed of 8, 10, 12 or 16 carbon atoms. Chen et aL (1980) reported a similar pattern of release of fatty acids during yeast autolysis in beer. However, there are no reports on the changes of lipid classes in sparkling wine during ageing on lees.

2.4235 Release and formation of volatiles

The accumulation of various esters and carbonyl compounds during ageing of sparkling wine is considered a major source of aromatic components in sparkling wine bouquet. Table 2.9 lists compounds reported by various authors as being linked to changes occurring during ageing of sparkling wine on lees. 54

In the majority of cases, the concentrations of volatile compounds in Table 2.9 increase with wine age. Silva et aj. (1987) found the concentration of diethyl succinate to range from about 2 to 5 mg/L in sparkling wine; after ageing for 750 days on lees, the concentration increased by an average of 107% in four different wines. For the same wines, the concentration 7-butyrolactone increased from about 12 to 14 mg/L (17 % increase) and 2-phenylethanol increased from about 30 to 34 mg/L (13% increase). Significantly, the same wines disgorged after 200 days ageing and stored without lees for an equal period of time contained 72, 28 and 5% less diethyl succinate, 7-butyrolactone and 2-phenylethanol, respectively.

However, it should be noted that a number of compounds actually decrease with time. Loyaux et al. (1981) showed that the concentrations of esters isoamyl butyrate and hexyl acetate decreased with time while Konovalova (1977) and Silva et aj. (1987) noted a decrease in the concentration of isoamyl acetate during ageing. Significantly, Silva et a\. (1987) demonstrated the presence of intracellular esterase activity up to 300 days following the secondary fermentation which may account for the loss of these compounds during ageing.

Higher alcohols may also decrease during ageing, although their loss is usually attributed to an improvement in quality (Rodopulo et aL, 1969, 1975). Elevated concentrations of iso-butanol and iso-pentanol are suggested to impart a 'rough' quality to sparkling wine (Rodopulo et a]., 1975). Konovalova (1977) showed that isoamyl alcohol decreased during three months ageing in the bottle, a trend which has since been confirmed by Chung (1986). 55

Table 2.9 Volatile compounds reported to undergo significant variation in concentration in sparkling wine during ageing on lees.

Class of Name of compound(s) References compound Esters Diethyl succinate Rodopulo et ai. (1969) Phenylethyl acetate Avakyants and Shakarova (1973) Ethyl lactate Rodopulo et ai- (1975) Octyl acetate Mikhailenko et a[. (1978) Ethyl linoleate Loyaux et aL (1981) Phenylethyl caproate Molnar etai- (1981) Hexyl acetate Versini and Margheri (1982) Ethyl palmitate Montedoro (1986) Ethyl stearate Cologrande and Silva (1986) Isoamyl caproate Silva etaj. (1987) Isoamyl acetate Postel and Ziegler (1991b) Isoamyl butyrate Etievant (1991)

Higher alcohols Isoamyl alcohol Konovalova (1977) 2-phenylethanol Molnar et aL (1981) Chung (1986) Silva et a\. (1987)

Carbonyl Benzaldehyde Rodopulo et ai. (1969) Vitispirane Loyaux etai. (1981) 3-ionone Molnar etaL (1981) Butan-2,3-dione

Terpenoid Linalool Molnar etai. (1981) a-terpenol Chung (1986) Cis-, trans-farnesol

Lactones Dimethyl-4,5-tetrahydrofuran Feuillat (1981) -2,3 dione 5-ethoxy butyrolactone Margheri et aL (1984) 7-butyrolactone

The long period of ageing will also allow opportunity for important secondary reactions to occur such as esterification of fatty acids with ethyl alcohol. This may be the source of large esters such as ethyl palmitate and ethyl stearate in 56 aged wine. It is apparent that incubation at low temperatures is required to encourage these favourable reactions (Kelly-Treadwell, 1988). Molnar et aL (1980a) have attempted to accelerate this activity and the process of yeast autolysis by using higher temperatures of storage. Unfortunately, yeast-like off- flavours were produced suggesting that higher temperatures encourage other less-favourable reactions to occur.

2.43 Influence of ageing on yeast lees upon sparkling wine quality

Sparkling wine quality may be defined in terms of aroma, flavour and bubble size and persistence. The release of intracellular components of yeast through autolysis is believed to have a positive impact upon these aspects of sparkling wine quality (Bidan et aL, 1986). Additionally, it is important to consider the occurrence of secondary reactions between the products of autolysis and other constituents of the sparkling wine (Markides, 1986; Kelly-Treadwell, 1988). Both phenomena form part of the slow, but important process which occurs during ageing of sparkling wine on lees.

2.431 Effect upon sparkling wine aroma

Yeast autolysis is believed to have an important influence on the development of sparkling wine aroma through the formation of various volatile compounds. The most important groups of compounds involved in the formation of sparkling wine aroma include esters, higher alcohols, carbonyl compounds, terpenes and lactones (Bidan et aL, 1986). Most of these compounds are considered to arise directly from yeast autolysis (Avakyants and Shakarova, 1973; Feuillat et aL, 1980; LoyauxetaL, 1980; Molnar etaL, 1981; Chung, 1986; Postel and Ziegler, 1991b). 57

However, the importance of secondary reactions in the development of aroma should not be discounted. Of particular note is the formation of esters between .ethanol and various acidic compounds. For example, ethanol may react with fatty acids originating from lipid breakdown to form ethyl esters (Feuillat and Charpentier, 1993) such as ethyl caprylate, -caproate, -palmitate and -stearate. Ethanol may also react with organic acids such as lactic and to form ethyl lactate and diethyl succinate which have been shown to increase with ageing on lees (Cologrande and Silva, 1986; Postel and Ziegler, 1991b).

Finally, amino acids may also undergo secondary reactions to form aromatic compounds. Feuillat (1981) attribute the formation of two lactones, dimethyl-

4,5-tetrahydrofuran-2,3-dione and 5-ethoxy butyrolactone to secondary reactions involving threonine and glutamic acid, respectively. Another amino acid, methionine, is also believed to be involved in the formation of vitispirane which has a floral odour and is a characteristic of aged wines.

2.432 Effect upon sparkling wine flavour

Many of the aroma compounds such as esters, higher alcohols and carbonyl compounds described in Section 2.431 also affect sparkling wine flavour (Rodopulo et aL, 1969). Rodopulo et aL (1975) describes ethyl linoleate as having a light sunflower flavour. Rodopulo et aj- (1969) describe high boiling point esters and various aldehyde compounds such as heptanal and caprylic and caproic aldehydes as imparting a pleasant flavour and fruity aroma.

The evolution of nitrogen compounds as a result of autolysis during ageing of sparkling wine may also influence flavour. Sparkling wines with higher concentrations of nitrogen have rated higher in terms of quality (Francesco and

Margheri, 1973). However, although breakdown products of protein autolysis such as amino acids and peptides may influence the flavour of foods (Solms,

1969; Kiramura et aL, 1969), this effect has not been directly implicated in 58 sparkling wines. Similarly, nucleic acids and their various breakdown products are believed to contribute to the flavour of other beverages such as beer (Masschelein, 1986). Leroy et aL (1990) studied the release of nucleic acids during ageing of sparkling wine and found that the greatest increase occurred during the first five months of storage. As with the influence of protein nitrogen, the impact of nucleic acids upon the flavour of sparkling wine is inferred rather than directly implicated. However, there is little doubt that these compounds do contribute to the overall flavour of the final product (Leroy et aL, 1990).

2.433 Effect upon the formation and stability of bubbles (bead)

The formation and stability of foam in sparkling wines is an important indice of quality. Generally speaking, high quality sparkling wines tend to retain carbon dioxide in solution and produce small sized bubbles with persistent foam after pouring into the glass (Brissonet and Maujean, 1991). Numerous products of yeast autolysis have been shown to directly affect foam character in sparkling wines including proteins, polysaccharides and lipids (Dubinchuck et aL, 1980; Razmadze et aL, 1980; Razmadze, 1985; Codrington, 1986; Brissonet and Maujean, 1991; Brissonet and Maujean, 1993; Feuillat and Charpentier, 1993; Dussaud et aL, 1994).

The positive effects of increased concentrations of nitrogenous compounds in sparkling wines on foam formation were noted in early studies by Razmadze et aL (1980). Codrington (1986) showed that addition of bovine serum albumin to sparkling wine decreased bubble size and that similar results could also be obtained by adding yeast autolysate. Brissonet and Maujean (1991) found that proteins present in sparkling wine are closely associated with foam formation. These authors demonstrated that significant quantities of protein were lost from the sparkling wine when the foam was separated from the residual wine. The proteins were characterised by Brissonet and Maujean (1993) using SDS-PAGE 59

electrophoresis and affinity chromatography. Five proteins fractions were isolated with molecular weights of 13 900, 19 400-26 900, 35 700 and 60 000 daltons. The proteins were also found to hydrophobic rather than hydrophilic in nature.

Polysaccharides originating from the yeast cell wall are believed to contribute to the stability of foam in sparkling wine (Feuillat and Charpentier, 1993).

Brissonet and Maujean (1991) studied the effects of polysaccharides on formation of foam in sparkling wine and found that these compounds decrease

bubble size and enhance the persistence of the foam following pouring from the bottle. Similar effects of polysaccharides on foam stability have been reported

in beer (Roberts et aL, 1978).

The effect of lipids on foam formation has been studied by Dubinchuck et aL (1980), Razmadze (1985) and Dussaud et aL (1994). In general, lipid material has a negative effect on foam formation, although Dussaud et al. (1994) showed that, in the presence of high ethanol concentration (as in sparkling wines), this effect is largely negated. However, when the alcohol concentration of the sparkling wine was decreased to 5% (v/v) and lower, foam destruction became evident. This would explain the well documented destructive effects of lipids associated with brewing, considering the lower alcohol content of beer

(Hollemans et aL, 1991; Clark et aL, 1994).

2.5 Effect of carbon dioxide upon yeast growth during fermentation of sparkling wine

The evolution of carbon dioxide (CO2) during the secondary fermentation of sparkling wines plays a major role in developing the characteristic bubbly nature of the product. Carbon dioxide is produced in a deliberately closed 60 system so that approximately 5 to 6 atmospheres (5 to 6 x 105 Pa) of pressure (at 10°C) develops by the end of the fermentation (Markides, 1986). Under these conditions, the concentration of CO2 dissolved in wine has been reported to be about 12 g/L (Amerine et aL, 1980). Given that significantly lower levels of CO2 have been reported to influence the production of various flavour-active compounds during beer fermentation (Norstedt et aL, 1975; Posada et aL,

1977; Arcay-Ledezma and Slaughter, 1984; Knatchbull and Slaughter, 1987;

Slaughter, 1989), it would be reasonable to assume that such effects would also be observed in sparkling wine. There are, however, few published data on the influence of CO2 upon the metabolic processes of wine yeast during sparkling wine fermentation. In order to understand the response of yeast under these conditions and the impact upon product quality, it is necessary to review the published effects of CO2 on microbial metabolism.

2.51 The effect of carbon dioxide on the metabolism of microorganisms

The inhibitory effect of CO2 upon the metabolism of microorganisms has been the theme of considerable study because of its potential use as a food presen/ative. The reviews of Daniels et- aL (1985), Dixon and Keil (1989) and

Jones (1989) detail the use of CO2 for this purpose and its inhibitory effects upon the metabolism of microorganisms.

The mechanism by which CO2 exerts an inhibitory effect has not been conclusively determined, although many theoretical models have been suggested. These have been discussed by Jones and Greenfield (1982), Dixon and Kell (1989) and Slaughter (1989) and are summarised in Table 2.10. Hydrostatic pressure in itself is not sufficient to explain the inhibitory effects of

CO2. Chen and Gutmanis (1976) showed that inhibition of in

S. cerevisiae at a pressure of 2.90 x 10^ Pa of CO2 was not observed when nitrogen at the same pressure was utilised. For the purposes of food 61 preservation, much higher hydrostatic pressures ( > 303 x 10^ Pa ) must be used to inhibit microorganisms such as S. cerevisiae (Thom and Marquis, 1984;

Ison and Gutteridge, 1987; Haas et aj., 1989; Hoover et aL , 1989; Lin et aL,

1992). Thus, in terms of the environment of sparkling wine, pressure is significant only in that it has a major influence on the concentration of CO2 dissolved in solution.

Table 2.10 Proposed mechanisms for CO2 inhibition of microbial growth and metabolism.

Mechanism Description Displacement of oxygen Inhibition of growth due to decreased availability of oxygen

Internal pH Internal acidification following passage of CO2 (aq) into the cell

Cell membrane Impairment of membrane-mediated functions by CO2 (aq) dysfunction ('narcosis') and disruption of phospholipids and membrane charge potential by HCO3' (aq)

Inhibition of cytoplasmic Induction or repression of enzyme synthesis - enzyme synthesis predominantly carboxylation and decarboxylation reactions

Inhibition of enzymatic - CO2 (aq) inhibition of decarboxylation reactions activity - HCO3" (aq) inhibition of specific enzymes at a general anion-sensitive site on enzyme

The displacement of oxygen as a mechanism to explain the inhibitory effects of

CO2 on microorganisms has long been discounted because anaerobic bacteria are also inhibited (Killefer, 1930) and replacement of CO2 with 100% nitrogen did not give the same degree of inhibition as CO2 alone (Callow, 1932).

Other explanations for the inhibitory properties of CO2 depend upon its passage into the cell and thus its chemical properties in aqueous solution. 62

Other explanations for the inhibitory properties of CO2 depend upon its passage into the cell and thus its chemical properties in aqueous solution. Knoche (1980) and Slaughter (1989) have reviewed the possible chemical species of CO2 in water:

CO2 + H20 <-> H2CO3 <-> HCO3- + H+ CO32- + 2H+

The presence of each species in water is greatly dependent upon the pH of the solution. At wine pH of approximately 3.0, CO2 exists primarily as dissolved

CO2 gas as illustrated in Figure 2.2 (Daniels et ad., 1985).

80 -

70 -

60 -

40 -

30 -

20 -

10 -

0 2 4 6 8 10 12 14 pH

Figure 2.2 Fraction of CO2 (□), HCO3" (■), CC^- (0) and H2CO3 (A) in water as a function of pH (Daniels et al. 1985).

This fact is of major consequence in sparkling wines as carbon dioxide in the form of CO2 (aq) has been shown to penetrate into cells 30 times faster than oxygen (Krough, 1919). This effect in sparkling wines is accentuated by several factors. Firstly, CO2 binds to complex organic molecules such as proteins by the formation of carbamates through reaction with non-ionised 63

Additionally, the lower temperature of storage of sparkling wine (~ 15°C) increases CO2 solubility (Wolfe, 1980).

Internal acidification of the cell cannot totally account for the observed inhibitory effects of CO2. Becker (1933) showed that other acids producing equal internal acidification were not able to inhibit growth to the same degree as carbon dioxide. Similar results were obtained by Eklund (1984) using weak acids such as sorbate and benzoate, although the general form of inhibition was similar to CO2.

Carbon dioxide impairment of membrane function may occur either by disruption of the lipid component of the cell membrane (Sears and Eisenberg,

1961), thereby affecting closely associated membrane proteins, or by direct effect on the membrane proteins themselves (Dixon and Kell, 1989; Jones,

1989). Termed narcosis or anaesthesia, these effects are summarised in Table

2.11.

Not surprisingly, the inhibitory effects of carbon dioxide are aggravated by other membrane disturbing agents such as ethanol (Jones and Greenfield, 1982). This is an important consideration in sparkling wine production where the secondary fermentation is conducted in the presence of approximately 10% (v/v) ethanol resulting from the primary fermentation. Table 2.11 Reported theories explaining the anaesthetic or narcotic effect of carbon dioxide upon biological membranes (Dixon and Kell, 1989) DC £ Q o s h- SZ c 0 0 O CD 0 0 O c 0 O o 0 0 0 s if o o ■«5l to ’ X TO x sz O < -Q 1 ■g Q) o k_ ®l > O c 0 o c 0 k_ Q. o i_ 0 CD o c 0 o c 0 c 0 X Q 0 0 E 0 o 3 Q. CM l sz s X _0 _g .E TO TO o> CO X to •c 1 x ‘ x ■4-4 > 0 0 0 CD 0 6 0 0 9 13 o > 0 E 0 k_ 0 o c > CL o 0 0 0 >% c 0 O O 0 0

. V- H •£ SZ TO X ■a c o c E 0 E k_ 0 c 0 O 0 0 3 0 u. 0 O 0 C 13 — TO SI CD CO □ .E X •9 _g Q_ x iz 0 0 0 0 0 c c 0 O 3 C c o C 0 O) CD CD 0 Q_ 0 3 3 0 E Q_ c 0 0 0 0 c 0 o

— _0 TO TO -9 .E ■o .E .E x ■4-4 ■4 x ’ 4-< x ■4 CD 0 C C 0 Q. 0 O 0 0 0 c E 0 0 c o c o 0 k_ 3 0 E 0 c 0 E 0 0 0 CL o > — — » »

TO .E -a .E •g sz o O I- x sz 0 k_ O C CD O) 0 c 0 E 0 o 3 3 0 0 3 CM

.E *0 JO 3 .E "O •g 0 E 0 E k_ 0 c 0 o 0 0 0 > c o 0 3 CD 3 c o E 0 0 Q. be x TO JZ sz .E t 5= ■O TO TO O •4 ■ JO CL 4 0 cel $ q 0 k_ 0 0 0 E 0 E 0 c 0 0 0 c 0 0 0 0 k_ 0 0 0 3 O 0 0 £ 0 c — V o — — » »

03 I CO 0 JO TZ TO s . 0 k 0 c 0 X 0 E 0 >* 0 CL k_ 0 0 c E 0 0 0 c o

- — . TZ TO X X) TO x X ‘ -4 4-4 O k_ 0 O 0 c 0 CL k- O 0 C 0 c E 0 E 0 > 0 0 0 CL o — 1 *

V4_ X .E X X * X X TO ■4 -4 ■*-> ■« 0 CL 0 0 k_ 0 C O k_ 0 O L. CD 0 CL 0 O CL O 0 O Q- E CL 0 0 > 0 0 — — — — 1 4 * » »

interact with phospholipids. 64 65

Other proposed sites of inhibition by CO2 are the cytoplasmic enzymes (Jones and Greenfield, 1982; Dixon and Kell, 1989; Jones, 1989; Slaughter, 1989). In this theory, carbon dioxide is suggested to induce or repress the synthesis of enzymes, or to act upon the enzymes themselves. An example is cited by Dixon and Kell (1989) where the production of the enzyme 1,5- bisphosphate carboxylase (RuBPCase) in Rhodospirillum rubrum was dramatically increased by lowering the partial pressure of CO2 from 0.05 to

0.02 (Sarles and Tabita, 1983; Tabita et aL, 1985). Slaughter (1989) suggests that carbamate formation may play a role in this effect.

For detailed descriptions of enzyme systems affected by carbon dioxide, the reader is directed to Jones and Greenfield (1982). These authors propose that the bicarbonate ion (HCO3") exerts regulation in enzymes which contain a general anion-sensitive site, whereas the CO2 (aq) species have been implicated as an inhibitor in decarboxylation reactions which are equilibrium sensitive. The major decarboxylation reactions most likely affected by CO2 (aq) inhibition are summarised in Figure 2.3. Clearly, biosynthesis of nucleic acids, amino acids and lipids would be directly affected by CO2 inhibition.

Notably, enzyme activities not necessarily involved in carboxylation or decarboxylation reactions have also been reported to be inhibited or activated by CO2 or HC03". Of particular note is the suggestion by Mitsuda et aL (1980) that carbon dioxide causes a transient inactivation of hydrolytic enzymes associated with autolysis in fish. This may be an influencing factor delaying the process of yeast autolysis in sparkling wine where a latent period of several months is observed between the end of the secondary fermentation and the onset of autolysis (Feuillat and Charpentier, 1982; Leroy et aL, 1990). 66

glucose

glucose-6-P

fructose-6-P -----^-►G16P------6PG Other amino r NADPH-, NADP * acids OAA 4 NADP nadph-N-»- co2 e.g. threonine HCO3 i ADP i ribulose-5-P isocitrate.*— citrate Pyr J ATP i ^nadh2 Biosynthesis NADH£ CoA nucleotides ♦ NAD amino acids NAD- Acetyleiyi CoA others EtOH HCO: °x°g,uy*Asp k NH- MMCoA '3 ; A*oaa glut ^ K-*CO- Other amino acids e.g. proline Lipids Fatty acids

Figure 2.3 Major carboxylation reactions ( * ) involving carbon dioxide that may be affected by the presence of CO2 (aq) within the yeast cell (adapted from Jones and Greenfield, 1982)

2.52 Inhibition of yeast by carbon dioxide

2.521 Physiological effects

In general, the combined effects of carbon dioxide decrease metabolic activity in microorganisms (King and Nagel, 1975). This is reflected as a decrease in cell yield and growth rate of S. cerevisiae in baker's yeast production (Norton and Krauss, 1972; Chen and Gutmanis, 1976), beer fermentation (Posada et aL, 1977; Arcay-Ledezma and Slaughter, 1984; Nakatani et aL, 1991;

Knatchbull and Slaughter, 1987) and in continuous culture fermentations (Kunkee and Ough, 1966; Cahill et aJ., 1980; Kuriyama et aL, 1993).

Norton and Krauss (1972) related decreased cell yield to a lower occurrence of cell division. As mentioned earlier, the controlling influence of carbon dioxide concentration in solution is pressure; the range of pressures reported 67 to inhibit cell division in S. cerevisiae is 0.5 x 10^ to 3.9 x 10^ Pa carbon dioxide (Jones and Greenfield, 1982). This range has been confirmed by Lumsden et aL (1987) in brewing yeast, although these values may vary according to fermentation conditions and yeast strain (Slaughter, 1989).

Suppression of DNA replication is not the cause of inhibited cell division

because Norton and Krauss (1972) noted continued DNA synthesis despite

no further increase in cell numbers. Lumsden et aL (1987) showed that while cell division was inhibited in S. cerevisiae under 2.9 x 10$ Pa of CO2, DNA

continued to be produced and doubled in size within 24 hours. Yeast cells increased in volume, indicating that cellular material was still being synthesized in preparation for cell division. An increase in cell volume was

also observed by Slaughter et ai. (1987). It has been suggested that CO2 was inhibiting the production of chitin required for bud formation (Lumsden et

aL, 1987). Slaughter (1989), however, considers that this may not be the full explanation for increase in cell volume as growth in unexposed cells was too slow to account for increased cellular material.

Other cellular components affected by carbon dioxide include RNA, protein and lipids. Slaughter et aL (1987) found that RNA and protein contents of yeast decreased in response to 2.9 x 10^ Pa of CO2. Castelli et aL (1969) showed that an increase in CO2 pressure from 0.18 x 105 to 0.56 x 105 Pa caused an increase in the proportion of unsaturated fatty acids.

Carbon dioxide may also decrease the viability of S. cerevisiae during fermentation (Slaughter, 1989). Knatchbull and Slaughter (1987) showed that viability of S. cerevisiae decreased from 93% to 87% and then to 80% under

0, 0.5 and 1 x 105 Pa CO2 pressure, respectively, after 120 hours fermentation of wort. After 24 hours of fermentation in a malt medium, 68

fermentation of wort. After 24 hours of fermentation in a malt medium, Slaughter et aL (1987) showed that the viability of S. cerevisiae decreased

from about 85% under no CO2 pressure to about 45% under 1.97 x 10^ Pa

CO2 pressure.

2.522 Effect of carbon dioxide on yeast metabolic activities during fermentation

While CO2 exerts an influence upon yeast growth at the range of pressures

discussed previously, it appears that the glycolytic pathway is unaffected

(Norton and Krauss, 1972; Slaughter, 1989). These observations suggest that carbon dioxide is responsible for uncoupling yeast growth from ethanol production during fermentation (Jones and Greenfield, 1982; Dixon and Kell, 1989). Chen and Gutmanis (1976) showed that ethanol production by S. cerevisae is unaffected by up to 4.0 x 105 Pa of carbon dioxide pressure, while biomass production was reduced. Similar results have been found with Zymomonas mobilis (Nipkow et aL, 1985; Veeramallu and Agrawal, 1986). However, Nipkow et a]. (1985) found that the rate of glucose uptake in

Z mobilis increased when the CO2 partial pressure was decreased from 1.460 to 95 mbar. Similar results were found with S. cerevisiae during cultivation of yeast under CO2 pressure for use in continuous sparkling wine production (Cahill et aL, 1980).

The uptake of amino acids, however, is greatly affected by C02- The pattern of uptake has been observed to be dependant on CO2 pressure and is illustrated Table 2.12. 69

Table 2.12 Influence of carbon dioxide on uptake of amino acids by yeast.

Carbon dioxide Observed effect Reference pressure (Pa)

0.5 - 1.0 x 10b No effect on Glu, Asp, Thr or Lys Knatchbull and Slaughter (1987) Reduced uptake of Val, Leu and lieu

2.0 x 105a No effect on Thr, Lys, Leu and lieu Slaughter et a). (1987) Reduced uptake of Arg and Glu

Increased uptake of Gly, Val and Ala

Negligible uptake of Ser, Phe, Asp, Tyr and Trp a Data represents intial uptake of amino acids from medium.

Similarly, carbon dioxide affects the production of secondary metabolites such as esters, higher alcohols, fatty acids and glycerol which may impact upon the sensory qualities of a fermented product (Jones and Greenfield, 1982). This has been exploited by the brewing industry to control ester and fusel oil formation during fermentation (Norstedt, et aL, 1975; Slaughter, 1989). These effects are summarised in Table 2.13.

These observations are of relevance in sparkling wine production because of the build-up of CO2 pressure during the secondary fermentation. The implications of this phenomenon in sparkling wines is discussed in the following section. 70

Table 2.13 Effect of carbon dioxide on production of metabolites during yeast fermentation.

Secondary metabolite Observed effect References Production of glycerol Reduced glycerol production Kuriyama ei ai. (1993)

Production of Increase in acetaldehyde production Kumada ei ai. (1975) acetaldehyde Knatchbull and Slaughter (1987) Kruger at ai- (1992)

Production of acetate Reduced formation of ethyl acetate, Norstedt et ai- (1975) esters iso-amyl acetate and phenyl ethyl Knatchbull and acetate Slaughter (1987) Kruger at ai- (1992) RengerMai- (1992)

Production of fatty Increased production of C10 and Norstedt et ai. (1975) acids C-J2 fatty acids Posada at ai. (1977)

Production of Cr and Cr fatty acids unaffected

Production of ethyl Production of ethyl esters of Cr, C10 Norstedt at ai- (1975) esters of fatty acids and C-|2 maximum at 1 atmosphere Posada at ai- (1977)

Production of ethyl hexanoate unaffected

Production of higher Reduced production of n-propanol, iso­ Posada etai- (1977) alcohols butanol, iso-amyl alcohols, Arcay-Ledezma and 2-phenylethanol Slaughter (1984) Knatchbull and Slaughter (1987) Slaughter et ai- (1987) Renger et ai. (1992)

Production of Increased production of and Arcay-Ledezma and carbonyls 2,3-pentanedione and their precursors. Slaughter (1984) 71

2.523 Effect of carbon dioxide on yeast growth and metabolism during secondary fermentation of sparkling wines

The effect of carbon dioxide pressure upon yeast growth and fermentative performance during sparkling wine production has been investigated by Kunkee and Ough (1966) and Cahill et aL (1980). Kunkee and Ough (1966) studied the growth of S. cerevisiae under 0, 2.5 and 5 atmospheres of CO2

pressure for the purposes of devising a system of continuous production of sparkling wine. Increasing pressure of carbon dioxide decreased yeast growth and fermentation rates. Supplementation of the culture with nutrients (yeast extract) stimulated cell growth, although not fermentation rate. It should be noted, however, that yeast were added to bottles and pressurised over several days from commercial CO2 supplies, as opposed to the gradual increase in CO2 pressure over 4-6 weeks observed during sparkling wine production in bottles.

Cahill et ai. (1980) studied the effectiveness of adaptation of the yeast culture to low levels of CO2 pressure (0, 0.3 and 0.6 atmospheres) for use in a two stage continuous sparkling wine fermentation system. The first stage was maintained under 0.3 atmospheres CO2 pressure and operated with a relatively high fermentation and dilution rate. The second stage was operated at 5 atmospheres of CO2 pressure with a low fermentation and dilution rate. Under steady state conditions, higher utilisation of glucose by the pressurised yeast was observed and explained as an increased need for maintenance energy due to the inhibitory effects of CO2 pressure. Yeast that were grown under continuous fermentation conditions of 0.3 atmospheres CO2 pressure also demonstrated increased fermentation rates when transferred to bottle fermentations. 72

The literature, however, contains no data concerning the evolution of flavour- active secondary metabolites in sparkling wines in response to developing

CO2 pressures in bottle fermentations. Caution, however, must be employed in direct comparisons between the published data on brewing and any results to be obtained in sparkling wines. The published data refers exclusively to yeast metabolism observed under immediate exposure to various pressures of carbon dioxide. In sparkling wines, however, the process differs in that

CO2 pressure develops slowly over 4-6 weeks, thus allowing the yeast an opportunity to adapt to the changing environment. Cahill et aL (1980) have already shown that enhanced fermentative ability may be obtained in continuous sparkling wine production if the yeast inoculum is adapted to base wine over several weeks. Additionally, the secondary fermentation begins under conditions of little or no carbon dioxide pressure - it would, therefore, be unlikely that the effects of carbon dioxide pressure on initial amino acid uptake as reported by Slaughter et aL (1987) and Knatchbull and Slaughter (1987) would be observed. However, it is possible that production of the secondary metabolites (particularly towards the end of the secondary fermentation) will show the established effects of increased carbon dioxide pressure as discussed earlier. Such effects would impact upon the final quality of the sparkling wine and may be open to manipulation by the winemaker in a fashion similar to that used in the brewing industry.

2.6 Killer yeasts in the wine industry

The concept of killer yeast was first reported in Saccharomyces cerevisiae by

Bevan and Makower (1963) to describe the phenomenon where one yeast strain (killer) kills another strain (sensitive) through the secretion of a toxic compound (killer toxin). Strains which were immune to these toxins and did not kill other yeast were termed neutral strains. Subsequent to this report, 73 killer/sensitive strains were discovered which produced killer toxins to which they were immune, but were also sensitive to toxins of other killer strains (Woods et aL, 1974). Killer activity has since been demonstrated in other yeast genera such as Candida, Cryptococcus, Debaryomyces, Hansenula, Hanseniaspora, Kluyveromyces, Kloeckera, , Rhodotorula and Trichosporon (Philliskirk and Young, 1975; Stumm et aL, 1977; Rogers and

Bevan, 1978; Shimizu, 1993). The occurrence, characterisation and activity of killer yeasts, in general, have been reviewed by Young (1987).

Killer yeasts belonging to the genus Saccharomyces have been discovered or utilised in various beverage fermentations such as brewing (Philliskirk and Young, 1975; Young, 1981), sake production (Imamura et aL, 1974) and winemaking (Kitano et aL, 1984; Tredoux et aL, 1986; van Vuuren and Wingfield, 1986). Wild killer yeasts associated with winemaking have been known to produce slow or stuck fermentations (Kitano et aL, 1984; Shimizu et aL, 1985a; van Vuuren and Wingfield, 1986; Heard and Fleet, 1987; Radler and Knoll, 1988; Benda, 1989). Killer activity has also been detected in commercial strains of wine yeasts (Barre and Biron, 1982, Shimizu e\ aL, 1985b; Thornton, 1986; Thornton and Brunker, 1989). The positive and negative aspects of killer yeasts in winemaking have been reviewed by van Vuuren and Jacobs (1992) and Shimizu (1993).

2.61 Screening procedure for isolation of killer yeast

A simple assay for killer yeasts has been described by van Vuuren and

Jacobs (1992). Routine screening of yeast strains is carried out on a nutrient agar medium containing methylene blue (MBNA) buffered at pH 4.2 - 4.7. Sensitive strains of yeast may be spread on the surface of the agar or added to the molten agar before pouring into the petri-dish. Sensitive cells are usually added at concentrations of 4 x 10^ cells/mL while they are in the 74 exponential phase of growth for increased sensitivity to the toxin (Woods and Bevan, 1968). Strains of yeast to be assayed for killer activity are streaked onto the surface of the set agar and the plates incubated at 18° - 20°C. Strains are classified as killers when the streak of growth is surrounded by a clear zone of dead cells which stain blue. The methylene blue is necessary to distinguish between true killer activity and growth inhibition due to mating pheromones (Young, 1987).

2.62 Classification of killer yeasts

Killer yeasts are classified on the basis of properties of the secreted toxin

(Shimizu, 1993). According to van Vuuren and Jacobs (1992), toxin properties may be assessed by one of several ways : (a) by determination of the spectrum of activity against specific sensitive strains; (b) by assay of activity against mutants resistant to certain toxins; and (c) by assay of the cross-reactivity of toxin producing organisms (interaction between killer strains). Using cross-reactivity between killer strains, Young and Yagiu (1978) classified killer yeasts into 10 groups, K1 - K10. A further killer strain of Candida glabrata was discovered by Bussey and Skipper (1975) and added to this classification system as K11 (Wickner, 1979). Wingfield et a|- (1990), however, claimed that the K2 and K3 killer types were the same by demonstrating that the K3 killer is a mutant of the K2 yeast. Shimizu (1993) has summarised the types of killer toxins from different yeast strains (Table 2.14). 75

Table 2.14 Comparison of killer toxins isolated from different killer strains3

Killer strain Chemical Molecular Optimum pH Reference composition weight (kD*3) for activity Saccharomyces Protein 20 4.6-4.8 Bostian et aL cerevisiae (K1) (1984)

Saccharomyces Glycoprotein 16 4.2 Pfieffer and Radler cerevisiae (K2) (1984)

Saccharomyces Glycoprotein 16 5.8 Pfieffer and Radler cerevisiae (KT28) (1984)

Saccharomyces Protein 20 5.7 Goto et aL (1990) cerevisiae (KHR)

Kluyveromyces Glycoprotein 180 4.0-8.0 Stark el ai. (1990) lactis

Pichia kluyveri Glycoprotein 19 3.8-4.0 Middelbeek et ai. (1979)

Hansenula mrakii Protein 10.7 4.0-9.0 Ashida et aL (1983)

Hansenula Protein 11-12 3.5-7.0 Henschke (1979) saturn us

Candida spp. Glycoprotein >200 3.8-5.8 Yokomori el aL SW-55 (1988)

Pichia farinosa Protein 25 2.0-4.0 Suzuki and Nikkuni (1989)

Hansenula Glycoprotein 300 - Kagiyama et aL anomala (1988)

Hanseniaspora Protein 18 4.0-4.5 Radler etai. (1990) uvarum a Adapted from Shimizu (1993) b Monomer

2.63 Molecular biology of killer activity

The molecular biology of killer activity in yeast has been discussed in reviews by Young (1987), van Vuuren and Jacobs (1992) and Shimizu (1993). Killer activity is determined genetically by one of three ways: (a) by double stranded 76 ribonucleic acid (dsRNA); (b) linear DNA plasmids; or (c) chromosomal genes.

