BLBS102-c33 BLBS102-Simpson March 21, 2012 14:5 Trim: 276mm X 219mm Printer Name: Yet to Come
33 Biochemistry of Beer Fermentation 639overproduction of hydrogen sulfide, depending upon the
proportions of threonine and methionine carried forward
from primary fermentation.- Heating up the green beer to a high temperature (90◦C)
and hold it there for a short period (ca. 7–10 min) to de-
carboxylate all excretedα-acetohydroxy acids. To avoid
cell autolysis, yeast cells are removed by centrifugation
prior to heating up. The vicinal diketones can be further re-
duced by immobilized yeast cells in a few hours (typically
at 4◦C) (see further). - Adding the enzymeα-acetolactate decarboxylase(Godt-
fredsen et al. 1984, Rostgaard-Jensen et al. 1987): This
enzyme decarboxylatesα-acetolactate directly into ace-
toin (see Fig. 33.3). It is not present inS. cerevisiae,but
has been isolated from various bacteria such asEnter-
obacter aerogenes,Aerobacter aerogenes,Streptococcos
lactis, Lactobacillus casei, Acetobacter aceti,andAceto-
bacter pasteurianus. It has been shown that the addition
ofα-acetolactate decarboxylase fromL. caseican reduce
the maturation time to 22 hours (Godtfredsen et al. 1983,
1984). An example of a commercial product is Maturex L
from Novo Nordisk (Denmark) (Jensen 1993). Maturex L
is a purifiedα-acetolactate decarboxylase produced by a
genetically modified strain ofBacillus subtilis, which has
received the gene fromBacillus brevis. The recommended
dosage is 1a 2 kg per 1000 hL wort, to be added to the`
cold wort at the beginning of fermentation. - Using genetic modified yeast strains:
a. Introducing the bacterialα-acetolactate decarboxylase
gene into yeast chromosomes (Fujii et al. 1990, Suihko
et al. 1990, Blomqvist et al. 1991, Enari et al. 1992,
Linko et al. 1993, Yamano et al. 1994, Tada et al. 1995,
Onnela et al. 1996). Transformants possessed a very
highα-acetolactate decarboxylase activity that reduced
the diacetyl concentration considerably during beer
fermentations.
b. Modifying the biosynthetic flux through the ILV path-
way by partially deactivation ofILV2. Spontaneous
mutants resistant to the herbicide sulfometuron methyl
have been selected. These strains showed a partial in-
activation of theα-acetolactate synthase activity and
some mutants produced 50% less diacetyl compared to
the parental strain (Gjermansen et al. 1988).
c. Increasing the flux ofα-acetolactate acid isomerore-
cuctase activity encoded by theILV5gene (Dillemans
et al. 1987). Sinceα-acetolactate acid isomerore-
cuctase activity is responsible for the rate-limiting
step, increasing its activity reduces the overflow ofα-
acetolactate. A multicopy transformant resulted in a
70% decreased production of vicinal diketones (Villa-
neuba et al. 1990), whereas an integrative transformant
gave a 50% reduction (Goossens et al. 1993). A tan-
dem integration of multipleILV5copies resulted also
in elevated transciption in a polyploidy industrial yeast
strain (Mithieux and Weiss 1995). Vicinal diketones
production could be reduced by targeting the mito-
chondrial Ilv5p to the cytosol (Omura 2008).
SECONDARY FERMENTATION
During the secondary fermentation or maturation of beer, several
objectives should be realized:
Sedimentation of yeast cells
Improvement of the colloidal stability by sedimentation of
the tannin–protein complexes
Beer saturation with carbon dioxide
Removal of unwanted aroma compounds
Excretion of flavor-active compounds from yeast to give
body and depth to the beer
Fermentation of the remaining extract
Improvement of the foam stability of the beer
Adjustment of the beer color (if necessary) by adding color-
ing substances (e.g., caramel)
Adjustment of the bitterness of beer (if necessary) by
adding hop productsIn the presence of yeast, the principal changes that occur are
the elimination of undesirable flavor compounds, such as vicinal
diketones, hydrogen sulfide, and acetaldehyde, and the excretion
of compounds enhancing the flavor fullness (body) of beer.Vicinal DiketonesIn traditional fermentation lagering processes, the elimination
of vicinal diketones required several weeks and determined the
length of the maturation process. Nowadays, the maturation
phase is much shorter since strategies are used to accelerate
the vicinal diketones removal (see the preceding text). Diacetyl
is used as a marker molecule. The objective during lagering is
to reduce the diacetyl concentration below its taste threshold
(<0.10 mg/mL).Hydrogen SulfideSulfite and hydrogen sulfide are intermediates in the biosynthe-
sis of the sulfur-containing amino acids methionine and cysteine
(Van Haecht and Dufour 1995, Duan et al. 2004). Hydrogen sul-
fide plays an important role during maturation. Inorganic sulfate
is taken up by the yeast cells via a permease. Subsequently,
it is reduced to sulfide via the intermediates adenylyl sulfate,
phosphoadenylyl sulfate and sulfite (see Fig. 33.4). H 2 S and
SO 2 , which are not incorporated in S-containing amino acids,
are excreted by the yeast cell during the growth phase (Ryder
and Masschelin 1983, Thomas and Surdin-Kerjan 1997). The
excreted amount depends on the used yeast strain, the sulfate
content of the wort and the growth conditions (Romano and
Suzzi 1992). Methionine causes decreased production of sulfur
compounds by feedback inhibition, while threonine increases
the production (Thomas and Surdin-Kerjan 1997). H 2 S and SO 2
can also be formed from the catabolism of S-containing amino
acids (Dual et al. 2004). The production of H 2 S and SO 2 de-
pends on the yeast strain, sulfate content in the wort, and growth
conditions. The production of H 2 S could be reduced by the ex-
pression of cystathione synthase genes fromS. cerevisiaein a
brewing yeast strain (Tezuka et al. 1992).