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Yeast autolysis in sparkling wine a review

HERV ALEXANDRE1 and MICHLE GUILLOUX-BENATIER Institut Universitaire de la Vigne et du Vin Jules Guyot, UMR INRA-Universit de Bourgogne 1232, rue Claude Ladrey, BP27877, 21078 DIJON Cedex- France 1 Corresponding author: Dr Herv Alexandre, facsimile +33 3 8039 6265, email rvalex@u-bourgogne.fr Abstract Sparkling wine produced by the traditional mthode champenoise requires a second in-bottle alcoholic fermentation of a base wine, leading to the sparkling wine. This second fermentation is followed by prolonged ageing in contact with yeast cells (lees). The autolysis of yeast occurs during the ageing of sparkling wines. During this process, the yeast releases different compounds that modify the organoleptic properties of the wine. The ageing period is required to give these wines their roundness and characteristic aroma and avour. Autolysis products also inuence the foaming properties of sparkling wine. Yeast autolysis is characterised by the hydrolysis of intracellular biopolymers by yeast enzymes activated after cell death. This results in the release of low molecular weight products. This article reviews the recent advances in understanding the yeast autolysis mechanism, the factors affecting autolysis, the nature of the released compounds and their effects on sparkling wine quality. Keywords: sparkling wine, yeast, autolysis, mthode champenoise Introduction Sparkling wine manufactured by the traditional or mthode champenoise requires two successive fermentations. The rst fermentation transforms grape must into base wine. The essence of the champenoise method is the second fermentation, which takes place in the bottle and increases the alcohol content and internal bottle pressure (up to 57 atmospheres). After this second alcoholic fermentation, the wine is aged on yeast lees. Ageing on lees lasts for at least nine months, depending on the legislation of the wine-producing country. The ageing on yeast lees for Champagne and Champagne millsim lasts one and three years respectively. Autolysis of the yeast occurs during this prolonged contact. Yeast autolysis is a slow process associated with cell death, and involves hydrolytic enzymes that act to release cytoplasmic (peptides, fatty acids, nucleotides, amino acids) and cell wall (mannoproteins) compounds into the wine. Low ageing temperature causes a low death rate and low enzymatic reaction rates, explaining the slowness of the process. During ageing on yeast lees, the organoleptic and foam properties of the wine are modied, reecting changes in the wine composition. In this review, we focus on yeast autolysis in sparkling wine, which differs from yeast autolysis during ageing of still wine (Charpentier and Feuillat 1993, FornaironBonnefond et al. 2001). Yeast autolysis in sparkling wine production has been the subject of many studies. The lees present in still wine during ageing are composed of tartaric acid salts, organic residues and microorganisms, whereas sparkling wine lees are mainly composed of yeast with any technological coadjuvant (riddling aids), such as bentonite, that helps the occulation and the elimination of yeast lees at the end of ageing. In-bottle sparkling wine ageing on lees usually lasts longer than still wine ageing, and autolysis occurs under pressure (6 atmospheres). For still wine, malolactic fermentation usually takes place during ageing on yeast lees, whereas for sparkling wine, malolactic fermentation, when desired, occurs before bottle ageing. The commercial yeasts that carry out the rst and second alcoholic fermentation are different. Yeasts for the rst fermentation are selected for their high fermentation speed and low acid production, as well as other desirable properties, whereas yeasts for the second fermentation are selected for other technological properties (MartinezRodriguez et al. 2001c). Yeast strains are selected for their ability to grow at low temperatures and under pressures in a medium containing at least 10% (v/v) ethanol, as well as having desirable occulating or agglutinating ability. For sparkling wine production, the yeast strain is also selected for its autolytic capacity and its ability to inuence foam quality. Yeast autolysis mechanisms Yeast autolysis can be considered a lytic event in the cells. This is an irreversible process caused by intracellular yeast enzymes. Autolysis generally takes place at the end of the stationary phase of growth and is usually associated with cell death (Babayan and Bezrukov 1985). Babayan et al. (1981) proposed four steps of yeast autolysis. First, the cell endostructures degrade, releasing vacuolar proteases in the cytoplasm. Second, the released proteases are initially inhibited by specic cytoplasmic inhibitors, and are then activated due to degradation of these inhibitors.