2.631 Killer activity encoded by dsRNA systems

In S. cerevisiae, killer characteristics (type K1 and K2) are encoded by intracellular dsRNA which are encapsulated by protein into virus-like particles

(VLP) (Herring and Bevan, 1974, 1977; Hopper et aL, 1977). Two major groups of dsRNA have been characterised, the larger of which is termed the

' L ' dsRNA and the smaller, ' M ' dsRNA (Young, 1987). Each group is further sub-divided into smaller species depending on homology studies. The reader is directed to van Vuuren and Jacobs (1992) for detailed descriptions of each species of dsRNA.

Saccharomyces cerevisiae strains that exhibit killer activity contain both L and M forms of dsRNA (Young and Yagiu, 1978; Wickner, 1981; Tipper and Bostian, 1984). However, it is the M dsRNA which confers killer characteristics to a particular yeast strain (Fink and Styles, 1972; Bevan et aL, 1973; Young and Yagiu, 1978; Tipper and Bostian, 1984). The M dsRNA is believed to encode for the production of the killer toxin precursor which is subsequently processed into the active form. The L dsRNA encodes for production of a RNA polymerase and viral coat proteins for both VLP (Hopper et aL, 1977; Bostian et aL, 1980; Tipper and Bostian,

1984). The presence of the M dsRNA also confers immunity to killer strains of S. cerevisae (Bostian et aL, 1984; Hannig and Leibowitz, 1985).

Expression of killer activity in K1 and K2 killer yeast is dependant upon the activity of several chromosomal genes. These systems have been outlined in

Table 2.15 (Shimizu, 1993). 77

Table 2.15 Chromosomal genes responsible for expression of killer activity in dsRNA killer systems3

Chromosomal gene(s) Function(s) References MAK, SKI Maintenance and replication Wickner (1986) of dsRNA

KEX1 Processing of killer toxin Bussey et aL (1983) (carboxypeptidase)

KEX2 Processing of killer toxin Bussey et aL (1983) (endopeptidase)

SEC Secretion of toxin Wickner and Leibowitz (1976)

END Endocytosis Al-Aidroos and Bussey (1978)

KRE Binding of toxin Bussey et aL (1983)

VPL Vacuolar protein localisation Chvatchko et aL (1986)

REX Expression of resistance Rothman and Stevens (1986)

3 Summarised from Shimizu (1993)

2.632 Killer activity encoded by linear DNA plasmids

The existence of linear double-stranded DNA encoding for killer activity has only been demonstrated with the yeast, Kluyveromyces lactis (Young, 1987).

Gunge et aL (1981) characterised two plasmids, pGKL1 and pGKL2, with functions analogous to dsRNA particles in S. cerevisae. Production of the killer toxin (precursor consisting of a, p and 7 subunits) and conferring of immunity is encoded by the pGKL1 molecule, while the function of pGKL2 is to maintain pGKL1 (Hishinuma et ai., 1984; Stark and Boyd, 1986). As 78 observed in S. cerevisae, expression of the toxin is controlled by the chromosomal gene, KEX1, which processes the a p protoxin into the a and p subunits of the active killer toxin (Wesolowski Mai-, 1988).

2.633 Killer activity encoded by chromosomal genes

The existence of chromosomal genes encoding for killer toxins in

S. cerevisiae (KHR and KHS) was demonstrated by Kitano e\ aL, (1988).

Similar conclusions have been reached about many non-Saccharomyces killer yeast as researchers have failed to isolate non-nuclear genes encoding for killer toxin production (Young, 1987). Table 2.16 indicates possible chromosomal locations of genes encoding for killer toxin production in non- Saccharomyces species.

Table 2.16 Species of non-Saccharomyces yeast where chromosomal genes are implicated in the production of killer activity.

Yeast Reference Candida glabrata Sriprakash and Batum (1984)

Candida spp. SW-55 Yokomori e\ a\. (1988)

Pichia kluyveri Starmer e\a\. (1987)

Pichia farinosa Suzuki and Nikkuni (1989)

Hansenula anomala Kagiyama e\ aL (1988)

Hanseniaspora spp. Shimizu et aL (unpublished work) 79

2.64 Mode of action of killer toxin

The mode of action of killer toxins has been most extensively reviewed in the S. cerevisiae K1 system (Bussey et aL, 1990; Polonelli et aL, 1991). The first stage of the process involves the binding of the toxin to a cell wall receptor (Al-Aidroos and Bussey, 1978; Bussey et aL, 1979) which has been shown to be a (1-»6)-p-D-glucan for both K1 and K2 killer toxins (Bussey, 1981;

Hutchins and Bussey, 1983). In contrast, the KT 28 toxin of S. cerevisiae strain 28 binds to a mannoprotein cell wall receptor (Schmitt and Radler,

1987). Binding of the toxin to the cell wall receptor is an energy-independent step which is complete in 3 minutes at 20°C and pH 4.7 (Bussey et aL, 1979). Studies utilising spheroplasts of S. cerevisae indicate that cell wall receptors determine the specificity of the toxin. When applied to whole cells, the K1 toxin is only effective against strains of S. cerevisiae and Torulopsis glabrata (Bussey and Skipper, 1975; Young and Yagiu, 1978). When applied to spheroplast preparations, however, the spectrum of activity was extended to yeast belonging to the genera Candida, Kluyveromyces and Sch wanniomyces.

Following binding to the cell wall, the toxin is then inserted into the cell membrane via an energy-dependant step where it disturbs proton permeability and leads to efflux, a lowering of intracellular pH, metabolic inhibition, loss of ATP and finally cell death (De la Pena et aL,

1980, 1981). Bussey et aL (1990) postulate that the killer toxin upsets the energised membrane state, although whether this occurs by affecting a component of the proton pump or by forming a protein channel is not yet clear. However, Kagan (1983) found direct evidence for the formation of a protein channel in the lipid bilayer of the cell membrane through the action of a killer toxin from Pichia kluyveri. The toxin from this organism has been shown to exert physiological effects similar to the K1 toxin upon sensitive 80 strains of S. cerevisiae. These channels provided a pathway for the leakage of major cations such as K+ and H+ in addition to dissipating ionic gradients such as the proton gradient required for transport processes across the cell membrane. However, it is still not clear whether this process occurs with the K1 or K2 toxins of S. cerevisiae.

2.65 Occurrence of killer yeasts in the wine industry

Killer yeasts involved in winemaking may originate as wild strains from the surfaces of grape berries and winery equipment, or from culture collections that are commercially available to the winemaker (van Vuuren and Jacobs,

1992). The occurrence and impact of killer yeast in the wine industry has been reviewed by Shimizu (1993).

The incidence of wild killer strains of S. cerevisiae in wineries shows considerable regional variation (Table 2.17).

Table 2.17 Occurrence of wild killer strains of Saccharomyces cerevisiae in various winemaking countries

Country Incidence observed in Reference isolated strains (%) Australia 21 Heard and Fleet (1987) Spain 21 CansadoetaL (1989) Brazil >27 Pasqual et aj. (1990) Italy 12-83 Rosini et aL (1988) France 83 Cuinier and Gros (1983)

In addition to killer strains of S. cerevisiae, the influence of non- Saccharomyces killer yeast on wine fermentations should also be considered due to their ability to grow to high concentrations during the early stages of wine fermentations (Fleet, 1990). Radler and Knoll (1988) showed that a killer strain of Hanseniaspora uvarum delayed the complete fermentation of a 81 grape juice by a sensitive strain of S. cerevisiae by 10-20 days. Similar effects on wine fermentations have been observed with killer strains of Pichia fermentans and Hansenula anomala (Benda, 1989). A summary of non- Saccharomyces killer yeast reported to kill strains of S. cerevisiae is given in Table 2.18.

Table 2.18 Wild non-Saccharomyces killer yeast reported to kill strains of Saccharomyces cerevisiae

Yeast species Reference Candida krusei Radler (1980)

Hansenula anomala Rosini (1983, 1985) Nomoto et aL (1984)

Pichia fermentans Pfieffer and Radler (1984)

Pichia kluyveri Zorg et ah (1988)

Hanseniaspora uvarum Radler and Knoll (1988) Zorg et aj. (1988) Radler etaj. (1990)

Heard and Fleet (1987), however, were unable to demonstrate the reverse situation ie. killer strains of S. cerevisiae were unable to kill strains of

C. krusei, C. pulcherrima, Kl. apiculata and H. anomala isolated from wine.

Due to strain variation, a larger survey of wine yeasts would need to be examined to state whether or not this effect applies to all killer strains of S. cerevisiae.

Killer strains of S. cerevisiae have been found in culture collections of wine yeasts which are used commercially to conduct fermentations. This has become a desirable trait in wine yeast because of the perceived advantages 82

of preventing growth of wild sensitive yeast strains and immunity against killing from wild killer yeast (Shimizu, 1993).

2.66 Factors affecting the activity of killer toxin during winemaking

The activity of killer toxin during vinification is profoundly affected by the wine environment. The most active killer toxin in such conditions is the K2 toxin

because it retains activity at the low pH values of wine (Shimizu et aL, 1986).

Table 2.19 summarises the factors, in addition to pH, which affect the efficacy of the K2 toxin in wine.

Table 2.19 Factors affecting the activity of the K2 killer yeast toxin in wine.

Factor Effect on toxin activity References pH Decreases with decreasing pH Ciolfi (1984) below 4.0 Shimizu et aL (1986) Heard and Fleet (1987)

Ethanol Decreases with increasing Shimizu (unpublished work) ethanol concentration

Temperature Decreases rapidly above 25°C Shimizu (unpublished work)

Fining agents Bentonite binds with toxin and Barre (1980) reduces activity Radler and Schmitt (1987)

Relative Killer strains with vigorous Radler (1988) fermentative growth are more likely to ability of strains dominate the fermentation

Ratio between In general, toxin activity Heard and Fleet (1987) killer and increases as the proportion of Petering et aL (1991) sensitive yeast killer yeast increases Van Vuuren and Jacobs (1992) Shimizu (1993) 83

Of the factors listed in Table 2.19, the greatest variation in influence on the efficacy of killer toxin appears to be the ratio of killer to sensitive cells at the start of fermentation. This point is illustrated in Table 2.20 which shows the reported ratios of killer to sensitive yeast found to cause sluggish wine fermentations in various wine-producing countries.

Table 2.20 Minimum ratio of killer : sensitive strains of Saccharomyces

cerevisiae found to cause stuck or sluggish wine fermentations3

Country Ratio Reference Killer Sensitive Australia 1 1 Heard and Fleet (1987) 1 2 Petering et aL (1991)

France 1 50 Barre (1980)

Germany 1 1000 Radler (1988)

Japan 25 1 Seki eta). (1985)

Japan 100 1 Shimizu et aL (1985b)

South Africa > 1 40 Tredoux et aL (1986)

South Africa 1 500 van Vuuren and Wingfield (1986)

Former USSR 1 20 Tyurina et a]. (1986)

a Adapted from van Vuuren and Jacobs (1992)

Jacobs and van Vuuren (1991) suggest that the wide variation in killer to sensitive ratios (Table 2.20) is dependent on several factors, including the amount and activity of the toxin secreted by the killers, the relative growth rate

of the killer compared to the sensitive strain, and the susceptibility of sensitive 84 strains. Such observations reflect the influence that genetic variation has upon the activity of killer yeast in winemaking.

If wine fermentations are to be conducted with sensitive strains of S. cerevisiae, Radler and Schmitt (1987) recommended that protein- adsorbing materials such as bentonite or yeast cell walls should be added to inactivate any killer toxin produced by wild killer yeast.

2.67 Impact of killer yeast on wine fermentations

The most widely reported problems associated with wild killer yeast strains of S. cerevisiae have involved slow or stuck fermentations where the wild killer inactivates the inoculated strain of S. cerevisiae (van Vuuren and Wingfield,

1986; Heard and Fleet, 1987; Radler, 1988; Jacobs and van Vuuren, 1991). Notably, these observations have not been restricted to killer strains of S. cerevisiae as protracted fermentations have also been obtained with non- Saccharomyces species of yeast such as Hanseniaspora uvarum (Radler and Knoll, 1988) and Pichia fermentans and Hansenula anomala (Benda, 1989). Fermentations infected with wild killer yeast have been reported to contain elevated levels of acetaldehyde, fusel oils, lactic acid and acetic acid which can depreciate wine quality (Benda, 1985; van Vuuren and Jacobs, 1992).

Recently, research has been directed towards positive applications of the killer phenomenon in wine fermentations. As mentioned earlier, killer yeast with desirable winemaking properties may be used to kill wild sensitive yeast with poor vinification properties. These yeasts will also possess immunity against killer activity of any wild yeast (Shimizu, 1993). Researchers have utilised genetic manipulation to cross-breed potent strains of killer yeasts with desirable winemaking strains to further improve their winemaking performance. Hara et a]. (1981) cross-bred a killer yeast isolated from sake 85 fermentation with a winemaking yeast to control a film-forming spoilage yeast in wine fermentations. Transfer of killer traits is accomplished with yeast containing dsRNA plasmids or linear DNA plasmids encoding for killer activity (Shimizu, 1993). Such procedures are usually performed by protoplast fusion and may be of two types the first is where only the killer plasmids are transferred without changing the basic genetic material of the wine strain

(Yokomori et a!., 1989), and the second involves the formation of a true hybrid with an intermediate genetic karyotype (Shimizu et aL, 1986; Yokomori et aL,

1989, 1990). Such genetically modified yeast have already been successfully utilised in commercial wine fermentations (Yokomori et aL, 1990). 86

3.0 Development of Methods

3.1 Measurement of protein release by yeasts in sparkling wine

3.11 Introduction

Reliable estimation of the concentration of soluble protein in wines has proved difficult to achieve because of the complex chemical nature of the wine, itself. This problem is reflected in the wide variation of values reported for the protein content of wine. These values range from those as little as 1.5 mg/L (Yokotsuka et aL, 1977) to as high as 840 mg/L (Somers and Ziemelis,

1973). Although there is natural variation in the protein content of wines, the discrepancy in reported values is due, in part, to the range of different methods used to determine the protein concentration and the degree of interference from other wine components. As an example, the Kjeldahl method for measurement of total nitrogen, although regularly utilised in the brewing industry to estimate the protein content of beer (Anonymous, 1992), cannot be applied directly to wine to determine soluble protein because wines contain significant amounts of non-protein nitrogen (Koch and Sajak, 1959; Anelli, 1977).

To avoid this interference, many authors have attempted to first isolate the protein by precipitation and then measure the concentration of the partially purified material. Chemicals such as ethanol, ammonium sulphate, trichloroacetic acid, phosphotungstic acid and phosphomolybdic acid have been used to precipitate wine protein (Somers and Ziemelis, 1973). Following precipitation, various methods have been used to determine the protein content of the isolated fraction. These include a modified Kjeldahl procedure (Koch and Sajak, 1959), a colourimetric procedure based on the original Lowry et aL (1951) method (Bayly and Berg, 1967), acid hydrolysis and amino acid determination of the isolated fractions (Anelli, 1977), and dye- 87 binding with amido black (Yokotsuka et aL, 1978). However, Bensadoun and Weinstein (1976) showed that, at very low concentrations (such as that occurring in grape juice and wine), protein is not quantitatively precipitated and that other compounds can be precipitated which interfere with the protein assays (Ferenczy, 1966). Additionally, measurement of protein by amino acid analysis following acid hydrolysis is inaccurate because some amino acids such as tryptophan are destroyed (Modra, 1989).

A method for the direct measurement of various protein fractions in wine was suggested by Somers and Ziemelis (1973) which involved isolation of the

protein fractions by gel chromatography. Protein concentration was calculated from a calibration line devised from a regression analysis of absorbance at 280 nm of the column eluate versus protein content. Protein content, however, was determined from analysis of total nitrogen of the

isolated column eluate which was assumed to be free from non-protein nitrogen.

An alternative method for the rapid analysis of soluble protein in wine is based on binding of Coomassie brilliant biue G-250 (Ngaba-Mbiakop, 1981;

Hsu and Heatherbell, 1987 a,b; Hsu Mai-, 1987; Murphy eta}., 1989). It is a modification of the original Bradford (1976) method based on the shift from 465 to 595 nm of the absorbance maximum of Coomassie brilliant blue upon binding to protein (Sedmak and Grossberg, 1977).

However, as discussed by Modra (1989), the Coomassie brilliant blue assay is subject to interference by phenolic compounds (Compton and Jones, 1985; Mattoo et aL, 1987). Modra (1989) studied the chemistry associated with interference by phenolic compounds of the Coomassie brilliant blue assay in a Gordo grape juice. The author concluded that the assay was subject to 88 minimal interference by monomeric phenols and that polymeric phenols also did not to interfere with the assay provided these compounds were bound to protein. However, once the wine protein became saturated with polymeric phenols, the presence of free polyphenolic compounds produced significant interference. The author concluded that the assay was suitable for grape juice (in this case, Gordo grape juice) and wines where all the polymeric phenols are bound to protein. Murphy et aj. (1989) investigated the possible interference of this method from phenolic compounds in the assay of several white wines (Gewurztraminer, White Reisling and ). Although these authors found a delayed reaction in binding of the dye to protein (possibly due to hindrance at binding sites by high molecular weight phenols already bound to wine proteins), they did not find significant interference from the free phenolic component of the wines. They concluded that, in the presence of phenolic compounds, the Coomassie brilliant blue assay provides the most reliable results. Their findings support work done by Robinson (1979) who evaluated several methods of protein determination in plant extracts containing polyphenolic material and found the Coomassie brilliant blue assay to be the most reliable method.

It would appear, therefore, that the Coomassie brilliant blue assay is the most acceptable method for determining soluble protein in wines in the presence of phenolic compounds. For the present study, a method was required that will reliably indicate changes in the soluble protein content of sparkling wines due to autolysis of yeast during storage of the product. Possible interference by phenolic components in the wine may simply be determined by spiking the wine with increasing amounts of protein. According to Modra (1989), if free polyphenolics are present, a non-linear response to the added protein would occur. The objective of this section of work, therefore, was to confirm that the 89

Coomassie brilliant blue assay is a reliable method for detecting and quantifying increases in soluble protein during the storage of sparkling wines.

3.12 Materials and Methods (Murphy et aL, 1989)

3.121 Soluble protein assay

Commercial wine samples from wineries located at Wagga and Nuriootpa

(Section 4.21) were clarified by passage through a 0.45 pm filter and 4.8 mL

of the filtrate incubated with 1.2 mL of Coomassie brilliant blue dye reagent (Bio-Rad Laboratories, Richmond, CA.). The mixtures were vortexed and then incubated at room temperature before absorbance was measured at 595 nm. Absorbance was measured in a 1 cm path length cuvette on a Shimadzu

UV-120-02 spectrophotometer. The blank contained 4.8 mL of distilled water mixed with 1.2 mL of the dye reagent. Standard curves for estimation of soluble protein content were prepared using bovine serum albumin (BSA) obtained from Calbiochem (product number 12567).

3.122 Rate of formation and stability of protein-dye complex

To investigate the effect of grape phenolic compounds on the formation of the protein-dye complex, the rate of colour development and stability of the colour was determined over 60 minutes at 5 minute intervals for the Wagga and Nuriootpa commercial wines. Reagents and absorbance measurements were as described in the soluble protein assay procedure. The optimal incubation time for the assay was taken as the time required for maximum colour development to occur. 90

3.123 Linearity of response of method to standard protein addition

As indicated by Modra (1989), the presence of free grape polyphenolic compounds in wine can be expected to cause a non-linear response to protein addition in the assay. To test this effect, Wagga and Nuriootpa wines were filtered through a 0.45 pm filter and 1.9 mL samples of wine spiked with 0.1 mL of 0, 10, 20, 30 and 40 pg/mL of BSA to simulate total protein increases of 0, 0.5, 1.0, 1.5 and 2.0 pg/mL. To this mixture was added 0.5 mL of dye reagent and the solution vortexed and incubated at room temperature for 35 minutes (as indicated by the results of Section 3.131.).

3.13 Results and discussion

3.131 Rate of formation and stability of protein-dye complex

Figure 3.11 shows that colour development of the Coomassie brilliant blue assay for protein in the Wagga and Nuriootpa sparkling wines was a delayed process requiring at least 35 minutes for completion. Murphy et ai- (1989) encountered a similar phenomenon in protein assays of Gewurztraminer,

White Reisling and Chenin Blanc wines where 55 minutes at room temperature were required for maximum colour development.

The authors suggested that the delayed binding process was due to hindrance at the binding sites by high molecular weight phenols already bound to the wine protein. The quicker response time found in the sparkling wines used in this study suggests that their phenolic contents are less than those in the wines examined by Murphy et aL (1989). It should be noted, though, that the colour development of standard BSA with Coomassie brilliant blue is complete within 2 minutes and is stable for up to one hour (Murphy et 91 aL, 1989). To simplify the control assay, therefore, standard BSA may be incubated for the same period of time as the commercial wines.

0.25

B 0.23

in 0.21 -

0.19 -

'B 0.17 -

0 10 20 30 40 50 60 70 Incubation time (minutes)

Figure 3.11 Rate of formation and stability of colour complex in two commercial sparkling wines assayed for soluble protein using the Coomassie brilliant blue assay ( Wagga □, Nuriootpa A commercial sparkling wines ).

3.132 Response of dye method to addition of known concentrations of protein

Samples of Wagga and Nuriootpa wines were incubated with increasing amounts of BSA to observe whether grape phenolics present in the wines interfered with the assay. According to Modra (1989), the presence of free polyphenolic compounds can be expected to result in a non-linear response of the assay to protein addition. Figure 3.12 shows that each commercial wine gave a linear response to addition of known concentrations of standard protein. However, Figure 3.12 also shows that the Wagga sparkling wine (Figure 3.12a) exhibited a lower unit increase in absorbance in comparison to standard BSA alone. This is probably due to competition at the binding sites 92

by phenolic compounds already bound to wine proteins (Murphy et aj., 1989). This does not detract from the utility of the method, though, as a correction factor for calculation of increases in protein concentration in the Wagga sparkling wine may be estimated from the curve in Figure 3.12a. This is summarised in Table 3.11.

0.07

0.06 es' 0.05 to LOCTn 0.04 ^QJ 0.03 g

0.02 con 0.01 <

0.00 0 12 Added standard protein (ug/ml)

0.34 0.07 0.06 s G S 0.32 0.05 in CJN LO 0.04 QJ 0.30 0.03

0.02 < 0.28 0.01 < 0.27 0.00 0 12 Added standard protein (ug/ml)

Figure 3.12 Colour response of (a) Wagga and (b) Nuriootpa sparkling wines to added bovine serum albumin (BSA) as measured by the Coomassie brilliant blue assay (Commercial sparkling wines Wagga □ and Nuriootpa A, and standard bovine serum albumin 0 ) 93

Table 3.11 Increase in absorbance at 595 nm in response to additions of standard protein (BSA) in two Australian sparkling wines using the Coomassie brilliant blue assay.

Amount of standard Absorbance (595 nm) protein (BSA) added (pg/mL) Standard BSA in Wagga sparkling Nuriootpa distilled water wine sparkling wine 0.5 0.012 0.010 0.018 1.0 0.028 0.021 0.036 1.5 0.056 0.032 0.051 2.0 0.068 0.044 0.066

Regression analysis of the data in Table 3.11 for sparkling wines gives increases of 0.022 and 0.033 absorption units for 1 pg BSA added per mL of

Wagga and Nuriootpa wines, respectively. This compares with an increase of 0.035 absorption units for BSA in distilled water.

It may be concluded, therefore, that the Coomassie brilliant blue assay can be applied to reliably indicate increases in protein content in the sparkling wines as a result of yeast autolysis during storage of the product. For the

Wagga sparkling wine, however, the application of a correction factor is required in order to account for a lowered response of the assay to added protein. 94

3.2 Methods for the determination of nucleic acids released during fermentation and ageing of sparkling wines

3.21 Introduction

The enzymatic degradation of nucleic acids during yeast autolysis and release of the breakdown products into the extracellular environment are well known phenomena (Hough and Maddox, 1970; Trevelyan, 1978a; Halasz and

Lasztity, 1991; Charpentier and Feuillat, 1993). Autolytic degradation of yeast nucleic acids generates small molecular weight products such as guanosine monophosphate which have recognised flavour enhancing properties (Benaiges et aL, 1990). The production of these compounds is thought to contribute to the flavour of fermented foods and beverages such as beer (Ziegler and Piendl, 1976; Qureshi et a]., 1979) and wine (Leroy et aL, 1990). As the production of sparkling wines involves lengthy storage times on lees (at least 6 months), there is the possibility that the yeast deposit will undergo autolysis and release nucleic acids and their breakdown substances that will contribute to the overall flavour of the product.

The nucleic acid content of wines is an area which has received iittie attention in the literature. Somers and Ziemelis (1972) reported a 'total nitrogenous bases' concentration of 85 mg/L in a Reisling wine which included nucleotidic compounds. These authors utilised a gel chromatography method with continuous monitoring of the eluate to separate various UV-absorbing fractions of the wine. The nucleic acid bases guanine, cytosine and uracil were identified by paper chromatography following perchloric acid hydrolysis of the nucleotidic fraction. However, the authors used spectral profiles (265, 280 and 320 nm) as a means of separating the various fractions and could only provide a semi-quantitative estimate of the nucleic acid content of the wine. Leroy et aj. (1990), in a study of yeast autolysis during the ageing of sparkling wine, 95 reported a concentration of about 400 mg/L for total nucleic acids in a wine base prior to the secondary fermentation. Unfortunately, details of the method used to measure the dissolved nucleic acid concentration of the sparkling wine were not provided.

It was decided, therefore, that a new method would be developed for the measurement of nucleic acids in wines. An important consideration in such a study is the origin of these nucleotidic compounds. In sparkling wines, the major sources of nucleic acids are the grape, the primary fermentation and the yeast deposit upon which the product is aged following the secondary fermentation. However, any increases in the concentration of nucleic acids in sparkling wines during the ageing period can only come from the yeast deposit. In yeast, RNA represents approximately 95% of the total nucleic acids (Mounolou, 1971; Trevelyan, 1978b). Thus, it can be assumed that RNA forms the predominant source of nucleic acids that could be released during sparkling wine ageing.

Classical procedures for assaying RNA have been based on its extraction from microorganisms followed by either (i) measurement of UV absorption of the bases, (ii) colourimetric determination of the sugar (ribose) by the orcinol method, or (iii) colourimetric determination of the phosphorus content.

However, the accuracy of such methods is determined by the presence of interfering compounds which may be extracted with the nucleic acid material. These procedures and their limitations have been described by Herbert et a]. (1971). Measurement of the ribose sugar by the orcinol procedure, for example, is subject to interference by other sugar moieties, while measurement of the UV absorbance of the nucleic acid bases is subject to interference by various amino acids found in proteins. Thus, these methods have been developed with the strategy to minimise the presence of these interfering 96 compounds. As mentioned already, they are based on initial extraction of nucleic acids from the microorganisms and so direct application of these methods to sparkling wine is not valid since they cannot eliminate interfering species that would be present in aqueous systems. In wines, for example, the presence of pentose sugars of non-nucleic acid origin (xylose and ) occur at concentrations of approximately 375 mg/L (Franta et a|M 1986).

Fluorometric analysis of RNA and DNA involving the binding of various dyes such as ethidium bromide (Le Pecq and Paoletti, 1966; Le Pecq and Paoletti,

1967; Abdel-Rahman et aL, 1975; Boer, 1975; Karsten and Wollenberger, 1977), ethidium homodimer (Markovits et aL, 1979; Moyer et aL, 1990), dansylprotamine (Nakamura et aL, 1984) and thiazole orange homodimer and oxazole yellow homodimer (Rye et aL, 1993 a,b) have been described. Although these methods show greatly improved sensitivity, they would experience interference from chlorophyll in sparkling wines since chlorophyll has fluorescent properties. Additionally, some of these dyes are toxic to humans and show different responses to DNA and RNA. Ethidium bromide, for example, is carcinogenic and demonstrates a two fold increase in fluorescence for DNA over RNA (Le Pecq and Paoletti, 1967).

Due to the presence of various interfering species, several authors when determining the nucleic acid content in aqueous systems have attempted to isolate the nucleic acid fraction before measurement. The majority of this work has been developed for measurement of DNA in marine environments and is summarised in Table 3.21. 97

Table 3.21 Published methods for measurement of nucleic acid released into the external environment.

Method of nucleic acid isolation Basis of detection Reference

Cation exchange, lyophilization Fluorescent measurement of Minear (1972) and gel chromatography to isolate DNA using 3,5-diaminobenzoic the high molecular weight fraction acid (DABA)

BaSC>4 precipitation and UV spectroscopy of nuclear Pillai and hydrolysis of precipitate base for determination of DNA Ganguly (1972)

Ethanol precipitation (48 hours), Fluorescent measurement of Deflaun et a\. dialysis against water (48 hours), DNA using Hoechst 33258 dye (1986) dialysis against salt buffer (24 hours)

Binding of nucleic acid onto Colourimetric determination of Hicks and hydroxyapatite and elution with a RNA using the orcinol reagent Riley (1980) strong salt solution at 609C for pentose measurement

Cetyltrimethylammonium (CTAB) Isocratic HPLC measurement Breter et aL precipitation and acid hydrolysis of thymine for the (1977) of isolate determination of DNA

Precipitation of nucleic acids with Fluorometric measurement of Karl and Bailiff CTAB DNA using DABA and (1989) colourimetric measurement of RNA using orcinol reagent

Of the procedures listed in Table 3.21, the method of Minear (1972) could not be used as it was designed for isolation of high molecular weight DNA only and could not be adapted for RNA. The method of Pillai and Ganguly (1972) was impractical because BaS04 treatment precipitates polysaccharides as well as nucleic acids that would interfere with subsequent measurement of ribose, while the method of Deflaun et al. (1986) is inconvenient and time 98 consuming (5 days per sample) and measures only DNA. Therefore, the isolation of nucleic acid material with hydroxyapatite and cetyltrimethylammonium bromide (CTAB) appeared the most applicable to nucleic acid analysis in sparkling wines. RNA can then be quantified by reaction of its ribose content with orcinol using the method of Herbert et aL (1971).

Measurement of nucleic acid components by HPLC also merits investigation because of the high specificity offered by this method. HPLC has the potential to measure nucleotides, nucleosides and nucleobases that would be produced by autolytic breakdown of RNA and DNA (Ohta et aL, 1971).

Alternatively, HPLC may form the basis of a method for the quantitative measurement of total nucleic acids through the assay of guanine. In the analysis of DNA in seawater, Breter et aL (1977) extracted nucleic acid by CTAB precipitation before acid hydrolysis with to breakdown the nucleic material to the nucleobase form. Thymine (specific to DNA) generated in this reaction was then detected by HPLC. In a modification of this concept, sparkling wine samples may be hydrolysed and the resultant formation of guanine analysed as a marker for RNA concentration. As RNA forms about

95% of total nucleic acid in yeasts (Mounolou, 1971; Trevelyan, 1978a), measurement of guanine is essentially a measure of RNA content.

In summary, the options for measurement of nucleic acids in wines include :

(i) isolation and separation of nucleic acids from wine using hydroxyapatite or CTAB followed by assay of the ribose content of the isolated fraction by the method of Herbert et aL (1971), or 99

(ii) use of HPLC to assay individual breakdown products of nucleic acids, or total nucleic acid content by measuring the guanine content of wine following acid hydrolysis.

This section investigates the utility of the approaches detailed above. The recovery of standard yeast RNA from sparkling wine using hydroxyapatite and

CTAB was examined and compared to measurement of nucleic acid breakdown products using HPLC.

3.22 Materials and Methods

3.221 The orcinol procedure for RNA determination - interference from non-RNA sugars

In the orcinol procedure of Herbert etaj. (1971), sample (1.0 mL) is mixed with freshly prepared orcinol reagent (iii) (3.0 mL) in a 20 mL test-tube with a teflon- lined . Orcinol reagent (iii) is made up of 4 volumes of reagent (i) (0.90 g FeCl3.6H20 in 1 L concentrated HCI) mixed with 1 volume of reagent (ii) (1.0 g orcinol in 100 mL distilled water).

The mixture is heated in a boiling water bath for 20 minutes, cooled to room temperature and made to 15 mL with n-butanol before reading the absorbance at 672 nm on a Shimadzu UV-120-02 spectrophotometer. Standard RNA is made up in distilled water using RNA extracted from Saccharomyces cerevisiae (Boehringer Mannheim 109223).

Interference from the presence of non-RNA sugars (principally hexoses contained in polysaccharide material originating from or microorganisms) can be detected by observing the absorption spectra (500- 100

700 nm) of wine samples treated with the orcinol procedure (Herbert et aL, 1971). Colour complexes formed by the reaction of RNA-ribose give a single peak at 672 nm while complexes formed in the presence of contaminating hexoses give an additional peak in the range 550-570 nm. This possibility was tested in the Wagga sparkling wine (359 days storage) and compared with pure RNA (60 pg/mL) and the hexose sugar, mannose (a component of yeast cell-wall polysaccharides).

