29 Biochemistry and Fermentation of Beer 671the synthesis pathway of isoleucine, leucine, and va-
line (ILV pathway) (see Fig. 29.3) and are thus also
linked to amino acid metabolism (Nakatani et al.
1984) and the synthesis of higher alcohols. They
impart a “buttery,” “butterscotch” aroma to alcoholic
drinks. Two of these compounds are important in
beer: diacetyl (2,3-butanedione) and 2,3-pentane-
dione. Diacetyl is quantitatively more important than
2,3-pentanedione. It has a taste threshold around
0.10–0.15 mg/L in lager beer, approximately 10
times lower than that of pentanedione (Wainwright
1973).
The excreted -acetohydroxy acids are overflow
products of the ILV pathway that are nonenzymati-
cally degraded to the corresponding vicinal dike-
tones (Inoue et al. 1968). Tetraploid gene dosage
series for various ILVgenes have been constructed,
and the obtained yeast strains were used to study the
influence of the copy number of ILVgenes on the
production of vicinal diketones (Debourg et al.
1990, Debourg 2002). It was shown that the ILV5
activity is the rate-limiting step in the ILV pathway
and is responsible for the overflow (Fig. 29.3). The
nonenzymatic oxidative decarboxylation step is the
rate-limiting step in the conversion of -acetolactate
to 2,3-butanediol and proceeds faster at a higher
temperature and a lower pH (Inoue and Yamamoto
1970, Haukeli and Lie 1978). The produced amount
of -acetolactate is very dependent on the yeast
strain used. The production increases with increas-
ing yeast growth. For a classical fermentation, 0.6
ppm -acetolactate is formed (Delvaux 1998). At
high aeration, this value can be increased to 0.9
ppm, and even to 1.2–1.5 ppm in cylindroconic fer-
mentations tanks.
Yeast cells posses the necessary enzymes (reduc-
tases) to reduce diacetyl to acetoin and further
to 2,3-butanediol, and 2,3-pentanedione to 2,3-
pentanediol. These reduced compounds have much
higher taste thresholds than the diacetyl diketones
and have no impact on the beer flavor (Van Den
Berg et al. 1983). The reduction reactions are yeast-
strain dependent. The reduction occurs at the end of
the main fermentation and during the maturation.
Sufficient yeast cells in suspension are necessary to
obtain an efficient reduction. Yeast strains that floc-
culate early during the main fermentation need a
long maturation time to reduce the vicinal diketones.
Diacetyl can be complexed using SO 2. These com-
plexes cannot be reduced, but diacetyl can again be
liberated at a later stage by aldehydes. This situation
is especially applicable to yeast strains that produce
a lot of SO 2. Worts, which are produced using many
adjuncts, can be low in free amino acid content.
These worts can give rise to a high diacetyl peak at
the end of the fermentation.Figure 29.3.The synthesis and reduction of vicinal diketones in S. cerevisiae.