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Third, intracellular polymer components hydrolyse, with the hydrolysis products accumulating in the space restricted by the cell wall. Finally, the hydrolytic products are released when their molecular masses are low enough to cross pores in the cell wall. Although this general yeast autolysis process may be valid for most autolysis processes, natural autolysis is different from induced autolysis. Induced autolysis is widely used in industrial applications, such as for the production of a yeast extract used as a avour enhancer or for production of intracellular enzymes (Breddam and Beenfeldt 1991, Kollar et al. 1993, Zambonelli et al. 2000). Yeast autolysates are also added to growth culture media as they are rich in vitamins and amino acids. Autolysis in industrial processes can be induced by physical inductors (rise in temperature, alternate freezing and thawing, and osmotic pressure), chemical inductors (pH, detergents, and antibiotics), or biological inductors (aeration and starvation). The autolysis process can be very fast, from 48 h to 72 h, depending on the inducer. Natural autolysis, however, takes much longer. This is especially true in wines, in which the autolytic conditions pH 3 to 4, ageing temperature of 15C, and the presence of ethanol (12% v/v) are far from the ideal of 45C at pH 5. These differences result in different autolysates, and have been the focus of studies on the autolytic process in wine (Charpentier and Feuillat 1993, Connew 1998). Yeast autolysis in the production of sparkling wines only starts two to four months after completion of the secondary fermentation (Charpentier and Feuillat 1993, Todd et al. 2000). Promotion of yeast autolysis has been done using a mixture of killer and sensitive yeast for the secondary fermentation; under these conditions a rapid death of sensitive yeast cells occurs in the presence of killer strains (Todd et al. 2000). Biochemical and morphological changes Hydrolytic enzymes play a major role in autolysis, and of all the enzymes involved the activities of proteases have been the most extensively studied. Lurton et al. (1989) used specic proteases inhibitors to show that in acidic conditions, protease A was the principal enzyme involved in proteolysis during autolysis in a model wine system, despite there being numerous proteolytic enzymes present in yeast. It was suggested that protease A activity may be responsible for 80% of the nitrogen released during autolysis under optimum conditions. Using a pep4 mutant deleted for protease A, Alexandre et al. (2003) showed that protease A was responsible for 60% of the nitrogen released during autolysis in wine. These results suggest that other acidic proteases may also be involved in the proteolytic process. Consistent with this, Komano et al. (1999) and Olsen et al. (1999) have identied other acidic proteases (Yapsin proteases). A study in wine showed that the proteolytic activity of yeast increased six-fold after sugar exhaustion and that protease activity decreased when yeast cell autolysis started (Alexandre et al. 2001). This proteolytic activity

