clarification.
1.7.7 Using Yeast Mannoproteins
It is well known that wine, especially red wine, naturally contains macromolecules that act as protective colloids (Section 9.4.2). At concentrations present in wine, these substances tend to hinder tartrate crystallization, but do not completely inhibit it (Section 3.6.5). Little research has been done into isolating these crystallization inhibitors in wine and making use of their stabilizing properties. On the contrary, for many years, major efforts were made to eliminate these colloids, by drastic fining and filtration, as they reduce the effectiveness of physical stabilization treatments, especially cold stabilization.
It is known, however, that the traditional practice of barrel‐aging white wines on yeast lees for several months often gives them a high level of tartrate stability, so cold stabilization is unnecessary (Section 5.6.4). Although, in practice, this phenomenon is very widespread, very little mention of it has been made until now in enology theory. Thus, in Bordeaux, most dry white wines aged on the lees are not stable in March after their first winter but become stable by June or July without any further treatment. When the same wines are not aged on the lees, they must be systematically cold stabilized to protect them from tartrate crystallization. As it was known that white wines are enriched with mannoproteins released by the yeast during lees aging, it was reasonable to suppose that these macromolecules contributed to the tartrate stabilization of wine.
Yeast mannoproteins were first found to have a certain inhibiting effect on tartrate crystallization in a model medium by Lubbers et al. (1993). However, these experiments used mannoproteins extracted by heat in alkaline buffers, under very different conditions from those accompanying the spontaneous enzymatic release of mannoproteins during lees aging. Furthermore, the effectiveness of mannoproteins extracted by physical processes in preventing tartrate precipitation has not been established in most wines, despite demonstrations in a model medium.
The discovery of the crystallization‐inhibiting effect of mannoproteins extracted by the enzymatic treatment of yeast walls (Dubourdieu and Moine‐Ledoux, 1994) adds a new dimension to this subject. The mannoprotein preparations are obtained by digesting yeast walls with an industrial preparation of β‐(1,3)‐ and β‐(1,6)‐glucanases (Glucanex™), authorized in winemaking as a clarifying enzyme to improve filterability of wines made from botrytized grapes (Sections 3.7.2 and 11.5.2). These preparations inhibit tartrate crystallization in white, red, and rosé wines, whereas the same dose (25 g/hl) of heat‐extracted mannoproteins does not have this stabilizing effect (Moine‐Ledoux and Dubourdieu, 1995).
The inhibiting effect of mannoproteins extracted from yeast on tartrate crystallization is not due to compound MP32, the invertase fragment responsible for protein stabilization in wine (Section 5.6.4) (Dubourdieu and Moine‐Ledoux, 1996). The mannoproteins in question are more highly glycosylated, with an average molecular weight of approximately 40 kDa. They have been purified (Moine‐Ledoux et al., 1997) from the same mannoprotein preparations obtained by the enzymatic treatment of yeast walls.
Furthermore, it has been demonstrated that these mannoproteins are covalently bonded with glucan (Moine‐Ledoux and Dubourdieu, 1999). They remain in the cell walls treated simultaneously with sodium dodecyl sulfate (SDS) (which cuts the hydrogen bonds) and β‐mercaptoethanol (which breaks SH bonds) (Figure 1.21). These two treatments do not affect glycosidic bonds.
The presence of peak 2, corresponding to elution of the mannoprotein responsible for tartrate stabilization, confirms that the bond is covalent. Some of the mannoproteins that share covalent bonds with glucan also have a special type of glycosylation, leading to a glycosylphosphatidylinositol (GPI) anchor. The use of a mutant strain (FBYII), deficient in GPI‐anchored mannoproteins when cultured at 37°C (FBYII‐37), showed that the mannoproteins responsible for tartrate stabilization had this type of glycosylation. Two types of mannoprotein extracts were obtained by enzyme hydrolysis of yeast cell walls (FBYII), cultured at 24°C or 37°C.
FIGURE 1.21 HPLC analysis of molecular‐sieved mannoprotein extract obtained by enzyme digestion of cell walls treated simultaneously with SDS and β‐mercaptoethanol.
HPLC analysis of these two extracts (Figure 1.22) showed that peak 2 was absent when the cell walls came from yeast cultured at 37°C, i.e. deficient in GPI‐anchored mannoproteins. These results (1) show that the mannoproteins responsible for tartrate stabilization are GPI‐anchored and (2) explain why they are only extractible by enzymatic digestion.
FIGURE 1.22 HPLC analysis of molecular‐sieved mannoprotein extract obtained by enzyme digestion of (a) FBYII‐24 and (b) FBYII‐37 yeast cell walls, cultured at 24 and 37°C, respectively.
An industrial preparation (Mannostab™) has been purified from yeast cell wall mannoprotein. It is a perfectly soluble, odorless, flavorless, white powder. This product has been quite effective (Table 1.20) in preventing tartrate precipitation in white wine samples taken before the normal cold stabilization prior to bottling. Initial results show that Mannostab inhibits potassium bitartrate crystallization at doses between 15 and 25 g/hl. However, in certain wines in Table 1.20 (1996 white Bordeaux and 1996 white Graves), larger quantities apparently reduced the stabilizing effect. A similar phenomenon has been reported with a protective colloid used to prevent protein precipitation (Pellerin et al., 1994). The dose of Mannostab necessary to stabilize a wine must be determined by preliminary testing. It is very clear that the use of excess amounts of this additive is ineffective.
The addition of this product could replace current stabilization methods (Moine‐Ledoux et al., 1997). With this in mind, its effectiveness has been compared with that of two other tartrate stabilization methods: continuous contact cold stabilization and the addition of metatartaric acid (Table 1.21). This comparison was carried out by measuring spontaneous crystallization after the addition of KHT (Section 1.6.4). The values obtained indicate the effectiveness of protective colloids, even if they do not necessarily correspond to the instability temperatures. The addition of 15 g/hl of Mannostab to wine 2 or 25 g/hl to wine 1 produced the same spontaneous crystallization temperature, i.e. a stability comparable to that obtained by continuous cold stabilization (Table 1.21). The addition of metatartaric acid, however, considerably reduced the crystallization temperature.
TABLE 1.20 Tartrate Stabilization of Various Wines by Adding Mannostab
| Wines | Mannostab (g/hl) | |||
|---|---|---|---|---|
| 0 | 15 | 20 | 25 | 30 |
|
|