3.222 Removal of polysaccharides in sparkling wines by two phase extraction (Kirby, 1956)

In an attempt to remove polysaccharides that caused interference with the analysis of ribose by the orcinol method, a two phase liquid extraction method described by Kirby (1956) was applied to commercial and model sparkling wines. This procedure had originally been developed to reduce the polysaccharide contamination of nucleic extracts of animal tissue. Sparkling wine (10 mL) was mixed with 10 mL of 2.5 M K2HPO4,0.5 ml_ 33.3% H3PO4 and 10 mL of 2-methoxyethanol. The mixture was shaken thoroughly and allowed to separate into 2 layers (upper phase : lower phase, 5:1).

Polysaccharides precipitated and formed a cloudy layer at the interface, the nucleic acids being contained in the upper layer. Samples of the upper layer were tested for interference of RNA estimation by polysaccharides using the orcinol procedure (Section 3.221).

3.223 Isolation of nucleic acids from sparkling wines using hydroxyapatite

The recovery of standard RNA and RNA from commercial sparkling wine was assessed using the method of Hicks and Riley (1980). Hydroxyapatite (Bio-

Rad) was prepared according to company specifications and packed into a 101 glass tube (1 cm diameter) to a height of 5 cm. The recovery of RNA was determined by first diluting a 2 mL aliquot of standard RNA (200 pg/mL) to 100 mL with 0.005M (pH 6.8) phosphate buffer and allowing this to pass through the column at the rate of 3 mL/min. The 0.005M phosphate buffer was a 1/100 dilution of a 0.5M phosphate buffer that was prepared by adding 0.5M KH2PO4 to 500 mL of 0.5M K2HPO4 until the pH was 6.80 ± 0.02. The column was then washed with 20 mL of 0.005M phosphate buffer (discarded) and immersed in a waterbath at 60gC. Following thermal equilibrium, the column was eluted with 20 mL of 0.5M phosphate buffer at a flow rate of 0.3 mL/min.

The eluate, containing the nucleic acid fraction, was tested for RNA by the orcinol procedure. Although the original method of Hicks and Riley (1980) specified adjustment of sample pH to 6.8 with the 0.5M phosphate buffer, the low pH and natural buffering capacity of sparkling wine required the use of 0.5M KOH (4.7 mL in 20 mL sparkling wine) for this purpose. The pH-adjusted sparkling wine (20 mL) and sparkling wine spiked with 350 pg of standard RNA

(0.5 mL of 700 pg/mL RNA) were diluted to 200 mL with 0.005M phosphate buffer and applied to a column of hydroxyapatite as above to assess recovery of RNA from sparkling wine.

In order to improve the recovery of RNA, the method was also modified by decreasing the concentration of the washing buffer from 0.005 to 0.001 M.

3.224 Isolation of nucleic acids from sparkling wines using CTAB a) Method of Karl and Bailiff (1989)

The recovery of RNA using cetyltrimethylammonium bromide (CTAB) was initially assessed using the method of Karl and Bailiff (1989). Sparkling wine (2 mL diluted to 30 mL, pH adjusted to 7.00 with 0.5 M KOH) was mixed with 2 mL of CTAB solution (0.5 g CTAB in 100 mL 0.5M NaCI), shaken gently and 102

frozen at -20gC. After thawing, the precipitate was filtered onto Whatman GFF filters, dried at 60gC for 1 hour and then vortexed with 3 mL 1M HCI to redissolve the nucleic acid material. The concentration of RNA in the extracted material was then determined by the orcinol procedure. Recovery of standard RNA added to Nuriootpa and Wagga wines (300 pg and 100pg, respectively) was also determined. Wine pH was adjusted to 7.0 with 0.5M

KOH following dilution of 2 mL wine to 30 mL with distilled water. It was

necessary to decolourise the wine prior to analysis because pigments were

also extracted with CTAB and interfered with the subsequent determination of

orcinol readings. Pigments were removed using a Water's Sep-pak C^g cartridge; the cartridge was washed sequentially with 2 mL methanol, 2 mL distilled water and then the wine sample passed through. No loss in nucleic acid material occurred during this step (determined separately).

The method was also modified by increasing the concentration of the original CTAB solution from 0.5 g to 1.25 g/100 mL of 0.5M NaCI.

b) Methods of Sclafani and Wechsler (1981) and Sibatani (1970)

The efficiency of extraction of standard yeast RNA using CTAB was assessed

using two further methods. In the first method (Sclafani and Wechsler, 1981), 1 mL of standard RNA solution (300 pg/mL) was diluted to 37 mL with distilled water, and then 2 mL of 0.25M EDTA (pH 6.0) and 1 mL of CTAB solution (1% w/v in 0.025M EDTA, pH 6.0) were added. In the second method (Sibatani,

1970), 1 mL of standard RNA solution (300 pg/mL) was diluted to 22 mL and

10 mL of 0.2 M EDTA (pH 6.0) and 8 mL of CTAB solution (0.8% w/v in 0.025M

EDTA, pH 6.0) added. The solutions were frozen overnight at -209C, thawed, and the filtered precipitate redissolved in 1 M HCI. The HCI solution was used to redissolve the precipitate in preference to 0.1M NaOH as it gave higher recoveries (determined separately). 103

3.225 Measurement of nucleic acid breakdown products by HPLC

(a) Separation of ribonucleotides, ribonucleosides, deoxyribonucleotides, deoxyribonucleosides and bases by HPLC (Zhao, Todd and Fleet, 1994)

Breakdown of nucleic acids during yeast autolysis will ultimately result in the formation of nucleotides, nucleosides and nuclear bases (Hough and

Maddox, 1970). Measurement of these components may then be used as a marker of nucleic acid breakdown during autolysis. A method was therefore developed to separate a mixture of 21 nucleotides, nucleosides and bases in sparkling wine and in simple defined media.

In this procedure, wine samples were prepared for analysis by filtering through a 0.45 pm membrane filter (Millipore, Sydney, Australia). The HPLC system consisted of : a Bio-Rad HPLC Gradient Processor System which included two Bio-Rad model 1330 HPLC pumps and a Bio-Rad Automation Interface with an Apple model lie computer; a Waters U6K injector and a Waters model 440 Absorbance Detector with two wavelengths of 245nm and 280nm. Retention times and peak areas or peak heights were measured with a Waters 745 Data Module. The column was a Waters Resolve C-|0 5p

Radial-Pak cartridge (8mm x 100mm I.D.) equipped with a Waters RCM-100 cartridge holder. Packing material of the cartridge was a silica based reversed-phase C13 HPLC matrix of 5-pm spherical beads.

Separation was performed at room temperature using a two solvent gradient program. Solvent A was a solution of 0.02M K2HP04 with pH adjusted to

6.30 using concentrated potassium hydroxide solution. Solvent B was a solution of methanol in distilled, de-ionised water (60% v/v). Prior to use, the eluents were filtered through a membrane filter, pore size 0.45 pm (Millipore,

Sydney, Australia), and degassed by vacuum and sonification for 5 min. 104

Before injection of samples, the column was equilibrated with solvent A for 5 min at a flow rate of 3 mL/min. This step served two purposes. Firstly, it washed out residuals from previous injections and secondly, it stabilised the baseline of the system for the next injection.

After injection of the sample (20 pi), elution was started with 100% solvent A and 0% solvent B. The ratio of solvent B in the elution system was increased linearly from 0% to 40% over 18 min. Solvent B was then increased to 100% over 5 min. and the column was then flushed with 100% solvent B for a further 2 min. in order to remove strongly absorbed compounds. The system was automatically returned to its starting condition (ie. 100% solvent A and 0% solvent B) over 5 min. by computer control. The elution of one sample took 35 minutes with the solvent flow rate being maintained at 3 mL/min throughout the program.

Standards of individual nucleotides, nucleosides and nucleic acid purine and pyrimidine bases (as listed in Table 3.22) were purchased from United States Biochemical Corporation (Cleveland, Ohio, USA). Stock solutions of standards (1% w/v) were made up by dissolving individual compounds in de-ionised water and were stored at -20°C. Working solutions of standards were made by diluting the stock solutions to 0.01% (w/v). 105

Table 3.22 Nucleotides, nucleosides and purine and pyrimidine bases separated by HPLC by the method of Zhao et aL (1994)

Class of component Name of component Retention time (min.)a Purine and pyrimidine bases Adenine 11.85 ±0.05 Cytosine 2.14 ±0.02 Guanine 7.07 ± 0.03 Uracil 2.64 ±0.02 Thymine 6.45 ±0.05

Nucleosides Adenosine 14.68 ±0.05 Cytidine 3.94 ± 0.03 Guanosine 9.55 ±0.04 Uridine 5.25 ± 0.03 Deoxyadenosine 16.18 ± 0.08 Deoxycytidine 5.73 ± 0.03 Deoxyguanosine 10.81 ±0.05 Thymidine 11.26 ±0.05 Nucleotides 5'-AMP 3.41 ±0.02 5'-CMP 1.00 ± 0.02 5'-GMP 1.67 ±0.02 5'-UMP 1.15 ± 0.02 5'-dAMP 6.10 ±0.03 5'-dCMP 1.34 ± 0.02 5'-dGMP 3.65 ±0.02 5'-dTMP 3.04 ±0.02

a Data indicate average retention times ± variation taken from 10 analyses

(b) Acid hydrolysis of nucleic acid material and measurement of

breakdown products by HPLC (guanine-HPLC) (Todd, Zhao and Fleet,

1995)

In the procedure, samples of standard RNA or wine (1 mL) were lyophilised in a 10 mL screw-cap test-tube and then hydrolysed with 1 mL of 2M HCIO4 in a

boiling waterbath for 60 minutes. To prolong the life of the HPLC column, the

pH of the hydrolysate was adjusted to within the range pH 3.0 to 8.0 by using a 106 combination of 0.1 g Na2CC>3 and 0.2 mL 0.75 M EDTA (tetra Na+ form) or, more simply, by addition of NaHCC>3 until evolution of CO2 gas ceased. Prior to injection, samples were filtered through a 0.45 pm membrane filter

(Millipore, Sydney, Australia).

HPLC was performed with Waters-Millipore Instrumentation (Milford, MA) consisting of Model 510 pumps, a U6K sample injector and a Model 660 gradient controller. Separation was achieved on a 4pm Nova-pak C-j q column (100 x 8 mm I.D.) eluted isocratically at room temperature with KH2PO4 (0.05M, pH 6.3) containing 1 mL/L of triethylamine at a flow rate of 1 mL/min. To ensure protection of the column, an in-line stainless steel filter and a guard-pak containing the Nova-pak C-iq packing material was used. Following triplicate runs (one sample), the column was flushed with methanol using a gradient program (0-100% over 10 minutes) and washed for 15 minutes to remove strongly retained compounds and to maintain reproducible retention times between samples. After reversal of the gradient program, the column was re-equilibrated with the 0.05 M KH2PO4 elution buffer for 10 minutes before the next injection. The gradient program was used to avoid precipitation of the salt in the elution buffer during the change over between solvents. Mobile phases were prepared using HPLC-grade solvents and were filtered and degassed prior to use. Guanine was detected by a Waters 490 spectrophotometer at 254 nm and the data processed on a Waters Baseline 810 chromatography workstation connected to an IBM-compatible AT computer.

Stability of guanine formed by hydrolysis of RNA with HCIO4 was assessed by hydrolysing a sample of a model sparkling wine from 0.5 - 3.0 hours. Purity of the guanine peak was determined by comparing the ratio of peak areas of standard guanine to sample guanine at 254 and 280 nm (Qureshi et aj., 1979). 107

Recovery of guanine from the model sparkling wine was also determined. A standard curve for calculation of RNA concentration was prepared from standard yeast RNA (Boehringer Mannheim 109223) and compared to a similar curve prepared by the orcinol procedure. Finally, the described method was compared with the orcinol method of Herbert et aj. (1971) for estimating the concentration of cellular RNA in strains of Debaryomyces hansenii,

Kloeckera apiculata and Saccharomyces cerevisiae.

3.23 Results and Discussion

3.231 Estimation of RNA in sparkling wines by the orcinol procedure - interference by non-RNA sugars (polysaccharides)

Figure 3.21 shows the absorption spectra from 500-700 nm of the ribose-RNA colour complexes for the two commercial (Wagga and Nuriootpa) and one model sparkling wines tested for RNA by the orcinol procedure (Figure 3.21b). Peaks attributable to interference from hexose sugars occurred at about 530- 550 nm and were also observed in similarly treated samples of pure hexose sugars (glucose and mannose, 100 pg/mL, Figure 3.21a). Although reacting to a lesser extent than , hexoses do contribute to an increase in absorbance at 672 nm which is the absorption maximum for the ribose-orcinol complex. Figure 3.21 indicates that direct application of the orcinol procedure for determination of RNA in sparkling wines is not possible without appreciable interference from non-RNA sugars. 108

0.60

0.50

0.40 -

0.20

0.10

0.00

Wavelength

0.60

0.50 -

0.40 -

0.20 -

0.00 —

Wavelength

Figure 3.21 Absorption spectra (500-700 nm) of sugar-orcinol complexes for (a) standard RNA, glucose and mannose (100 pg/mL) and (b) samples of commercial (Wagga and Nuriootpa) and a model sparkling wine tested for RNA by the orcinol procedure of Herbert et a\. (1971). Standard RNA (□), glucose (0) and mannose (A); commercial (Wagga, ■, and Nuriootpa, ▲) and a model (♦) sparkling wines.

3.232 Removal of polysaccharides by two phase extraction (Kirby, 1956)

The two phase extraction procedure of Kirby (1956) was applied to two commercial wines and a model sparkling wine for extraction of polysaccharide material. The method was unable to remove polysaccharides in the commercial wines as evidenced by the absence of a cloudy precipitate between the two phases. When applied to the model sparkling wine, however, 109 a white precipitate was observed indicating the successful removal of polysaccharide material. An aliquot of the upper layer containing the nucleic acid material was then tested by the orcinol procedure and the absorption spectrum of the orcinol complex scanned for interference by hexose sugars as in Section 3.231 (Figure 3.22).

0.70 0.60

0.30 0.20

0.00

Wavelength

Figure 3.22 Absorption spectra (500-700 nm) of the sugar-orcinol complex of samples of standard RNA (50 pg/mL), glucose (100 pg/mL) and a model sparkling wine before and after treatment by the Kirby procedure for removal of polysaccharides. Standard RNA (□), glucose (0) and samples of a model sparkling wine before (A) and after (▲) processing by the Kirby procedure.

Figure 3.22 shows that while a large amount of polysaccharide material was removed by the Kirby procedure, a significant amount remained which would interfere with the RNA determination by the orcinol method and give higher contents than actually present. 110

3.233 Comparison of methods for extraction of nucleic acids from sparkling wines using hydroxyapatite and CTAB

Table 3.23 summarises the efficiency of extraction of RNA from standard solutions and standard amounts of RNA added to sparkling wine samples using hydroxyapatite columns and CTAB, by various methods. The recovery of

RNA from standard solutions by these methods ranged from 45-78 % and RNA from spiked sparkling wines from 69-83 %. These values were compared with the estimation of RNA in sparkling wines using HPLC for measurement of nucleic acid breakdown products (Section 3.234). 73 73 £ J0 4 'c O I 1 o < CD 0 CO X > CL 0 0 0 X c £ z O 0 E — < 0 — E E o E — 0 X 0 o c cn

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CO *♦ _0 ‘ 73 § CO _C 73 CD 00 73 X z < ■4 CO O O o 0 0 O E c 0 o 0 0 0 C 0 L. — — * a A fter extraction, the RNA was m easured using the orcinol m ethod of Herbert et aj. (1971) 111 112

3.234 Analysis of nucleic acid concentrations in wine by HPLC.

(a) Separation of ribonucleotides, ribonucleosides, deoxyribonucleotides, deoxyribonucleosides and bases by HPLC (Zhao et aL, 1994)

The HPLC procedure was applied to samples of commercial sparkling wine (Figure 3.23a) and a mixture of pure compounds listed in Table 3.22 (Figure 3.23b). The method was successful in separating 20 out of 21 compounds and has been useful in determining the nature of nucleic acid breakdown products during yeast autolysis in a simple defined media (Zhao et aL, 1994). However, the separation system was unable to fully resolve the peaks of interest in a commercial sparkling wine due to the complex nature of the product (Figure

3.23a). This was indicated by comparing the ratio of peak areas (or peak heights) at 254 nm and 280 nm in the commercial wines to that obtained for the pure chemical - this ratio is a constant for each compound (Qureshi et aL, 1979). None of the peaks in Figure 3.23a showed similar ratios to the corresponding pure compound in Figure 3.23b, indicating that these compounds were not contained in the standard and/or were contaminated with impurities. To improve peak resolution, the nucleic acid materia! from the commercial wine may be isolated prior to hydrolysis to reduce the occurrence of interfering peaks. Alternatively, the HPLC elution procedure may be modified to obtain improved separation of peaks. Further developmental work is therefore required before this procedure may be reliably applied to measure specific nucleic acid breakdown products in sparkling wines.

The potential exists for this system to be used for analysis of nucleic acid breakdown products in model wine systems. However, time constraints associated with the completion of this project did not allow this possibility to be pursued. 113

TIME (mm)

Figure 3.23 Chromatograms of (a) commercial sparkling wine and (b) a standard mixture of nucleotides, nucleosides and purine and pyrimidine bases.

Peaks: 1 = 5'-CMP; 2 = 5'-UMP; 3 = 5'-dCMP; 4 = 5'-GMP; 5 = cytosine; 6 = uracil; 7 = 5'-dTMP; 8 = 5'-AMP; 9 = 5'-dGMP; 10 = cytidine; 11 = uridine; 12 = deoxycytidine; 13 = 5'-dAMP and thymine; 14 = guanine; 15 = guanosine; 16 = deoxyguanosine; 17 = thymidine; 18 = adenine; 19 = adenosine; 20 = deoxyadenosine 114

(b) Acid hydrolysis of nucleic acid material and measurement of RNA by guanine using HPLC (guanine-HPLC) (Todd et a}., 1995)

Hydrolysis by perchloric acid degrades nucleic acids into purine bases and pyrimidine nucleotides (Loring, 1955). Figure 3.24 shows a standard curve constructed of the purine base, guanine, liberated from yeast RNA versus RNA concentration. The method shows a linear response equivalent to the orcinol procedure in the concentration range 0-100 pg/mL of yeast RNA.

0.25

0.20

0.15

0.10 -

0.05

0.00 0 20 40 60 80 100 RNA concentration. (ug/mL)

Figure 3.24 RNA standard curve from 0-100 pg/mL as determined by the orcinol (□) and guanine-HPLC (A) procedures.

The guanine-HPLC procedure was also compared to the orcinol method for determination of RNA extracted from various yeast genera (Saccharomyces,

Debaryomyces and Kloeckera). Table 3.24 shows that the guanine-HPLC procedure for the determination of RNA in yeast correlates closely with values obtained by the standard orcinol method. 115

Table 3.24 Concentration of RNA in different yeast species as determined by the orcinol and guanine-HPLC procedures

Yeast species RNA (% cell dry weight)3 Orcinol Guanine-HPLC

Saccharomyces cerevisiae 5A 10.1 9.9 Saccharomyces cerevisiae EC-1118 10.0 9.1 Saccharomyces cerevisiae X2180 7.4 7.2

Debaryomyces hansenii 2BCAS7 5.5 5.1

Debaryomyces hansenii TASDh9 5.1 4.4

Kloeckera apiculata 610 7.1 6.7

Kloeckera apiculata 401 4.5 4.4 a Data are means of triplicate assays

A potential source of variation in the measurement of guanine for RNA determination is the differences in base composition of RNA between organisms. It has been shown, however, that the molar ratio of guanine in ribosomal RNA is relatively constant between different organisms (Lava- Sanchez et aL, 1972). The molar ratio of guanine was found not to vary by more than 5% in 23-28s and 16-18s ribosomal RNA in selected fungal genera including Saccharomyces, Rhizopus and Aspergillus spp. As ribosomal RNA represents approximately 85% of total RNA in yeast (Phaff et aL, 1978), variation from differences in base composition can be assumed to be negligible. This assumption is supported by the results shown in Table 3.24.

To test the efficiency of recovery of RNA from sparkling wines by the guanine-

HPLC procedure, a model sparkling wine was spiked with 20 pg/mL of standard RNA and the assay performed for guanine. The recovery of the 116 added RNA was determined to be 95.5% and 98.4%, giving an average of 97.0% recovery. This is clearly superior to the recoveries of RNA obtained when utilising either hydroxyapatite or CTAB to extract the nucleic acid before assay (see Table 3.23).

An important variable in the guanine-HPLC procedure is the hydrolytic release of guanine from RNA by the concentrated HCIO4. Table 3.25 indicates that release of guanine from standard RNA and RNA in a model sparkling wine is essentially complete within 30 and 60 minutes, respectively, and is stable for at least 90 minutes of heating at 1009C.

Table 3.25 Effect of time of hydrolysis on the recovery of guanine from RNA as measured by the guanine-HPLC procedure

Source of RNA Recovery (%) after hydrolysis at 100gC for: 10 min. 20 min. 30 min. 60 min. 90 min.

Standard RNA in water3 93 98 101 100 100

RNA in model sparkling wine .b -b 97 100 100 a 50 pg/mL b Not determined

Figure 3.25 shows examples of HPLC elution profiles (254 and 280 nm) of hydrolysates of standard yeast RNA (Figure 3.25a), a model sparkling wine obtained after 562 days ageing on lees (Figure 3.25b) and a commercial sparkling wine (Nuriootpa) obtained after 709 days ageing on lees (Figure

3.25c). Guanine has an elution time of approximately 8.0 - 8.5 minutes.

Unfortunately, guanine could not be determined in the commercial sparkling wines by this method as the peaks corresponding to guanine were found to be contaminated by other unknown chemical species. This was indicated by 117 comparing the ratio of peak areas (or peak heights) at 254 nm and 280 nm in assays for the commercial wines to those obtained in standard yeast RNA - this ratio is constant for guanine (Qureshi et aL, 1979). Thus, the ratio of peak areas (or peak heights) at 254 nm and 280 nm in the commercial wine (Figure 3.25c) should correspond to approximately 1.3 as in the standard RNA and model sparkling wine chromatograms (Figures 3.25a,b). The 254 : 280 nm ratio for peaks found with the commercial sparkling wine was, however, 0.6.

These observations indicated that the method could not be reliably used to determine the evolution of RNA in the complex media of commercial sparkling wines. Nevertheless, the guanine-HPLC procedure could be utilised for measuring nucleic acid release during the secondary fermentation of the model wine system. The method has also been successfully used to measure the

RNA content of yeast and the kinetics of RNA release from yeast during autolysis in a defined buffer (Todd et aL, 1995).

As mentioned earlier, to use this procedure for analysis of nucleic acids in complex media such as commercial sparkling wines, various options must be evaluated - the nucleic acid material from the commercial wine may be isolated prior to hydrolysis to reduce the occurrence of interfering peaks, or the HPLC elution procedure modified to obtain improved separation of peaks, or both. These evaluations are major studies and could not be undertaken within the time frame of the project. — Guanine (200nm)

Guanine (25Cnm)

0 U 0 12 16 20

Guanine (25i.nm)

Guanine (20 Onm)

0 U 0 12 16 20

Guanine (200nm)

— Guanine (25^.nm)

0 U 0 12 16 20 TIME (min.)

Figure 3.25 HPLC chromatograms of hydrolysates of (a) standard yeast RNA (20 |jg/mL), (b) model sparkling wine (562 days ageing) and (c) Nuriootpa commercial sparkling wine (709 days ageing). 119

3.235 Summary

The following conclusions can be drawn from this section in relation to methods for the measurement of nucleic :

(i) the use of hydroxyapatite and CTAB to isolate nucleic acids from sparkling wines was unsuccessful. This conclusion is based on results in Table 3.23 indicating poor recovery of standard yeast RNA from standard solutions and sparkling wine using these adsorbents with a variety of published methods.

(ii) the use of HPLC for measurement of individual breakdown products of nucleic acids (nucleotides, nucleosides and nucleobases), although successful in studying yeast autolysis in buffers (Zhao et ah, 1994), was unable to resolve such compounds in commercial sparkling wines owing to the complex nature of the product. There is, however, potential use for this method in studying the degradation of nucleic acids in model wine systems.

(iii) measurement of guanine by HPLC following acid hydrolysis of model wine samples was successful in estimating totai nucieic acid content (guanine-HPLC procedure). However, guanine could not be resolved by HPLC in the commercial product owing to the presence of numerous interfering compounds, and thus measurement of the nucleic acid content of commercial sparkling wines remains to be perfected. 120

3.3 Isolation and analysis of lipids from sparkling wines

3.31 Introduction

The lipid content of sparkling wines is a topic where relatively little research has been published. This, in part, may be due to difficulties encountered in the application of effective experimental procedures. Analysis is complicated by the fact that lipids are present in very dilute concentrations. Troton et ai. (1989a) reported that the total content in a sparkling wine ranged between approximately 1-2 mg/L. With such low concentrations, efficient extraction is required to detect the material with any degree of precision. Similarly, reliable separation of extracted lipid classes is required if meaningful analysis of the constituent fatty acids of these compounds is to be attempted.

The objective of this section was to compare various methods of lipid analysis in order to maximise the precision of a study of changes to lipids that occur during the storage of sparkling wines. Two aspects of lipid analysis were assessed - efficiency of extraction of lipid material from the sparkling wine and separation of lipid classes in preparation for analysis of their fatty acid profile.

Published methods for the extraction of total lipids from wine and wine yeasts have been based upon the extraction procedure of Folch et ai. (1957) using chloroform-methanol (2:1) (Piton et a[., 1988; Troton et a[., 1989 a,b). However, this procedure was originally developed for extraction of lipids from animal tissues, so its adoption for extraction of lipids from sparkling wine may not necessarily be appropriate. Lipid extraction procedures have been discussed by Arnold (1981a) and Rose and Veazey (1988) who suggest that the variable results reported in the literature for lipids in yeast may, in part, be 121 attributed to inefficient extraction. These authors suggest that many workers adopt such extraction procedures without first checking the efficiency of the process.

An alternative method for lipid extraction is a modification of the Folch et aL (1957) procedure described by Bligh and Dyer (1959). The method is based on the hypothesis that optimum extraction of lipid occurs when a mixture of chloroform and methanol is mixed with the aqueous phase of the sample to yield a monophasic solution. Isolation of the organic phase containing the extracted lipid is then achieved by dilution with chloroform and water. The method has been shown to be superior to the Folch wash for extraction of lipid material in fish (de Koning et ai-, 1985). It has also been applied to the extraction of total lipids in rat livers (Hamilton and Comai, 1984) and, more significantly, yeast autolysates (Babayan et a}., 1981) and in milk (Christie et aL, 1987) which are both high water content matrices like sparkling wines. This method was, therefore, compared with the Folch wash which has been used previously for the extraction of total lipids from sparkling wine (Piton et aL, 1988; Troton et aL, 1989a).

Published methods for the separation of lipid classes extracted from wine and wine yeasts for analysis of their fatty acid composition have been based on preparative thin layer chromatography (TLC) (Piton et aL, 1988; Troton et aL,

1989 a,b). Alternative methods for separation of lipid classes include adsorption chromatography onto silicic acid, florisil or alumina, partition chromatography and HPLC. Another method described by Kaluzny et aL

(1985) describes the separation of lipid classes by adsorption onto amino- propyl columns. Lipid classes were washed off the column by application of small volumes of solvent mixtures which exploited slight differences in the bonding of the lipids to the solid phase. The method was shown to be more 122

rapid, give greater purity and recovery (> 95%) of lipid classes and was less sensitive to sample overload than TLC in analysis of standard lipid mixtures and extracts of bovine adipose tissue. This method was, therefore, compared with preparative TLC for its efficiency in the fractionation of lipids extracted from sparkling wines.

3.32 Materials and Methods

3.321 Extraction of lipid material

Sparkling wines were initially concentrated from 100 mL to 10 mL using rotary evaporation under vacuum at 40°C. Two methods for lipid extraction from the wine concentrate were compared; the first was the standard Folch wash by extraction with chloroform-methanol (2:1) as described by Troton et aL (1989a), and the second was a modified Folch wash as described by Bligh and Dyer (1959).

(a) Troton et aL (1989a)

The 10 mL of concentrated wine was extracted with 40 mL of chloroform- methanol (2:1) and the chloroform layer containing the lipid separated and evaporated to dryness. Total lipids were then resuspended in 0.3 mL of chloroform for analysis by thin layer chromatography (TLC).

(b) Bligh and Dyer (1959)

Chloroform-methanol (1:2) (37.5 mL) was added to concentrated sparkling wine (10 mL) and the mixture was thoroughly shaken in a 100 mL separating funnel. Chloroform (12.5 mL) was added to the mixture which was again thoroughly shaken. Finally, distilled water (12.5 mL) was added and the 123 mixture shaken and allowed to separate into two layers. The lower chloroform layer containing extracted lipid material was then passed through filter paper containing anhydrous sodium sulphate, collected, and evaporated to dryness. Total lipids were resuspended in 0.3 mL of chloroform for analysis by TLC.

(c) Comparison of extraction procedures

Resuspended lipids extracted by the two methods were applied (15 pL) to silica gel 60 TLC plates (Merck-5721) along one centimetre bands and then developed using the 2 x 1 dimension solvent system of Bitman et af. (1981). The first stage (chloroform-methanol-acetic acid 98:2:1) was allowed to run to within 3 cm from the top of the plate before development with the second stage (n-hexane-ether-acetic acid 94:6:0.2) to the top of the plate. Following development, the separated lipid classes were sprayed with copper acetate 3% (w/v) in phosphoric acid 8% (v/v) reagent and the spots on the chromatogram were visualised by heating in an oven at 180°C for 20 minutes (Hedegaard and Jensen, 1981). The separated lipid classes from both extraction methods were compared visually.

3.322 Separation of lipid classes by thin layer chromatography (TLC)

Separation of standard and extracted lipids on 20 cm x 20 cm silica gel 60

(Merck-5721) TLC plates was compared using three different solvent systems as summarised in Table 3.31. 124

Table 3.31 Solvent systems for the separation of standard lipid preparations and lipids extracted from wine.

Reference Solvent system Procedure Sahasrabudhe 1. Ether--ethanol-acetic acid Plate air dried between (1979) (40:50:2:0.2) solvent systems

2. n-hexane-ether (96:4)

Bitman et a|. 1. Chloroform-methanol-acetic acid (a) Solvent 1 developed (1981) (98:2:1) to within 3 cm from top of plate

2. n-hexane-ether-acetic acid (b) Plate air dried (94:6:0.2) between solvent systems

Troton et aL 1. Benzene-ether-ethyl acetate-acetic Single stage separation (1989a) acid (80:10:10:0.2) system

TLC plates spotted with lipid standards (triglyceride, 1,3 diglyceride, 1,2 diglyceride, monoglyceride, palmitic acid and phosphatidylserine (obtained from Sigma Chemicals) and lipid extracted from sparkling wine were

developed using the solvent systems described in Table 3.31. Separation of the lipid classes on the plates was visualised with copper acetate as already described and the efficacies of the different solvent systems compared. The solvent system giving the greatest separation between lipid classes was

selected for the routine analysis of lipids.

3.323 Separation of lipid classes with amino-propyl columns

A system allowing separation of neutral and polar lipids using an amino-propyl

bonded phase mini-column has been described by Kaluzny et aj. (1985). The procedure involves loading extracted lipid onto the mini-column and then sequentially eluting each lipid class with appropriate solvents. The method 125 was applied to separation of lipid class standards and lipid material extracted from a sparkling wine by the method of Bligh and Dyer (1959).

Dried, extracted lipid material was redissolved in 0.5 mL chloroform and passed through a mini-column (mini-column 1) (Activon, amino-propyl phase

Bond-Elut) previously equilibrated by washing twice with 2 mL amounts of n- hexane. The solvents used to elute the various lipid classes are listed in

Table 3.32.

Table 3.32 Solvents used to elute different lipid classes from Bond-Elut mini­ columns.

Solvent Description Volume added Lipids extracted

A Chloroform-2-propanol (2:1) 4 mL All neutral lipids

B Acetic acid (2%) in diethyl ether 4 mL Polar lipids

C Methanol 4 mL Free fatty acids

D n-Hexane 4 mL Cholesterol esters

E Diethyl ether (1%), methylene 6 mL Triglycerides

chloride (10%) in n-hexane

F Ethyl acetate (5%) in n-hexane 8 mL + 4 mL Cholesterol

G Ethyl acetate (15%) in n-hexane 4 mL Diglycerides

H Chloroform-methanol (2:1) 4 mL Monoglycerides

Neutral lipids were eluted from mini-column 1 with solvent A; the eluted material was dried and made up with 0.2 mL n-hexane. Mini-column 1 was then sequentially eluted with solvent B and solvent C to isolate the free fatty acids and polar lipids, respectively.

The neutral lipid fraction from mini-column 1 was applied to mini-column 2

(previously equilibrated with n-hexane as described already) and eluted with 126 solvent D to isolate the cholesterol ester fraction. Mini-column 3 (equilibrated as before) was then placed under mini-column 2 and solvent E allowed to pass through both columns to obtain the triglyceride fraction. Solvent F was passed through both columns to obtain the cholesterol fraction. The mini­ columns were then separated and mini-column 2 washed sequentially with solvent G and solvent H to obtain the diglyceride and monoglyceride fractions, respectively. Collected fractions were dried and made up with 0.2 mL chloroform for banding (15 pL) onto TLC plates and developed and visualised as before.