also depends on the temperature and pH during ageing. Sato et al. (1997) reported that in wines at pH 3 conserved at 10C the intracellular protease activity decreased after three months ageing, whereas the activity decreased considerably during the rst two months in the same wine stored at 20C. Under the same conditions, a very low extracellular protease activity was measured, explaining the slowness of the process. In sparkling wine, proteolytic activity decreases during active bottle fermentation and in the following months, whereas after ageing for nine months following fermentation the intracellular proteolytic activity greatly increases (Feuillat and Charpentier 1982). During Champagne ageing, Leroy et al. (1990) reported that proteolytic activity may also vary depending on the yeast strain. During autolysis, the yeast cell wall degrades, however very few studies have investigated the enzymes involved in cell wall degradation during autolysis in wine. This degradation has been seen in microscopic studies and from studies on cell wall composition during autolysis. The cell wall of Saccharomyces cerevisiae may account for between 20 and 30% of the cell dry mass. It primarily comprises mannoproteins and -glucans (8590% of cell wall dry mass). The inner layer of the cell wall is composed of glucans in which the mannoproteins are embedded and cover this glucan layer (Klis et al. 2002). Glucanases have been shown to be involved in yeast cell wall degradation (Arnold 1972, Notario 1982, Charpentier and Freyssinet 1989). -Glucanases, classied as endo- and exoglucanases, hydrolyse the -O-glycosidic links of the -glucan chains, which leads to the release of glucose, oligosaccharides and mannoproteins trapped in the cell wall or covalently bound to -(16) and -(13) glucans. The kinetics of the -glucanase activity during autolysis and the enological parameters affecting this activity are unknown in sparkling wine. The action of these enzymes has been deduced from the released compounds. The yeast cell walls release amino acids during autolysis. This reects the proteolytic activity that may be occurring in the cell wall (Hien and Fleet 1983). Cell wall degradation during autolysis releases both amino acids and macromolecules (see below). Charpentier and Freyssinet (1989) and Feuillat et al. (1989) showed that cell wall degradation could be summarised as follows. First, glucans are hydrolysed by glucanases, thus releasing mannoproteins trapped or covalently linked to the glucans. Second, the glucans are released due to either residual activities of cell wall glucanases or solubilised glucanases in the medium. Finally, the protein fraction of the mannoproteins is degraded by proteolysis. Microscopy has also been used to study the changes taking place in the cell wall of yeast. Although proteases and glucanases degrade the cell wall, there is no break-down of the cell wall (Vosti and

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Joslyn 1954, Avakyants 1982). The cell wall of yeast grown in a synthetic medium for 24 h is thick and smooth and can be easily distinguished from the plasma membrane. After autolysis, the yeast cells are much smaller and have wrinkles or folds and ridges (Avakyants 1982, Charpentier et al. 1986, Kollar et al. 1993). These wrinkles are thought to be due to plasmolysis, with the increased vacuole size due to solubilisation of the cytoplasmic content supporting this suggestion (Martinez-Rodriguez et al. 2001a). In these studies, the structural and ultrastructural changes occurring in yeast cells during autolysis were compared in a model wine system and in sparkling wines. After 24 h of incubation in a model wine system, the yeast cells were found to have lost most of their cytoplasmic content and have a large vacuole, whereas yeasts aged for 12 months still had most of their cytoplasmic content and have a small vacuole. This shows that the autolysis conditions during ageing of sparkling wine or champagne are not optimal. Many different events occur during yeast autolysis (Figure 1), although the process leading to autolysis is not completely understood. Immediately after the second alcoholic fermentation yeast cells are elongated and ovoid. The cell wall is thick and smooth. Inside the cell a large vacuole is surrounded by spherical bodies (Figure 1a). Between three and six months (Figure 1b), the cell and vacuole are smaller. Spherical bodies are distributed throughout the vacuole. The cell wall is rough, small wrinkles or folds can be seen. Between nine and 12 months (Figure 1c), the cell appears to have collapsed, explaining its small size. The cell wall remains unbroken, with many ridges and folds, nevertheless the yeast cells have lost most of their cytoplasmic content. The fate of the plasma membrane during this process is not clear. During yeast ageing, the biochemical changes are as follows: at the start there is an excretion or passive exorption (Morfaux and Dupuy 1966) of amino acids. After three to six months, the medium continues to be enriched in amino acids due to peptide and protein hydrolysis, and there is a signicant increase in polysaccharides from the cell wall. Plasma membrane degradation starts, with lipids being released into the medium. From nine to 12 months, the amino acid concentration decreases and peptide and protein release dominates. Cell wall polysaccharides, lipids and ribonucleotides increase slightly. Recently, autophagy was shown to play a possible role in the release of yeast compounds into the wines (Cebollero et al. 2005). Autophagy is a degradation pathway activated by nitrogen or carbon starvation. It is characterised by the formation of autophagosomes containing intracellular structures including mitochondria, which are carried to the vacuole and degraded, as reviewed by Huang and Klionsky (2002). Cebollero et al. (2005) used a yeast mutant defective in the autophagic or the Cvt pathways to show that autophagy does take place under wine production conditions. Therefore, genes related to autophagy are good candidates for studying the molecular basis of autolysis or for the genetic engineering of wine yeast.

