Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon
and acetoin. Their formation mechanisms are described in the following paragraphs.
2.3.4 Formation and Accumulation of Acetic Acid by Yeasts
Acetic acid is the principal volatile acid in wine. It is produced in particular during bacterial spoilage (acetic acid spoilage and lactic acid spoilage) but is always formed by yeasts during fermentation. Beyond a certain limit, which varies depending on the wine, acetic acid has a detrimental sensory effect on wine quality. In healthy grape must with a moderate sugar concentration (less than 220 g/l), S. cerevisiae produces relatively small quantities (100–300 mg/l), varying according to the strain. However, under certain winemaking conditions, even without bacterial contamination, yeast acetic acid production can be abnormally high and becomes a problem for the winemaker.
The biochemical pathway for the formation of acetic acid in wine yeasts has not yet been clearly identified. The hydrolysis of acetyl‐CoA can produce acetic acid. Pyruvate dehydrogenase produces acetyl‐CoA beforehand by the oxidative decarboxylation of pyruvic acid. This reaction takes place in the mitochondrial matrix but is limited under anaerobic conditions. Aldehyde dehydrogenase can also form acetic acid by the oxidation of acetaldehyde (Figure 2.11). This enzyme, whose cofactor is NADP+, is active during alcoholic fermentation. The NADPH thus formed can be used to synthesize lipids. When pyruvate dehydrogenase is repressed, this pathway forms acetyl‐CoA through the action of acetyl‐CoA synthetase. Under anaerobic conditions in a model medium, yeast strains producing the least amount of acetic acid have the highest acetyl‐CoA synthetase activity (Verduyn et al., 1990).
FIGURE 2.11 Acetic acid formation pathways in yeasts. 1, pyruvate decarboxylase; 2, alcohol dehydrogenase; 3, pyruvate dehydrogenase; 4, aldehyde dehydrogenase; 5, acetyl‐CoA hydrolase; 6, acetyl‐CoA synthetase.
The acetaldehyde dehydrogenase in S. cerevisiae has five isoforms, three located in the cytosol (Section 1.4.1) (Ald6p, Ald2p, and Ald3p) and the remaining two (Ald4p and Ald5p) in the mitochondria (Section 1.4.3). These enzymes differ by their specific use of the NAD+ or NADP+ cofactor (Table 2.2).
Remize et al. (2000) and Blondin et al. (2002) studied the impact of the deletion of each gene and demonstrated that the NADP‐dependent cytoplasmic isoform coded by ALD6 played a major role in the formation of acetic acid during the fermentation of dry wines, while the ALD5 mitochondrial isoform was also involved, but to a lesser extent (Figure 2.12).
TABLE 2.2 Isoforms of Acetaldehyde Dehydrogenase in S. cerevisiae (NavarroAvino et al., 1999)
| Chromosome | Gene | Location | Cofactor |
|---|---|---|---|
| XIII | ALD2 | Cytosol | NAD+ |
| XIII | ALD3 | Cytosol | NAD+ |
| XV | ALD4 | Mitochondria | NAD+ and NADP+ |
| V | ALD5 | Mitochondria | NADP+ |
| XVI | ALD6 | Cytosol | NADP+ |
Practical winemaking conditions likely to lead to abnormally high acetic acid production by S. cerevisiae are well known. As is the case with glycerol formation, acetic acid production is closely dependent on the initial sugar level of the must, independent of the quantity of sugars fermented (Table 2.3). The higher the sugar content of the must, the more acetic acid (and glycerol) the yeast produces during fermentation. This is due to the yeast's mechanism for adapting to a medium with a high sugar concentration: S. cerevisiae increases its intracellular accumulation of glycerol to counterbalance the osmotic pressure of the medium (Blomberg and Alder, 1992). This regulation mechanism is controlled by a cascade of signal transmissions leading to an increase in the transcription level of genes involved in the production of glycerol (GPD1), but also of acetate (ALD2, ALD3, and ALD4) (Attfield et al., 2000; Erasmus et al., 2003; Pigeau and Inglis, 2005). Acetate formation plays an important physiological role in the intracellular redox equilibrium by regenerating reduced equivalents of NADH. Thus, it is clear that an increase in acetate production is inherent to the fermentation of high‐sugar musts. However, Bely et al. (2003) demonstrated that it was possible to reduce acetate production by supplying more NADH to the redox equilibrium process. This may be done indirectly by stimulating biomass formation, which generates an excess of NADH during amino acid synthesis (Bakker et al., 2001). Assimilable nitrogen in the must plays a key role in this stimulation process. Thus, in high‐sugar musts, acetate production is inversely correlated with the maximum cell population (Figure 2.13), which is, in turn, related to the assimilable nitrogen content of the must. It is strongly recommended to monitor the assimilable nitrogen content of botrytized musts and to supplement them with ammonium sulfate, if necessary. The optimum assimilable nitrogen concentration in this type of must to minimize acetic acid production is approximately 190 mg/l (Figure 2.14). The best time for adding nitrogen supplements is at the very beginning of fermentation, as later additions are less effective and may even increase acetate production. Indeed, in view of the unpredictable increase in acetic acid production that sometimes occurred in botrytized musts supplemented with ammonium sulfate, many enologists had given up the practice entirely. It is now known that, provided the supplement is added at the very beginning of fermentation, adjusting the assimilable nitrogen content to the optimum level (190 mg/l) always minimizes acetic acid production in botrytized wines. Furthermore, Bely et al. (2005) demonstrated that direct inoculation with industrial preparations of active dry yeast leads to greater acetate production than does a yeast starter with a preculture period of 24 hours in a botrytized grape must diluted by half. This preculture period enables S. cerevisiae to adapt to the osmotic stress. Bely et al. (2008) showed that the species Torulaspora delbrueckii, which is a highly osmotolerant yeast, can be used in combination with S. cerevisiae to minimize acetic acid production during the making of wines from botrytized grapes.