3.324 Fatty acid profile of isolated lipid classes (Takakuwa and Watanabe, 1981)

Lipid fractions on TLC plates were scraped off and placed into teflon capped tubes for conversion to methyl esters. Four and half millilitres of 2% (v/v)

H2SO4 in methanol was added to the tubes and the air displaced by nitrogen before capping tightly and heating overnight at 60°C. Fatty acid methyl esters were extracted with 3x5 mL n-hexane before drying by rotary vacuum evaporation. The methyl esters were made up with 0.2 mL iso-octane for analysis by GLC. Lipid fractions on TLC plates were visualised indirectly by iodine gas staining of surrounding lanes containing the same lipid material. Lines were drawn across the plate from visualised bands of lipid to indicate the location of unvisualised lipid fractions. Lipids were protected from oxidation by iodine by covering with plastic wrap and sealed with masking tape. Visualisation using other non-destructive methods such as spraying with 2',7'-dichlorofluorescein and viewing under UV (Piton et aL, 1988; Troton et aL, 1989a) were unsuccessful due to the low concentration of lipid material extracted from the model wine (Section 4.212). Recovery of methyl esters by extraction with n-hexane was quantified by addition of a known amount of 127 internal standard (C-| 70 methyl ester) to the reaction tube prior to methylation.

Samples of lipid classes obtained by the mini-column procedure were collected and dried in teflon-capped tubes before treatment as already described to produce methyl esters.

Methyl esters were analysed by gas chromatography (Varian 3300) through a

J&W FFAP capillary column (30m x 0.25 mm x 0.25pm). The operating conditions for the gas chromatograph are listed in Table 3.33.

Table 3.33 Operating conditions of Varian 3300 gas chromatograph for the analysis of methyl esters of fatty acids.

Parameter Setting Injection mode Split Temperature program 180°C for 18 minutes and then 10°C/min. to 220°C before holding for 5 minutes. Injector temperature 220°C FID detector temperature 250°C Column head pressure 8 psi

Fatty acid methyl ester standards were obtained from the Sigma Chemical

Company.

3.33 Results and Discussion

3.331 Extraction of lipid material from sparkling wines

Figure 3.31 shows a comparison between lipid material extracted from a commercial sparkling wine by the method of Bligh and Dyer (1959) and Troton et a[. (1989a). Bands corresponding to various lipid classes appear 128 more intense in the Bligh and Dyer (1959) extract, indicating a more efficient extraction of lipid material than the method of Troton et aL (1989a).

The method of Bligh and Dyer (1959) was originally developed to extract lipids from fish and has been successfully adapted to extract lipids from yeast autolysates (Babayan et aL, 1981) and milk (Christie et aL, 1987). An essential part of the procedure is the formation of a monophase in the first step by mixing a volume of sample with a precisely determined amount of chloroform-methanol (1:2). This monophase gives greater extraction of lipid material than the biphasic method of Troton et aL (1989a). Routine extraction of lipid material from sparkling wines was therefore performed using the method of Bligh and Dyer (1959). 129

Figure 3.31 Comparison between the method of (a) Bligh and Dyer (1959) and (b) Troton et aL (1989a) for the extraction of lipids from a commercial sparkling wine. [ PL - Phospholipids; MG - Monoglycerides; FFA - Free fatty acids; 1,2 DG - 1,2 Diglycerides; 1,3 DG - 1,3 Diglycerides; TG - Triglycerides]

3.332 Separation of lipid classes by thin layer chromatography

Figure 3.32 shows a comparison of three different solvent systems for the TLC separation of lipid classes extracted from a commercial sparkling wine. 130

Of the systems examined, the 2 x 1 dimension TLC procedure of Bitman et aL (1981) gave the best separation of standard lipids and extracted lipids. Of particular note was the improved separation of the triglyceride fraction from the heavier bands located at a slightly lower Rf value. In comparison, the method of Sahasrabudhe (1979) gave limited separation of these bands while the solvent system of Troton et aL (1989a) failed to give a satisfactory separation.

Figure 3.32 Separation of standard lipids and lipids extracted from commercial sparkling wines by TLC using the solvent systems of (a) Sahasrabudhe (1979), (b) Bitman et aL (1981) and (c) Troton et aL (1989a). [ PL - Phospholipids; MG - Monoglycerides; FFA - Free fatty acids; 1,2 DG - 1,2 Diglycerides; 1,3 DG -1,3 Diglycerides; TG - Triglycerides]

However, even with the superior resolving power of the Bitman et aL (1981) system, separation of the bands was insufficient for subsequent accurate 131 processing. When applied to lipid extracted from a model sparkling wine, though, complete separation of bands was achieved (Figure 3.33). This procedure was therefore used for the separation of lipid classes extracted from a model sparkling wine.

Figure 3.33 Comparison of separation of standard and extracted lipids in (a) a commercial sparkling wine and (b) a model sparkling wine (using the solvent system of Bitman et ajL (1981). [ PL - Phospholipids; MG - Monoglycerides; FFA - Free fatty acids; 1,2 DG - 1,2 Diglycerides; 1,3 DG - 1,3 Diglycerides; TG - Triglycerides]

3.333 Isolation of lipid classes on amino-propyl mini-columns

Figures 3.34a and 3.34b show the separation of lipid class standards and lipids contained in a model wine using amino-propyl mini-columns and the solvent systems of Kaluzny et a]. (1985). Separation between lipid classes 132 was readily achieved although there was some contamination of the polar lipid fraction with free fatty acids. The system did not separate 1,2 and 1,3 diglycerides but collected them as a fraction of total diglycerides. Figure 4b also shows that the heavy bands located at a slightly lower Rf value than triglycerides in lipids extracted from sparkling wines and separated by TLC (as discussed in Section 3.332) were collected in the triglyceride fraction and, thus, would interfere in any fatty acid analysis. There was also an indication that a small amount of triglyceride contamination occurred in all the collected fractions. This may be due to plasticisers present in the mini-column and which were elutable with the organic solvents. Mini-columns without any added lipid extract were eluted with the solvent system of Table 3.32 and gave the same contamination of the various fractions. Methylation of these fractions indicated the presence of free fatty acids which would interfere with any analysis (Figure 3.35). It was concluded that the problem of contaminating lipid material eluting from the mini-column itself would have to be solved before quantitative studies of lipids could be achieved. The additional problem of fraction contamination of other classes (eg. free fatty acids present in the polar lipid fraction) would also have to be investigated by careful adjustment of the solvent solutions given in Table 3.32. On the basis of these results, preparative TLC was chosen as the method for separation of lipid classes in wines. 133

Figure 3.34 Lipid content of fractions obtained from Bond Elut mini-columns used to separate (a) standard lipid classes and (b) lipids extracted from a model sparkling wine. [ PL - Phospholipids; MG - Monoglycerides; FFA - Free fatty acids; 1,2 DG - 1,2 Diglycerides; 1,3 DG - 1,3 Diglycerides; TG - Triglycerides] 134

/> 5 --UJ—_

0 10 20 30 40 T I ME (min.)

Figure 3.35 GLC chromatograms of methylated free fatty acids obtained from the elution of (a) triglyceride and (b) free fatty acid fractions from a Bond-Elut mini-column without added lipid and (c) standard fatty acid methyl esters.

M-c8:0 2-c10:0 3- c12:0 4- c14:0 5-c16:0 6- c17:0 7-c18:0 8. C-|8:1 9- C-i8:2 l 135

3.4 Method to distinguish between populations of killer and sensitive strains of Saccharomyces cerevisiae in mixed culture

3.41 Introduction

Studies of the growth behaviour of killer and sensitive populations of Saccharomyces cerevisiae during mixed culture are complicated by the need for methods to reliably differentiate between the two types of strains of the same species. Several methods to distinguish between the two types of strains have been reported in the literature and are summarised in Table 3.41.

Table 3.41 Methods used to differentiate between killer and sensitive strains of Saccharomyces cerevisiae during mixed culture.

Method of strain identification Reference

Selection of strains exhibiting different growth rates on plating Barre (1984) media enabling differentiation based on colony size

Use of auxotrophic or respiratory mutants of killer strains with Hara el ai- (1980); specific plating media to differentiate them from sensitive strains Hara el al. (1981); Sekielai- (1985)

Differentiation between killer and sensitive strains on the basis Rosini (1985) of H?S production on specific plating media

Selection of killer and sensitive strains that show different Heard and Fleet colony morphology on plating media (1987)

Direct assay of colonies on plates for killer activity Longo et af. (1990)

Use of killer strain marked with 8-galactosidase gene from Petering el al. (1991) Escherichia coli and specific plating medium containing substrate to detect expression of fl-galactosidase.

Selection of killer and sensitive strains which show differing Jacobs el al. (1988); tendencies to absorb an indicator dye from a modified Jacobs and van Wallerstein medium (M-WLA) Vuuren (1991)

Of the methods listed in Table 3.41, the method of Heard and Fleet (1987) offers simplicity and speed of differentiation. However, this method depends on screening strains of S. cerevisiae for killer or sensitive properties and 136 further screening these strains for differences in colony morphology on the enumeration medium, malt extract agar. Unfortunately, only a small proportion of strains exhibit the latter property. The method of Jacobs et aL (1988) and Jacobs and van Vuuren (1991) offers similar simplicity and rapidity of differentiation. Distinction between killer and sensitive strains is based on colony colour resulting from absorption of the indicator dye, bromocresol green, from the plating medium. Killer and sensitive strains are screened for their ability to absorb differing amounts of the dye giving different coloured colonies on the plating medium, a property much more widespread than the colonial morphological differences required by the method of Heard and Fleet (1987).

However, to ensure the accuracy of population counts (particularly the death kinetics of the sensitive yeast strain), a variety of other dyes were assessed for similar properties to bromocresol green as basis for the development of another plating medium to confirm counts obtained on modified Wallerstein agar (M-WLA).

3.42 Materials and Methods

3.421 Choice of agar and indicator dye

In order to simplify preparation of the plating media, commercially available media which support the growth of yeasts were chosen for evaluation. These media should also be colourless prior to addition of the dye to minimise interference between the background colour and dye colour. Media selected for trial on this basis included plate count agar (PCA - Oxoid CM 463) and potato dextrose agar (PDA - Oxoid CM 139). A further medium already 137 containing a dye, dichloran rose bengal chloramphenicol agar or DRBC

(Oxoid CM 727), was also evaluated.

Indicator dyes were selected on the basis of the pH at which they change colour. In the modified Wallerstein medium (M-WLA), for example, the starting pH of the agar is 5.5 at which bromocresol green is a blue colour.

However, as the yeast colony grows, release of acid causes a decrease in pH so that the agar turns green. This change in background colour increases the ease of strain differentiation.

Using these criteria, the matching of agar and dyes for differentiation of killer and sensitive strains is shown in Table 3.42.

Table 3.42 Modified media evaluated for use in differentiation of killer and sensitive strains of Saccharomyces cerevisiae in mixed culture.

Dye and Initial pH Initial colour Colour of Media concentration of agar of agar agar after (%w/v) plate growth Potato dextrose agar Congo red 5.6 red violet (0.01)

Potato dextrose agar Bromophenol blue 5.6 purple purple/grey (0.005)

Plate count agar Bromocresol purple 7.0 blue/violet grey (0.005)

Dichloran rose bengal Rose bengal 5.6 pink pink3 chloramphenicol agar (0.0025) a DRBC was selected for evaluation as it is a commercially prepared medium already containing a dye. This dye, however, is not an indicator dye so it does not change colour during growth of the colony. 138

3.422 Assessment of media for distinguishing killer and sensitive strains of Saccharomyces cerevisiae.

Killer (Tyr 303 and AWRI 3A) and sensitive (AWRI 5A and S-1) strains of S. cerevisiae were plated onto the media listed in Table 3.42 and incubated at 30°C for 48-72 hours. A successful medium was taken to be one where the killer strains could be readily distinguished from the sensitive strains on the basis of colony colour.

3.423 Recovery of killer and sensitive strains of Saccharomyces cerevisiae on potato dextrose containing 0.005% bromophenol blue (M- PDA)

The recovery of killer (Tyr 303 and AWRI 3A) and sensitive (AWRI 5A and

S-1) strains of S. cerevisiae on M-PDA was compared with modified

Wallerstein medium (Jacobs et aL, 1988; Jacobs and van Vuuren, 1991) by spread inoculating dilutions of mixed cultures of combinations of Tyr 303 / AWRI 5A and AWRI 3A / S-1 in duplicate onto both media on five separate occasions. Following incubation at 30°C/48-72 hours, the number of colonies for each strain was counted and the average calculated. The Student's t-test was then applied to observe whether counts obtained on

M-PDA medium were significantly different from those on M-WLA medium.

3.43 Results and Discussion

3.431 Assessment of media for distinguishing killer and sensitive strains of Saccharomyces cerevisiae.

Of the media listed in Table 3.42, only the potato dextrose agar containing

0.005% bromophenol blue (M-PDA) successfully differentiated between the 139 killer and sensitive strains on the basis of colony colour. An example is provided in Figure 3.41a which demonstrates the ability of M-PDA to distinguish between the killer strain S. cerevisiae AWRI 3A (dark colonies) and sensitive strain S. cerevisiae S-1 (white colonies).

Figure 3.41 Colour reactions of killer (AWRI 3A) and sensitive (S-1) strains of S. cerevisiae on a) potato dextrose agar + 0.005% bromophenol blue, b) potato dextrose agar + 0.01% congo red, c) dichloran rose bengal chloramphenicol agar and d) plate count agar + 0.005% bromocresol purple.

These yeast strains were inoculated onto modified Wallerstein media

(M-WLA) and placed alongside the same strains on modified potato dextrose agar (containing 0.005% bromophenol blue; M-PDA) for comparison of colour reactions. Figure 3.42 shows that both media give clear and readily distinguishable colour reactions for the different strains. 140

Figure 3.42 Colour reactions of killer (AWRI 3A, dark colonies) and sensitive (S-1, light colonies) strains of S. cerevisiae on a) Potato dextrose agar + 0.005% bromophenol blue (M-PDA) and (b) Wallerstein medium + 0.01% bromocresol green (M-WLA).

The colour reactions for killer (AWRI 3A and Tyr 303) and sensitive (S-1 and AWRI 5A) strains of S. cerevisiae on M-WLA and M-PDA are summarised in Table 3.43.

On the basis of the colour reactions described in Table 3.43, the following combinations of strains of S. cerevisiae were utilised in subsequent experiments : (a) AWRI 3A and S-1 and (b) Tyr 303 and AWRI 5A. 141

Table 3.43 Colour reaction of colonies of killer and sensitive strains of Saccharomyces cerevisiae on differential media.

Yeast Medium strain Modified Wallerstein agar Modified Potato dextrose agar AWRI 3Aa Uniform medium to dark Uniform medium to dark grey green

Tyr 303a Medium green centre with Dark grey centre with white light green periphery periphery

S-1b Uniform light green Uniform white

AWRI 5Ab Uniform medium green Light grey centre with white periphery a Killer strains of S. cerevisiae b Sensitive strains of S. cerevisiae

3.432 Recovery of killer and sensitive strains of Saccharomyces cerevisiae on potato dextrose agar containing 0.005% bromophenol blue (M-PDA)

The recovery of mixed cultures of killer and sensitive strains of S. cerevisiae

(AWRI 3A + S-1 and Tyr 303 + AWRI 5A) on M-PDA was compared with counts obtained on M-WLA for five separate populations. These counts are summarised in Table 3.44. Table 3.44 Comparison of counts (cfu/mL) of individual strains of mixed cultures of killer and sensitive strains of Saccharomyces cerevisiae on modified Wallerstein agar (M-WLA) and modified potato dextrose agar (M-PDA). .£ n CVJ co o E c o -

"(0 H £•§ I O O ■z: 75 O t 2 f == .E ♦3 s q CD 0 CO 0 CO c “

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cvj co' t CVJ O) CVJ cd CVJ CD co' T- cvj in CO CO CO cvj CO o - - - l ' '

q CD ^ o CVJ CD in CVJ CD o O q < in o co cvj o 0 > 0 co CT)

CVJ CT) CVJ CT) O' CD I CD CVJ o' cvj oo oo CVJ CD cvj CD CO CO co in Tf CO CT) CT) s - '

co CO CT) o in o I in CD o o CO in CD o < CO CT) o in CO O) o s 0 0 > CO CT) -"

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CD o cvj t in < CO in 0 CO CT) 0 > - 1

O > 0 average 27.9______2 0 8______3 0 0______29J______2T0______2 0 4______3 ^ 0______303 142 143

Comparison of average counts obtained for individual strains in mixed cultures of killer and sensitive strains of S. cerevisiae showed no significant difference by the Student's t-test at the 95% confidence level. It was concluded that M-PDA may be utilised to follow the population kinetics of killer and sensitive strains of S. cerevisiae in mixed cultures with at least equal reliability to the published medium of M-WLA. 144

3.5 Development of a chemically defined wine (CDW)

3.51 Introduction

Base wines that are used in the production of sparkling wines are themselves products of a fermentation and thus show considerable heterogeneity in terms of chemical composition. Such heterogeneity is not only determined by the yeast and conditions used to produce the base wine, but most significantly by the grape. Interpretation of the chemical changes that occur during the secondary fermentation and ageing of sparkling wines will be influenced not only by the activities of the yeast, but also by activities of residual enzymes contributed by the grape. To eliminate these influences on the chemical changes that occur during the secondary fermentation and ageing, experiments were undertaken to develop a chemically defined wine medium.

This section is divided into two parts; the first part is the development of a chemically defined wine (CDW) based on data available in the literature, and the second gives a comparison between the secondary fermentation of the proposed CDW and two commercial sparkling wines.

3.52 Formulation of a chemically defined wine

The development of the chemically defined wine (CDW) was divided into three major sections :

(i) inclusion of major chemical components such as ethanol and organic acids,

(ii) addition of carbon and nitrogen sources required for yeast growth, and 145

(iii) comparison of yeast growth in CDW and a commercial sparkling wine during fermentation.

The basic formula for the CDW was a modification of the model wine described by Voilley et al- (1990) as shown in Table 3.51.

Table 3.51 Model wine composition (Voilley et aL, 1990)

Compound* Concentration (g/L)

Ethanol 100 Tartaric acid 4 Malic acid 3

Acetic acid 0.1

K2SO4 0.1

MgSC>4 0.025

Mineral water (Evian) -

Wine was adjusted to pH 3.0 with NaOH (1M)

The formula was modified by replacement of the Evian water with distilled water and addition of mineral and vitamin supplements according to the formulation of the chemically defined grape juice as described by Monk and Cowley (1984) and shown in Table 3.52. 146

Table 3.52 Vitamin and mineral supplements used in the formulation of a chemically defined winea (Monk and Cowley, 1984).

Component Concentration (pg/L)

Minerals

MnCI2.4H20 19.8

ZnCI2 13.6

FeCI2 3.2

CuCI2 1.4

H3BO3 0.6

Co(N03)2.6H20 2.9

NaMo04.2H20 2.4

KIO3 11.1

Vitamins

Myo- 1x105

Pyridoxine HCI 2000

Nicotinic acid 2000

Ca pantothenate 1000 Thiamin HCI 500

p-Aminobenzoic acid 200

Riboflavin 200 125

Folic acid 200 a Mineral supplement added as a 1/10 dilution and vitamins added undiluted.

The nitrogen source utilised was made up of casein amino acids supplemented with diammonium phosphate (DAP). Casein amino acids were added to an amount which approximated values given in the literature for wine bases used in sparkling wine production (Table 3.53). 147

Table 3.53 Concentrations of amino acids reported in various wine bases.

Cologrande and Feuillat and Waggaa Amino Acid Silva (1986) Charpentier (1982)

mg/L mg mg/L mg mg/L mg NHq/L NHVL NHVL Alanine 48.4 9.3 31.3 6.0 51.2 9.8 Arginine 156.3 61.1 68.1 26.6 85.2 33.3 Aspartic acid 19.5 2.4 20.5 2.6 20.0 2.6

- Glutamic acid - 32.1 3.7 46.2 5.3 Glycine 11.7 2.7 10.2 2.3 16.0 3.6 Iso-leucine 30.1 3.9 2.8 0.4 10.2 1.3 Leucine 4.8 0.6 14.8 1.9 19.7 2.6 Methionine 9.4 1.1 3.9 0.4 4.6 0.5 Phenylalanine 6.2 0.6 10.6 1.1 14.2 1.5 Serine 8.6 1.4 5.9 1.0 19.5 3.2

Threonine - - 11.0 1.6 14.5 2.1 Tyrosine 9.1 0.9 4.0 0.4 12.5 1.2 Valine 6.8 1.0 5.6 0.8 21.1 3.1 Proline 595.4 88.1 298.5 44.2 275.2 40.7 TOTAL13 310.9 85.0 220.8 48.8 334.9 70.1 a Determined during this project. b Excludes proline as this is not consumed during anaerobic fermentation (Pekur et ai., 1981).

Using the individual amino acid values given in Table 3.53 as a guide, casein amino acids (Oxoid L 41) were added to an amount (0.5 g/L) which gave approximately the same concentration of amino acids found in commercial base wines. Individual amino acids deficient in this additive were added separately as a supplement to obtain the final formulation described in Table

3.54.

As addition of DAP to the wine base is usual practice in Australia (Rankine,

1989), an amount of 250 mg/L was also added to the CDW. This addition is 148

done to avoid H2S production or even a which may occur

if the amino nitrogen content becomes limiting (Henschke and Jiranek, 1993).

Table 3.54 Amino acid formulation for addition to a chemically defined wine (CDW)

Concentration in CDW

Amino 0.5 g/L casein 0.5 g/L casein amino 0.5 g/L casein amino acid amino acids3 acids + supplement^ acids + supplement^ (mg/L) (mg/L) (mg NHVL) Alanine 8.7 48.7 9.3 Arginine 8.3 83.3 32.5 Aspartic acid 20.3 20.3 2.6 Glutamic acid 66.7 66.7 7.7 Glycine 5.7 5.7 1.3 Iso-leucine 9.2 9.2 1.2 Leucine 16.7 16.7 2.2 Methionine 6.9 6.9 0.8 Phenylalanine 11.4 11.4 1.2 Serine 5.2 5.2 0.8 Threonine 7.2 7.2 1.0 Tyrosine 7.9 7.9 0.7 Valine 16.9 16.9 2.5 Proline 27.8 277.8 41.1 TOTAL0 191.1 306.1 63.8 a Casein amino acids (Oxoid L41) b supplement consists of 40 mg/L alanine, 75 mg/L arginine and 250 mg/L proline. c Excludes proline as this is not consumed during anaerobic fermentation (PekuretaL, 1981).

The carbon source used for the secondary fermentation was glucose and was

added at the amount of 24 g/L for the production of approximately 5-6

atmospheres (500-600 kPa) headspace of pressure at 10gC (Amerine et aL,

1980). The final formulation of the CDW medium is shown in Table 3.55. 149

Table 3.55 Formulation of a chemically defined wine base3 used for model studies on the secondary fermentation and ageing of sparkling wine.

Component Concentration (per litre) Glucose 24 g Tartaric acid 4g Malic acid 3 g k2so4 0-1 g MgS04 0.025 g CaCI2b 0.044 g DAP 0.25 g Casein amino acids 0.5 g Alanine 0.04 g Arginine 0.075 g Proline 0.25 g Mineral stock (x100)c 1 mL Vitamin stock (x100)c 10 mL Acetic acid 0.1 g Ethanol 130 mL a CDW was adjusted to pH 3.0 with 5M NaOH. b CaCl2 is part of the mineral stock but is added separately due to its limited solubility. c Mineral and vitamin stock solutions as described by Monk and Cowley (1984).

Using the above formulation, a volume of CDW was made up and inoculated with a sparkling wine yeast strain. The kinetics of growth and death of the yeast over 4 months were then compared to that observed in commercial sparkling wines. 150

3.53 Materials and Methods

3.531 Commercial wine bases

A Chardonnay/Pinot Noir blend (48:52) from the Wagga region of New South Wales and a Chardonnay/Pinot Noir blend (35:65) from Nuriootpa in South Australia were tiraged under commercial conditions and stored at 12--159C for the fermentation (Chapter 4, Section 4.211).

3.532 Chemically defined wine (CDW)

Approximately 9 litres of CDW according to the formulation of Table 3.55 were made up and tiraged under commercial conditions in 500 mL bottles. These were stored and examined for viable counts over 4 months as for the commercial wines.

3.533 Yeast strains

For the Wagga and CDW wines, the commercial yeast strain Saccharomyces cerevisiae EC-1118 was utilised while the Nuriootpa wine was fermented with a Penfold's in-house S. cerevisiae strain (Chapter 4, Section 4.221)

3.534 Viable counts

For yeast counts, samples of wine from two bottles during various stages of the fermentation were diluted in 0.1% peptone and plated onto Malt Extract Agar (Oxoid CM 59) in triplicate. The plates were then incubated at 25gC for 3 days after which colonies were counted and viable population calculated.

Counts were expressed as averages of 2 counts sampled from duplicate bottles. 151

3.54 Evaluation of a chemically defined wine (CDW)

The kinetics of yeast growth and death during sparkling wine fermentation of two commercial base wines and a CDW are shown in Figure 3.51.

7.0 -

5.0 -

4.0 -

3.0 -

2.0 -

1.0 0 20 40 60 80 100120 Days at 12-15C

Figure 3.51 Changes in viable yeast population during the secondary fermentation of commercial base wines and a chemically defined wine (CDW). Wagga (0) and Nuriootpa (A) commercial base wines and a chemically defined wine (□).

Figure 3.51 shows that the inoculated yeast grew in each wine base from about 1x10^ cfu/mL to a maximum viable population of approximately 6x10^ cfu/mL during the secondary fermentation. After the alcoholic fermentation over the period 0-35 days, the yeast strains in the various wines exhibited an extended death phase lasting from about 35 days to 95-115 days which is a characteristic of sparkling wines following the secondary fermentation

(Chapter 4, Section 4.231). 152

In comparison to S. cerevisiae strain EC-1118 used in the CDW and Wagga wines, the Nuriootpa yeast strain died off relatively early at about 95 days which is probably a strain specific response. The general similarity in population kinetics of the yeast in the various base wines, however, indicated that the formulation of the CDW given in Table 3.55 would be suitable for use as a defined system for further studies on sparkling wine fermentations. The

CDW was used in later studies on the effect of CO2 pressure on yeast growth and metabolism during the secondary fermentation (Chapter 5). 153

4.0 Chemical Changes during the Fermentation and Ageing of Two Commercial and One Model Sparkling Wine

4.1 Introduction

The chemical changes that occur during during production of sparkling wines may be studied in two phases. The first stage involves the secondary fermentation where the yeast convert fermentable sugars (glucose, fructose and/or sucrose) to carbon dioxide. The second stage begins at the end of the secondary fermentation where the sparkling wine is allowed to age on lees until disgorgement. This stage corresponds to the period of yeast autolysis which involves more subtle chemical changes than during secondary fermentation and thus may be studied separately.

This section also contains data on the chemical changes that occur during fermentation and ageing of a model wine. As shown in Chapter 3 (Sections 3.2 and 3.3), the complex chemical nature of commercial wines do not allow components such as nucleic acids and lipids to be reliably measured. To facilitate such measurements, a chemically defined grape juice was inoculated with a wine yeast and allowed to undergo the primary fermentation to form a model wine base. This system does not contain various interfering compounds originating from grapes, and would therefore allow more accurate measurement of chemical species such as dissolved nucleic acids and lipids. 154

4.2 Materials and Methods

4.21 Base wines

4.211 Commercial samples

Two wine bases were processed for sparkling wine production under commercial conditions. The first was a Chardonnay/Pinot Noir blend (37:63) produced by the winery of Charles Sturt University, Wagga Wagga, NSW and the second was a Chardonnay/Pinot Noir blend (35:65) produced by Penfolds

Pty Ltd. in Nuriootpa, South Australia. Important winemaking characteristics of these wines are shown in Table 4.21.

Table 4.21 Characteristics of Wagga and Nuriootpa base wines utilised for commercial sparkling wine production.

Characteristic Waggaa Nuriootpa3 Ethanol (% v/v) 10.9 10.2 Residual sugar (g/L) 2.0 1.7 PH 2.98 3.18 Titratable acidity (g/L) 7.9 8.2 Sulphur dioxide (free/total) (ppm) 18/48 20/94 3 Information supplied by respective wineries.

These wine bases were processed and tiraged as part of the usual commercial operations at the respective wineries. Key processing adjustments made to the wines are outlined in Table 4.22. 155

Table 4.22 Processing adjustments to wine bases in preparation for tirage

Processing adjustment3 Wagga Nuriootpa Addition of fermentable sugar (g/L) 21.4 (sucrose) 24.2 (invert sugar) Addition of diammonium phosphate 1000 200 (DAP) (ppm) Addition of bentonite (ppm)*3 70 100

Addition of tannin (ppm) - 10

Addition of malic acid (g/L) 0.2 - 3 Both wines were also stabilised against tartrate precipitation (low temperature storage), b Added to assist flocculation of protein.

The concentration of free SO2 in the Wagga base wine was decreased to 0 ppm prior to tirage by the addition of H2O2; no such treatment was recorded for the Nuriootpa wine.

4.212 Sparkling wine produced from a fermented chemically defined grape juice ( model wine )

The chemical composition of commercial wine bases are variable due to differences in the grapes used. To minimise the influence of such variables and to assist interpretation and understanding of the changes that occur during sparkling wine production, a model system was devised using a chemically defined grape juice medium. The composition of the chemically defined grape juice was based on the formulation of Monk and Crowley

(1984) and is given in Table 4.23.

Seventeen litres of this medium were filter-sterilised, placed in a sterile 25 L carboy and fermented by inoculation with S. cerevisiae strain 'Chandon'. Fermentation was conducted at 25°C for 13 days. At the end of the fermentation, this model wine base was filter-sterilised by passage through a

0.45 pm membrane. The residual sugar content, ethanol content and pH 156

were 1.7 g/L, 10.1 % (v/v) and 3.4, respectively. In preparation for tirage,

glucose and DAP were added to 24 g/L and 0.5 g/L, respectively. No SO2

and flocculating material (eg. bentonite) were added. This model wine should

not be confused with the chemically defined wine (CDW) (Chapter 3, Section

3.5) used specifically for later studies on the effect of CO2 on yeast growth

and metabolism during the secondary fermentation (Chapter 5).

Table 4.23 Composition of a chemically defined grape juice medium3 used for the fermentation of a model wine (Monk and Crowley, 1984).

Component Concentration Component Concentration (per litre) (per litre)

Glucose*3 200 g Biotin 0.125 mg K Tartrate 5g Folic acid 0.2 mg L-Malic acid 3g Aspartic acid 350 mg MgS04.7H20 1.23 g Glutamic acid 500 mg

K2HP04 114 g Asparagine 150 mg

Citric acid 0.2 g Serine 400 mg

CaCl2-2H20 0.44 g Glutamine 200 mg MnCl2-4H20 198.2 pg Histidine 150 mg

ZnCI2 135.5 pg Glycine 50 mg

FeCI2 31.96 pg Threonine 350 mg

CuCl2 13.6 pg Arginine 750 mg

H3BO3 5.7 pg Alanine 100 mg

Co(N03)2-6H20 29.1 pg Tyrosine 20 mg

NaMoC>4.2H20 24.2 pg Methionine 150 mg

kio3 10.8 pg Valine 200 mg

Myo-inositol 100 mg Tryptophan 100 mg

Pyridoxine-HCI 2 mg Phenylalanine 150 mg

Nicotinic acid 2 mg Iso-leucine 200 mg

Ca Pantothenate 1 mg Leucine 300 mg Thiamin.HCI 0.5 mg Lysine 250 mg

p-Aminobenzoic acid 0.2 mg Proline 500 mg

Riboflavin 0.2 mg a Final pH was adjusted to 3.5 with 5 M NaOH. b Starter culture formulation contains only 100g glucose and includes 10 mg and 0.5 mL of ergosterol and Tween 80, respectively. 157

4.22 Preparation of yeast strains for inoculation into wine base (tirage)

4.221 Commercial sparkling wines

(a) Wagga

Freeze-dried commercial strain S. cerevisiae EC-1118 (160 g) was rehydrated for 15 minutes in water at 35°C before addition to 10 L of a mixture of wine base and water (1:1) containing 100g/L of sucrose, 1 g/L of

DAP and 12 ppm of nutriferm. This suspension was incubated at 25°C for 2 hours and then vigorously aerated for 4 hours before addition as a starter culture to the wine base at a level of 1.2x10^ cells/mL and 89% viability.

(b) Nuriootpa

A wet culture of S. cerevisiae (Penfold's in-house strain) was propagated in steps in a medium consisting of 95% wine base and 5% liquid invert sugar. This culture was then added at a level of 1.3x10^ cells/mL and 91% viability to the wine base. No further details of starter culture preparation were provided.

4.222 Model wine

A wet culture of the commercial S. cerevisiae strain EC-1118 was prepared in two stages. Initially, a loopful of the yeast was added to 3 test-tubes containing 10 mL of the starter culture formulation of the chemically defined grape juice medium outlined in Table 4.23 and incubated at 20°C for 24 hours. These cultures were then transferred to 3 x 500 mL flasks containing

250 mL of the same medium and incubated for 48 hours at 20°C. Flask contents were then pooled and 15 mL of the starter culture added to each bottle at a concentration of 4.4 x10^ cells/mL and 95 % viability. Each bottle had previously been filled with 475 mL of sterile model base wine. 158

4.23 Bottling, secondary fermentation and ageing

After inoculation, the Wagga and Nuriootpa wine bases were bottled under commercial conditions into 500 and 750 mL bottles, respectively. The model wine was pooled into a 25 L pressurised canister and sparged with sterile N2 gas for 15 minutes before dispensing in 500 mL bottles. Inoculation of the model wine with the starter culture occurred after bottling.

All bottles were then capped with crown seal caps and stored horizontally at

12°-15°C for the secondary fermentation and subsequent ageing on lees.

4.24 Analysis of sparkling wines during fermentation and ageing

Each wine was analysed for a range of properties at regular intervals during fermentation and ageing. Two bottles were opened at each sample time - each bottle was assayed separately for viable counts and the contents combined for chemical analyses.

4.241 Viable counts

Samples of wine were diluted in 0.1% peptone solution and 0.1 mL of the dilutions were spread inoculated over the surface of triplicate plates of Malt Extract Agar (Oxoid CM 59). Plates were incubated at 25QC for 3 days after which colonies were counted and viable population calculated.