Amino acids

a
Cell wall

Nucleus

Vacuole

Plasma membrane

b
Lipids Polysaccharides

Sugars

Spherical bodies Proteins and peptides

Ribonucleotide

Figure 1. Schematic representation of the morphological and biochemical changes in yeast during autolysis in sparkling wine. Immediately after the second alcoholic fermentation (a), between 3 and 6 months (b) and between 9 and 12 months (c).

Factors affecting autolysis The principal factors that may affect autolysis are pH, temperature, the presence of ethanol and the nature of the yeast strain. As pH and ethanol content effectively cannot be changed we will not consider these in this discussion. High temperatures, up to 60C, have been reported as favouring autolysis in a model wine system. Molnar et al. (1981) report that the optimal temperature for proteolysis in the champenoise method is between 10 and 12C.

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Table 1. The origin of different compounds released during yeast autolysis and their proven or potential impact on sparkling wine
Origin Compound type Proven or potential impact on sparkling wine References

Nucleoside Nucleotide Amino acid Peptide Protein Protein Lipids Glucan Mannoprotein

Flavouring agent

Leroy et al. (1990) Charpentier et al. (2005) Courtis et al. (1998) Feuillat and Charpentier (1982) Moreno-Arribas et al. (2000) Malvy et al. (1994) Polo et al. (1992) Gallart et al. (2002) Andres-Lacueva et al. (1997) Moreno-Arribas et al. (2000) Bertuccioli and Ferrari (1999)

Cell content

Aroma precursors Foam quality Sweet and bitter taste Sweet and bitter taste Foam quality Foam quality Foam quality Increase in mouthfeel

Cell wall

Autolysis varies greatly with the yeast strain. Suzzi (1990) compared the autolytic capacity of different strains and suggested that this criterion could be used to select yeasts. The autolytic capacity was evaluated by measuring the amino acids released by the yeast at different temperatures ten days after fermentation. Signicant differences were observed in the autolytic capacity of three strains. Therefore, the yeast strain affects the amount of nitrogen released in the medium, which could be potentially useful for sparkling wine production (MartinezRodriguez et al. 2001b). Martinez-Rodriguez et al. (2001c) suggested that a yeast strain with good autolytic capacity would produce better quality sparkling wine than yeast having a low autolytic capacity, and that autolytic capacity together with foam analysis should be used for selecting yeast for sparkling wine production. Nunez et al. (2005) recently conrmed that the autolytic capacity of yeast was important for the quality of sparkling wine. They used a mutant having accelerated autolysis to show that the second fermentation of wines with this mutant improved the foaming properties versus a control strain. Similar results with this mutant were also obtained when the ageing period was reduced from nine to six months, which could reduce production costs. Yeast autolysis compounds and their impact on sparkling wine quality The autolysis of yeast during ageing in sparkling wine releases different compounds into the medium, which modify the physical and organoleptic properties of sparkling wine. Table 1 summarises these changes. Evolution of nitrogen compounds at different stages of mthode champenoise production of sparkling wines Numerous studies have investigated the changes in nitrogen composition during the ageing of wine with yeasts. Nitrogen release is thought to reect the autolytic activity

