Vdk At Peak Fermentation Is

a-Acetohydfoxybutyrate —^—► 2,3-Pentanedione NADH—

Acetoin 2,3-Dihydroxy-isovalerate

NAD*^-2,3-Dihydroxy-3-methylvalerate

2,3-Butanediol

2,3-Pentanediol a-Keto-isovalerate a-keto-3-methylvalerate

Fig. 3.17 Pathways leading to the formation and dissimilation of the vicinal diketones, diacetyl and 2,3-pentanedione.

An alternative synthetic route has been suggested (Chuang & Collins, 1968, 1972). As a result of studies with radiolabeled substrates these authors concluded that in S. cerevisiae, acetoin and diacetyl arose from condensation reactions between hydro-xyethylamine pyrophosphate and acetaldehyde and hydroxyethylamine pyrophosphate and acetyl-CoA, respectively. Although the evidence supported the existence of this pathway the majority of work performed by others suggests that it is likely to be of minor importance and that most diacetyl arises via the route depicted in Fig. 3.17. Thus, all the enzymes in the pathway have been identified and characterised. Mutant strains lacking acetohydroxy acid synthase, and which therefore cannot generate acetolactate, do not form diacetyl. Furthermore, supplementation of non-growth media with valine or a-ketoisovaletate does not increase diacetyl formation, indicating that the reverse pathway is apparently not operative (Wainwright, 1973).

The biochemistry of VDK and the influence of fermentation conditions can be considered in three phases: steps leading to the formation of a-acetohydroxy acids, factors affecting the spontaneous decarboxylation of a-acetohydroxy acids and the subsequent reduction of VDK. As discussed later in this section, the evidence suggests that under most circumstances the rate-determining step for VDK formation is the non-enzymic decarboxylation of a-acetohydroxy acids. Furthermore, the subsequent yeast catalysed reduction of VDK is usually very rapid. Therefore, analysis of fermenting worts is usually referred to as 'total VDK' and represents the sum of a-acetohydroxy acids and diacetyl. Practically, this requires subjecting samples to a heat treatment, which ensures that all a-acetohydroxy acids are converted to VDK prior to analysis. The relative proportions of each of these formed in a laboratory high-gravity lager fermentation are shown in Fig. 3.18. In this experiment samples were removed and total VDK determined using a standard heat treatment and analysis by gas chromatography. Simultaneously samples were removed under conditions that minimised decomposition of a-acetolactate (high pH and anaerobiosis and no heat treatment). Analysis of these samples provided a value for free diacetyl and by difference, a-acetolactate. It may be seen that the bulk of the total VDK was actually precursor. In fact, it was likely that the measured free diacetyl concentration was over-estimated since some decomposition of a-acetolactate during handling was inevitable.

Fig. 3.18 Proportions of diacetyl and a-acetolactate formed during the course of a 2-litre stirred high-gravity (1060) wort fermentation (Box & Boulton, unpublished data).

The formation of a-acetohydroxy acids is intimately related to amino acid metabolism. In this respect the total wort amino acid content and amino acid spectrum are influential, as are parameters which regulate yeast growth and by inference effect amino acid utilisation. Nakatani et al. (1984a) presented data from a large number of fermentations performed under a variety of conditions. They concluded that at high levels of wort free amino nitrogen (FAN) the VDK profile took the form of an extended peak, which appeared early in fermentation. At low wort FAN levels, the profile was transformed into two peaks. The first was small and coincided with the appearance of the broad single peak seen in the high FAN condition, the second peak was sharper and larger. It was demonstrated that a correlation existed between the maximum achieved total diacetyl concentration (T-VDKmax) and the minimum value of wort free amino nitrogen (FANmin). The hyperbolic relationship was described by the following equation:

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