4.242 Glycerol, glucose, fructose and sucrose

The concentrations of these compounds were determined by HPLC with detection by refractive index. The HPLC system consisted of a Waters-

Millipore (Milford, MA) Model 510 pump, a U6K injector and Model 730 data module; detection was performed with an ERMA refractive index detector 159

(ERC-7512) set at x8 sensitivity. Separation of the compounds was performed on a Waters-Millipore 8 x 100 mm Silica column eluted isocratically at room temperature with acetonitrile.water (75:25) containing SAM 1 additive (# 10873) at a flow rate of 4 mL/min. Concentrations of compounds were calculated against standard solutions of either glycerol, glucose, fructose and sucrose (1% w/v).

4.243 Ethanol

The concentration of ethanol was determined by HPLC with detection by refractive index. The HPLC system consisted of a Waters-Millipore (Milford, MA) Model 510 pump, a U6K injector and a Waters Baseline 810 chromatography workstation connected to an IBM-compatible AT computer for data processing; detection was performed with an ERMA refractive index detector (ERC-7512) set at x8 sensitivity. Separation was conducted on a Waters-Millipore 8 x 100 mm Dextropak column eluted isocratically at room temperature with water at a flow rate of 1 mL/min. Concentration of ethanol was calculated with a standard solution of ethanol (10% w/v).

4.244 Headspace pressure

The development of headspace pressure was measured in the model wine using a WIKA pressure gauge capable of determining pressure in the 0-800 kPa range as shown in Figure 4.21. 160

WIKA gauge

rubber seal spike for penetration of crown cap

Figure 4.21 WIKA gauge for the measurement of developing CO2 pressure in bottles.

Pressure could not be measured in the commercial sparkling wines because the crown caps utilised contained a thick cork seal which could not be penetrated by the WIKA gauge.

4.245 Organic acids (Davis et al., 1986)

The concentrations of organic acids in wine samples were determined by HPLC consisting of the following instrumentation from Waters-Millipore (Milford, MA) : Model 501 pump, U6K injector, Model 441 variable wavelength detector set at 210 nm and a Model 745 data module. Separation was achieved on an Aminex non-exclusion cation-exchange column (Bio-Rad

HPX-87H) eluted isocratically at 65°C with 0.07% phosphoric acid at a flow rate of 0.5 mL/min. Wine samples were decolourised prior to injection using a

Waters-Millipore Sep-pak C-|q cartridge in the following manner: the cartridge was sequentially washed with 2 mL methanol, 2 mL distilled water and then 4 mL wine sample, the first 2 mL of which was discarded. Identity of the organic acids and their concentrations were determined by reference to the elution of solutions of standard concentrations of citric, tartaric, malic, succinic, lactic, acetic, propionic and formic acids (0.1%). 161

4.246 Soluble protein (Murphy et aL, 1989)

Wine samples from the Wagga and Nuriootpa wineries were passed through a 0.45 pm filter and 4.8 mL incubated with 1.2 ml_ of Coomassie brilliant blue dye reagent (Bio-Rad Laboratories, Richmond, CA.). The mixtures were vortexed and then incubated at room temperature for 35 minutes before absorbance was measured at 595 nm (Chapter 3, Section 3.1). Absorbance was measured in a 1 cm pathlength cuvette on a Shimadzu UV-120-02 spectrophotometer. The blank contained 4.8 mL of distilled water mixed with

1.2 mL of the dye reagent. Standard curves for estimation of soluble protein were prepared using bovine serum albumen (BSA) obtained from Calbiochem (product number 12567). Samples of the model wine were treated in a similar manner except that incubation at room temperature was conducted for 10 minutes before reading absorbance at 595 nm. Further incubation was not required because of the absence of polyphenolic compounds which delay binding of the dye to protein (Chapter 3, Section 3.1).

4.247 Amino acids

Amino acids in wine samples were determined by HPLC and post-column derivatisation with ninhydrin. HPLC was performed with Waters-Millipore

(Milford, MA) instrumentation consisting of two pumps for solvent delivery to the column (Model 510), one pump for post-column delivery of ninhydrin (Eldex), a manual injector (Model U6K), an automated solvent gradient controller (Model 680), a column temperature controller (Temperature Control Module) for maintaining the column at 43°C and the ninhydrin reactor chamber at 110°C, and a detector (Model 440) for measurement of absorbance at 436 nm and 546 nm. Separation was achieved on a Waters-Millipore (Milford, MA) physiological amino acid column (4.6 x 120 mm) packed with a 5 pm spherical cation exchange resin in the lithium form 162

(ANW P150). Data were processed on a Waters Baseline 810 chromatography workstation connected to an IBM-compatible AT computer. Lithium buffers A (pH 2.51) and B (pH 10.08) were used for gradient elution of the column and were prepared according to the directions of the Water's Millipore instruction manual. The multi-step gradient program used to control the mobile phase mixture during each run is shown in Table 4.24.

Table 4.24 Gradient program for HPLC analysis of amino acids in wines

Time3 Flow rate Solvent present Curve profile (minutes) (mL/minute) (%) A B

0 0.4 100 0 - 32 0.4 68 32 5 50 0.4 50 50 5 60 0.4 23 77 7 87 0.4 0 100 8 100 0.4 0 100 6 101 0.4 100 0 6 a Total run time was 120 minutes

Samples of wine were prepared for analysis by filtration through an Amicon YM5 ultrafilter to remove proteins of molecular weight 5000 or greater. Amino acids in samples were quantified by comparison with peak areas of the same amino acids in a standard solution (500 pmole/L). Standard amino acids were obtained from Sigma Chemicals. The total amino nitrogen content was determined by calculating the sum of the amino nitrogen content of each individual amino acid measured by HPLC. 163

4.248 Total Nucleic Acids (RNA)

Filter-sterilised samples of the model wine were assayed for total nucleic acids (RNA) by the guanine-HPLC method outlined in Chapter 3, Section 3.2.

4.249 Lipids

Details of methods used to isolate and analyse the changes in lipid material

(triglycerides, 1,3 diglycerides, 1,2 diglycerides, monoglycerides, free fatty acids and polar lipids) during storage of the model wine are given in Chapter

3, Section 3.3. A brief outline of the procedures is given in Table 4.25.

Table 4.25 Outline of methods used to extract and analyse the lipid component of the model wine.

Step Comments Reference Sample Sterile-filtered wine (100 mL) concentrated Troton et aL preparation to 10 mL by rotary evaporation at 40°C (1989)

Extraction of Monophasic organic solvent extraction Bligh and Dyer lipids (1959)

Separation of Preparative thin layer chromatography Bitman et aL iipid classes (1981)

Fatty acid profile Methylation of lipid classes and analysis by Takakuwa and GLC Watanabe (1981)

4.250 Volatiles

Volatiles were analysed by a modification of the method of Gelsomini et aL

(1990). Sparkling wine (6 mL) and internal standard solution (0.2 mL containing 1000 mg/L n-dodecanol and nonanoic acid) were pipetted into a 10 mL capacity Extube Chem Elut column (Analytichem Int.) and allowed to stand at room temperature for 10 minutes. Volatile components were then eluted with 16 mL, 12 mL and 12 mL volumes of freshly redistilled diethyl 164 ether which were allowed to pass through the column over 5 minute intervals. The organic extracts were then pooled and then concentrated to approximately 0.5 mL in a vigreaux column at 45°C. The concentrated extract was then analysed by GLC in a Varian 3300 gas chromatograph. Details of the GLC program are given in Table 4.26.

Table 4.26 Operating conditions of Varian 3300 gas chromatograph for the analysis of volatiles extracted from sparkling wines.

Parameter Setting Injection mode Split Temperature program 50°C for 10 minutes and then 6°C/min. to 200°C before holding for 10 minutes. Injector temperature 220°C FID detector temperature 250°C Column head pressure 8 psi

Presumptive identification of compounds was performed by matching of retention times of sample peaks to that in the standard. Positive identification of peaks was performed by computerised gas chromatography mass spectrometry (GC-MS) on a Hewlett-Packard HP5890 GC interfaced with a VG Quattro mass spectrometer (Hewlett-Packard). Working conditions were as follows : column, DB-Wax (30 m x 0.25 mm ); oven temperature program,

50°-200°C at 3°C/min.; carrier gas, helium at an inlet pressure of 70 kPa. Mass spectrometry was performed with the ionisation voltage of 70 eV and photomultiplier energy of 450 V. Scan rate was 1 sec/delay and the vacuum was maintained at 1.33 x 10“4 Pa with the ion source temperature at 180°C. Mass spectra (MS) were recorded and processed with an on-line desktop Intel 486 computer. Individual components in the wine were identified by comparing the MS data with those in the National Institute of Standards and Technology (NIST, USA) Mass Spectral Database held in the computer library. 165

Quantitation of major wine higher alcohols and esters was performed using the n-dodecanol internal standard and fatty acids by the nonanoic acid internal standard (Edwards and Beelman, 1990). A calibration standard containing a mixture of volatile compounds in 10% (w/v) ethanol and the internal standards were subjected to the same extraction procedure and the resulting chromatogram used to calculate the response factor for each compound relative to the internal standard.

The relative recovery of each volatile compound using solid phase extraction

on the Ex-tube was determined by recovery from a sample of sparkling wine

spiked with the compound of interest (Edwards and Beelman, 1990). These

recoveries are shown in Table 4.27.

Table 4.27 Relative recoveries3 of volatile compounds from a sparkling wine

Compound Relative recovery (%) Ethyl acetate 99.7 n-Propanol 96.0 Iso-butanol 92.7 Iso-amyl acetate 95.0 Iso-amyl alcohol 88.9 Ethyl hexanoate 100.0 Ethyl octanoate 100.5 Phenyl acetate 85.0 2-Phenyl ethanol 101.1 Octanoic acid 143.7 Decanoic acid 128.4 a Relative recovery = Ca where Cs = concentration of compound in spiked wine Cj = concentration of compound in unspiked wine Ca = concentration of compound added to unspiked wine 166

4.3 Results

4.31 Commercial sparkling wines

4.311 Changes in yeast population during fermentation

Figure 4.31 shows the kinetics of growth and death of S. cerevisiae during the secondary fermentation in two commercial sparkling wines. The yeast increased from an initial population of approximately 1x10^ cfu/mL to a maximum viable population of approximately 6x10^ cfu/mL at 8 to 10 days, thereafter maintaining this population until about 30 days. From 30-100 days for the Nuriootpa wine and 30-120 days for the Wagga wine, the yeasts then exhibited an extended death phase. No viable yeast cells were detected in either wine after 100-120 days.

0 20 40 60 80 100120 Days at 12-15C

Figure 4.31 Changes in yeast population during the fermentation of two commercial sparkling wines (Wagga □, Nuriootpa A) 167

4.312 Utilisation of sugars during fermentation

Figures 4.32a and 4.32b show the utilisation of fermentable sugars during the fermentation of two commercial sparkling wines. The sparkling wine from the Wagga winery contained glucose, fructose and sucrose, each at an initial concentration of about 0.6%. The concentration of sucrose decreased rapidly during fermentation, causing a slight increase in the initial concentrations of fructose and glucose. By 6 days, most of the sucrose had been utilised and, thereafter, the concentration of glucose and fructose decreased. Glucose was utilised slightly faster than fructose and was depleted by day 30. The concentration of fructose was depleted by day 37.

The wine produced at the Nuriootpa winery contained both glucose and fructose (no sucrose) each at an initial concentration of about 1.2%. Both sugars were rapidly utilised during the first 15 days; thereafter, residual amounts were depleted over the next 15 days for glucose and 22 days for fructose. 168

0 10 20 30 40 Days at 12-15C

0 10 20 30 40 Days at 12-15C

Figure 4.32 Changes in the concentration of sugars during fermentation of (a) Wagga and (b) Nuriootpa commercial sparkling wines. (Fructose □, glucose A, sucrose 0 )

4.313 Changes in concentration of ethanol during fermentation and ageing

Figures 4.33a and 4.33b show the kinetics of ethanol production during fermentation and ageing of two commercial sparkling wines. 169

Figure 4.33 Changes in concentration of ethanol during (a) fermentation and (b) ageing of two commercial sparkling wines. (Wagga □, Nuriootpa A)

During the active fermentation of reducing sugars (0-40 days), the ethanol concentration in the Wagga and Nuriootpa wines increased by 1.38 and 1.49% (v/v), respectively (Figure 4.33a). Following this, the concentration of ethanol remained relatively constant until the end of storage at about 950 days (Figure 4.33b).

4.314 Changes in concentration of glycerol during fermentation and ageing

Changes in the concentration of glycerol during the fermentation and ageing of two commercial sparkling wines are given in Figures 4.34a and 4.34b. The initial concentration of glycerol was 0.66% and 0.53% (w/v) in the Wagga and Nuriootpa wines, respectively (Figure 4.34a).

During the secondary fermentation, the concentration of glycerol in the

Wagga wine increased by 0.02% (w/v) and by 0.03% (w/v) in the Nuriootpa wine. Figure 4.34b shows that the concentration of glycerol increased by 170 about 0.04% (w/v) during ageing from 100-500 days in the Wagga wine and then remained stable until the end of storage at about 950 days. The concentration of glycerol in the Nuriootpa wine progressively decreased by 0.03% (w/v) from 200-950 days of ageing.

0.70 - 0.70 -

a 0.65 c 0.65

£ 0.60 £ 0.60

9 0.55 - 9 0.55 -

0 20 40 60 80 100120 250 500 750 1000 Days at 12-15 days Days at 12-15C

Figure 4.34 Changes in concentration of glycerol during (a) fermentation and (b) ageing of two commercial sparkling wines. (Wagga □, Nuriootpa A)

4.315 Changes in the concentration of organic acids during fermentation and ageing

The changes in concentration of organic acids during the fermentation of the two commercial sparkling wines are summarised in Figures 4.35a and 4.35b.

The concentrations of formic, acetic and citric acids in both wines were about 0.1% (w/v) at tirage and remained static during the secondary fermentation (Figure 4.35a). The concentration of lactic acid was about 0.65% and 0.30% at tirage in the Wagga and Nuriootpa wines, respectively, and remained 171 stable during the secondary fermentation. The concentration of malic acid at tirage was about 0.25% and 0.90% in the Wagga and Nuriootpa wines, respectively. The concentration of malic acid decreased during the active sugar fermentation (0-40 days) in both the Wagga (15% decrease) and Nuriootpa (27% decrease) wines.

0.80 0.80 -

c 0.60 - 0.60 -

040 ----- ♦

■9 0.20 0.20 -

0.00 |

Days at 12-15C Days at 12-15C

Figure 4.35 Changes in concentration of organic acids during fermentation of (a) Wagga and (b) Nuriootpa commercial sparkling wines. (Citric □, lactic ■, formic 0, malic A, acetic ▲ and succinic acids ♦)

Figure 4.36 illustrates the separation of organic acids in the Wagga sparkling wine during fermentation. 172

Figure 4.36 Separation of organic acids in sparkling wine by HPLC. (a) Wagga sparkling wine at tirage, (b) Wagga sparkling wine after 37 days fermentation and (c) standard mixture of organic acids (0.5 % w/v).

[1. Citric acid 2. Tartaric acid 3. Malic acid 4. Succinic acid 5. Lactic acid 6. Formic acid 7. Acetic acid 8. 9. and X, Y = unknown] 173

None of the acids exhibited any significant change in concentration (< 5%) during storage on lees for up to 950 days (Figures 4.37a, 4.37b).

0.8 - £ 0.8

e a 0.6 - o '■5 £2c a; -4----♦ C uo 0.2 -x^Vyyyyw—

0.0 100 300 500 700 900 100 300 500 700 900 Days at 12-15C Days at 12-15C

Figure 4.37 Changes in concentration of organic acids during ageing of (a) Wagga and (b) Nuriootpa commercial sparkling wines. (Citric □, lactic ■, formic 0, malic A, acetic ▲ and succinic acids ♦)

4.316 Changes in the concentration of soluble protein during fermentation and ageing

The changes in soluble protein concentration in two commercial sparkling wines during fermentation and ageing are shown in Figures 4.38a and 4.38b.

At tirage, the protein content was approximately 5.3 pg/mL for both wines; it then increased to about 7.2-7.7 pg/mL by the end of the secondary fermentation, after which it decreased slightly in both wines for about 90 days

(Figure 4.38a). Thereafter, the protein content of the wines increased progressively during ageing on lees until 950 days (Figure 4.38b). The commencement of this phase of protein release was approximately 40-50 days following complete death of the viable yeast population (Figure 4.31). 174

During this phase, the protein concentration increased by approximately 40% in the Wagga wine and 12% in the Nuriootpa wine.

-r io

jr io -

0 200 400 600 8001000 Days at 12-15C Days at 12-15C

Figure 4.38 Changes in the concentration of soluble protein during (a) fermentation and (b) ageing of two commercial sparkling wines on lees. (Wagga □, Nuriootpa A)

4.317 Changes in the concentration of amino acid nitrogen during fermentation and ageing

The changes in concentration of total amino acid nitrogen during fermentation and ageing of the two sparkling wines are given in Figures 4.39a and 4.39b.

The amino acid nitrogen content in the Wagga and Nuriootpa wines decreased initially by 4.0 mg/L (0-10 days) and 4.7 mg/L (0-15 days), respectively (Figure 4.39a). This was followed by a minor release of amino nitrogen from 10 to 50 days in the Wagga wine (2.4 mg/L) while in the

Nuriootpa wine the amino nitrogen concentration remained relatively stable.

From about 150 days, the total amino nitrogen content increased by 6.5 mg/L in the Wagga wine and by 4.7 mg/L in the Nuriootpa wine, respectively. 175

80

o 70 - o 70 -

1= 60-

100 350 600 850 1100 Days at 12-15C Days at 12-15C

Figure 4.39 Changes in concentration of total amino nitrogen during (a) fermentation and (b) ageing of two commercial sparkling wines. (Wagga □, Nuriootpa A)

Table 4.31 summarises the changes in concentrations of individual amino acids in the Wagga sparkling wine from about 250-950 days ageing on lees.

The increase in amino nitrogen content in the Wagga sparkling wine between

250-974 days is reflected by increases in the contents of individual amino acids. Table 4.31 shows that the largest increase in amino acids occurred between about 250-600 days. The amino acids arginine, glutamic acid, glycine, aspartic acid, threonine, histidine and methionine increased by 10% or more during this period. 176

Table 4.31 Changes in concentration of amino acids during storage of the Wagga sparkling wine on lees.

Concentration (pmolar) in Wagga wine during ageing (days) Amino acid 250 440 % 600 % 975 % increase3 increase*3 increase0

Alanine 608 672 +10.5 661 -1.7 634 -4.2

Arginine 476 502 +5.5 533 +6.2 538 +1.1 i

Glycine 238 263 +10.5 264 +0.3 252 oo

Valine 186 191 +2.7 184 -3.8 184 0

Glutamic acid 171 185 +8.2 223 +20.5 220 -1.4

Leucine 163 171 +4.9 154 -11.1 156 +1.2

Serine 152 167 +9.8 166 -0.6 169 +1.8

Aspartic acid 103 112 +8.7 133 +18.8 141 +6.0

Threonine 91 108 +18.7 133 +23.1 136 +2.3

Iso-leucine 91 95 +4.3 90 -5.5 90 0

Tyrosine 91 92 +1.1 97 +5.4 93 -4.3

Phenylalanine 87 94 +8.0 90 -4.4 90 0

Histidine 50 56 +12.0 70 +25.0 74 +4.2

Methionine 22 27 +22.7 31 +14.8 31 0

a Increase relative to 250 days b Increase relative to 440 days c Increase relative to 600 days

Figure 4.310 illustrates the HPLC separation of amino acids in the Wagga

sparkling wine during ageing on lees. 177

0 20 to 60 00 100-120 Time (min.)

Figure 4.310 Separation of amino acids during sparkling wine ageing on lees by HPLC. (a) Wagga sparkling wine after 359 days ageing, (b) Wagga sparkling wine after 440 days ageing and (c) standard mixture of amino acids (500 pmolar).

[ 1. Aspartic acid. 2. Threonine. 3. Serine. 4. Glutamic acid. 5. Proline. 6. Glycine. 7. Alanine. 8. Valine. 9. Methionine. 10. Iso-leucine. 11. Leucine. 12. Tyrosine. 13. Phenylalanine. 14. Histidine. 15. Tryptophan. 16. Arginine. X = unknown; Y = 7 - amino butyric acid; Z = ammonia] 178

The changes in concentration of individual amino acids from about 150-950 days ageing in the Nuriootpa wine are summarised in Table 4.32.

Table 4.32 Changes in concentration of amino acids during storage of the Nuriootpa sparkling wine on lees.

Concentration (pmolar) in Nuriootpa wine during ageing ______(days)______Amino acid 305 515 % 709 % 920 % increase3 increase^ increase0

Alanine 711 714 +0.4 716 +0.3 712 -0.6

Glutamic acid 396 410 +3.5 402 -2.0 399 -0.8

Glycine 243 263 +9.5 263 0.0 260 -1.2

Serine 204 217 +6.4 221 +1.8 225 +1.8

Valine 201 233 +15.9 233 0.0 233 0.0

Aspartic acid 160 166 +3.8 173 +4.2 178 +2.9

Threonine 159 165 +3.8 165 0.0 169 +2.4

Arginine 143 142 -1.0 142 0.0 142 0.0

Leucine 139 148 +6.5 148 0.0 160 +8.1

Phenylalanine 86 90 +4.7 89 -1.2 90 +1.1

Tyrosine 81 85 +4.9 87 +2.4 88 +1.1

Iso-leucine 79 79 0 82 +3.8 88 +7.3

Histidine 62 63 +1.6 63 0.0 63 0.0

Methionine 30 34 +13.3 33 -3.0 36 +9.1

a Increase relative to 305 days b Increase relative to 515 days c Increase relative to 709 days

Table 4.32 shows that the increase in concentration of individual amino acids

was generally less than that observed in the Wagga wine (usually less than

10%). The greatest increase in amino acids, however, occurred between

about 300-500 days of ageing with glycine, serine, valine, leucine and

methionine all registering increases in the range 6.0 - 16.0%. 179

4.318 Changes in concentration of volatiles during fermentation and ageing

Figure 4.311 illustrates the separation of volatiles extracted from the Wagga sparkling wine.

TIME (min) 2.0

Figure 4.311 Separation of major wine volatiles in sparkling wine by GLC. (a) Wagga sparkling wine at tirage and (b) standard mixture of wine volatiles.

[ 1. Ethyl acetate 2. n-Propanol 3. Iso-butanol 4. Iso-amyl acetate 5. Iso­ amyl alcohol 6. Ethyl hexanoate 7. Ethyl octanoate 8. Phenyl acetate 9. 2-Phenylethanol 10. Dodecanol 11. Octanoic acid 12. Nonanoic acid 13. Decanoic acid; X = unknown and Y = Acetic acid. ] 180

Changes in content of high concentration volatiles (ethyl acetate and iso-amyl alcohol) in the two wines are shown in Figures 4.312a and 4.312b.

IT 80 150 -

■S' 130 -

B 50 -

80

0 250 500 750 1000 250 500 750 1000 Days at 12-15C Days at 12-15C

Figure 4.312 Changes in the concentration of (a) ethyl acetate and (b) iso-amyl alcohol during fermentation and ageing of two commercial sparkling wines. (Wagga □, Nuriootpa A)

The concentration of ethyl acetate at tirage was approximately 90 and 25 mg/L in the Wagga and Nuriootpa wines, respectively. Between tirage and 80 days, ethyl acetate decreased in the Wagga wine by 18 mg/L after which there was a gradual increase of 10 mg/L over storage to 950 days. The concentration of ethyl acetate in the Nuriootpa wine increased during fermentation and ageing from 25-60 mg/L, which represents a 140% rise in concentration.

The concentration of iso-amyl alcohol increased during fermentation in both

Wagga and Nuriootpa wines by 11 mg/L and 36 mg/L, respectively (Figure

4.312b); thereafter, the concentration of iso-amyl alcohol remained constant in the Wagga wine but increased substantially in the Nuriootpa wine by approximately 50 mg/L (50% increase). 181

Changes in the concentrations of other, more dilute volatiles are shown in Figure 4.313 (esters) and Figure 4.314 (higher alcohols). Figure 4.313a shows that ethyl hexanoate and ethyl octanoate increased by about 0.5 mg/L during fermentation and then remained relatively constant during storage on lees in the Wagga wine. Iso-amyl acetate decreased during fermentation and storage until it became undetectable by about 750 days. The concentration of ethyl hexanoate and ethyl octanoate increased by about 0.5 mg/L during the fermentation of the Nuriootpa wine (Figure 4.313b). From about 60-375 days, ethyl octanoate decreased in concentration by approximately 50% and then became constant until the end of storage. The concentration of ethyl hexanoate increased slightly from 60-500 days and then became stable until

950 days. The concentration of iso-amyl acetate increased by about 0.8 mg/L during the fermentation and then steadily decreased until it became undetectable at about 700 days.

Figure 4.313 Changes in the concentration of esters during fermentation and ageing of (a) Wagga and (b) Nuriootpa commercial sparkling wines. (Iso-amyl acetate □, ethyl hexanoate A, ethyl octanoate ♦) 182

Figure 4.314 shows that the concentration of n-propanol increased during fermentation of both the Wagga (7 mg/L) and Nuriootpa (15 mg/L) wines. The concentration of n-propanol in the Wagga wine remained relatively stable thereafter until the end of storage while it increased from 400 days onwards in the Nuriootpa wine. The concentration of iso-butanol increased during fermentation by 2 mg/L and 11 mg/L in the Wagga and Nuriootpa wines, respectively. Following 40 days, the concentration of iso-butanol remained steady (Wagga) or increased from 500 days onwards (Nuriootpa).

The production of 2-phenylethanol during fermentation for both wines was relatively small (less than 1 mg/L); thereafter, the concentration of

2-phenylethanol remained constant (Wagga) or increased slightly from 400 days onwards (Nuriootpa).

o o 0 250 500 750 1000 0 250 500 750 1000 Days at 12-15C Days at 12-15C

Figure 4.314 Changes in concentration of higher alcohols during fermentation and ageing (a) Wagga and (b) Nuriootpa commercial sparkling wines, (n-propanol □, iso-butanol A, 2-phenylethanol ♦) 183

4.32 Sparkling wine produced from a model wine

4.321 Changes in yeast population during fermentation

Figure 4.315 presents the kinetics of growth and death of S. cerevisiae in the model wine (produced from a fermented chemically defined grape juice) during the secondary fermentation. The yeasts grew from an initial population of approximately 4x106 cfu/mL to 6x106 cfu/mL over 10 days.

Thereafter, yeast viability decreased, with no viable yeast cells being detected in the wine after 120 days.

CT 7.0 -

6.0 -

c 5.0 -

4.0 -

0 20 40 60 80 100120 Days at 12-15C

Figure 4.315 Changes in yeast population during the secondary fermentation of a sparkling wine produced from a model wine.

4.322 Utilisation of sugars and production of CO2 during fermentation

The wine contained glucose at an initial concentration of about 2.3%. The glucose was rapidly consumed during the first 15 days of fermentation and the residual amount was depleted over the next 20 days (Figure 4.316). 184

Utilisation of glucose was accompanied by CO2 production with a maximum of 528 kPa being attained upon exhaustion of glucose after 34 days (Figure

4.316).

e o ca 300 G CL) G o QJ 100

0 10 20 30 40 50 Days at 12-15C

Figure 4.316 Changes in glucose concentration and carbon dioxide headspace pressure during fermentation of model wine (Glucose □, CO2 pressure in bottle A).

4.323 Changes in concentration of glycerol and ethanol during fermentation and ageing

Figures 4.317a and 4.317b show changes in the concentrations of glycerol and ethanol during secondary fermentation and ageing of the wine. The concentration of ethanol increased from an initial value of 10.13% to 11.80% (v/v) during the alcoholic fermentation (0-34 days). Thereafter, the concentration of ethanol decreased slightly to about 11.44 % (v/v) between

35-140 days and then remained constant until the end of storage at 550 days 185

(Figure 4.317b). The initial concentration of glycerol in the wine was 0.49% (w/v) which increased to 0.51% (w/v) during the secondary fermentation. Between 140-550 days, the concentration of glycerol increased by 0.02% (w/v).

.2 12 - 0.55 - 0.55

- 0.50

0.45

Days at 12-15C Days at 12-15C

Figure 4.317 Changes in the concentration of ethanol and glycerol during (a) secondary fermentation and (b) ageing of a model wine. (Ethanol □, glycerol A)

4.324 Changes in concentration of organic acids during fermentation and ageing

Changes in the concentration of organic acids during the secondary fermentation and ageing of a wine produced from a model wine are presented in Figures 4.318a and 4.318b.

The concentration of organic acids remained constant during the secondary fermentation (Figure 4.318a) with the exception of malic acid which increased slightly between 0-10 days (0.02%) and was followed by a decrease between 186

10-28 days (0.03%). Figure 4.318b shows that no significant changes in the concentration of the organic acids occurred during ageing.

> 0.5 - > 0.5 -

0.3

S3 0.2 - to 0.2 -

0.0 50 200 350 500 650 Days at 12-15C Days at 12-15C

Figure 4.318 Changes in the concentration of organic acids during (a) secondary fermentation and (b) ageing of a model wine. (Citric □, lactic ■, succinic ♦, malic A, acetic A and formic acids 0)

4.325 Changes in concentration of soluble protein during fermentation and ageing

Figure 4.319 shows the changes in soluble protein content during the secondary fermentation and ageing.

The soluble protein concentration of the wine was about 8.6 pg/mL at tirage

(Figure 4.319a). During fermentation the soluble protein concentration increased by 2.13 pg/mL until 85 days of storage where after it decreased slightly until 139 days. This was followed by a period of increase in protein concentration from 139-300 days (1.63 pg/mL) after which it remained constant until the end of storage at 550 days (Figure 4.319b). 187

is 10 - i 11-

100 200 300 400 500 600 Days at 12-15C Days at 12-15C

Figure 4.319 Changes in concentration of soluble protein during the (a) secondary fermentation and (b) ageing of a model wine.

4.326 Changes in concentration of amino acid nitrogen during fermentation and ageing

Figures 4.320a and 4.320b give the changes in total amino acid nitrogen content during the fermentation and ageing of the wine.

The concentration of total amino nitrogen decreased by about 8.7 mg/L between 0-28 days fermentation (Figure 4.320a). This was followed by an increase in total amino nitrogen (2.6 mg/L) between 28-85 days after which the concentration remained relatively stable until about 250 days. From 250-

550 days, the amino nitrogen content increased by about 7.1 mg/L (Figure 4.320b) 188

^ 155

o3 150 g 150 -

.5 145 c 145

^ 140 hn 140 100 200 300 400 500 600 Days at 12-15C Days at 12-15C

Figure 4.320 Changes in concentration of total amino nitrogen during (a) secondary fermentation and (b) ageing of a wine prepared from a model wine.

The changes in concentrations of individual amino acids during the period 250-550 days are summarised in Table 4.33. In general, the greatest increases in individual amino acids were between 5.0 - 12.5% and included iso-leucine, alanine, threonine, arginine, glycine, leucine, histidine and tyrosine. Notably, the concentration of tryptophan decreased during storage, and was reduced by 50% between 240 and 575 days of storage. 189

Table 4.33 Changes in concentration of amino acids during storage of a sparkling wine prepared from a model wine.

Concentration (pmolar) in the wine during ageing (days) Amino Increase3 Increase^ acid 240 375 (%) 575 (%)

Glutamic acid 719 713 -0.8 725 +1.7

Valine 716 716 0.0 747 +4.3

Iso-leucine 696 711 +2.2 754 +6.0

Alanine 585 601 +2.7 616 +2.5

Serine 528 508 -3.9 523 +3.0

Threonine 481 508 +5.6 517 +1.8

Arginine 454 477 +5.1 482 +1.0

Glycine 389 414 +6.4 408 +1.5

Leucine 368 379 +3.0 398 +5.0

Histidine 339 358 +5.6 353 -1.4

Phenylalanine 332 340 +2.4 359 -5.6

Methionine 263 267 +1.5 276 +3.4

Aspartic acid 214 223 +4.2 221 -0.9

Tyrosine 56 56 0.0 63 +12.5

Tryptophan 14 10 -40.0 7 -42.9 a Increase relative to 240 days b Increase relative to 375 days

4.327 Changes in concentration of total nucleic acids (RNA) during fermentation and ageing

The initial concentration of total nucleic acids in the wine was 59 pg/mL

(Figure 4.321). The greatest increase in nucleic acid content of the wine occurred during the fermentation and death phase of the yeast, which covered 0-140 days (18%). Subsequently, there was a small but progressive increase in concentration until the end of storage at 550 days (7%). 190

Days at 12-15C

Figure 4.321 Changes in the concentration of total nucleic acids during the fermentation and ageing of a model wine.

4.328 Changes in concentration of lipids during fermentation and ageing

Lipid material was extracted from the wine and the isolated lipid classes

(triglycerides, 1,3 diglycerides, 1,2 diglycerides, monoglycerides, free fatty acids and polar lipids) analysed for their fatty acid profile. Figure 4.322 shows changes observed in the total fatty acid composition of these lipid classes during storage of the wine. The concentration of triglycerides and diglycerides (Figure 4.322 a,b and c) increased between 0-250 days and then decreased. The concentration of monoglycerides decreased sharply after about 100 days (Figure 4.322d). Changes in the concentration of free fatty acids followed the same general pattern as triglycerides and diglycerides 191

(Figure 4.322e). The concentration of polar lipids remained relatively constant during ageing of the wine (Figure 4.322f)

Methylation of the lipid classes described in Figure 4.322 indicated the presence of Ci2:0> ^14:0> Cl6:0> ^18:0 and Cl8:1 fatty acids. The changes in concentration of these fatty acids within each lipid class during fermentation and ageing of the wine are shown in Figure 4.323.