of yeast, especially the proteolytic activity. In the sparkling wine process, amino acids are released into the medium during bottle fermentation. After the available glucose has been exhausted, the levels of amino acids in wine increases (Feuillat and Charpentier 1982). This excretion or passive exorption as described by Morfaux and Dupuy (1966) should not be confused with autolysis. The autolysis of yeast begins only after three to nine months. The time before autolysis starts varies greatly, depending on base wine composition, ageing time and yeast strain (Moreno-Arribas et al. 1996). Different studies agree that the level of total amino acids increases before the level of free amino acids increases. This shows that peptides are rst released into the medium and are then degraded into amino acids. Moreno-Arribas et al. (1996) studied the evolution of different nitrogen fractions during sparkling wine ageing following the champenoise method. Between three and nine months after tirage, they observed no differences in the concentration of free amino acids irrespective of the grape variety. After nine months, the concentration of free amino acids increased, indicating the start of autolysis. These results have been recently conrmed by Nunez et al. (2005). The peptide content uctuates during ageing, reaching a maximum after 12 to 15 months of ageing with yeast and then decreasing. This behaviour may be due to the initial release of peptides that are subsequently degraded. Moreno-Arribas et al. (1996) also showed that the distribution of free amino acids is very different from the distribution of amino acids in peptides and proteins this has has been conrmed in other studies (Moreno-Arribas et al. 1998a, b, Guilloux-Benatier and Chassagne 2003) The amount of peptides released during the autolysis of yeast during sparkling wine ageing is highly variable and dependent on grape variety and ageing time (Moreno-Arribas et al. 1998b). The nature of peptides also changes with ageing time, being smaller as the ageing time

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increases (Martinez-Rodriguez and Polo 2000). The amino acid composition of the peptides present in sparkling wines has also been investigated (MorenoArribas et al. 1996, 1998a, b). These studies showed that the yeast was the origin of the peptides. Indeed, the proteins from various musts differ. As these proteins are the substrates of yeast proteases, we would expect to get different wine peptides. Bartolom et al. (1997) showed that the amino acid composition of peptides from different varieties of sparkling wines aged with the same yeast over 26 months was the same. The fact that threonine and serine have a major presence in peptides from sparkling wine is also consistent with yeast being the origin of peptides, as these amino acids are not found in base wines as the major free amino acids (Usseglio-Tomasset and Bosia 1990, Acedo et al. 1994, Moreno-Arribas et al. 1998b). Threonine and serine are involved in glycosidic linkages between proteins and mannans in the cell wall (Klis et al. 2002). Curiously, protein concentration and composition during autolysis in sparkling wine has been little studied. This may be explained by amino acids being considered as good markers for following autolysis. The evolution of protein content during autolysis seems to depend on the yeast strain. Leroy et al. (1990) compared two different yeast strains and found that the protein content remained stable during the rst nine months for one strain, whereas it decreased greatly from the end of the second fermentation for the other strain. A similar recent study (Nunez et al. 2005) showed that the protein and polypeptide levels increased during the first three months and then decreased. This was attributed to protease activity. The protein and peptide content then increased again after six months. Protein concentration has been reported to be stable for 90 days after the secondary fermentation and increased slightly, by 8 to 13%, thereafter (Todd et al. 2000). Impact of nitrogen fractions on sparkling wine quality Amino acid enrichment of the medium may improve the aroma potential of sparkling wines. Amino acids are the precursors of some aroma compounds by deamination or decarboxylation reactions (Feuillat and Charpentier 1982). Of the lactones, 3-hydroxy-4,5-dimethyl-2(5H)furanone, so-called sotolon (with a green nut or curry odour), slowly increases in sparkling wine during ageing. Pham et al. (1995) reports that sotolon comes from threonine that is transformed into -ketobutyric acid which then reacts with acetaldehyde. Some peptides have sweet and bitter tastes (Polo et al. 1992) and the surfactant properties of peptides are also thought to play a role in wine. Thus, sparkling wine peptides may play a role in foam stability similar to that in beer. A positive correlation has been reported between polypeptide molecular mass, hydrophobicity and foam stabilising activity in beer (St John Coghlan et al. 1992, Onishi and Proudlove 1994). Moreno-Arribas et al. (1998a) stated that the hydrophobicity of the characterised peptides could account for the the foam properties of sparkling wine.

