3A Sparkling Wines and Yeast Autolysis 69
Without its protection the protoplast could lyse, since the inner osmotic pressure is
high compared to the environmental pressure.
During autolysis, the glycoproteinsand polysaccharides in the cell wall are
hydrolyzed. Lysis of the cell wall has been studied by microscopy and ultramis-
croscopy methods, also studying the products obtained after cell wall autolysis.
However, the enzymes involved in cell wall autolysis have been less investigated
than other autolytic enzymes, such as proteases, and the kinetics of glucanase activ-
ity is unknown in sparkling wines (Alexandre and Guilloux-Benatier 2006). Char-
pentier and Freyssinet (1989) have suggested the following mechanism for lysis of
the wall inSaccharomyces cerevisiae: in the early stage of the process, glucanases
act on glucans, releasing mannoproteins inserted or covalently linked to glucans.
Later, these enzymes release the glucans into the wine. Mannoproteins and other
polymerized compounds are, finally, degraded by proteolytic enzymes.
Microscopic observation of the yeast cell under autolysis has revealed that
although glucanases and proteases degrade the wall, there is no break down of the
cell wall. The yeast cell wall retains its shape during autolysis, a so the variation
in optical density of the medium cannot be related with the degree of autolysis,
as in the case of bacteria. Although microscopy is used less to investigate yeast
autolysis in sparkling wines than studies based on analysing the products released
into the medium, several researchers have used different microscopic techniques to
observe the changes taking place in the yeast cell wall (Fumi et al. 1987b; Mart ́ınez-
Rodr ́ıguez et al. 2001b, 2004; Piton et al. 1988; Takeo et al. 1989). Since the natural
autolysis that takes place in wines is a long-lasting process, model systems are com-
monly used to study this phenomenon (Feuillat and Charpentier 1982; Hernawan
and Fleet 1995; Mart ́ınez-Rodr ́ıguez and Polo 2000b) in order to obtain results
in shorter periods of time. Structural and ultrastructural changes occurring during
autolysis have been compared in model wines and in sparkling wines. Structural
observations have revealed that yeast cells in fermentation are elongate, ovoid and
present a large vacuole containing a number of spherical bodies, located mainly
on the edges of the vacuole. However, after 24 h of induced autolysis in a model
wine, the volume of cells is much smaller, due to solubilization of the cytoplasmic
content that takes place during induced autolysis (Mart ́ınez-Rodr ́ıguez et al. 2001b;
Mart ́ınez-Rodr ́ıguez et al. 2004). The observation of cells which have been aged
in wine for 12 months shows the presence of more spherical bodies than in cells
isolated from a model wine. As the spherical bodies can be considered as intermedi-
ates in the authopagy process (Takeshige et al. 1992; Cebollero and Gonz ́alez 2007),
these findings indicated that, after 24 h induced autolysis in a model wine yeast, a
higher degree of autolysis has been reached than after 12 months of aging in wine.
Ultrastructural observations, using different scanning microscopy techniques, of the
yeast cell under autolysis have revealed the presence of wrinkles or folds on the wall
which are mostly longitudinal. These wrinkles are due to plasmolysis and do not
appear in yeast isolated during fermentation. Low Temperature Scanning Electron
Microscopy (LTSEM) has shown three-dimensional images of empty yeast cells
that have lost most of their cytoplasmic contents as a result of autolysis after 24
h of incubation in a model wine (Fig. 3A.1) (Mart ́ınez-Rodr ́ıguez et al. 2001c).