The concentration of C-|6:0> ^18:0 and ^18:1 fatty acids in triglycerides and diglycerides (Figures 4.323a, 4.323b and 4.323c) followed a similar pattern during storage - an increase between 0-250 days followed by a decrease until the end of storage. The only exception to this trend was the slight decrease in concentration of C-|6:0 and C-|q q between 0-100 days in 1,2 diglycerides. The concentrations of all fatty acids in the monoglyceride fraction (with the exception of Ci6:0) increased slightly during 0-100 days whereupon they stabilised for the rest of the storage period. A relatively large increase in

Ci6:0 occurred during 0-100 days which was followed by a decrease of similar proportions from 100 - 250 days (Figure 4.323d). During this reduction, the concentration of C-j6:0 increased as the free fatty acid in the wine (Figure 4.323e). The concentration of C-| g:0 and C-| Q: 1 in the free fatty acid fraction decreased from 250 and 100 days ageing, respectively. The concentration of fatty acids in the polar lipid fraction decreased during 0-

100 days storage and then stabilised until the end of storage at 550 days (Figure 4.323f). The only exception to this trend was a gradual increase in the concentration of C-j 8:0 from 100 days until 550 days. The changes in concentration of C-)2:0 and ^14:0 fatty acids in all lipid classes were minimal. 192

Days at 12-15C Days at 12-15C

Days at 12-15C Days at 12-15C

Days at 12-15C Days at 12-15C

Figure 4.322 Changes in concentration of (a) triglycerides, (b) 1,3 diglycerides, (c) 1,2 diglycerides, (d) monoglycerides, (e) free fatty acids and (f) polar lipids during storage of a model wine. 193

g 10 -

Days at 12-15C Days at 12-15C

20 -

g 10 -

Days at 12-15C Days at 12-15C

20 - 20 -

g 10 -

Days at 12-15C Days at 12-15C

Figure 4.323 Changes in fatty acid composition of (a) triglycerides, (b) 1,3 diglycerides, (c) 1,2 diglycerides, (d) monoglycerides, (e) free fatty acids, and (f) polar lipids from lipids extracted from a wine prepared from a model wine. [C-|2:0 c14:0 0> c16:0 ■> c18:0 A and c18:1 Al 194

4.329 Changes in concentration of volatile components during fermentation and ageing

Figure 4.324 shows the changes in concentrations of high content volatiles (ethyl acetate and iso-amyl alcohol) during the fermentation and ageing of the wine.

60 -

50 -

40 -

30 -

Days at 12-15C

Figure 4.324 Changes in concentration of ethyl acetate (□) and iso-amyl alcohol (A) during the fermentation and ageing of a model wine.

The concentration of ethyl acetate decreased slightly (5 mg/L) during fermentation and was followed by an increase in concentration (17 mg/L) from 40-300 days storage; thereafter, the concentration of ethyl acetate remained relatively constant. The concentration of iso-amyl alcohol showed minimal change (less than 10 mg/L) during fermentation and ageing of the wine. 195

Figure 4.325 gives the changes in concentration of low concentration esters and fusel alcohols during the fermentation and ageing of the wine.

Figure 4.325 Changes in concentration of low level (a) esters and (b) higher alcohols during the fermentation and storage of a model wine. [ esters : Iso-amyl acetate □, ethyl hexanoate A and ethyl octanoate 0 higher alcohols : n-propanol ■, 2-phenylethanol A and iso-butanol ♦]

Iso-amyl acetate and ethyl hexanoate increased by about 0.1 mg/L during 0-

40 days. This was followed by a decrease of about 0.2 mg/L of iso-amyl acetate, whereas the concentration of ethyl hexanoate remained relatively constant upon further storage. The concentration of ethyl octanoate was about 1 mg/L at tirage and gradually decreased by about 50% during ageing.

The concentration of fusel alcohols increased by 1-3 mg/L (Figure 4.325b) during fermentation of the wine; thereafter, the concentration of these compounds was relatively constant. 196

4.4 Discussion

The unique properties of sparkling wines develop as a consequence of the secondary fermentation and ageing processes. Detailed studies of the microbiological and chemical changes that occur during these stages should provide a better understanding of the overall operation, with the potential benefits of process optimisation and improved control over product quality. This chapter has provided detailed information about the microbiological and chemical changes that occur during the secondary fermentation and ageing of two commercial sparkling wines and a model sparkling wine.

4.41 Growth and survival of yeasts during sparkling wine fermentation

Investigation of the kinetics of growth and death of yeast during sparkling wine fermentation was a major objective in this project because of its primary role in affecting the onset of yeast autolysis (Arnold, 1981b). Data obtained with two commercial wines and the model system indicated the following basic features of yeast growth and death during the secondary fermentation :

(a) rapid onset of growth with essentially no lag phase being evident,

(b) growth of the yeasts to a maximum viable population of about 6 x 106 cells/mL within 8 to 10 days,

(c) a stationary phase of 15 to 20 days where the population of viable yeast remained relatively constant, and

(d) the onset of a death phase at about 30 days, which lasted for about 65 to

80 days until no viable cells could be detected.

There is little published information on the growth kinetics of the yeast during the secondary fermentation. Bidan et aj. (1986) provided a generalised 197 graph showing changes in the viable and total yeast population up to 180 days following tirage. The viable population increased from 1x10® cells/mL to a maximum of about 4.5 x 10® cells\mL over 35 days, and then decreased to less than 10® cell/mL by 80 days. The data of this Chapter are in general agreement with this report, but also highlight two additional features. First, the cells rapidly enter the stationary phase; its duration of about 25 to 30 days, compared with the relatively short exponential phase, means that about

50 to 65% of the secondary fermentation (as denoted by consumption of sugar) is conducted by the cells in the stationary phase. Second, the death phase is protracted and may last twice as long as the phase of active fermentation. As noted later, autolysis is not detected until the cells have completed the death phase.

The maximum viable population of about 5-6x10® cells/mL is a common feature of the three fermentations examined in this study and that reported by Bidan et aJ. (1986). In two of these wines fermented with the same yeast strain (Wagga and model wines), this final population was found irrespective of the population of the yeast added at tirage (Wagga, 1 x 10® cells/mL, Figure 4.31; model wine, 4 x 10® cells/mL, Figure 4.315). This observation suggests that some common factor is limiting population development. This is unlikely to be a shortage of sugars, since analyses indicated that they are still present when the stationary phase is reached (Figures 4.32a and 4.316).

Similarly, nitrogen added at tirage (di-ammonium phosphate) would ensure that it is present in excess amounts. As will be discussed in more detail later in Chapter 5, the increasing concentration and pressure of CO2 is likely to be a limiting property. The growth limiting effects of CO2 on yeast has been well established (Jones and Greenfield, 1982; Dixon and Kell, 1989; Slaughter,

1989). At 10 days, the internal bottle pressure in the model wine was about 300 kPa at 15°C (Figure 4.316); Lumsden et aJ. (1987) reported total 198 inhibition of cell growth in S. cerevisiae at 390 kPa. However, strain variation in resistance to C02 within a yeast species can be expected; additionally, Lumsden et aL (1987) conducted fermentations at 25°C where the higher temperature would decrease the concentration of C02 in solution.

The extended death phase varied from about 30 to 100 (Nuriootpa wine) or 30 to 120 days (Wagga and model wines). Both the Wagga and model wines were fermented with S. cerevisiae strain EC-1118, which may explain the similarity in death kinetics in these wines compared with the earlier death of the yeast used in the Nuriootpa wine (S. cerevisiae, Penfold's in-house strain). Similar kinetics of death were observed by Bidan et aL (1986) in sparkling wines, although they did not attach any significance to the length of this phase. It is not clear what carbon source the yeast population is using for survival as exhaustion of sugars occurs at about 30 to 40 days (Figures 4.32 and 4.316). However, it may be a situation where the yeast are maintaining themselves by using storage polysaccharides built up during the secondary fermentation.

4.42 Chemical changes during sparkling wine production

4.421 Sugars, ethanol, glycerol and carbon dioxide

The patterns of sugar utilisation and concomitant production of ethanol and carbon dioxide observed during the secondary fermentation of the commercial and model wines was consistent with data published by Bidan et aL (1986).

The kinetics of glucose and fructose utilisation in the commercial wines initially differed because of the presence of sucrose in the Wagga wine (compare Figures 4.32a and 4.32b). The glucose and fructose concentration 199 in the Wagga wine increased from 0 to 5 days due to the action of yeast invertase. Following this stage, consumption of the remaining monosaccharides was similar for both commercial wines and followed first order kinetics as predicted by Merzhanian and Kozenko (1972). Glucose was consumed at a faster rate than fructose in both commercial wines, a characteristic that has been noted for fermentations conducted by many, but not all, strains of S. cerevisiae using mixtures of these two monosaccharides

(D’Amore et aL, 1989). In the model wine, fermentable sugar was added as glucose only. Although present at twice the concentration, the kinetics of glucose utilisation in the model wine was similar to that found for the Nuriootpa wine. These results indicate that the carbon source added at tirage may be in the form of sucrose, invert sugar, or glucose only, without any apparent effect on the kinetics of yeast growth and survival.

As indicated by the kinetics of sugar utilisation and ethanol and CO2 production (Figures 4.32, 4.33, 4.316 and 4.317), a significant proportion of carbohydrate metabolism (50 to 65%) occurred during the stationary phase (about 8 to 35 days) when the cells were mostly in the non-proliferating form. This observation has not been reported previously for the secondary fermentation of sparkling wines, although in recent years it has been recognised that a good proportion of the primary alcoholic fermentation of wines occurs when the yeast cells have entered the stationary phase (Bisson,

1993).

The secondary fermentation generated about 540 kPa of carbon dioxide pressure in the bottles, or 88 kPa of C02 pressure (0.87 atmospheres) / 4g glucose (Figure 4.316) which compares with the value of 1 atmosphere of

C02 pressure (1 atmosphere = 101.3 kPa) for the equivalent amount of sugar quoted by Bidan et aL (1986). 200

Based on the stoichiometry of sugar fermentation into ethanol by the EMP pathway, 24 g/L of sugar should yield about 1.4% (v/v) ethanol (Monteiro, 1991) and this was found within the range for the three wines (Figures 4.33a and 4.317) and that reported by Bidan et aj. (1986). Little change in ethanol concentration is expected after the secondary fermentation, although the slight decrease observed in the model wine may reflect the occurrence of secondary reactions such as esterification with fatty acids to produce esters

(Etievant, 1991).

Glycerol is normally produced as a significant secondary metabolite during fermentation of glucose (Bisson, 1993) and this is reflected in the small amounts (0.02 to 0.03%) produced during the secondary fermentation of the sparkling wines (Figures 4.34 and 4.317). Glycerol was produced at a slower rate than ethanol with the concentration increasing in the wine gradually over 60 to 70 days. Factors which may affect glycerol production include temperature of fermentation, inoculation level, aeration, presence of sulphite, and differences between yeast strains (Rankine and Bridson, 1971; Ough et aL, 1972; Gardner et aL, 1993), but such influences were not evident during the secondary fermentation. The slight difference (0.01%) observed between the Wagga and Nuriootpa wines, however, could be due to strain variation. Of the other factors, temperature could not play a role since all wines were stored at the same temperature. Inoculation level had no effect as the model wine contained about four times the initial viable population of the Wagga wine. Sulphite concentration in the Wagga wine was 66 mg/L compared with none in the model wine, but both wines showed similar increases (0.02%) in the amounts of glycerol during the secondary fermentation. Aeration of the culture before tirage was also not likely to play a role in glycerol production as the Wagga and model wines produced the same amount of glycerol despite the same yeast strain being exposed to different conditions (Section 201

4.22). The yeast used for the Wagga wine was an active dried yeast (prepared aerobically) and vigorously aerated before use, while the yeast inoculated into the model wine was prepared semi-aerobically in flasks. Theoretically, the glycerol production by such yeast should differ to reflect the different enzyme systems induced by the availability of oxygen during preparation (Bisson, 1993).

For the Wagga and model wines, glycerol concentration increased during the ageing period (100 to 550 days) and may arise from the enzymatic hydrolysis of lipids into their constituent fatty acids and glycerol backbone. Both of these wines were produced by the same strain of S. cerevisiae. Similar transformations have been suggested by Piton et aj. (1988) in studies of lipid changes in sparkling wine yeast during ageing on lees. In contrast, the concentration of glycerol decreased in the Nuriootpa wine during ageing suggesting its participation in secondary reactions such as the formation of acetal compounds by reaction with acetaldehyde (Williams and Strauss, 1978; Etievant, 1979). Notably, the secondary fermentation of this wine was conducted with a different strain of S. cerevisiae compared with the Wagga and model wine, and thus might account for the difference observed.

4.422 Organic acids

The concentrations of organic acids in the base wines (0 day data in Figure

4.35) were consistent with values reported in the literature (Radler, 1993).

The lower concentration of malic acid in the Wagga wine (2.5 g/L) compared to the Nuriootpa wine (9.1 g/L) wine suggests it may have undergone a partial malolactic fermentation prior to its use in the sparkling wine fermentation.

With the exception of malic acid, no significant changes in the concentration of organic acids in the commercial (Figures 4.35 and 4.37) and model wines 202

(Figure 4.318) were observed during secondary fermentation and ageing. The concentration of malic acid decreased during the secondary fermentation by 15% and 27% in the Wagga and Nuriootpa commercial wines, respectively. In the model wine, malic acid decreased by 7% after an initial increase of 6% at the start of fermentation. Such deacidification could impact upon flavour by softening the palate of the wine. According to Radler (1993), wine strains of S. cerevisiae may metabolise 3 to 45% of malic acid during fermentation depending on the strain. Malate is initially oxidatively decarboxylated to pyruvate by the NAD-dependant malic enzyme which, in turn, is decarboxylated to acetaldehyde and then reduced to ethanol. Some malic acid may also be transformed to succinate via fumarate (Kuczynski and

Radler, 1982), although this does not appeared to have occurred in these wines as a concomitant increase in succinic acid concentration was not observed. The increase in malic acid concentration during the initial stages of fermentation of the model wine could originate from the reduction of oxaloacetate produced from phosphoenolpyuruvate (Radler, 1993).

Unless organic acids are substantially involved in secondary reactions such as esterification, no changes in their concentration would be expected during ageing, and this was eventually the conclusion from Figures 4.37 (commercial wines) and 4.318b (model wine). Similar findings have been reported by Postel and Ziegler (1991a) for the tartaric acid, malic acid, lactic acid, citric acid and pyruvic acid during ageing of sparkling wine for 2 years.

4.423 Nitrogenous compounds

Changes in the concentrations of nitrogenous compounds were followed by measuring amino acid nitrogen, protein and nucleic acids in the sparkling wines. 203

The variation in concentration of amino acid nitrogen during fermentation and ageing of the commercial and model wines followed four distinct phases :

(a) a decrease in concentration during the first 10 to 25 days,

(b) a slight increase in concentration during 20 to 50 days (commercial wines) or 28 to 85 days (model wine),

(c) no change in concentration during 50 to 150 days (commercial wines ) or

80 to 250 days (model wine), and

(d) an increase in concentration during 150 to 600 days (Wagga wine), or

150 to 400 days (Nuriootpa wine), or 230 to 550 days (model wine).

Similar patterns of change in amino acid nitrogen concentration have been recorded in the literature (Feuillat and Charpentier, 1982; Bidan et aL, 1986).

The concentration of amino acid nitrogen decreased by about 4 mg/L in the commercial wines (Figure 4.39) and by 8.7 mg/L in the model wine (Figure 4.320) during the secondary fermentation. The difference in nitrogen demand between the commercial and model wines is probably a function of the differences in composition of the respective media. Flenschke and Jiranek (1993) discussed the factors affecting amino nitrogen uptake during wine fermentation and indicated that a decreasing environmental pH will decrease the efficiency of amino acid permeases of yeast, and would therefore lower amino acid uptake. This may have been an important factor as the nitrogen demand of the yeast in the wines examined increased with pH; the nitrogen demands of Wagga, Nuriootpa and model wines were 4.0, 4.7 and 8.7 mg/L, respectively, and the wines had pH values of 2.98, 3.18 and 3.4, respectively, at tirage. 204

The uptake of amino acid nitrogen during fermentation was followed by a small release of amino acids (< 2.6 mg/L) in all the wines which is regarded as a passive release mechanism triggered by glucose exhaustion and not related to autolysis (Charpentier et ai., 1986). Following a latent phase during ageing up to 150 - 250 days, the amino acid nitrogen concentration of all wines increased by 4.7 to 7.1 mg/L (4.5 to 12.8%). This compares to an increase of 24.5% in amino acid concentration during a similar phase described by Feuillat and Charpentier (1982) and is believed to be a function of proteolysis during yeast autolysis (Leroy et aL, 1990). Notably, this coincides with the release of protein nitrogen (Figures 4.38 and 4.319).

However, there was noticeable variation in the identity of individual amino acids which increased significantly during the autolytic phase (Table 4.41).

This result is typical of published data where numerous discrepancies exist between authors with regards to which amino acids increase during the autolytic phase (Chapter 2, Section 2.4231, Table 2.8). The work of Tzvetanov and Bambalov (1994) suggests that a contributing factor to this observation may be differences in yeast strain used in the studies. These authors studied the effect of yeast strain upon changes in amino acid nitrogen content of sparkling red wines during ageing on lees. Secondary fermentations were conducted with 16 yeast strains over 36 months with 5 strains giving an increase in amino acid nitrogen (ranging from 0.9 to 7.2%),

8 strains giving a decrease in amino acid nitrogen (ranging from -1.4 to

-5.4%), and 3 strains having no effect on amino acid nitrogen content. 205

Table 4.41 Amino acids found to increase (> 5 %) during the yeast autolytic phase in two commercial and one model sparkling wine

Amino acid Sparkling wine Wagga Nuriootpa Model Alanine / / Glutamic acid / Glycine / // Aspartic acid / Threonine / / Histidine / / Methionine / / Valine / Serine / Iso-leucine / Leucine / / Phenylalanine / Arginine / / Tyrosine /

However, since the Wagga and model wines were fermented with the same yeast strain, it can be surmised that other factors such as differences in media pH and ethanol content, temperature of storage, and growth conditions of the yeast starter culture would have affected the extent and identity of amino acid release during the autolytic phase (Bidan et a]., 1986; Kelly-

Treadwell, 1988). Of these factors, temperature may be discounted as all wines were stored at the same temperature. However, the pH of the model wine was significantly higher than that of the Wagga wine (pH 3.4 compared with 2.98) which is more favourable to amino acid release during autolysis (Vosti and Joslyn, 1954a). Similarly, the lower ethanol content of the model wine compared with the Wagga wine (10.2% compared to 10.9%) might have favoured more extensive autolysis (Sugimoto, 1974). Finally, Vosti and Joslyn (1954a) showed that aeration of cultures during preparation has the effect of increasing the optimum pH for autolysis of yeast. This could have 206 been a contributing factor to the lesser amino acid release in the Wagga wine during autolysis. The yeast culture for the Wagga wine was an active dried yeast (almost certainly prepared under aerobic conditions) that was vigorously aerated for 4 hours prior to addition to the wine base; in comparison, the yeast culture for the model wine was prepared under semi- aerobic conditions in a shaking culture. This aspect of sparkling wine production requires further research because of the widespread use of dried cultures, particularly in the Australian wine industry.

The protein content of the wines at tirage ranged from 7.2 to 8.6 pg/mL. Due to wide variation in methods and technical difficulties associated with measuring proteins in wines (Chapter 3, Section 3.1), values of 1.5 (Yokotsura et aL, 1977) to 840 pg/mL (Somers and Ziemelis, 1973) have been reported. However, Murphy et aL (1989), utilising the procedure adopted in this study, reported values approximately 10 times higher than those found in this study. It should be noted that both commercial wines had undergone routine treatments (bentonite addition) prior to tirage to guard against protein precipitation during storage. These treatments are designed to remove as much protein from the base wine as possible (Amerine et aL, 1980). Similarly, the model wine contains no grape proteins and thus contains less soluble protein than a commercial wine.

For the commercial and model wines, changes in protein content followed similar patterns of increase, decrease and increase. Initially, there was a definite increase in protein concentration corresponding to yeast growth and activity during the secondary fermentation (days 0 to 35). In all cases, the protein content increased by about 2 to 3 pg/mL. This release of protein is typical of growing yeast cultures (Kokitkar et aL, 1990) and was also observed during growth of killer and sensitive strains of S. cerevisiae in Chapter 6. 207

The phase of increase was followed by a period of about 100 days where the protein content decreased slightly (by about 0.5 pg/mL). This period corresponds with the phase of marked cell death and, possibly, could be explained as representing an early phase of yeast autolysis where living cells respond to the presence of protein (secreted by a proportion of cells undergoing autolysis) by releasing exocellular proteolytic enzymes for its degradation and utilisation as a nutrient (Hough and Maddox, 1970).

The final phase (from about 200 days until the end of ageing) was characterised by a marked increase in soluble protein concentration (1.5 to 3.5 pg/mL, depending on the wine) and probably represents protein release by autolysis as, by this stage, all the yeast cells are dead. This observation would correspond to the process of yeast autolysis described by Feuillat and Charpentier (1982) who suggested that autolysis of the yeast lees began between 6 to 12 months of ageing. In contrast, Leroy et a|. (1990) found that autolysis did not begin until 15 months ageing on lees. However, many factors such as temperature, starter culture preparation, pH and ethanol affect the kinetics of autolysis in sparkling wine. Additionally, as explained already, Tzvetanov and Bambalov (1994) showed that yeast strain influences the occurrence and extent of nitrogen release during the ageing of sparkling wine on lees.

In summary, the release of amino acid and protein nitrogen during ageing is consistent with the theory that, following a latent phase lasting about 200 to 250 days, yeast cells in sparkling wine undergo autolysis resulting in a net release of these compounds into the wine. However, further research is required to determine exactly how these compounds affect wine quality. For example, in what manner does autolysed yeast protein influence foaming character and how do environmental parameters influence these reactions. 208

The breakdown of nucleic acids, particularly RNA, is a distinctive reaction of yeast autolysis (Hough and Maddox, 1971; Trevelyan, 1977, 1978a). Given that sparkling wines are aged for a considerable period of time with about 107 yeast cells/mL presumably undergoing autolysis, then release of nucleic acids and their degradation products can be expected. However, as noted already (Chapter 3, Section 3.2), major difficulties are associated with detecting changes in the concentration of nucleic acid degradation products in wine as a consequence of yeast autolysis. The grape itself contains considerable amounts of nucleic acid material that complicate specific assays for degradation products of yeast autolysis. Also, some yeast autolysis probably occurs during the primary fermentation giving additional background nucleic acid products. These sources may also contribute other compounds

(eg. polysaccharides) which interfere with assays for nucleic acids. A further complication is the need for method(s) that give specific detection of RNA and DNA in the wine, as well as degradation products of nucleotides, nucleosides and bases. As noted in Chapter 3 (Section 3.2), dye-binding and colourimetric methods for specific DNA and RNA assays do not work in wines due to interference from grape components such as chlorophyll and polysaccharides. A HPLC method for the separation of nucleotides, nucleosides and bases was unsuccessful when applied to wine because of various interfering substances associated with the complex nature of the matrix (Chapter 3, Section 3.234a). Similarly, a further HPLC method for the assay of guanine as a measure of total nucleic acids was not successful in commercial wines due to various interfering compounds (Chapter 3, Section

3.234b). However, the method was successful when applied to the relatively defined medium of the model wine. For the same reason, the model wine should lend itself to analysis for individual degradation products by the previously described HPLC procedure. However, due to the late development of this specialised analytical procedure within the time frame of 209

this project, insufficient time was available to pursue this analysis and this goal remains an area for further study.

The changes in concentration of nucleic acids in the model wine followed two distinct phases. The first phase encompassed the secondary fermentation and death phase of the yeast (0 to 120 days) where the nucleic acid content of the wine increased by 18%. The second phase of release (120 days to end of ageing at about 550 days) was characterised by a small, but

continuous enrichment of the wine with nucleic acid material (7%). Similar

results were recorded by Leroy et aL (1990) who found that the largest enrichment in nucleic acids in commercial sparkling wine (about 10%)

occurred during the first 3.5 months (100 days). This was followed by another large increase in nucleic acid content (about 9%) from 6 to 9 months (200 to

280 days). However, over equivalent periods of ageing (20 months or 600 days), the total percentage increase in nucleic acid content of the model wine (24%) and wines studied by Leroy et aL (1990) (20%) were relatively similar. The work of Leroy et aL (1990) also indicates that nucleic acid release from autolysing cells is essentially complete by 20 months and is supported by the observations with the model wine.

In summary, the picture of nucleic acid degradation in yeast and release into

wine during ageing is still incomplete although, clearly, the data obtained in this study indicate that some degradation does occur. The relative

degradation of DNA to RNA is not known, although it is probably not significant due to the low content of DNA compared with RNA (about one

'twentieth; Mounolou, 1971). Before a precise description of the changes of

DNA and RNA in sparkling wines can be proposed, more research is needed

to (i) develop methods for the specific quantitative assay of DNA and RNA in

wines and (ii) develop methods for the quantitative assay of nucleic acid degradation products. The second point is particularly important as it is well 210 known that the degradation products of nucleic acids have flavour potentiating properties (Peppier, 1982; Benaiges et aL, 1990) and are likely to impact on sparkling wine quality.

4.424 Volatiles

The two main volatiles found in the sparkling wines were ethyl acetate (25 to

90 mg/L) and iso-amyl alcohol (68 to 108 mg/L) (Figures 4.312 and 4.324).

These concentrations lie within the values reported in wines (Etievant, 1991).

There were variations between the wines in the behaviour of these two volatiles. Most noticeably, there were strong increases in the concentration of both ethyl acetate and iso-amyl alcohol during the secondary fermentation and ageing of the Nuriootpa wine. For the Wagga and model wines the changes were more subtle, with a small decrease in ethyl acetate during the secondary fermentation followed by an increase during ageing while the concentration iso-amyl alcohol did not change significantly. According to Etievant (1991), changes in ester concentration during ageing are due to the following reaction :

ester + H2O <-> acid + alcohol

Acetate esters (including ethyl acetate) may increase or decrease during ageing according to the conditions of the wine. However, increases in concentration of ethyl esters usually occur because of secondary reactions involving ethanol (esterification).

Of the esters found at lower concentrations in the wines, there was a consistent decrease in the concentration of iso-amyl acetate during storage

(Figures 4.313 and 4.325a). In the case of the commercial wines, the concentration of this ester decreased to non-detectable levels by 750 days.

Cologrande and Silva (1986) and Silva et aj. (1987) found that the 211 concentration of iso-amyl acetate decreased by 50 to 60% in ageing sparkling wines. Silva et aj. (1987) suggested that this decrease was due to the presence of esterase activity that was detected in yeast lees up to 300 days after the secondary fermentation. This explanation may also be the reason for the decrease in concentration of ethyl octanoate in the Nuriootpa and model wines during ageing.

The concentration of higher alcohols may increase during ageing in wine through the hydrolysis of esters as outlined above (Etievant, 1991).

Alternatively, some higher alcohols may be formed through oxidation reactions. Simpson (1978), for example, suggests that 2-phenylethanol may increase in ageing wine through the oxidative deamination of phenylalanine. However, aside from small increases during the secondary fermentation, the content of low concentration higher alcohols (Figures 4.314 and 4.325b) in the wines did not vary greatly during ageing. This lack of change is in general agreement with the work of Silva et aj. (1987), except for the small increase in n-propanol in the latter stages of storage of the Nuriootpa wine.

In summary, the concentration of major wine volatiles did not vary greatly during ageing of the wines, although reversible reactions involving esterification may have influenced changes in concentration of ethyl acetate and iso-amyl alcohol and esterase activity, iso-amyl acetate. Future research should concentrate on determining the factors influencing such changes, particularly in encouraging the more favourable reactions such as ester formation. 212

4.425 Lipids

Lipid degradation is a significant event occurring during yeast autolysis (Arnold, 1981b) with the various degradation products, such as fatty acids, likely to influence wine quality (Piton et aL, 1988; Troton et a}., 1989b).

As discussed earlier (Chapter 3, Section 3.3), there were major problems in developing reliable procedures for the quantitative assay of lipids in commercial sparkling wines. However, aside from the technical difficulties involved, lipids in sparkling wines originate not only from yeast autolysis during ageing, but also from the grape itself. In a relative context, lipids arising from autolysis during ageing would contribute only a small fraction of the total lipid present, further complicating any quantitation of their changes during ageing. For these reasons, the study of lipid changes during ageing of sparkling wines in this project was confined to the model wine.

The concentration of lipid classes in the model wine (10 to 70 fig/L) was about 10% of that reported by Troton et aL (1989a) for commercial wines. However, these authors were studying lipid changes in commercial sparkling wines which would include lipids originating from the grape. The model wine lacks these lipids and at tirage would contain only lipids excreted from yeast during the primary fermentation.

There were two distinct phases in evolution of the various lipid classes during sparkling wine fermentation and ageing. The first phase involved an enrichment in neutral lipids and fatty acids in the wine between tirage and

250 days storage (with the exception of monoglycerides, which decreased in concentration after 100 days). The second phase (from about 250 to 550 days) involved a decrease in concentration all lipid classes in the wine, with the exception of monoglycerides and fatty acids which stabilised in the wine 213 after 250 and 400 days, respectively. The concentration of polar lipids decreased slightly between 0 and 400 days, and then increased until the end of storage (Figure 4.332).

During the first phase, there was an increase in the concentration of neutral lipids in the wine which may represent an autolytic release of these compounds from the yeast. The subsequent decrease in concentration of these compounds during the second phase may indicate their complete hydrolysis to form fatty acids and glycerol. Significantly, glycerol concentration increased in the wine during this period between 140 to 550 days (Figure 4.317b). Although one might expect a concomitant increase in the concentration of fatty acids as well, this may be masked by the occurrence of secondary reactions to form ethyl esters; notably, the concentration of fatty acids were observed to stabilise between 400 to 550 days against the general trend of decrease in other lipid components.

According to Piton et aL (1988) and Troton et aj. (1989a), yeast accumulate triglycerides at the expense of polar lipids during storage of sparkling wine over 10 years by the following pathway : polar lipids -> diglycerides (+ fatty acids) -> triglycerides

This theory was based on the observation that triglycerides gradually accumulated in large vesicles within the cell and were accompanied by a decrease in polar lipids originating from cellular membranes. This process may be occurring in the yeast after 250 days storage since the concentration of triglyerides and diglycerides decreased in the wine and did not appear to be replenished by further releases from the cells.

In general, the change in concentration of long chain fatty acids (C16:o, C18:0 and C18:i) followed a similar pattern as the lipid classes. Notably, the 214 dramatic decrease in concentration of C16:0 from about 100 to 250 days in the monoglyceride fraction was accompanied by a large increase in C16:o as the free fatty acid during the same period. The initial increase in C12:o. C16:o anc* C18:1 fatty acids between tirage and 100 days is similar to results obtained by Chen et aj. (1980) who studied ethanol-induced autolytic release of free fatty acids by brewer's yeast over three days at 25°C; as the concentration of ethanol increased from 10% to 15%, the concentration of C10:0, C12;0, C14;0>

Cie:o C16:1, C18:0, C1B: 1 and C24;i fatty acids increased in the autolysate. The presence of such fatty acids in sparkling wine will lead to the formation of their ethyl esters (Etievant, 1991). Such transformations are suggested to play a role in the occurrence of secondary reactions which modify the sensory properties of the final product over an extended period of time (Piton et a]., 1988; Troton et af., 1989 a,b).

The results of this chapter confirm that yeast autolysis is a process which occurs over a lengthy period of time during ageing of sparkling wine. A primary cause for its slow occurrence may be the extended death phase of the yeast lasting up to 2.5 times the active fermentation. It may be monitored, mainly, by changes in the concentration of nitrogen compounds (amino acids, proteins and nucleic acids), although other, more dilute, chemical species such as lipids and volatiles also contribute. Future directions for research lie in developing and/or improving analytical techniques for measuring compounds such as those only measured in a model system in this project

(nucleic acids, lipids). Additionally, research is required to quantify the sensory impact these chemicals have on wine quality and the factors which influence their release with the long-term aim of improved control over this process in sparkling wine production. 215

5.0 Influence of Carbon Dioxide Pressure on Growth and Metabolism of Yeast During Fermentation of Sparkling Wines

5.1 Introduction

The production of carbon dioxide during the secondary fermentation to form bubbles is the most characteristic feature of sparkling wines. The secondary fermentation takes place in specialised bottles which are designed to withstand the pressure resulting from the CO2 produced by this fermentation.

This pressure may reach 500-600 kPa at 10°C (Markides, 1986).

However, in addition to the aesthetic considerations, carbon dioxide also exerts an influence on yeast metabolism. This aspect has received little attention in the context of sparkling wine production, although the general effects of carbon dioxide on microbial metabolism are well documented (Jones and Greenfield, 1982; Daniels et. aL, 1985; Slaughter, 1987; Dixon and Kell, 1989).

Apart from a slight stimulatory effect at low concentrations of 10-15 mM

(Slaughter, 1989), carbon dioxide at higher concentrations is generally inhibitory to yeast growth and metabolism. In sparkling wine production, the concentration of carbon dioxide may reach approximately 275 mM (12 g/L) at 15°C at the end of the secondary fermentation (Amerine et aL, 1980) At these concentrations, the effect of carbon dioxide is inhibitory upon yeast growth and metabolism. It has been shown in continuous sparkling wine production that carbon dioxide reduces growth rate and cell yield while increasing the lag phase of yeast growth (Cahill et aL, 1980). Kunkee and Ough (1966) determined that carbon dioxide pressure decreased the fermentation rate, although yeast adapted to the base wine achieved a higher fermentation rate than non-adapted yeast. However, apart from the effects of 216 carbon dioxide upon cell yield and fermentative performance, there are few data in the literature on the effect of carbon dioxide on the products of metabolism during the secondary fermentation in sparkling wines.

In contrast, the influences of carbon dioxide upon the production of various flavour volatiles (acetaldehyde, higher alcohols, fatty acids, esters and vicinal diketones) and uptake of their precursors, such as amino acids, during brewing have been reported. These effects have been reviewed by Slaughter

(1989) and include reduced production of volatiles (except vicinal diketones and various fatty acids) and a modified absorption pattern of amino acids. Similar effects may occur in sparkling wines, particularly towards the end of the secondary fermentation, and would be expected to impact upon the sensory properties of the wine .

