670 Part VI: Fermented Foods
vesicles are small organelles in which certain neutral
lipids are metabolized or stored. This indicates that
fruity esters are possibly by-products of these pro-
cesses.
BIOSYNTHESIS OFORGANICACIDS
Important organic acids detected in beer include ace-
tate, lactate, succinate, pyroglutamate, malate, cit-
rate, -ketoglutarate and -hydroxyglutarate (Coote
and Kirsop 1974). They influence flavor directly,
when present above their taste thresholds, and by
their influence on beer pH. These components have
their origin in raw materials (malt, hops) and are
produced during the beer fermentation. Organic acids
that are excreted by yeast cells are synthesized via
amino acid biosynthesis pathways and carbohydrate
metabolism. Especially, they are overflow products
of the incomplete Krebs cycle during beer fermenta-
tion. Excretion of organic acids is influenced by
yeast strain and fermentation vigor. Sluggish fer-
mentations lead to lower levels of excretion. Pyru-
vate excretion follows the yeast growth: maximal
concentration is reached just before the maximal
yeast growth, and the pyruvate is next taken up by
the yeast and converted to acetate. Acetate is synthe-
sized quickly during early fermentation and is later
partially re-used by the yeast during yeast growth.
At the end of the fermentation, acetate is accumulat-
ed. The reduction of pyruvate results in the produc-
tion of D-lactate or L-lactate (most yeast strains pro-
duce preferentially D-lactate). The highest amount
of lactate is produced during the most active fermen-
tation period.
The change in organic acid productivity by dis-
ruption of the gene encoding fumarase (FUM1)has
been investigated, and it has been suggested that
malate and succinate are produced via the oxidative
pathway of the TCA cycle under static and sake
brewing conditions (Magarifuchi et al. 1995). Using
a NAD-dependent isocitrate dehydrogenase gene
(IDH1, IDH2)disruptant, approximately half of the
succinate in sake mash was found to be synthesized
via the oxidative pathway of the TCA cycle in sake
yeast (Asano et al. 1999).
Sake yeast strains possessing various organic acid
productivities were isolated by gene disruption (Ari-
kawa et al. 1999). Sake fermented using the aconi-
tase gene (ACO1)disruptant contained a two-fold
higher concentration of malate and a two-fold lower
concentration of succinate than that made using the
wild-type strain. The fumarate reductase gene
(OSM1)disruptant produced sake containing a 1.5-
fold higher concentration of succinate, whereas the
-ketoglutarate dehydrogenase gene (KGD1) and
fumarase gene (FUM1) disruptants gave lower suc-
cinate concentrations. In S. cerevisiae, there are two
isoenzymes of fumarate reductase (FRDS1 and
FDRS2), encoded by the FRDSand OSM1genes,
respectively (Arikawa et al. 1998). Recent results
suggest that these isoenzymes are required for the
reoxidation of intracellular NADH under anaerobic
conditions, but not under aerobic conditions (Eno-
moto et al. 2002).
Succinate dehydrogenase is an enzyme of the
TCA cycle and is thus essential for respiration. In S.
cerevisiae, this enzyme is composed of four non-
identical subunits, that is, the flavoprotein, the iron-
sulfur protein, the cytochrome b 560 , and the ubi-
quinone reduction protein encoded by the SDH1,
SDH2, SDH3, and SDH4genes, respectively (Lom-
bardo et al. 1990, Chapman et al. 1992, Bullis and
Lamire 1994, Daignan-Fournier et al. 1994). Sdh1p
and Sdh2p comprise the catalytic domain involved
in succinate oxidation. These proteins are anchored
to the inner mitochondrial membrane by Sdh3p and
Sdh4p, which are necessary for electron transfer and
ubiquinone reduction, and constitute the succinate:
ubiquinone oxidoreductase (complex II) of the elec-
tron transport chain. Single or double disruptants of
the SDH1, SDH1b(which is a homologue of the
SDH1gene), SDH2, SDH3,and SDH4genes have
been constructed, and it has been shown that the
succinate dehydrogenase activity was retained in the
SDH2 disruptant and that double disruption of
SDH1and SDH2or SDH1bgenes is necessary to
cause deficiency of succinate dehydrogenase activi-
ty in sake yeast (Kubo et al. 2000). The role of each
subunit in succinate dehydrogenase activity and the
effect of succinate dehydrogenase on succinate pro-
duction, using strains which were deficient in succi-
nate dehydrogenase, have also been determined. The
results suggested that succinate dehydrogenase ac-
tivity contributes to succinate production under
shaking conditions, but not under static and sake
brewing conditions.BIOSYNTHESIS OFVICINALDIKETONESVicinal diketones are ketones with two adjacent car-
bonyl groups. During fermentations, these flavor-
active compounds are produced as by-products of