Handbook of Enology: Volume 1. Pascal Ribéreau-Gayon
For high glucose concentrations—for example, in grape must—S. cerevisiae only metabolizes sugars by the fermentative pathway. Even in the presence of oxygen, respiration is impossible. Discovered by Crabtree (1929) on tumor cells, this phenomenon is known by several names: catabolite repression of respiration by glucose, the inverted Pasteur effect, and the Crabtree effect. Yeasts manifest the following signs during this effect: a degeneration of the mitochondria, a decrease in the proportion of cellular sterols and fatty acids, and a repression of the synthesis of Krebs cycle mitochondrial enzymes and constituents of the respiratory chain. With S. cerevisiae, there must be at least 2 g of glucose per liter for the Crabtree effect to occur. The catabolite repression exerted by glucose on wine yeasts is very strong. In grape must, at any level of aeration, yeasts are forced to ferment because of the high glucose and fructose concentrations. From a technological viewpoint, yeasts consume sugars via the respiratory pathway during the industrial production of dry yeast, but not in winemaking. If must aeration helps the alcoholic fermentation process (Section 3.7.2), the fatty acids and sterols synthesized by yeasts, which proliferate in the presence of oxygen, are responsible, not respiration.
Saccharomyces cerevisiae can metabolize ethanol via the respiratory pathway in the presence of small quantities of glucose. After alcoholic fermentation, oxidative yeasts develop in a similar manner on the surface of wine (Sections 14.5.2 and 14.5.3) as part of the process of making certain specialty wines (Sherry and vin jaune from Jura in France).
2.3.2 Regulation Between Alcoholic Fermentation and Glyceropyruvic Fermentation: Glycerol Accumulation
Wines contain about 8 g of glycerol per 100 g of ethanol. During grape must fermentation, about 8% of the sugar molecules undergo glyceropyruvic fermentation and 92% undergo alcoholic fermentation. The fermentation of the first 100 g of sugar forms the majority of the glycerol, after which glycerol production slows but is never nil. Glyceropyruvic fermentation is therefore more than just an inductive fermentation that regenerates NAD+ when acetaldehyde, normally reduced into ethanol, is not yet present. Alcoholic fermentation and glyceropyruvic fermentation overlap slightly throughout the fermentation process.
Pyruvic acid is derived from glycolysis. When this molecule is not used by alcoholic fermentation, it participates in the formation of secondary products. In this case, a molecule of glycerol is formed by the reduction of dihydroxyacetone.
Glycerol production therefore equilibrates the yeast endocellular oxidation–reduction potential or NAD+/NADH balance. This “relief valve” eliminates surplus NADH, which appears at the end of the synthesis of amino acids, proteins, and the oxidations that generate secondary products.
Some winemakers place too much importance on the sensory role of glycerol. This compound has a sugary taste similar to glucose. In the presence of other constituents of wine, however, the sweetness of glycerol is practically imperceptible. For the majority of tasters, even well trained, the addition of 3–6 g of glycerol per liter to a red wine is not discernible. Therefore, the pursuit of winemaking conditions that are more conducive to glyceropyruvic fermentation has no enological interest. On the contrary, the winemaker should favor a pure alcoholic fermentation and should strive to minimize glyceropyruvic fermentation. The production of glycerol is accompanied by the formation of other secondary products, derived from pyruvic acid, whose increased presence (such as carbonyl function compounds and acetic acid) decreases wine quality.
2.3.3 Secondary Products Formed from Pyruvate by Glyceropyruvic Fermentation
When a molecule of glycerol is formed, a molecule of pyruvate is also formed. The latter cannot be transformed into ethanol following its decarboxylation into acetaldehyde. Under anaerobic conditions, oxaloacetate is the means of entry of pyruvate into the cytosolic citric acid cycle. Although the mitochondria are no longer functional, the enzymes of the citric acid cycle are present in the cytoplasm. Pyruvate carboxylase (PC) catalyzes the carboxylation of pyruvate into oxaloacetate. The prosthetic group of this enzyme is biotin; it serves as a CO2 transporter. The reaction makes use of an ATP molecule:
Under these anaerobic conditions, the citric acid cycle cannot be completed since the succinate dehydrogenase activity requires the presence of FAD, a strictly respiratory coenzyme. The chain of reactions is therefore interrupted at succinate, which accumulates (Figure 2.7) up to levels of 0.5–1.5 g/l. The NADH generated by this portion of the citric acid cycle (from oxaloacetate to succinate) is reoxidized by the formation of glycerol from dihydroxyacetone.
Under anaerobic conditions, α‐ketoglutarate dehydrogenase has a very low activity; some authors therefore believe that the oxidative reactions of the citric acid cycle are interrupted at α‐ketoglutarate. In their opinion, a reductive pathway of the citric acid cycle forms succinic acid under anaerobic conditions:
Bacteria have a similar mechanism. Camarasa et al. (2003) demonstrated that this is the main pathway found in S. cerevisiae yeast under anaerobic conditions. Furthermore, additional succinate is formed during alcoholic fermentation in a glutamate‐enriched medium. Glutamate is deaminated to form α‐ketoglutarate, which is oxidized into succinate.
Among secondary products, compounds with a ketone function (pyruvic acid and α‐ketoglutaric acid) and acetaldehyde predominantly bind with sulfur dioxide in wines made from healthy grapes. Their excretion is significant during the yeast proliferation phase and decreases toward the end of fermentation. Additional acetaldehyde is released in the presence of excessive quantities of sulfur dioxide in must. A high pH and high fermentation temperature, anaerobic conditions, and a deficiency in thiamine and pantothenic acid increase the production of ketoacids. Adding thiamine to must limits the accumulation of ketone compounds in wine (Figure 2.10).
FIGURE 2.10 Effect of thiamine addition on pyruvic acid production during alcoholic fermentation (Lafon‐Lafourcade, 1983). I, control must; II, thiamine‐supplemented must.
Other secondary products of fermentation are also derived from pyruvic acid: acetic acid, lactic