Читать книгу Handbook of Enology: Volume 1 - Pascal Ribéreau-Gayon - Страница 58

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.

FIGURE 2.12 Acetate production by strains of S. cerevisiae (V5) following deletion of different gene coding for isoforms of acetaldehyde dehydrogenase (Blondin et al., 2002).

TABLE 2.3 Effect of Initial Sugar Concentration of the Must on the Formation of Secondary Products of the Fermentation (Lafon‐Lafourcade, 1983)

Initialsugar (g/l)	Fermentedsugar (g/l)	Secondary products
Aceticacid (g/l)	Glycerol(g/l)	Succinicacid (g/l)
224	211	0.26	4.77	0.26
268	226	0.45	5.33	0.25
318	211	0.62	5.70	0.26
324	179	0.84	5.95	0.26
348	152	1.12	7.09	0.28

FIGURE 2.13 Correlation between volatile acidity production and the maximum cell population in high‐sugar botrytized musts.

FIGURE 2.14 Effect of the yeast‐assimilable nitrogen content in must (with or without ammonium supplements) on the production of volatile acidity (initial sugar content: 350 g/l).

In wines made from botrytized grapes, certain substances in the must inhibit yeast growth (Volume 2, Section 3.7.2) and increase the production of acetic acid and glycerol during fermentation. Botrytis cinerea secretes these “botryticine” substances (Ribéreau‐Gayon et al., 1952, 1979). Fractional precipitation with ethanol partially purifies these compounds from must and culture media of B. cinerea. These heat‐stable glycoproteins have molecular weights between 10,000 and 50,000. They are composed of a peptide (10%) and a carbohydrate part containing mostly mannose and galactose as well as some rhamnose and glucose (Dubourdieu, 1982). When added to healthy grape must, these compounds provoke an increase in glyceropyruvic fermentation and a more‐or‐less significant excretion of acetic acid at the end of fermentation (Figure 2.15). The mode of action of these glycoproteins on yeasts has not yet been identified. The physiological state of yeast populations at the time of inoculation seems to play an important role in the fermentation of botrytized grape must. Commercial dry yeast preparations are much more sensitive to alcoholic fermentation inhibitors than yeast starters obtained by preculture in healthy grape must.

FIGURE 2.15 Effect of an alcohol‐induced precipitate of a botrytized grape must on glycerol and acetic acid formation during the alcoholic fermentation of healthy grape must (Dubourdieu, 1982). 1, Evolution of acetic acid concentration in the control must; 2, evolution of acetic acid concentration in the must supplemented with the freeze‐dried precipitate; 1′, evolution of glycerol concentration in the control must; 2′, evolution of glycerol concentration in the must supplemented with the freeze‐dried precipitate.

Other winemaking factors favor the production of acetic acid by S. cerevisiae: anaerobic conditions, very low pH (<3.1) or very high pH (>4), certain amino acid or vitamin deficiencies in the must, and excessively high temperature (25–30°C) during the yeast growth phase. In red winemaking, temperature is the most important factor, especially when the must has a high sugar concentration. In hot climates, the grapes should be cooled when filling the tanks. The temperature should not exceed 20°C at the beginning of fermentation. The same procedure should be followed during thermovinification immediately following the heating of the grapes.

In dry white and rosé winemaking, excessive must clarification can also lead to the excessive production of volatile acidity by yeast. This phenomenon can be particularly pronounced with certain yeast strains. Therefore, must turbidity should be adjusted to the lowest possible level that enables a complete and rapid fermentation (Sections 3.7.3 and 13.5.3). The input of lipids made available to yeasts via solid sediments (grape solids), in particular long‐chain unsaturated fatty acids (C18:1 and C18:2), greatly influences acetic acid production during white and rosé winemaking.

The experiment in Figure 2.16 illustrates the important role of lipids in acetic acid metabolism (Delfini and Cervetti, 1992; Alexandre et al., 1994). The volatile acidity of three wines obtained from the same Sauvignon Blanc must was compared. After filtration but before yeast inoculation, must turbidity was adjusted to 250 Nephelometric Turbidity Units (NTU) by three different methods: reincorporating fresh grape solids (control), adding cellulose powder, and supplementing with a lipid extract (using methanol–chloroform) that contained the same quantity of grape solids adsorbed on the cellulose powder. The volatile acidities of the control wine and the wine that was supplemented with a lipid extract of grape solids before fermentation are identical and perfectly normal. Although the fermentation was normal, the volatile acidity of the wine made from the must supplemented with cellulose (therefore devoid of lipids) was practically twice as high (Lavigne, 1996). Supplementing the medium with lipids appears to favor the penetration of amino acids into the cell, which limits the formation of acetic acid.

FIGURE 2.16 Effect of the lipid fraction of grape solids on acetic acid production by yeasts during alcoholic fermentation (Lavigne, 1996).

During the alcoholic fermentation of red or slightly clarified white wines, yeasts do not continuously produce acetic acid. The yeast metabolizes a large portion of the acetic acid secreted in must during the fermentation of the first 50–100 g of sugar. It can also assimilate acetic acid added to must at the beginning of alcoholic fermentation. The assimilation mechanisms are not yet clear. Acetic acid appears to be reduced to acetaldehyde, which favors alcoholic fermentation to the detriment of glyceropyruvic fermentation. In fact, the addition of acetic acid to a must lowers glycerol production but increases the formation of acetoin and 2,3‐butanediol. Yeasts seem to use the acetic acid formed at the beginning of alcoholic fermentation (or added to must) via acetyl‐CoA in their lipid synthesis pathways.

Certain winemaking conditions produce abnormally high amounts of acetic acid. Since this acid is not used during the second half of the fermentation, it accumulates until the end of fermentation. When refermenting a tainted wine, yeasts can lower volatile acidity by metabolizing acetic acid. The wine is incorporated into freshly crushed grapes at a proportion of no more than 20–30%. The wine should be sulfited or filtered before incorporation to eliminate bacteria. The volatile acidity of this mixture should not exceed 0.6 g/l expressed as H₂SO₄. The volatile acidity of this newly made wine rarely exceeds 0.3 g/l expressed as H₂SO₄. The concentration of ethyl acetate decreases simultaneously.