Fig. 3.12 Patterns of formation of important groups of flavour active yeast metabolites during the course of a high-gravity (1060) lager fermentation. The fermentation conditions were as described in Fig. 3.9. Apart from present gravity, scales are arbitrary. (PG, present gravity; esters, the sum of ethyl acetate, and isoamyl acetate; higher alcohols; the sum of propanol, butanol, 3-methylbutanol and 2-methylbutanol; FAN, free amino nitrogen; VDK, free diacetyl plus a-acetolactate).
with uptake of other wort nutrients, many of which also potentially join the sugar catabolising pathways at the level of pyruvate. Since yeast growth extent during fermentation is relatively modest and there is no opportunity for complete oxidation of sugars, it is perhaps not surprising that many of the partial oxidation products are excreted from yeast cells, to appear as potential flavour-active beer components.
Meilgaard (1975) reported the presence of some 110 organic or short-chain fatty acids in beer. Many of these are derived from wort; however, the concentrations of some are modulated during the course of fermentation indicating their participation in yeast metabolism. Organic acids in general have sour flavours and they contribute to the lowering of pH that occurs during fermentation. In addition to sourness, individual organic acids are reported to have characteristic flavours. For example, succinate has a salty/bitter taste (Whiting, 1976). Short-chain fatty acids have a negative impact on beer flavour. For example, caproic and caprylic acids have unpleasant 'goat-like' aromas.
Organic acids include pyruvate (100-200 ppm), citrate (100-150 ppm), malate (3050 ppm), acetate (10-50 ppm), succinate (50-150 ppm) lactate (50-300 ppm) and 2-oxoglutarate (0-60 ppm) (Coote & Kirsop, 1974; Whiting, 1976; Klopper et al., 1986). The comparatively wide ranges of organic acids arising in various beers are in part due to variability in wort composition but are mainly a consequence of yeast strain-specific differences. The majority of organic acids are derived directly from pyruvate or from the branched tricarboxylic acid cycle which is characteristic of the repressed, anaerobic physiology of brewing yeast during fermentation (Wales et al., 1980). Their excretion into beer can be explained by the lack of any mechanism for further oxidation, the need to maintain a neutral intracellular pH and the fact that they are not required for anabolic reactions. There is some evidence that levels of some organic acids fluctuate during the course of fermentation. For example, it has been reported that the formation of extracellular pyruvate, which occurs during active fermentation, may be re-assimilated and replaced by acetate (Coote & Kirsop, 1973, 1974 and see Fig. 3.10). Presumably this reflects changes in carbon flux through glycolysis during the course of fermentation. The contribution that they make to lowering the pH during fermentation could confer a selective advantage in mixed microbial populations.
Two oxo-acids, a-acetolactate and a-acetohydroxy acids, which are excreted by yeast during brewing fermentation, are of particular importance to beer flavour. These are the precursors of diacetyl and 2,3-pentanedione, respectively. The significance of these is discussed in Section 3.7.4.
On the whole fatty acids are undesirable components of beers both from the point of view of taste and their potential for adversely affecting foam performance. Chen (1980) reported that nearly 90% of the free fatty acids in worts were accounted for by palmitic (16:0), linoleic (18:2), stearic (18:0) and oleic (18:1). In beers 75-80% of fatty acids were caprylic (8:0), caproic (6:0) and capric (10:0). In three beers that results were given for there was a net increase in the total fatty acid contents of worts and beers of between 13 % and 6 5 % (5-7 mg 1 1 in wort increasing to 6-12 mg 1 1 in beers). The author suggested that these results indicated that long-chain fatty acids were assimilated by growing yeast and incorporated into structural lipids. The shorter-chain saturated fatty acids in beers were released as by-products of de novo lipid synthesis.
This result would infer that any change in fermentation conditions, which promoted the extent of yeast growth, would also favour increased levels of short-chain fatty acids found in beer. Thus, higher temperature, increased wort oxygenation and possibly elevated pitching rates would all be expected to be effective in this respect. However, caution must be exercised in the interpretation of such results. Short-chain fatty acids, particularly C8-Ci4, are toxic to yeast cells (Nordstrom, 1964). This is probably due to non-specific detergent effects disrupting cell membranes, and therefore it is unlikely that they would be excreted into beers. Possibly free fatty acids could be excreted if there was a restricted supply of coenzyme A. However, it is more plausible that short-chain fatty acids appear via an autolytic mechanism or through ethanol induced membrane leakage. Such a possibility has been proposed for the formation of 'yeasty' or stale off-flavours developing during beer maturation (Masschelein, 1986).
More than 40 higher alcohols have been identified in beers (Engan, 1981). Those that have organoleptic importance because they occur at concentrations above flavour thresholds are: n-propanol, iso-butanol, 2-methylbutanol and 3-methylbutanol. Their contribution to beer flavour is by a general intensification of alcoholic taste and aroma and by imparting a warming character. In addition, the aromatic alcohol 2-phenylethanol, which has a rose/floral aroma, is considered a desirable character (Meilgaard, 1974). Apart from being of significance to beer flavour and aroma in their own right the higher alcohols have the important secondary role of providing precursors for ester synthesis. Typical concentrations of higher alcohols in beers are of the order of 100-200 mg 1 1. Usually, 3-methylbutanol and 2-methylbutanol are the most abundant. In addition to these, glycerol may be found in beers at concentrations of the order of 1-2 gl 1 (Quain & Duffield, 1985).
Higher alcohols may be synthesised by two metabolic routes. First, de novo synthesis from wort carbohydrates via pyruvate, the anabolic route, and, second, as by-products of amino acid assimilation, the catabolic or Ehrlich pathway (Ayrapaa, 1965, 1967a, b, 1968). In both cases, the immediate precursors are 2-oxo (a-keto) acids. These are decarboxylated to form an aldehyde which is then reduced to the corresponding alcohol. In the anabolic route the 2-oxo acid derives from pyruvate or acetyl-CoA as part of amino acid biosynthetic pathways. In the catabolic route, the 2-oxo acid is formed by transamination of an amino acid (Fig. 3.13). Biosynthesis of higher alcohols important to beer flavour and their relation to amino acid metabolism is shown in Fig. 3.14.
II a-Ketoacid from transamination
R - C - COOH or from de novo synthesis a-Ketoacid decarboxylase
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