Various studies have attempted to determine the compounds in wine that affect the quality of the foam (Brissonet and Maujean 1991, 1993, Malvy et al. 1994, Andrs-Lacueva et al. 1996). Most studies investigated base wine and the results are therefore difcult to extrapolate to sparkling wines because autolysis causes important changes during the champenoise method. MorenoArribas et al. (2000) found a positive correlation between foam characteristics and most of the free amino acids and proteins, conrming the results of Malvy et al. (1994). However, no relationship was found between foam characteristics and the concentration of wine peptides. Polysaccharides Glucanase and protease activity results in the release of polysaccharides during autolysis in sparkling wines. These macromolecules contain mainly glucose (74%) and mannose (26%). The mannose/glucose ratio increases during autolysis possibly due to mannoprotein release after glucan degradation. Indeed, mannoproteins are trapped in the glucan layer although the very low mannosidase activity does not explain the increase in mannose concentration (Freyssinet et al. 1989). The concentration of polysaccharides in wines varies greatly and depends on how the polysaccharide content is measured. Charpentier (2000) reported the level of polysaccharides in a sparkling wine increased from 366 mg/L in the base wine to 602 mg/L after nine months of ageing. There is much evidence that mannoproteins from the yeast cell wall play a key role in wine stability and in the organoleptic properties of sparkling wine. Mannoproteins have been shown to reduce haze formation (Ledoux et al. 1992, Dupin et al. 2000), presumably by competing with wine proteins for unknown factors. It is hypothesised that as the concentration of these unknown factors decreases due to the presence of the mannoproteins, the particle size of the proteins decreases and the turbidity consequently decreases (Dupin et al. 2000). Mannoproteins also prevent the precipitation of tartaric salt (Lubbers et al. 1993, Gerbaud et al. 1997, MoineLedoux et al. 1997). Mannoproteins affect the crystal growth rate, by sticking to the growth sites of the crystal, blocking growth of the crystal lattice (Gerbaud et al. 1997). The effect of colloids (macromolecules) on foam quality has also been investigated (Brissonnet and Maujean 1991). Material that precipitates in ethanol has been found among the compounds present in the foam. This suggests the presence of macromolecules. MorenoArribas et al. (2000) showed the importance of neutral polysaccharides on the foam quality of sparkling wines. The optimum time of ageing for the best and most stable foam appears to be 18 months. However after 18 months, foam quality decreases and this phenomenon is accompanied by an increase in monomeric compounds such as fructose, most likely due to hydrolysis of plant components by yeast enzymes released during autolysis (Andreas-Lacueva et al. 1997). Finally, mannoproteins are thought to contribute to the mouthfeel of the wine. Bertuccioli and Ferrari (1999) dened an index to evalu-

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ate the body of wine. They showed that mannoproteins increase the Body Index. Mannoproteins also inuence the intensity and the persistence of the aroma of wine (Lubbers et al. 1994). Lipids Lipids are important components of sparkling wines because they are a large source of avour compounds (Forss 1969) and also inuence foam stability. Therefore, the evolution of the lipid content in sparkling wine has been the focus of several studies. During the second fermentation, lipid content increases (Troton et al. 1989). After bottle ageing in contact with yeasts, the lipid content increases further and qualitative changes also occur (Piton et al. 1988). During ageing, the concentration of polar lipids decreases whereas the concentration of neutral lipids (i.e. mono-, di- and triglycerides) increases. However, there are data concerning the evolution of phospholipids and sterols during sparkling wine ageing. Experiments in a model wine system showed that the levels of triacylglycerols, 1,3diacylglycerols, 2-monoacylglycerols, free fatty acids, sterol esters and sterols increase after two days of autolysis and then decrease, probably due to yeast hydrolytic enzymes (Pueyo et al. 2000). No phospholipids were released into the medium, conrming previous results from Hernawan and Fleet (1995), and it was suggested that any phospholipids are degraded. There have been conicting results concerning the inuence of lipids on foam. Maujean et al. (1990) found that the addition of octanoic and decanoic acids reduces foam stability, whereas Dussod et al. (1994) reported that the addition of a lipid mixture did not affect the foam. Furthermore, Pueyo et al. (1995) found that linolenic and palmitoleic acid were the best indicators of foam stability. The inuence of fatty acids on wine foaming has been reinvestigated (Gallart et al. 2002). It was found that free fatty acids such as C8, C10 and C12 acids were negatively related to the quality of the foam, whereas the ethyl esters of hexanoic, octanoic, and decanoic acids were positively related. Nucleic acids Although the degradation of proteins during autolysis has been extensively researched, the hydrolysis of RNA and DNA has been less studied. RNA and DNA make up 5 to 15% and 0.1 to 1.5% of the cell dry weight, respectively. During autolysis, DNA from a brewing and baking Saccharomyces cerevisiae strain was almost completely degraded (Hough and Maddox 1970, Suomalainen 1975). However, Trevelyan (1978) found no decrease in DNA content during the autolysis of bakers yeast. The extent of DNA degradation during autolysis appears to depend on the yeast species (Hernawan and Fleet 1995). The very low levels of DNA detected in the autolysate reects DNase activity. DNA degradation requires several active enzymes and leads to oligonucleotide, nucleotide and nucleoside degradation products. The predominance of deoxyribonucleotides in the autolysate indicates that endo- and exonucleases are primarily involved in the

