672 Part VI: Fermented Foods
There are several strategies that can be chosen to
reduce the vicinal diketones during fermentation:
- Since the temperature has a positive effect on
the reduction efficiency of the -acetohydroxy
acids, a warm rest period at the end of the main
fermentation, and a warm maturation are
applied in many breweries. In this case, tem-
perature should be well controlled to avoid
yeast autolysis. - Since the rapid removal of vicinal diketones
requires yeast cells in an active metabolic con-
dition, the addition of 5–10% Krausen (con-
taining active, growing yeast) is a procedure
that gives enhanced transformation of vicinal
diketones (N.N. 2000). However, this proce-
dure can lead to overproduction of hydrogen
sulphide, depending upon the proportions of
threonine and methionine carried forward from
primary fermentation. - Heating up the green beer, beer obtained after
the primary fermentation, to a high temperature
(90°C) and holding it there for a short period
(ca. 7–10 minutes) to decarboxylate all excret-
ed -acetohydroxy acids. To avoid cell autoly-
sis, yeast cells are removed by centrifugation
prior to heating. The vicinal diketones can be
further reduced by immobilized yeast cells in a
few hours (typically at 4°C) (see further). - Adding the enzyme-acetolactate decarboxy-
lase (Godtfredsen et al. 1984, Rostgaard-Jensen
et al. 1987). This enzyme decarboxylates-
acetolactate directly into acetoin (see Fig. 29.3).
It is not present inS. cerevisiae, but has been
isolated from various bacteria such as
Enterobacter aerogenes,Aerobacter aerogenes,
Streptococcus lactis, Lactobacillus casei,
Acetobacter aceti,andAcetobacter pasteuri-
anus. It has been shown that the addition of-
acetolactate decarboxylase fromLactobacillus
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 purified-acetolactate decarboxylase pro-
duced by a genetically modified strain of
Bacillus subtilisthat has received the gene from
Bacillus brevis. The recommended dosage is
1–2 kg per 1000 hL wort, to be added to the cold
wort at the beginning of fermentation.
5. Using genetic modified yeast strains:
a. Introducing the bacterial -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 high -
acetolactate decarboxylase activity, which
reduced the diacetyl concentration consider-
ably during beer fermentations.
b. Modifying the biosynthetic flux through the
ILV pathway. Spontaneous mutants resistant
to the herbicide sulfometuron methyl have
been selected. These strains showed a par-
tial inactivation of the -acetolactate syn-
thase activity, and some mutants produced
50% less diacetyl compared with the
parental strain (Gjermansen et al. 1988).
c. Increasing the flux of -acetolactate acid
isomeroreductase activity encoded by the
ILV5gene (Dillemans et al. 1987). Since
-acetolactate acid isomeroreductase activi-
ty is responsible for the rate-limiting step,
increasing its activity reduces the overflow
of -acetolactate. A multicopy transformant
resulted in a 70% decreased production of
vicinal diketones (Villaneuba et al.1990),
whereas an integrative transformant gave a
50% reduction (Goossens et al. 1993). A
tandem integration of multiple ILV5copies
also resulted in elevated transciption in a
polyploid industrial yeast strain (Mithieux
and Weiss 1995).
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 coloring substances (e.g., caramel),