Fig. 3.14 Biosynthetic routes for synthesis of some higher alcohols important to beer flavour and aroma.
Although the proportion of wort carbon which appears in beers as higher alcohols is modest compared to ethanol it is still ostensibly profligate. Since yeasts, in common with all cells, are never wasteful of metabolic resources it suggests that there must be a reason for higher alcohol formation. Quain and Duffield (1985) argued that the appearance of these metabolites could be a manifestation of cellular redox control. Under conditions of brewery fermentation where respiratory function is precluded the cell requires a means of re-oxidising NADH, which is generated by glycolysis, and reactions which utilise pyruvate other than ethanol formation. With regard to the latter, it is usually assumed that pyruvate dehydrogenase is the significant route for NAD + formation. In fact, it is more likely that acetaldehyde dehydrogenase channels carbon flow from pyruvate to acetyl-CoA. The cytosolic form of this enzyme is apparently NADP linked (Dickinson, 1996).
Whichever routes predominate, it remains true that yeast must maintain an appropriate redox balance. It is generally considered that the redox-balancing role is performed by glycerol formation (Fig. 3.3). Quain and Duffield (1985) suggested that higher alcohol formation could represent a further 'fine tuning' mechanism for regenerating NAD +. Evidence for this premise was provided by the observation that washed yeast cells suspended in buffer and provided with a supply of energy but no nitrogen source generated 3-methylbutanol when supplemented with the precursor, isovaleraldehyde. This phenomenon was associated with suppression of glycerol formation apparently indicating that formation of the higher alcohol relieved the requirement for the redox-balancing role of the former metabolite. In further experiments, addition of acetoin, a product of diacetyl metabolism which also arises via an NADH-dependent dehydrogenase (Section 3.7.4), suppressed both glycerol and higher alcohol formation.
Glycerol contributes to the body and mouth-feel of beers. Typical concentrations are 1-2 gl 1. Although it may have a role in redox control (Oura, 1977), it is also reported to function as an osmoprotectant (Edgely & Brown, 1983; Blomberg & Adler, 1989; Mager & Varela, 1993). Thus, elevated glycerol concentrations may be induced by cultivating yeast in a medium with low water activity. The phenomenon may be distinguished from the effects due to increased availability of nutrients in that it still occurs if the water activity is reduced by addition of non-metabolisable solutes. The generalised stress response of yeast has been discussed previously (Section 184.108.40.206). High osmotic pressures constitute such a stress and indeed subjecting yeast to heat shock has been shown to enhance glycerol production (Omori et al., 1996, 1997). This also suggests that glycerol production is not solely implicated in redox control and adds further weight to the premise that higher alcohol formation may provide supplementary redox control mechanisms.
Control of higher alcohol formation during fermentation can be accomplished in three ways. First, by choice of an appropriate yeast strain, second, by modification to wort composition, and, third, by manipulation of fermentation conditions. Several authors have concluded that the yeast strain is the most significant factor. For example, Szlavko (1974), Engan (1978) and Romano et al. (1992) all observed variability in higher alcohol concentrations between individual strains cultivated under identical conditions greater than that seen with the same strain cultivated under a variety of different conditions. It has been reported that ale strains produce greater concentrations of higher alcohols compared to lager strains (Hudson & Stevens, 1960). Manipulation of the concentrations of individual higher alcohols is possible via genetic modification of yeasts. For example, Rous and Snow (1983) noted that mutants of Montrachet wine yeasts which were auxotrophic for specific branched chain amino acids produced reduced concentrations of the corresponding higher alcohols.
Wort composition influences higher alcohol formation, in particular the amino nitrogen content. This is of great significance in the case of high-gravity brewing where adjuncts may alter the ratio of carbon to nitrogen. As a general rule any change in fermentation conditions which increases the extent of yeast growth produces a concomitant increase in higher alcohol concentration. Thus, excessive wort amino nitrogen tends to elevate higher alcohol concentrations. However, very low amino nitrogen also produces excessive higher alcohols (Szlavko, 1974). In this instance it may be surmised that lack of nitrogen will promote enhanced synthesis of amino acids and that this will stimulate the anabolic higher alcohol synthetic route.
High wort oxygen concentration also favours increased yeast growth and this also enhances higher alcohol production. Increase in temperature has a similar effect (Barker et al., 1992). The effects of temperature are puzzling. This parameter influences yeast growth rate but not growth extent, and therefore it should not affect yields of metabolites which are growth-related. A similar effect is observed with respect to esters. In this case it has been suggested that alterations in membrane fluidity and diffusion rates from the cell might be implicated (Peddie, 1990). Control of excessive higher alcohol concentration in beer can be achieved by applying top pressure during fermentation. Thus, Rice et al. (1976) observed an inverse correlation between the concentrations of higher alcohols, other volatiles and yeast growth extent and applied pressure. It was demonstrated that this could be used to produce beers with similar volatile spectra fermented at 15°C with no applied pressure and at 22°C with 8 psig top pressure. The applied pressure had no effect on fermentation rate and therefore the advantage of using the higher temperature was retained.
Esters comprise possibly the most important set of flavour-active beer components which arise as a result of yeast metabolism. In the region of 100 distinct esters have been identified in beers (Drawert & Tressl, 1972; Meilgaard, 1975; Engan, 1981). Esters impart floral/fruity flavours and aromas to beers. Those whose concentrations in beer are considered crucial to product quality include ethyl acetate ('fruity/solvent'), isoamyl acetate ('banana/apple'), isobutyl acetate ('banana/fruity'), ethyl caproate ('apple/aniseed') and 2-phenylethylacetate ('roses/honey/apple'). Concentrations in beers are typically less than 1 ppm for minor components and 10-20ppm for ethyl acetate (Table 3.4). Ethyl esters are the most abundant presumably because ethanol is the most readily available substrate.
Formation of esters by reactions between free fatty acids and alcohols is unlikely since the rates of such esterification would be too slow to account for the kinetics of their appearance during fermentation. Instead esters may potentially arise via two routes. First, from reactions between an alcohol, either ethanol or a higher alcohol
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