degradation process. Zhao and Fleet (2003) reported that up to 55% of the total DNA was degraded during autolysis, releasing 3-and 5 deoxyribonucleotides. Even under optimum autolytic conditions, some parts of the DNA are resistant to autolytic degradation. However, studies of DNA degradation in oenological conditions are still needed. It is expected that the presence of ethanol, and the lower pH and temperature would result in a much lower DNA degradation. More than 95% of the total content of nucleic acid within yeast cells is RNA. Zhao and Fleet (2005) suggested that RNA degradation is a key reaction of yeast autolysis. They determined several autolytic conditions and showed that up to 95% of cell RNA was degraded, releasing mainly 3-, 5- and 2-ribonucleotides. The conditions for forming the two avour-enhancing nucleotides, 5-AMP and 5-GMP, were 50C at pH 7.0 and 40C at pH 4.0 respectively. Although these are far from the optimal conditions for sparkling wine production, the degradation of nucleic acids and the release of nucleotides in sparkling wine during autolysis can affect the organoleptic properties of the wine (Courtis et al. 1998). Although RNAse activity has been shown to occur during autolysis in Champagne (Leroy et al. 1990), the level of nucleic acids reported should be interpreted with caution. The results were from spectrophotometric observations, but nucleotides in wine are present in complex mixture with organic acids, phenolic compounds, peptides etc. that can interfere with the measurement. Recently our colleagues have unequivocally identied monophosphate nucleotides in Champagne wine (Aussenac et al. 2001, Charpentier et al. 2005), including three monophosphate nucleotides (5-UMP, 5-GMP and 5-IMP) in Champagne aged on lees for 8 years. The concentration of the nucleotide monophosphates ranged from 50 g/L to 500 g/L, which is considerably different from that previously reported (Courtis et al. 1998). In the food industry, monophosphate nucleotides are well recognised as avour compounds, but further studies are needed to evaluate the impact of nucleotides on wine avour. Volatile compounds Volatile compounds released during yeast autolysis have been less well studied than non-aroma compounds. The few existing studies have shown that many compounds are released, some having low perception levels. Chung (1986) reported in a model wine system (12% v/v ethanol, pH 3.5) that autolysis of Saccharomyces cerevisiae at 1520C or 3540C releases many different volatile compounds after four to six months. Esters are the major family of volatile compounds released during autolysis, both qualitatively and quantitatively. Short chain (C3C4) and medium chain (C6C12) acyl esters with characteristic fruity odours appear at the beginning of yeast autolysis and then decrease. Long chain acyl esters have also been identied in model wine and sparkling wine (Molnar et al. 1981). Terpenic alcohols and higher alcohols are also released during autolysis. Geraniol, -terpineol, citronellol and farnesol have all been identied. These compounds

