clear that lactic acid bacteria have transformed substrates other than malic acid.
FIGURE 2.17 Acetoin, diacetyl, and 2,3‐butanediol formation by yeasts under anaerobic conditions. TPP, thiamine pyrophosphate; TPP‐C2, active acetaldehyde.
Yeasts also make use of pyruvic acid to form acetoin, diacetyl, and 2,3‐butanediol (Figure 2.17). This process begins with the condensation of a pyruvate molecule and a molecule of active acetaldehyde bound to TPP, leading to the formation of α‐acetolactic acid. The oxidative decarboxylation of α‐acetolactic acid produces diacetyl. Acetoin is produced by either the non‐oxidative decarboxylation of α‐acetolactic acid or the reduction of diacetyl. The reduction of acetoin leads to the formation of 2,3‐butanediol; this last reaction is reversible.
From the start of alcoholic fermentation, yeasts produce diacetyl, which is rapidly reduced to acetoin and 2,3‐butanediol. This reduction takes place in the days that follow the end of alcoholic fermentation, when wines are conserved on the yeast biomass (de Revel et al., 1996). Acetoin and especially diacetyl are strong‐smelling compounds that evoke a buttery aroma. Above a certain concentration, they have a negative effect on wine aroma. However, in wines that have not undergone malolactic fermentation, their concentration is too low (a few milligrams per liter for diacetyl) to have a sensory impact. On the other hand, lactic acid bacteria can degrade citric acid to produce much higher quantities of these carbonyl compounds than yeasts (Section 5.3.2).
FIGURE 2.18 Citramalic acid and dimethylglyceric acid.
Finally, yeasts condense acetic acid (in the form of acetyl‐CoA) and pyruvic acid to produce citramalic acid (0–300 mg/l) and dimethylglyceric acid (0–600 mg/l) (Figure 2.18). These compounds have little sensory impact.
2.3.6 Degradation of Malic Acid by Yeast
Saccharomyces cerevisiae partially degrades malic acid (10–25%) in the must during alcoholic fermentation. Different strains degrade varying amounts of this acid and degradation is more significant when the pH is low. Alcoholic fermentation is the main pathway degrading malic acid. The pyruvic acid resulting from this transformation is decarboxylated into acetaldehyde, which is then reduced to ethanol. Malic enzyme is responsible for the transformation of malic acid into pyruvic acid (Figure 2.19). This oxidative decarboxylation requires NAD+ (Fuck and Radler, 1972). This malo‐alcoholic fermentation lowers wine acidity significantly more than malolactic fermentation does.
Schizosaccharomyces differs from wine yeasts. The alcoholic fermentation of malic acid is complete in yeasts of this genus, which possess an active malate transport system. In S. cerevisiae, malic acid penetrates the cell by simple diffusion. Yet at present no attempts to use Schizosaccharomyces in winemaking to break down the malic acid in musts have been successful (Peynaud et al., 1964; Carre et al., 1983). First of all, the implantation of these yeasts in the presence of S. cerevisiae is difficult in a non‐sterilized must. Secondly, their optimum growth temperature (30°C), higher than for S. cerevisiae, imposes warmer fermentation conditions. Sometimes, the higher temperature adversely affects the sensory quality of wine. Finally, some grape varieties fermented by Schizosaccharomyces do not express their varietal aromas. The acidic Gros Manseng variety produces a very fruity wine when correctly vinified with S. cerevisiae, but has no varietal aroma when fermented by Schizosaccharomyces. To resolve these problems, some researchers have used non‐proliferating populations of Schizosaccharomyces enclosed in alginate balls. These populations degrade the malic acid in wines having already completed their alcoholic fermentation (Magyar and Panyik, 1989; Taillandier and Strehaiano, 1990). Although no sensory defect is found in these wines, the techniques have not yet been developed for practical use.
Today, molecular biology permits another strategy for making use of the ability of Schizosaccharomyces to ferment malic acid. It consists of integrating Schizosaccharomyces malate permease genes and the malic enzyme (Mae 1 and Mae 2) in the S. cerevisiae genome (Van Vuuren et al., 1996). The technological interest of a wine yeast genetically modified in this manner is not yet clear, nor are the risks of its proliferation in wineries and nature.
FIGURE 2.19 Decomposition of malic acid by yeasts during alcoholic fermentation.
2.4 Metabolism of Nitrogen Compounds
The nitrogen requirements of wine yeasts and the nitrogen supply in grape musts are discussed later (Section 3.4.2). The following section covers the general mechanisms of assimilation, biosynthesis, and degradation of amino acids in yeasts. The consequences of these metabolisms, which occur during alcoholic fermentation and affect the production of higher alcohols and their associated esters in wine, are also discussed.
2.4.1 Amino Acid Synthesis Pathways
The ammonium ion and amino acids found in grape must supply the yeast with nitrogen. The yeast can also synthesize most of the amino acids necessary for constructing its proteins. It fixes an ammonium ion on a carbon skeleton derived from the metabolism of sugars. The yeast uses the same reaction pathways as all organisms. Glutamate and glutamine play an important role in this process (Cooper, 1982; Magasanik, 1992).
FIGURE 2.20 Incorporation of the ammonium ion in α‐ketoglutarate catalyzed by NADP glutamate dehydrogenase (NADP‐GDH).
NADP+ glutamate dehydrogenase (NADP+‐GDH), a product of the GDH1 gene, produces glutamate (Figure 2.20) from an ammonium ion and an α‐ketoglutarate molecule. The latter is an intermediate product of the citric acid cycle. The yeast also possesses an NAD+ glutamate dehydrogenase (NAD+‐GDH), produced by the GDH2 gene. This dehydrogenase is involved in the oxidative catabolism of glutamate. It produces the inverse reaction of the previous one, liberating the ammonium ion used in the synthesis of glutamine. NADP‐GDH activity is at its maximum when the yeast is cultivated on a medium containing exclusively ammonium as its source of nitrogen. The NAD‐GDH activity, however, is at its highest level when the principal source of nitrogen is glutamate. Glutamine synthetase (GS) produces glutamine from glutamate and ammonium. This amidationrequires the hydrolysis of an ATP molecule (Figure