The objective of this chapter is to investigate the influence of carbon dioxide pressure on yeast viability and fermentation during sparkling wine fermentation. In particular, the effects of carbon dioxide upon yeast viability following the secondary fermentation and upon production of several flavour compounds and uptake of their precursors are examined. It may, therefore, be possible to manipulate the quality of sparkling wine by controlling the carbon dioxide concentration during the secondary fermentation - a practise which has been employed in the brewing industry (Slaughter, 1989).

5.2 Materials and Methods

5.21 Chemically defined wine (CDW)

Fermentations were conducted in a chemically defined wine (CDW) as formulated in Table 3.55 of Chapter 3, Section 3.5. Three trials were conducted in which differences in headspace pressure in the bottle were 217 obtained by adding different amounts of glucose to the CDW prior to tirage as outlined in Table 5.1. Table 5.1 also indicates the methods by which bottles maintained at 0 kPa headspace pressure in each trial were sealed.

Table 5.1 Addition of glucose to a chemically defined wine (CDW) for the production of CO2 headspace pressure during sparkling wine fermentation.

Approximate headspace pressure (kPa)

Glucose added (g/L) Trial 1 Trial 2 Trial 3 24 0a 0b 0C

12 260d 260d 260d 24 520d 520d 520d

30 _e _e 650d a In Trial 1, bottles were simply covered with foil caps in order to give no headspace pressure. b In Trial 2, following the end of fermentation (approximately 50 days) the headspace of the bottles were flushed with N2 gas and then the bottles sealed with crown caps. c In Trial 3, bottles were fitted with fermentation locks and the water seal spiked with 0.5 g ascorbic acid every 2 weeks during fermentation and storage, d Bottles sealed with crown caps at tirage to develop the appropriate headspace pressure. e Over pressurised bottles not prepared in this trial.

Nine litres of CDW were prepared and the appropriate amount of glucose

(Table 5.1) was added to generate the final pressure desired after bottle fermentation. The CDW was sterilised by passage through a 0.45 pm filter and approximately 475 mL placed in sterile 500 mL bottles prior to addition of the starter culture (tirage). 218

5.22 Preparation of yeast starter culture for inoculation into CDW

A wet culture of the commercial strain S. cerevisiae EC-1118 was prepared in two stages. Initially, a loopful of the yeast from a fresh slant culture was added to a 100 ml_ flask containing 30 mL of the chemically defined grape juice medium (starter culture formulation) outlined in Table 4.23 (Chapter 4, Section 4.212) and incubated at 20°C with shaking at 200 rpm for 48 hours. This culture was then transferred to a 1 litre flask containing 570 mL of the same medium and incubated at 20°C with shaking at 200 rpm for 24 hours.

Inoculum (5 mL) from this culture was added to each bottle before sealing and storage upright at 12°-15°C for the secondary fermentation.

5.23 Analysis of CDW during the secondary fermentation

Each sample of wine was analysed for a range of properties at regular intervals during the active fermentation and death phase of the yeast. Two bottles were opened at each sample point and each bottle assayed separately for viable counts and CO2 headspace pressure and the data averaged. Bottle contents were then combined and 200 mL were filter sterilised and frozen for chemical analyses. Chemical analyses were conducted on samples of CDW obtained in Trial 3 (Table 5.1) where the effects of headspace pressures of 0, 260, 520 and 650 kPa on yeast activity were investigated.

5.231 Measurement of viable counts

Samples of CDW from all three trials were diluted in 0.1% peptone and 0.1 mL of the dilutions were spread inoculated over the surface of triplicate plates of Malt Extract Agar (Oxoid CM 59). Plates were incubated at 25gC for 3 days after which colonies were counted and viable population calculated. 219

5.232 Determination of glucose, glycerol and ethanol

The concentrations of glucose, glycerol and ethanol were determined by HPLC with detection by refractive index as already described in Chapter 4, Sections 4.242 and 4.243.

5.233 Development of CO2 headspace pressure

The CO2 headspace pressure that developed in bottles sealed with crown caps was measured using a WIKA gauge as described in Chapter 4, Section 4.244.

5.234 Concentration of amino acid nitrogen

Amino acids in the CDW filtrates were determined by HPLC and post-column derivitisation with ninhydrin on a Beckman 6300 automated amino acid analyser. Samples of CDW (1 mL) were prepared for analysis by the addition of 30% sulfosalicylic acid (0.1 mL) and centrifugation to remove precipitated protein. Separation was achieved over 130 minutes on a Beckman ion- exchange column (4 mm x 100 mm) using the 4 elution buffers and column temperature gradient program as outlined in Table 5.2.

Table 5.2 Gradient program for the separation of individual amino acids in CDW on a Beckman 6300 amino acid analyser.

Time of run Column Time of run Elution buffer (minutes) temperature (°C) (minutes)

0-12 34 0-31 1

12-85 63 31-72 2

85-130 70 72-86 3

- - 86-130 4 220

Data were processed by Delta Data Systems chromatography software (Digital Solutions) and IBM-compatible AT computer. Amino acids in samples were quantified by comparison with peak areas of the same amino acids in a standard solution (Beckman). These analyses were performed in the amino acid analyser located within the Department of Biochemistry and Molecular Genetics, the University of NSW.

5.235 Measurement of volatiles

Volatiles were extracted from CDW filtrates and measured by GLC as outlined in Chapter 4, Section 4.250 using n-dodecanol as internal standard.

Due to the low concentration of volatiles in this medium, only higher alcohols n-propanol, 2-phenylethanol, iso-amyl alcohol and iso-butanol and the ester ethyl acetate were detected by this method.

5.236 Measurement of cellular RNA concentration

Yeast cells from the wines after 30 days of fermentation were assayed for RNA using the guanine-HPLC method outlined in Chapter 3, Section 3.225b. The content of ceiluiar RNA was calculated by dividing the RNA concentration by the population of yeast cells in each wine. The concentration of yeast cells in the wines at 30 days was determined using a haemocytometer. 221

5.3 Results

5.31 Effect of CO2 on viable population during secondary fermentation

Figures 5.1a, 5.1b and 5.1c show the kinetics of growth and death of

S. cerevisiae in response to CO2 pressure during sparkling wine fermentation.

Viable yeast population in trials 1 and 3 (Figures 5.1a and 5.1c) increased from an initial value of approximately 1x10® cfu/mL to about 6x10® cfu/mL over 10 days. In trial 2 (Figure 5.1b), the viable yeast population was added at about 6x10® cfu/mL and did not significantly increase beyond this level. Yeast populations began to decrease slowly from 20-40 days until the end of the alcoholic fermentation. No effect of CO2 on viable population was observed up to this point. However, following the alcoholic fermentation,

CO2 was seen to accelerate the death of the yeast in the pressurised bottles during the period 60-125 days after which no viable yeast were detected in any of the pressurised fermentations. Similar patterns of growth and survival were observed in two commercial and one model sparkling wine in Chapter 4 (Figures 4.31 and 4.315). In contrast, viable populations of 10® - 10® cells/mL were still recovered from the non-pressurised fermentations after

125 days. 222

Trial 1 7.0 -

5.0

3.0

Days at 12-15C

-J 8.0 Trial 2 'g 7.0

o 5.0

4.0

> 2.0

Days at 12-15C

Trial 3

2.0 -

Days at 12-15C

Figure 5.1 Effect of CO2 headspace pressure on viable yeast population during the secondary fermentation of a chemically defined wine (CDW) in three trials. [ No CO2 pressure 0,260 kPa CO2 pressure □ , 520 kPa CO2 pressure ▲ and 650 kPa CO2 pressure A ] 223

5.32 Changes in concentration of glucose and CO2 pressure during the secondary fermentation

The consumption of glucose and production of CO2 during the secondary fermentation of wines with different initial concentrations of glucose are shown in Figure 5.2.

As expected, the consumption of glucose was mirrored by production of CO2 during the sparkling wine fermentation. Approximately 22 kPa headspace pressure was produced per gram of glucose consumed. However, Figure

5.2a indicates that CO2 pressure influences the rate at which glucose is consumed in the first 15 days of fermentation (exponential phase). This effect is shown in Table 5.3 which summarises the development of CO2 pressure and the consumption of glucose in bottles under no CO2 pressure and bottles which eventually developed 260 kPa, 520 kPa and 650 kPa pressure. 224

_ 3.0

o 1.0

CJ 0.0 0 25 50 75 100 Days at 12-15C

^ 700

600 -

500 -

g 400 -

y 300 -

100 -

Days at 12-15C

Figure 5.2 Consumption of (a) glucose and development of (b) CO2 headspace pressure during the secondary fermentation of a chemically defined wine (CDW). [ No CO2 pressure 0,260 kPa CO2 pressure □ , 520 kPa CO2 pressure ■ and 650 kPa CO2 pressure A ] 225

Table 5.3 Effect of developing CO2 pressure on the rate of glucose consumption during the secondary fermentation of a chemically defined wine (CDW).

Days Developing CO2 headspace Glucose remaining in medium (g/L) at pressure (kPa) 12°-15°C 0a 260a 520a 650a 0a 260a 520a 650a

0 - - 24 12 24 30

6 65 68 67 20.1 9.2 19.0 24.3 Rate of glucose uptake (g/day) 0.65 0.47 0.83 0.95

15 180 230 210 14.9 3.0 10.9 17.2 Rate of glucose uptake (g/day) 0.61 0.60 0.87 0.85 a Refers to potential headspace pressure developed by the end of the secondary fermentation.

Table 5.3 indicates that glucose fermentation in the bottles which eventually developed 520 kPa and 650 kPa proceeded at a faster rate than in the non- pressurised and low pressure (260 kPa) bottles during the initial 15 days of fermentation.

5.33 Effect of CO2 pressure on production of glycerol and ethanol during the secondary fermentation

Figures 5.3a and 5.3b show the effect of CO2 headspace pressure on the production of glycerol and ethanol during the secondary fermentation of a chemically defined wine (CDW).

For the equivalent amount of available glucose (uncapped and capped

bottles containing 24 g/L glucose at tirage), approximately 10% more glycerol

was produced in bottles under no CO2 pressure than in C02-pressurised

bottles (Figure 5.3a). This indicates that CO2 has a suppressing effect on the production of glycerol during the alcoholic fermentation. 226

'P 0.05

^ 0.04

2 0.03

S o.oi

0 10 20 30 40 50 60 Days at 12-15C

Days at 12-15C

Figure 5.3 Effect of CO2 headspace pressure on the production of (a) glycerol and (b) ethanol during the secondary fermentation of a chemically defined wine. [ No CO2 pressure 0 , 260 kPa CO2 pressure □ , 520 kPa CO2 pressure ■ and 650 kPa CO2 pressure A ]

Figure 5.3b indicates that the initial rate of production of ethanol in bottles under no CO2 pressure was slower in comparison to the pressurised (520 and 650 kPa CO2) bottles. This is in agreement with the findings of Table

5.3 where glucose consumption in bottles under no CO2 pressure was observed to be slower than in the pressurised (520 and 650 kPa CO2) bottles. However, the final yield of ethanol in bottles containing equivalent 227 amounts of glucose (unpressurised and pressurised bottles containing 24 g/L of glucose) was not significantly different (about 1% difference)

5.34 Effect of CO2 pressure on the kinetics of amino acid nitrogen uptake and release during the secondary fermentation and storage

The effects of CO2 pressure on total amino nitrogen uptake during the secondary fermentation of a chemically defined wine are shown in Figure 5.4.

The development of CO2 pressure decreases the amino N requirement of the yeast. For the equivalent amount of glucose consumed, approximately 25 mg/L and 22.9 mg/L of amino nitrogen was consumed in the bottles under no

CO2 pressure and CC>2-pressurised bottles, respectively (Figures 5.4a and 5.4c). This represents a difference of about 8% which may be considered significant as the variation of the method was less than 1% (calculated from repeated injections of the same sample). Figure 5.4c also indicates that the pressurised fermentation proceeded at a faster rate which confirms the conclusions of Table 5.3 concerning slower glucose uptake by the unpressurised fermentation. 228

60 - 60 -

50 - 50

Days at 12-15C Days at 12-15C

60 - 60 -

50 - 50 -

Days at 12-15C Days at 12-15C

Figure 5.4 Effect of CO2 pressure on changes in total amino N during the secondary fermentation of a chemically defined wine containing final bottle pressures of (a) 0 kPa (0) (b) 260 kPa (A) (c) 520 kPa CO2 pressure (▲) and (d) 650 kPa CO2 pressure □ ).

Figures 5.4a and 5.4c also show that there was a greater efflux of amino N in the non-pressurised bottles following the secondary fermentation; the efflux of amino N was 6.06 mg/L and 0.89 mg/L for the non-pressurised and pressurised bottles, respectively, which contained the equivalent amount of glucose at tirage (24 g/L).

The general trends observed in Figure 5.4 are reflected in the patterns of uptake and release of individual amino acids. These data are summarised in Tables 5.4, 5.5, 5.6 and 5.7. 229

Table 5.4 Uptake and release of individual amino acids during secondary fermentation of a chemically defined wine under no CO2 pressure.

Amino acid Concentration (pmolar) in wine during secondary fermentation and ageing (days) 0 6 15 30 47 87 124 Aspartic acid 130 89 64 41 55 63 65 Threonine 74 46 31 20 23 24 23 Serine 100 58 35 14 17 17 18 Glutamic acid 258 205 167 106 128 133 130 Proline 2282 2198 2224 2121 2146 2146 2138 Glycine 61 58 61 60 71 77 78 Alanine 447 400 364 301 339 344 343 Valine 71 62 49 26 33 31 32 Methionine 31 14 5 2 1 1 3 Iso-leucine 50 31 18 6 8 7 7 Leucine 122 74 40 11 13 11 11 Tyrosine 28 24 20 13 16 16 16 Phenylalanine 38 25 13 5 6 6 8 NHc, 1146 1101 1098 1076 1091 1104 1128 Lysine 82 56 39 18 33 38 39 Histidine 30 23 21 15 21 23 24 Arginine 319 270 256 223 250 249 249

Table 5.5 Uptake and release of individual amino acids during secondary fermentation of a chemically defined wine under 260 kPa CO2 pressure3

Amino acid Concentration (pmolar) in wine during secondary fermentation and ageing (days) ______0 6 15 30 47 87 124 Aspartic acid 133 82 48 47 55 55 58 Threonine 90 49 21 21 21 21 21 Serine 83 40 24 19 21 19 13 Glutamic acid 263 195 136 131 132 116 120 Proline 2188 2152 2086 2049 2070 2028 2037 Glycine 61 59 59 62 64 64 66 Alanine 454 400 337 328 335 324 333 Valine 72 61 43 36 36 31 32 Methionine 32 13 3 2 2 2 2 Iso-leucine 49 29 13 8 8 6 6 Leucine 123 69 28 16 14 10 11 Tyrosine 27 24 20 18 18 17 18 Phenylalanine 37 26 13 9 9 8 8 nh3 1160 1116 1087 1093 1081 1090 1086 Lysine 82 50 24 23 30 27 29 Histidine 30 22 16 16 19 19 19 Arginine 317 259 228 233 239 236 245 3 Refers to pressure developed in the bottle by the end of the secondary fermentation 230

Table 5.6 Uptake and release of individual amino acids during secondary fermentation of a chemically defined wine under 520 kPa CO2 pressure3

Amino acid Concentration (pmolar) in wine during secondary fermentation and ageing (days) 0 6 15 30 47 87 124 Aspartic acid 132 76 45 51 55 62 62 Threonine 73 45 21 23 23 23 23 Serine 102 37 16 16 21 22 22 Glutamic acid 264 189 130 135 127 119 112 Proline 2279 2243 2145 2066 2054 1988 2002 Glycine 60 59 59 66 68 70 69 Alanine 446 385 326 336 339 332 332 Valine 73 60 39 37 34 32 29 Methionine 32 11 2 2 1 2 2 Iso-leucine 50 27 10 9 8 7 6 Leucine 123 62 21 20 17 13 11 Tyrosine 28 24 18 18 17 17 17 Phenylalanine 38 24 10 9 8 8 7 NH3 1158 1112 1081 1097 1067 1072 1087 Lysine 82 46 17 18 22 29 28 Histidine 30 21 16 18 19 21 20 Arginine 320 256 236 235 234 234 240 3 Refers to pressure developed in the bottle by the end of the secondary fermentation

Table 5.7 Uptake and release of individual amino acids during secondary fermentation of a chemically defined wine under 650 kPa CO2 pressure3.

Amino acid Concentration (pmolar) i n wine during secondary fermentation and ageing (days) 0 6 15 30 47 87 124 Aspartic acid 130 66 48 50 53 61 66 Threonine 74 35 25 21 21 21 21 Serine 101 32 16 22 22 23 28 Glutamic acid 259 172 137 130 123 128 138 Proline 2289 2219 2130 1998 2021 1959 1945 Glycine 61 57 58 66 70 71 71 Alanine 446 368 328 332 336 346 358 Valine 73 55 40 35 32 36 42 Methionine 32 8 3 2 2 2 2 Iso-leucine 50 22 12 9 7 9 12 Leucine 125 51 26 18 14 20 31 Tyrosine 28 23 19 18 17 19 20 Phenylalanine 38 21 11 9 8 11 14 NH3 1130 1075 1097 1101 1096 1106 1115 Lysine 83 38 20 16 19 30 38 Histidine 31 19 17 17 18 21 23 Arginine 326 247 247 242 241 245 250 3 Refers to pressure developed in the bottle by the end of the secondary fermentation 231

Analysis of the data for fermentations conducted under no CO2 pressure and under 520 kPa CO2 pressure at the end of the secondary fermentation (ie. equivalent glucose concentrations at tirage) indicates that CO2 pressure has an effect on amino nitrogen uptake during fermentation and efflux of amino nitrogen following fermentation. These effects are summarised in Table 5.8.

Table 5.8 Changes in concentration of individual amino acids in response to carbon dioxide pressure during the secondary fermentation of a chemically defined wine.

Amino acid Change in concentration Change in concentration during fermentation3 (%) following fermentationb (%) Unpressurised Pressurised Unpressurised Pressurised fermentation fermentation fermentation fermentation Aspartic acid -68.5 -65.9 58.5 37.8 Threonine -73.0 -71.2 15.0 9.5 Valine -63.4 -46.1 23.1 -25.6 Alanine -32.7 -26.9 14.0 1.8 Glutamic acid -58.9 -50.8 22.6 -13.8 Leucine -91.0 -82.9 0.0 -47.6 Iso-leucine -88.0 -80.0 16.7 -40.0 Serine -86.0 -84.3 28.6 37.5 Proline -7.1 -5.9 0.8 -6.7 Glycine -1.6 -1.7 30.0 16.9 Methionine -93.5 -93.8 -50.0 0.0 Tyrosine -53.6 -35.7 23.1 -5.6 Phenylalanine -86.8 -73.7 60.0 -30.0 NH3 -6.1 -6.6 4.8 -0.5 Lysine -78.0 -79.3 116.7 64.7 Histidine -50.0 -46.7 60.0 25.0 Arginine -30.1 -26.3 11.7 1.7 a Refers to change in concentration of amino acids between tirage and end of uptake at 30 days and 15 days for the unpressurised and pressurised bottles, respectively. b Refers to change in concentration of amino acids between the end of uptake at 30 days and 15 days for the unpressurised and pressurised bottles, respectively, and 124 days 232

In general, CO2 pressure decreases the demand for individual amino acids during the secondary fermentation and decreases the efflux of individual amino acids following the secondary fermentation. Notably, some amino acids went against the general trend and continued to be consumed in significant amounts (> 10%) by the pressurised yeast following the secondary fermentation. These included the amino acids glutamic acid, valine, iso-leucine, leucine and phenylalanine.

5.35 Effect of CO2 pressure on evolution of higher alcohols and ethyl acetate during the secondary fermentation

Figure 5.7 shows the effect of CO2 pressure on the formation of the higher alcohols (n-propanol, 2-phenylethanol, iso-amyl alcohol and iso-butanol) during the fermentation of CDW.

Carbon dioxide pressure had no effect on the production of higher alcohols, n-propanol, iso-amyl alcohol and iso-butanol (Figures 5.7b, 5.7c and 5.7d).

However, the production of 2-phenylethanol was depressed by CO2 pressures in the bottle (Figure 5.7a). 233

(a) 2-phenylethanol n-propanol

w> 3.0 -

S 1.0 -

Days at 12-15C Days at 12-15C

(c) iso-amyl alcohol iso-butanol

15

Days at 12-15C Days at 12-15C

Figure 5.7 Effect of CO2 pressure on the production of (a) 2-phenylethanol, (b) n-propanol, (c) iso-amyl alcohol and (d) iso-butanol during the secondary fermentation of a chemically defined wine. [ No CO2 pressure 0,260 kPa CO2 pressure A , 520 kPa CO2 pressure ▲ and 650 kPa CO2 pressure □ ]

The effect of CO2 pressure in the bottle on production of ethyl acetate is shown in Figure 5.8. Comparison of the amount of ethyl acetate formed in bottles containing equivalent concentration of glucose at tirage

(unpressurised and bottles developing 520 kPa pressure) Indicates that CO2 pressure has minimal effect on ethyl acetate production during the secondary fermentation. 234

40 -

30 -

Days at 20C

Figure 5.8 Effect of CO2 on the production of ethyl acetate during the secondary fermentation of a chemically defined wine. [ No CO2 pressure 0,260 kPa CO2 pressure A , 520 kPa CO2 pressure A and 650 kPa CO2 pressure □ ]

5.36 Effect of CO2 pressure on the RNA concentration in yeast during the secondary fermentation

The concentrations of RNA in yeast at tirage and after 30 days fermentation

in the bottle were compared to observe whether CO2 pressure affects RNA

levels in the yeast (Table 5.9). Increasing CO2 pressure over 260 kPa gives a decrease in the average RNA concentration of yeast cells after 30 days of fermentation. Additionally, yeast cells at tirage had approximately 2.8 to 3 times the RNA content compared with yeast after 30 days of fermentation,

irrespective of what pressure finally developed in the bottle. 235

Table 5.9 Influence of CO2 pressure on the average RNA content of yeast cells after 30 days of secondary fermentation in a chemically defined wine.

Days at 12°-15°C Total cell number CO2 pressure RNA concentration (x106/mL) (kPa) (x10'12 g/cell)

0 1.41 0 3.07

30 11.9 0 1.10

30 12.6 260 1.10

30 13.2 443 1.03 30 13.8 500 0.91

5.4 Discussion

The results of this Chapter have shown that the development of CO2 pressure during the secondary fermentation has the effect of (i) accelerating the death of the yeast cells on completion of the fermentation,

(ii) decreasing the consumption and subsequent efflux of amino acids during fermentation and storage of the wine, and (iii) it has the potential to influence the production of some volatile constituents of the wine. These findings are significant considering the extended death phase of the yeast following the secondary fermentation as reported in Chapter 4, and the important effect of secondary metabolites on wine flavour and aroma.

5.41 Effect of carbon dioxide on the growth and survival of yeasts

The kinetics of growth and survival of the yeast during the secondary fermentation of CDW (Figure 5.1) under carbon dioxide pressure were similar to those observed in Chapter 4 for the commercial and model wines.

Increasing CO2 pressures during the secondary fermentation (0 to 40 days) did not affect the maximum viable cell population. The lack of nutritional 236 factors (glucose, nitrogen) were not responsible for this effect since fermentation continued normally after cessation of growth (see next section). Alternatively, ethanol produced during the secondary fermentation may be inhibiting growth. This is supported by the observation that the higher initial population in Trial 2 would have produced ethanol at a higher rate resulting in inhibition of cell growth earlier than in the other trials. Although not evident from the data, the contribution of dissolved CO2 should not be discounted since super-saturation of the fermenting wine is possible under the low temperatures of storage (15°C). An improvement to the experimental design for future study would be to measure dissolved CO2 concentration in the bottle since the inhibitory effects of CO2 are due to its concentration in solution rather than the hydrostatic pressure itself (Chen and Gutmanis, 1976).

Following exhaustion of glucose, and during the period 40 to 120 days, yeast contained in pressurised bottles underwent faster death in comparison to yeast contained in unpressurised bottles. This result is consistent with the concept that the pressurised yeast are unable to sustain themselves for as long as unpressurised yeast following exhaustion of the sugars due to stress associated with the inhibitory effects of CO2. This is not suprising considering CO2 is well documented as increasing yeast mortality (Jones and

Greenfield, 1982; Dixon and Kell, 1989; Slaughter, 1989) . However, the data do not suggest that increasing CO2 pressures gave increasing rates of death as similar death rates were observed at 260, 520 and 650 kPa of pressure (Figure 5.1c).

As death of the cells is a prerequisite to yeast autolysis (Arnold, 1981b), it follows that yeasts maintained under CO2 pressure during ageing of sparkling wine will autolyse faster than those not stored under pressure (as 237 during storage of base wine on lees following the primary fermentation). However, this assumption requires confirmation in long term storage studies of sparkling wine as carbon dioxide has been reported to cause a transient inhibition of autolytic enzymes during storage of fish flesh (Mitsuda et aj., 1980).

5.42 Effect of carbon dioxide upon the chemical changes during secondary fermentation

The development of CO2 during the secondary fermentation (Figure 5.2b) followed first order kinetics as predicted by Merzhanian and Kozenko (1972).

Approximately 22 kPa CO2 pressure per gram of glucose was produced (90 kPa / 4g of glucose) which correlates closely to the 100 kPa / 4g of glucose reported by Bidan et aL (1986).

Notably, the consumption of glucose by yeast growing under pressure (bottles that were to develop 520 and 650 kPa pressure) was faster than yeast in the unpressurised bottles during the first 15 days. A similar effect has been observed in Zymomonas mobilis (Nipkow et aL, 1985) and S. cerevisiae under continuous fermentation (Cahill et aL, 1980). This phenomenon was explained by Cahill et aL (1980) as being due to an increased requirement for maintenance energy in response to the inhibitory effects of increasing CO2 concentration in solution. An increased requirement for maintenance energy may also play a role in the accelerated death phase observed in the pressurised yeast following the end of the secondary fermentation (Figure 5.1).

The production of glycerol by yeast was reduced by 10% in response to increasing CO2 pressure during fermentation (Figure 5.3a). A similar response has been noted in S. cerevisiae grown under continuous culture 238 where an increase of CO2 pressure from 44 to 195 kPa decreased glycerol production by 50% (Kuriyama et aj., 1993). This observation was explained as a response to increased intracellular CO2 levels. The authors suggested that elevated intracellular CO2 levels encourage the equilibrium of the pyruvate dehydrogenase enzyme system to produce pyruvate since CO2 is one of the major products of the reaction catalysed by this enzyme. However, the resulting increase in intracellular pyruvate concentration encourages pyruvate decarboxylase to produce acetaldehyde since this enzyme is relatively insensitive to inhibition by CO2 (Jones and Greenfield, 1982).

Acetaldehyde, in turn, is converted to ethanol by alcohol dehydrogenase.

The final step is suggested to depress glycerol production as it competes with glycerol-3-phosphate dehydrogenase for the oxidation of NADH.

The slower rate of production of ethanol in the first 15 days by yeast in the unpressurised bottles reflects a lower rate of glucose consumption compared to pressurised yeast (Table 5.3, unpressurised and 520 kPa bottles). Essentially there was no difference in the amount of ethanol produced by unpressurised and pressurised yeast in bottles containing equivalent amounts of glucose (about 1.3% higher in the pressurised bottles). This is in agreement with results reported for S. cerevisiae (Norton and Krauss, 1972;

Knatchbull and Slaughter, 1987) and Z.mobilis in batch culture (Veeramallu and Agrawal, 1986) suggesting that CO2 at these pressures is having no effect on (Slaughter, 1989).

Carbon dioxide pressure decreased the uptake of amino acid nitrogen during fermentation (Figure 5.4). Similar effects have been noted in the brewing literature (Kumada et a]., 1975; Knatchbull and Slaughter, 1987; Slaughter et a!., 1987). In an excess pressure of about one atmosphere of CO2, Kumada et aj. (1975) observed a decrease in amino nitrogen uptake during beer 239 fermentation. Knatchbull and Slaughter (1987) observed the same phenomena at about 0.5 and 1 atmosphere CO2 pressure, but found that individual amino acids were absorbed in varying amounts. These authors found that the uptake of Group A amino acids (glutamic acid, aspartic acid, asparagine, glutamine, serine, threonine, lysine and arginine) were little affected by the application of COg pressure, but that absorption of Group B amino acids (valine, methionine, leucine, iso-leucine and histidine) were significantly retarded. Similar results were obtained during uptake of amino acids in the secondary fermentation of the CDW (Table 5.8). Minimal difference in decreases in the concentrations of Group A amino acids occurred with an average of 65.8% and 63.0% being observed for the unpressurised and pressurised yeast, respectively. However, the difference in uptake of Group B amino acids was much greater with a decrease of

77.2% and 69.9% being observed for unpressurised and pressurised yeast, respectively. In particular, the absorption rate of Group B amino acids valine, leucine and iso-leucine has been cited by Knatchbull and Slaughter (1987) as being especially affected by increasing CO2 pressure. These authors provided valine as an example where its uptake was decreased by about

59% under 1 atmosphere of CO2 pressure. The absorption of valine, leucine and iso-leucine in the CDW was reduced by 17.3%, 8.1% and 8.0%, respectively, and accounted for the bulk of reduced absorption of group B amino acids in the model wine. The greater inhibition of valine uptake by

CO2 observed by Knatchbull and Slaughter (1987) is probably due to their experimental design where pressure was applied at the start of fermentation, whereas in sparkling wine the process is gradual over 40 days. Nevertheless, this phenomenon may impact on the formation of vicinal diketones and their precursors during fermentation. As an example,

Knatchbull and Slaughter (1987) described the formation of diacetyl and its precursor, a-acetolactate, during brewing where depressed absorption of 240 valine was implicated as being central to formation of increased levels of these compounds in wort. This process could possibly impact upon the flavour profile of sparkling wines via a similar mechanism.

Efflux of amino acids occurred at an earlier stage of the secondary fermentation in pressurised bottles (after 15 days) compared with unpressurised bottles (after 30 days). A similar effect was noted by Slaughter et aj. (1987) during fermentation of a malt medium by S. cerevisiae over 24 hours. These authors noted that yeast exposed to two atmospheres of CO2 pressure released amino acids after 4 hours while the control (unpressurised) yeast absorbed amino acids throughout the 24 hours. Slaughter et aL (1987) suggested that co-incidental increase in yeast size during fermentation under pressure may result in disruption of internal organisation of the cell. This could release vacuolar proteases that would hydrolyse cell protein in a process similar, presumably, to autolysis. The increase in internal amino acid concentration was then suggested to affect various amino acid permeases by feedback repression. The increased rate of initial absorption of some amino acids was ascribed to transport systems that were not repressed and operated with higher activity because of greater availability of energy that was not consumed by other amino acid transport systems. With continued proteolysis, the internal amino acid concentration would reach a level where leakage would occur through the plasma membrane which may already be weakened through the increase in size of the cell. Alternatively, it was suggested that some amino acids may only appear to be initially absorbed at a reduced rate due to leakage counteracting uptake during fermentation. In contrast, Feuillat and Charpentier (1982) describe this period of amino acid release as being a physiological response to decreasing sugar levels, and not due to autolysis. A clearer picture of the cause of this nitrogen release may be obtained by monitoring cell protein levels as suggested by Slaughter 241 et aL (1987). Extensive proteolysis and decrease in cellular protein concentration would indicate the occurrence of autolysis and remains a question for future investigation.

Table 5.8 also indicates that, in general, the efflux of amino acids following the secondary fermentation was greater in unpressurised bottles. This, however, may be a function of higher total numbers of cells in unpressurised bottles as CO2 is reported to inhibit yeast growth (Norton and Krauss, 1971;

Arcay-Ledezma and Slaughter, 1984; Lumsden et aL, 1987). This needs to be confirmed by further study which takes into account total cell numbers.

Interestingly, the amino acids glutamic acid, valine, leucine, iso-leucine, and phenylalanine continued to decrease following the secondary fermentation. The metabolic rationale for this observation is not clear considering the accelerated death of the yeast in the pressurised bottles, and needs further investigation.

Unfortunately, the storage period of the wine (125 days) was not long enough to observe the autolytic release of nitrogen compounds at about 150 to 250 days onwards as seen in the commerical and model wines of Chapter 4. It would be interesting to observe whether the accelerated death of the yeast in pressurised bottles would result in an earlier onset of autolysis. Such longer term storage trials of wine under various pressures remains an area for future research.

The volatiles detected and their kinetics of change in concentration in the

CDW were similar to that found in the commercial and model wines of Chapter 4. The only exception to this observation was the increase in concentration of ethyl acetate during the fermentation of the CDW, as compared to the Wagga and model wines. However, the decrease in ethyl 242 acetate concentration observed in the Wagga and model wines may be due to residual esterase activity from the primary fermentation, whereas the CDW was not the product of a primary fermentation.

Slaughter (1989), in a review of the brewing literature, reported that the application of CO2 pressure during brewing decreased the concentration higher alcohols and esters. However, of the volatile compounds detected in the CDW (2-phenylethanol, iso-amyl alcohol, n-propanol, iso-butanol and ethyl acetate), only 2-phenylethanol formation was depressed by developing

CO2 pressure. This observation may be a result of the relatively small amount of glucose being fermented in the secondary fermentation and analytical limitations in detecting the differences between the small amounts of volatile components. The mechanism of depressed production of higher alcohols in response to CO2 pressure is not clear. Higher alcohols originate from a-keto acids and are produced by the activities of pyruvate decarboxylase and alcohol dehydrogenase (Henschke and Jiranek, 1993). However, as mentioned already, these enzymes form a part of the glycolytic pathway which has been shown to be essentially unaffected by CO2 (Slaughter, 1989).

Table 5.9 indicates that fermentation in bottles to pressures above 260 kPa progressively decreased the RNA content of the yeast. This agrees with the findings of Lumsden et a[. (1987) who showed that an excess pressure of 282 kPa CO2 decreased the RNA content of S. cerevisiae and

Schizosaccharomyces pombe during fermentation. A possible mechanism for this observation is inhibition of the decarboxylation step of glucose-6-

phosphate to ribulose-5-phosphate for eventual conversion into nucleotides

(Chapter 2, Figure 2.3). Interestingly, unpressurised cells at 30 days fermentation had about one third the RNA content of the cells at tirage. 243

However, the cells at tirage were grown up under semi-aerobic conditions and harvested in the logarithmic phase of growth, an environment which favours high RNA contents in yeast (Polakis and Barteley, 1966). Additionally, the low temperature of storage of the bottles (about 12°-15°C) would encourage supersaturation of CO2 in solution which may also exert an effect.