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have low perception levels ranging from 100 to 300 g/L. Molnar et al. (1981) suggested that farnesol contributes greatly to the aromatic quality of sparkling wine and Loyaux et al. (1981) suggested nerolidol in Champagne. Among the higher alcohols, the rapid formation of isoamyl alcohol and 2-phenylethanol (rose odour) has been observed during autolysis in a model wine system (Chung 1986). About ten aldehydes have been measured and identied. 3-Methylbutanal is the most abundant, representing 40% of the total aldehydes, and may be formed through a mechanism involving isoamyl alcohol oxidation. Most of the aldehydes identied are present at levels close to or greater than the perception level in water. Aldehydes are described as having a grassy odour that negatively affects the organoleptic properties, although most disappeared during ageing (Chung 1986). Francioli et al. (2003) recently characterised the volatile compounds released during autolysis that could serve as age markers in sparkling wines. Acetates appeared to decrease during ageing, whereas diethylsuccinate, vitispirane and TDN (1,1,6-trimethyl-1,2-dihydro naphthalene) increased over time. Hexanol and 2-phenylethanol have been shown to be released during autolysis. Compounds such as vitispirane, TDN and diethylsucinate may be good age markers and can discriminate between young and aged sparkling wines. Riu-Aumatell et al. (2006) obtained similar results, reporting that some high molecular weight acetates, and ethyl and isoamyl esters are typical aroma compounds in young cavas (Spanish sparkling wine), whereas vitispirane, diethyl succinate, TDN, hexenol and ethyl acetate are typical aroma compounds in long-aged cavas. The origin of the post-fermentative aromas in bottle ageing in contact with lees has been studied. Enzymatic release (by yeast enzymes) from glycosidic precursors has been suggested as causing changes in aroma compounds, as C13 norisoprenoids including vitispirane can be derived from glycosidically-bound, carotenoid-derived megastigmane compounds (Riu-Aumatell et al. 2006). TDN may be a direct degradation product of carotene (Rapp 1998), although precursors linked to a sugar molecule have also been reported (Winterhalter 1991). Descriptive analysis is another way to characterise the effect of the mthode champenoise process on aroma. Changes in aroma occurring during production of sparkling wine varied for either individual wines or wines of different varieties. The proles of the base wines do not permit prediction of the sensory properties of the sparkling wines after 18 months of lees ageing (De La Presa-Owens et al. 1998). In this study the authors demonstrated that descriptive analysis of the base wine allows discrimination among different grape varieties like Chardonnay or Pinot Noir. However, after the secondary fermentation the sparkling wines were no longer differentiated by variety or colour. This study reported for the rst time that secondary fermentation together with lees ageing profoundly modify the aromatic prole of the wine.

Conclusion According to Zambonelli et al. (2000), the study of the autolysis of yeast has been so extensive that there is nothing more to uncover. However, this review has shown that several questions remain to be addressed. We have mainly focused on autolysis during sparkling wine ageing and the information available in that context. There have been extensive studies on yeast autolysis, although the different conditions used in these studies (fresh yeast, active dry yeast, temperature, pH, model wine system or wine) have led to contradictory results. Furthermore, not all the studies could be extrapolated to wine. For example, it is not known whether the autolysis of yeast following the fermentation of must and of yeast after fermentation of wine, such as for the champenoise method, is similar. Analytical studies on wine, and especially sparkling wine, have given a clear picture of the different compounds released during autolysis. However, the kinetics of release of certain compounds, such as nucleotides, nucleosides and lipids needs to be studied in more depth and to be correlated to enzyme activity. Currently, the molecular mechanisms responsible for the induction of autolysis, and what the signal transduction is, remain unknown. Understanding such mechanisms should increase our understanding of autolysis and may reveal potential targets for accelerating the process. Finally, the yeast origin of many aroma compounds needs to be proven. Volatile compounds that are characteristic of a long-aged Champagne may also be found in still wine that has not been aged on yeast lees. The impact of these compounds on the physical and organoleptic properties of sparkling wine is also poorly understood. Many changes occur during autolysis, making it difcult to attribute a specic compound to specic organoleptic changes. We are still unsure as to which components formed or released during ageing are odour-active compounds. Therefore, the effect of yeast autolysis on the organoleptic properties of wine should be re-evaluated using techniques such as gas chromatography-olfactometry (GC/O) complemented with sensory descriptive analysis.

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Manuscript received: 1 February 2006 Revised manuscript received: 25 April 2006