In general, the effect of developing carbon dioxide pressure on the growth and metabolism of the yeast reflects the results reported in the literature.

Principal amongst these effects are increased rate of fermentation, accelerated mortality following the secondary fermentation, altered pattern of amino acid uptake and release, and depressed production of some secondary metabolites and RNA by the yeast. Some of the results obtained were in variance to those reported in the literature (particularly with regards to production of secondary metabolites). However, it should be noted that in sparkling wine, carbon dioxide pressure is gradually produced from a relatively small amount of sugar over the 40 days of the secondary fermentation. In contrast, most of the reported literature refers to conditions where CO2 pressure is directly applied to a culture. Additionally, significantly greater amounts of sugar were generally fermented which would amplify any effects of carbon dioxide on growth of the yeast.

Therefore, while an increase in bottle pressure will accelerate cell death and

(presumably) autolysis, any production advantage gained must be balanced against the impact that CO2 will have on various sensory attributes such as the development of secondary metabolites during and after the secondary fermentation. 244

6.0 Promotion of Autolysis Through the Interaction of Killer and Sensitive Strains of Saccharomyces cerevisiae

6.1 Introduction

Production of sparkling wine by the ‘methode champenoise' involves secondary fermentation of a base wine in the bottle followed by a prolonged ageing period where the yeast remains in contact with the wine. During ageing, which may be 6 months to several years, the yeast cells undergo autolysis and release various intracellular components, such as proteins, nucleic acids, polysaccharides, lipids and their breakdown products, that improve sparkling wine quality (Feuillat and Charpentier, 1982; Markides,

1986; Kelly-Treadwell, 1988; Leroy et a]., 1990; Charpentier and Feuillat,

1993). However, sparkling wine production does not favour rapid yeast autolysis, which is a slow process that occurs over several years. Feuillat and Charpentier (1982) used amino acid release to show that, following a latent phase of several months, autolysis occurred over four years of storage of sparkling wine. A similar latent phase was observed during the ageing of two Australian sparkling wines for 18 months (Chapter 4, Section 4.317). The death phase of the yeast cells following the secondary fermentation was protracted over 2-3 months and, essentially, autolysis did not begin until this phase was complete. A possible method to accelerate the onset of autolysis and overcome this delay would be to provide a substantial pool of dead cells of yeast at the start of the secondary fermentation, or immediately after its completion.

A strategy to achieve such a pool of dead cells would be to exploit the killing effect of f<2 killer strains of Saccharomyces cerevisiae upon sensitive strains of the same species. Killer activity is characterised by the release of a toxin which is lethal to the sensitive yeast and was first reported by Bevan and 245

Makower (1963). Of the various types of killer yeasts, only the K2 killer strains are of relevance to the wine industry as their toxin shows stability in the pH range 2.9 - 4.9 (Shimizu et aL, 1985). The occurrence and significance of killer yeast in the wine industry have been reviewed recently by van Vuuren and Jacobs (1992) and Shimizu (1993).

Using a model system of yeast growth in a defined medium, the kinetics of cell death and enhanced protein release associated with the death of sensitive yeast in mixed fermentations of killer and sensitive strains of S. cerevisiae was investigated. Fermentations were conducted in yeast nitrogen base at pH 3.5 and in the same medium containing 10% (w/v) ethanol. Fermentations were also conducted under conditions where killer activity was absent; these included conducting the fermentation at pH 6.0 and the use of a cured killer strain where the dsRNA encoding for toxin production had been removed.

6.2 Materials and Methods

6.21 Yeast cultures

Thirty strains of Saccharomyces cerevisiae were obtained from the culture collection of the Department of Food Science and Technology, UNSW and nine strains came from the Australian Wine Research Institute (AWRI), Glen Osmond,

South Australia. All cultures were cultured and maintained on slants or plates of malt extract agar.

6.22 Initial screening for killer and sensitive yeast strains

Preliminary screening was performed by seeding malt extract agar with each strain of yeast (approximately 105 cells/mL of agar) and cross-streaking with 246 each of the other strains before incubation at 209C for 72 hours. Strains were recorded as possible killers by the production of clear zones of no growth in the seeded culture. Seeded cultures that gave such zones of no growth were considered as potential sensitive strains.

6.23 Confirmation of killer and sensitive characteristics

Confirmation of killer and sensitive characteristics of yeast strains was performed on glucose-yeast extract agar containing 0.003 % methylene blue, buffered at pH 4.3-4.7 with sodium citrate (Young, 1987). Killer activity was demonstrated by production of blue-stained clear zones in plates seeded with sensitive yeast strains. Methylene blue is added to the agar to distinguish between genuine killer activity (blue stain remains due to death of seeded strain) and growth inhibition (decolourised clear zones) due to sexual or other interactions between strains (Young, 1987).

6.24 Differentiation between killer and sensitive strains

Killer and sensitive strains were streaked onto two modified media, M-WLA (Wallerstein medium, Oxoid CM 309, containing 0.01% bromocresol green) and M-PDA (Potato Dextrose Agar, Oxoid CM 139, containing 0.005% bromophenol blue) which allow differentiation of strains based on their different uptake of the dyes. M-WLA medium has been used previously (Jacobs et aL, 1988; Jacobs and van Vuuren, 1991) as a means of differentiating killer and sensitive strains in mixed cultures. Two killer strains (Tyr 303 and AWRI 3A) that could be distinguished from two sensitive strains (S-1 and AWRI 5A) were selected for trials in model media (see Chapter 3, Section 3.4 for details on the development and evaluation of M-WLA and M-PDA medium for differentiation of killer and sensitive strains). 247

6.25 Strain interaction in the presence of killer activity

6.251 Strain interaction in Yeast Nitrogen Base (YNB) at pH 3.5

Cultures of either killer or sensitive strains of S. cerevisiae were prepared by adding a loopful of yeast to 40 mL YNB (without amino acids, containing 5% glucose) at pH 3.5 (adjusted with citric acid) and incubating at room temperature in an orbital shaker at 1500 rpm. After 24 hours, the population was determined using a haemocytometer and the cells concentrated by centrifugation and made up to a volume (2-5 mL) so that 1 mL of inoculum into 75 mL of culture medium gave a population of approximately 3-5 x 106 cells/mL. Experiments to determine strain interaction were conducted in 75 mL of YNB (without amino acids, containing 5% glucose) buffered with citric acid/phosphate buffer at pH 3.5. The cultures were incubated at 209C and samples were withdrawn daily for protein and population analyses.

6.252 Strain interaction in YNB containing 10% (w/v) ethanol at pH 3.5

Cultures of the killer strain S. cerevisiae Tyr 303 were adapted to growth in the presence of 10% (w/v) ethanol by three stages (Figure 6.1). The killer strain S. cerevisiae AWRI 3A was not utilised due to its poor growth in the presence of

10% (w/v) ethanol. Cells of the ethanol-adapted killer yeast were concentrated by centrifugation and made up to a volume (2-5 mL) so that an inoculum of 1 mL gave a population of approximately 3-5 x 106 cells/mL in the experimental medium. The sensitive strain of S. cerevisiae (AWRI 5A) was not adapted to ethanol (to minimise the chances of survival) and was grown up over 24 hours as in Section 6.251. The experimental culture was 75 mL of buffered YNB (without amino acids, 5% glucose) containing 10% (w/v) ethanol to simulate the presence of ethanol in a wine base. The fermentation was conducted at 20°C over 3 days and samples were withdrawn for population and protein analyses. 248

All mixed cultures were inoculated so that the ratio of killer cells to sensitive cells was 1:1 at the commencement of fermentation. All experiments and analyses were conducted in triplicate.

Loopful of Tyr 303

Shake at 1500 YNB (20 mL) rpm at room at pH 3.5 temperature for 24 h

4 mL aliquot

Incubate at YNB (36 mL) at pH 20C for 48 h 3.5 containing 5% (w/v) ethanol

v 4 mL aliquot

Incubate at YNB (36 mL) at pH 20C for 72 h 3.5 containing 10% (w/v) ethanol

v Fermentation broth : YNB (75 mL) at pH 3.5 containing 10% (w/v) ethanol.

Figure 6.1 Progressive adaptation of S. cerevisiae killer strain Tyr 303 to the presence of ethanol.

6.26 Strain interaction in the absence of killer activity

Fermentation trials were conducted in conditions where the K2 killer activity of

S. cerevisiae was absent. These conditions included fermentation in YNB

(without amino acids) at pH 6.0 and the use of the cured killer strain S. cerevisiae

AWRI 3A in which the dsRNA encoding for K2 toxin production had been removed (strain AWRI 3AMC). 249

6.261 Strain interaction in YNB at pH 6.0

Interaction between the killer and sensitive pairs S. cerevisiae AWRI 3A + S-1 and S. cerevisiae Tyr 303 + AWRI 5A were repeated as for Section 4.251 except the culture medium pH was buffered at 6.0 where the K2 toxin is inactive. Population kinetics and production of protein during fermentation were analysed as before.

6.262 Strain interaction in YNB at pH 3.5 with the cured killer strain S. cerevisiae AWRI 3AMC

Cured killer strain S. cerevisiae AWRI 3AMC (obtained from AWRI) is the killer strain S. cerevisiae AWRI 3A where the dsRNA encoding for production of the K2 toxin has been removed (Petering et aL, 1991). Strain interaction during mixed culture between the cured killer S. cerevisiae strain AWRI 3AMC and sensitive S. cerevisiae strain S-1 was conducted as for Section 4.251 at pH 3.5. Population kinetics and production of protein during fermentation were analysed as before.

6.27 Determination of viable populations of yeast cells

Culture samples were serially diluted in 0.1% peptone and then 0.1 mL spread inoculated in duplicate onto plates of M-WLA and M-PDA. The M-WLA medium and M-PDA medium allowed differentiation between killer and sensitive strains on the basis of colony colour. Population was expressed as averages of 4 counts (two each on M-WLA and M-PDA).

6.28 Protein determination

Protein was determined by the Bio-Rad Coomassie Brilliant Blue dye procedure using the micro-assay protocol with bovine serum albumen as standard. Sterile 250 culture medium (4.8 mL) was incubated in contact with the dye (1.2 mL) at room temperature for 5 minutes before reading the absorbance at 595 nm in a 1 cm cuvette on a Shimadzu UV-120-02 spectrophotometer. Significant increase in protein release in the mixed strain cultures over that of single strain cultures was used as a marker of autolysis (Postma et aL, 1990).

6.3 Results

6.31 Survey of Saccharomyces cerevisiae strains for killer and sensitive properties

Strains of S. cerevisiae exhibiting presumptive killer or sensitive properties (formation of clear zones on seeded malt extract agar plates) were confirmed as such by culturing on glucose-yeast extract agar containing 0.003% methylene blue (Young, 1987) as shown in Table 6.1. Strains which gave neutral reactions included : AWRI 8A, SIHA 5, 900, 633, Y1, HBM1, 2002 D11, 801, 507, 729, 6001,4012, 900 D-j ESY, 8003 D2 and 806.

The killer/sensitive strain combinations Tyr 303/AWRI 5A and AWRI 3A/S-1 were chosen for subsequent trials based on killer reactions shown in Table 6.1, and the ease with which these strains could be differentiated in mixed culture on the M-WLA and M-PDA agar plates (Chapter 3, Section 3.4). 251

Table 6.1 Killer and sensitive properties of strains of Saccharomyces cerevisiae obtained from the culture collection of the Department of Food Science and Technology, UNSW and the Australian Wine Research Institute.

Sensitive Killer strains strains Tyr 303 AWRI 3A V-1116 EC-1118 HB-350 3002 402 411 516 303 S-1 +++ +++ ++ ++ ++ + + + +++ ++ AWRI 5A +++ ++ ++ ++ + + - - ++ ++ 921 +++ NA +++ +++ + + - ++ +++ +++ 914 +++ NA ++ ++ ++ + - - ++ ++ 2180-1a ++ NA - ++ ++ + + + ++ ++ ATTC +++ - ++ ++ ++ + + + +++ ++ 100 +++ NA - ++ ++ +++ + + ++ +++ AWRI 92E ++ NA - - ++ - - - ++ - 806 + NA - + + NA NA NA + + SR 310 + NA NA - NA + + - NA + SR 310 dull +++ NA NA ++ NA +++ ++ + NA ++ SR 800 - NA + - NA - - -- - 2H-Y1 +++ NA ++ ++ +++ NA ++ + +++ +++

+++ strong reaction ++ medium reaction + weak reaction NA not assessed

6.32 Strain interaction in the presence of killer activity

6.321 Strain interaction in YNB at pH 3.5

Figure 6.2 shows the growth of killer and sensitive strains of S. cerevisiae in YNB as either single or mixed cultures. When cultured alone, the killer and sensitive strains grew from initial populations of 5x103 cfu/mL to about 5x107 cfu/mL in 24-

48 hours. The sensitive strains exhibited rapid loss in viability in mixed culture with the killer strains. No growth was observed and initial viable populations of

5x106 cfu/mL decreased to non-detectable levels (< 103 cfu/mL) within 2-3 days. The cell populations of the killer strains in the mixed culture increased to about

5x107 cfu/mL A more rapid killing effect was observed with the combination of strains S. cerevisiae AWRI 3A and S. cerevisiae S-1 than S. cerevisiae Tyr 303 and S. cerevisiae AWRI 5A. 252

The growth of the killer and sensitive strains either alone or in combination was accompanied by the release of protein into the medium (Figure 6.3). Most of the protein had been excreted into the medium by day 2, when maximum cell population had developed and exponential growth had ceased. The concentration of extracellular protein in the medium after 3 days was similar (about 3.0-3.5 pg/mL) for the four strains of S. cerevisiae when grown as single cultures. It is interesting to note that the large increase in protein concentration in the medium between days 1 and 2 was accompanied by only small increases in cell population (See Figure 6.2). Also, even after viable populations had stabilised (days 2-3), there was continued release of protein into the medium.

However, the concentration of extracellular protein was significantly increased in the mixed cultures of killer and sensitive strains (error bars on graphs indicate standard error of triplicate measurements). After 3 days, the concentrations of extracellular protein in the mixed cultures of S. cerevisiae strains Tyr 303 and 5A (3.75 pg/mL) and S. cerevisiae strains AWRI 3A and S-1 (4.75 pg/mL) were 20% and 29% higher than those found in single culture. 253

8.0 s p------a------a 7.5 AWRI 3A e»n 7.0 o 6.5 V eo 6.0 '■£ 5.5

Oh O 5.0 Oh o> 3 4.5 03 > 4.0 I I I 0 12 3 Days at 20 C

Days at 20 C

Days at 20 C Days at 20 C

Figure 6.2 Changes in viable population of killer and sensitive strains of S. cerevisiae during growth of single and mixed cultures in YNB at pH 3.5. Single cultures of killer S. cerevisiae ( AWRI 3A, (a); Tyr 303, (b) ); single cultures of sensitive S. cerevisiae ( S-1, (c); AWRI 5A, (d) ) and mixed culture of killer and sensitive S. cerevisiae strains ( AWRI 3A + S-1, (e); Tyr 303 + AWRI 5A,(f) ). 254

—o— AWRI 3A + S-l 5.0 —AWRI 3A ^ S-l A 4.0

1.0

Days at 20C

Tyr303+AWRI 5A 5.0 - Tyr 303 AWRI 5A 4.0 -

Days at 20C

Figure 6.3 Changes in the concentration of protein in the fermentation medium during growth of killer and sensitive strains of S. cerevisiae in single and mixed culture.

6.322 Strain interaction in YNB containing 10% (w/v) ethanol at pH 3.5

After adaptation (Figure 6.1), killer strain S. cerevisiae Tyr 303 in single culture gave weak growth in YNB containing 10% (w/v) ethanol (Figure 6.4a). The sensitive strain S. cerevisiae 5A did not grow in YNB containing 10% (w/v) ethanol and slowly died off (Figure 6.4b). However, its rate of death was greatly accelerated when cultured in combination with the killer strain S. cerevisiae Tyr

303 (Figure 6.4c). Both strains released extracellular protein when cultured singly, but this was significantly increased (error bars on graph indicate standard error of measurements) by 32% when grown in mixed culture (Figure 6.5). 255

6.5 n------Tyr 303 6.0

5.5 -

Days at 20 C

AWRI 5A

6.0

5.5 -

5.0

Days at 20 C

Tyr 303 6.0 AWRI 5A 13 5.5 -

Days at 20 C

Figure 6.4 Changes in viable population during growth of killer and sensitive strains of S. cerevisiae during growth of single and mixed cultures in YNB containing 10% ethanol at pH 3.5. Single cultures of (a) killer S. cerevisiae Tyr 303 ( □ ) and (b) sensitive strain S. cerevisiae AWRI 5A ( A ) and (c) both strains in mixed culture. 256

—o— Tyr 303+AWRI 5A -o— Tyr 303 1.2 - AWRI 5A

0.2 -

Days at 20 C

Figure 6.5 Changes in concentration of protein in the fermentation medium during growth with killer strain S. cerevisiae Tyr 303 and sensitive strain S. cerevisiae AWRI 5A in single and mixed culture.

6.33 Strain interaction in the absence of killer activity

6.331 Strain interaction in YNB at pH 6

Figure 6.6 shows the growth of killer and sensitive strains of S. cerevisiae in YNB medium at pH 6.0 as either single or mixed cultures. As observed in cultures grown at pH 3.5, all strains when cultured singly grew from an initial population of about 5x10^ cfu/mL to about 5x107 cfu/mL in 24-48 hours. However, due to the inactivity of the K2 toxin at pH 6.0, no losses in viability of sensitive S. cerevisiae strains was observed when killer and sensitive strains were cultured in combination. The sensitive S. cerevisiae strain 5A was even able to dominate growth of the killer strain S. cerevisiae Tyr 303 due to its faster growth rate under these conditions (Figure 6.6f). 257

8.0 8.0 6 (b) 3 7.8 7.8

bO o _] 7.5 7.5 Tyr 303 c o 7.3 7.3 P"" ■§ / Q-, 7.0 7.0 o D-, 6.8 6.8 qj d S 03 > 6.5 > 6.5 i I ~! 0 12 3 4 0 12 3 4 Days at 20C Days at 20C

AWRI 5A

Days at 20C Days at 20C

AWRI 3A Tyr 303

AWRI 5A

Days at 20C Days at 20C

Figure 6.6 Changes in population of killer and sensitive strains of S. cerevisiae during growth of single and mixed cultures in YNB at pH 6.0. Single cultures of killer S. cerevisiae ( AWRI 3A, (a); Tyr 303, (b) ); single cultures of sensitive S. cerevisiae ( S-1, (c); AWRI 5A, (d) ) and mixed culture of killer and sensitive S. cerevisiae strains ( AWRI 3A + S-1, (e); Tyr 303 + AWRI 5A,(f)). 258

In agreement with the findings for growth at pH 3.5, growth of the yeast strains at pH 6.0 was accompanied by release of protein into the medium (Figures 6.7a and 6.7b). Due to greater growth, increased concentrations of protein were released at pH 6.0 than at pH 3.5. Notably, however, protein release in the mixed cultures was not significantly greater than the corresponding single strain cultures. For the mixed S. cerevisiae culture of AWRI 3A + S-1, the protein release was actually less than the nearest single strain culture (strain S. cerevisiae AWRI 3A).

12 T — — AWRI 3A + S-1 —o— AWRI 3A g 10 - S-1 / 77

Days at 20C

Tyr 303 + AWRI 5A

■o— Tyr 303

^ 0 12 3 4 Days at 20C

Figure 6.7 Changes in concentration of protein in the fermentation medium during growth of killer and sensitive strains of S. cerevisiae in single and mixed culture in YNB at pH 6.0. 259

6.332 Strain interaction in YNB at pH 3.5 using a cured killer strain (S. cerevisiae AWRI 3AMC)

Growth curves of single and combined cultures of the cured killer strain S. cerevisiae AWRI 3AMC and sensitive strain S. cerevisiae S-1 are shown in Figure 6.8. When grown as single cultures (Figure 6.8a and 6.8b), both strains grew from about 5x106 cfu/mL to about 5x107 cfu/mL within 24-48 hours. The sensitive strain S. cerevisiae S-1 also grew in the presence of the cured killer strain S. cerevisiae AWRI 3AMC when both were cultured together (Figure 6.8c) indicating the absence of killer activity during growth.

Figure 6.9 shows the release of protein during growth of the cured killer strain S. cerevisiae AWRI 3AMC and sensitive strain S. cerevisiae S-1 in single and mixed culture. The protein release in the mixed culture was not significantly greater than the corresponding single strain cultures. This is consistent with a lack of killer activity and, thus, the enhanced protein release due to death of the sensitive strain S. cerevisiae S-1 as seen in Figure 6.3 is absent. 260

8.0 6 7.5 7.0 60 O 1 6.5 e o 6.0 5.5 D-, O 5.0 O-, 4.0 0 12 3 4 Days at 20C

7.0 - 6.5 4

6.0 - 5.5 - 5.0 - 4.5 -

Days at 20C

CT 8.0 7.5 - 7.0 - AWRI 3AMC + S-l

5.5 - 5.0 - Ji 4.5 - — 4.0

Days at 20C

Figure 6.8 Changes in viable population during growth of cured killer and sensitive strains of S. cerevisiae during growth of single and mixed cultures in YNB at pH 3.5. Single cultures of (a) cured killer S. cerevisiae AWRI 3AMC ( □ ) and (b) sensitive strain S. cerevisiae S-1 ( A ) and (c) both strains in mixed culture. 261

—O— AWRI 3AMC + S-l —O— AWRI 3AMC —S-l

Days at 20C

Figure 6.9 Changes in concentration of protein in the fermentation medium during growth with cured killer strain S. cerevisiae AWRI 3AMC and sensitive strain S. cerevisiae S-1 in single and mixed culture.

6.4 Discussion

By exploiting the sensitive interaction of particular strains of S. cerevisiae, this chapter has demonstrated a potential strategy for accelerating the onset of autolysis in sparkling wine fermentation.

The background studies for the selection of appropriate killer and sensitive strains of S. cerevisiae on the basis of efficient killer activity and differentiation of strains in mixed culture is described in Section 6.31. During growth in mixed culture in a model system, the killer effect was clearly indicated by a rapid loss in viability of the sensitive strains (Figure 6.2). Sensitive strains exhibited rapid loss in viability and decreased to non- detectable levels (< 103 cfu/mL) within 48 to 72 hours. Similar kinetics of death for killer and sensitive interactions have been reported by using killer strains isolated from Australian wineries (Heard and Fleet, 1987), although 262 numerous environmental factors can influence the efficacy of killer toxins during wine fermentations (van Vuuren and Jacobs, 1992; Shimizu, 1993). A significant factor for consideration in sparkling wine fermentation is the presence of ethanol as suggested by Figure 6.4 where the relative growth rates and survival kinetics of the two strains were clearly retarded by the presence of 10% (w/v) ethanol. In commercial sparkling wines, the individual and combined effects of ethanol, SO2 concentration, temperature, pH, relative growth rates of killer and sensitive yeast would be relevant factors to be considered in strain selection.

For the purposes of this study, protein release into the growth medium was used as a convenient assay or indicator of yeast autolysis. While the release of protein is an expected part of yeast growth (Kokitkar et aL, 1990) (as was observed in this project when the various strains of S. cerevisiae were grown individually), significantly higher amounts of protein were released when killer and sensitive strains were grown in mixed culture at pH 3.5 (Figure 6.3). Notably, the largest release in protein concentration between days 1 and 2 was accompanied by only a small increase in killer population. This observation suggests that autolysis of the sensitive strain was initiated during this period. The difference in the enhancement of protein release in the mixed cultures over the single cultures (20% for the Tyr 303 + AWRI 5A mixed culture, and 29% for the AWRI 3A + S-1 mixed culture) indicates that the amount of protein released by sensitive yeast during autolysis is strain specific. This is expected since differences between yeast strains with respect to protein release have also been observed during storage of sparkling wine on yeast lees (Tzvetanov and Bambalov, 1994). Notably, the protein released by the yeast during growth in the presence of ethanol was about 4 to 5 times less than that observed without ethanol (Figure 6.5).

However, the lower release of protein in this case is a consequence of lower growth (population) due to the inhibitory effects of the ethanol. Nevertheless, 263 in mixed culture, the killer strain accelerated the death of the sensitive strain and resulted in significantly higher release of protein than when either strain was cultured alone.

An alternative explanation for the elevated release of protein could be that lysis of the cells occur in response toxin-induced membrane damage. However, Bussey (1981) points out that toxin-treated cells do not lyse or contain large pores. Thus, macromolecules such as proteins are not lost from yeast as a direct result of toxin-mediated death, but may be released by subsequent autolytic events.

In order to verify that the extra protein released during mixed culture fermentations could be attributed to the killer-induced death and autolysis of the sensitive yeast, experiments were conducted under conditions where killer activity was absent. These conditions included conducting the fermentations at pH 6.0 where the toxin is inactive (Shimizu, 1993), and where the killer strain has been cured of the dsRNA fragment encoding for killer activity (strain AWRI 3AMC). In both experiments, the protein release in the mixed culture fermentations were not significantly greater than when the strains were cultured individually (Figures 6J and 6.9). This confirms the view that the extra protein release observed in the mixed culture fermentations (20% to 32%) was due to death of the sensitive strain resulting from the activity of the K2 toxin of the killer strains.

In conclusion, the results of this chapter indicate that interactions between killer and sensitive strains of S. cerevisiae may be potentially exploited in sparkling wine production by :

(a) preparing yeast starter cultures for tirage by growing mixed cultures of killer and sensitive strains so that a pool of dead yeast cells together with 264 viable killer cells could be inoculated with the wine base for the secondary fermentation, or

(b) inoculating the wine base with approximately equal populations of viable killer and sensitive strains, so that the sensitive interactions occur in the bottle as part of the secondary fermentation. With the former option, the sensitive interaction would have occurred during the starter culture preparation stage before inoculation into the base wine.

It should be noted, however, that the experiments reported in this Chapter were conducted in model systems over a relatively short period of fermentation. Further research is required to observe whether the killer- induced onset of autolysis will occur in grape wines under commercial conditions during long term storage at lower temperatures. These wines would then require sensory evaluation and comparison with wines prepared using the traditional procedure before the utility of the method can be fully assessed. 265

7.0 Conclusions

It was the intention of this project to gain more precise information about the microbiological and chemical changes that occur during the secondary fermentation and ageing of sparkling wines. The ageing period involves a lengthy autolytic reaction of yeasts and it was hoped that more precise information about the changes that evolve during the sparkling wine process might reveal prospects for accelerating production. The general conclusions of this study include :

1. Preliminary investigations indicated major obstacles to achieving the primary objective, principally because of difficulties associated with the availability of reliable methods for the quantitative measurement of key cellular constituents (eg. lipids and nucleic acids) that were likely to change during the ageing-autolytic phase. After a lengthy program of experimentation, it was concluded that suitable procedures were not available for the accurate measurement of lipids and nucleic acid components in commercial wines. An extensive project would be needed to develop such methods and was considered outside the scope of the present study. Since the main complication in these methods was the complexity of wine composition, it was decided to follow up changes in concentration of lipids and nucleic acids as a consequence of yeast autolysis using a model system consisting of a wine produced from a fermented chemically defined grape juice. This data were used to complement information gained from other analyses concerning yeast autolysis in commercial wines.

2. Growth of the yeast, S. cerevisiae, during the secondary fermentation of sparkling wines was characterised by a rapid increase in numbers from about

1x10$ viable cells/mL to a maximum viable population of about 5 - 6 x 10^ cells/mL over 8-10 days. Notably, the initial yeast population did not appear to influence maximum viable population achieved during this period. This 266 observation may be the result of a combined inhibitory effect of increasing ethanol and carbon dioxide concentration in the wine. The period of growth was followed by a stationary phase lasting until the end of the active fermentation, and was followed by an extended death phase of about 65 - 80 days, with no viable yeast being detected in the wines after 100 - 120 days. This pattern of population change was very reproducible and was also observed in other areas of the project using chemically defined wine media. It is not apparent which substance(s) the yeast are utilising to maintain themselves during this extended death phase but, possibly, they are surviving upon storage polysaccharides built up within the cell during earlier stages of the secondary fermentation.

3. During the secondary fermentation, the consumption of sugar and amino acid nitrogen, and production of ethanol, glycerol, carbon dioxide and volatiles was similar to that described in the literature. With the notable exception of malic acid (which decreased), the concentration of organic acids did not change during the secondary fermentation. The metabolism of malic acid by the yeast culture is a significant finding as this could impact upon wine flavour by reducing the acidity of the final product. Also, the yeast culture consistently released protein as a normal by-product of growth during the secondary fermentation. Similar releases were observed elsewhere in the project (Chapter 6) and appear to be typical of yeast growth during fermentation.

4. From about 200 days (the ageing phase of sparkling wine production), various chemical changes occurred in the wine which were indicative of yeast autolysis. The most significant changes included increases in the concentration of the nitrogen compounds, amino acids and protein. However, there was no distinct pattern with respect to the extent and identity of the amino acids released during this autolytic period. Similarly, differences were 267 observed in the amount of protein released into the wines during this time. These observations are typical of published data and may reflect the influences of other factors such as differences in wine composition, yeast strains and method used for preparation of the yeast for tirage.

For reasons presented already, changes in the concentration of nucleic acids were not followed in the commercial wines. However, the greatest change in the concentration of nucleic acids in the model wine occurred during the secondary fermentation and death phase of the yeast (0-130 days) and not during the autolytic period as indicated by protein and amino acid release.

5. Changes in the concentration of major volatile compounds varied among the wines. This was probably in response to variations in the occurrence of reversible reactions involving esterification due to differences in wine composition. The decrease in concentration of iso-amyl acetate to non- detectable levels during ageing may be due to residual esterase activity in the yeast pellet. Identification of the factors affecting the formation or degradation of volatile components in sparkling wines is an important area for further research because of the importance of aroma in determining wine quality.

The changes in the concentration of lipid compounds in the model wine followed two distinct phases. The first, an increase in neutral lipids and fatty acids, represented an autolytic release of lipids during the period. The second phase was defined by a decrease in all lipid classes except monoglycerides and fatty acids, and may represent hydrolysis of the neutral lipids to glycerol and fatty acids. Analysis of the free fatty acid content of the wine suggested that the pattern of release may be influenced by the ethanol content of the wine. Further research is required to resolve the analytical difficulties associated with lipid analysis in commercial wines in order to confirm the trends in lipid evolution found in the model wine. 268

6. The effects of carbon dioxide pressure on yeast growth and aspects of their metabolism were studied in Chapter 5. Analysis of population kinetics in bottles at varying internal pressures indicated that carbon dioxide pressure hastens death of the yeast following the secondary fermentation. However, there appears to be a minimum threshold above which increasing pressure in the bottle does not give any further increase in death rate.

7. Carbon dioxide pressure also impacted upon some chemical changes during the secondary fermentation. Initially, glucose consumption in pressurised bottles was faster than in non-pressurised bottles with a concomitant increase in the rate of ethanol and carbon dioxide production. This was probably due to a higher maintenance energy requirement by the yeast to withstand the inhibitory effects of increasing carbon dioxide concentration in the wine. This may explain the accelerated death of these yeasts following the secondary fermentation since they would exhaust any available carbon source at a faster rate than the unpressurised yeast.

8. Carbon dioxide was observed to decrease glycerol and 2-phenylethanol production and cellular RNA levels in the yeast during the secondary fermentation. Glycerol production may have been lowered because its biochemical pathway is less able to compete for the oxidation of NADH; however the mechanism to explain depressed production of 2-phenylethanol is unclear. Similar findings are reported in the brewing literature, with the production of other volatiles also being depressed. However, it should be noted that such differences may not have been observed in the chemically defined wine because of the relatively small amount of glucose consumed during the fermentation. The lower RNA content of the yeast cells is a notable observation since this may affect the extent of RNA release during and after the secondary fermentation. Considering the potential flavour- 269 enhancing properties of RNA degradation products, such an effect would be a worthy area for further research.

9. Carbon dioxide affected the kinetics of uptake and release of amino acids during, and after, the secondary fermentation. Initially, developing carbon dioxide pressure in the bottle decreased the nitrogen demand of the yeast, particularly for Group B amino acids valine, leucine and iso-leucine. Similar effects have been reported in the brewing literature and may impact upon production of flavour-active compounds such as vicinal diketones and their precursors. Efflux of amino acids during the secondary fermentation also occurred at an earlier stage in the pressurised bottles. It is not clear whether this was in response to earlier onset of autolysis. However, final total efflux of amino acid nitrogen was greater in the unpressurised bottles, although this may be due to higher total cell numbers.

10. In search of a strategy to accelerate the process of yeast autolysis in sparkling wines, mixed cultures of killer and sensitive strains of S. cerevisiae were grown together for the purpose of supplying a dead pool of yeast which would theoretically undergo immediate autolysis during, and after, the secondary fermentation. These interactions were conducted in Yeast Nitrogen Base at pH 3.5 (with and without the presence of 10% ethanol) and resulted in significantly greater protein release in mixed culture, thus suggesting the occurrence of autolysis of the sensitive strain. Notably, when fermentations were repeated in the absence of killer activity (at pH 6.0, and with the killer strain lacking the ability to produce the killer factor), no significant difference between mixed and single strain cultures was observed.

This indicates that autolysis of sensitive strain was indeed occurring in mixed culture in the presence of active killer toxin. An added advantage of this system is the presence of an actively growing killer strain which would still conduct the secondary fermentation. Further research involving fermentation 270 trials in wine and grape juice, and long-term storage of such samples are required to determine whether this activity will occur under commercial winemaking conditions. Additionally, sensory trials are required to determine whether the quantity of protein and associated products released during the autolysis of the sensitive strain will have a significant impact on sparkling wine quality. 271

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