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33 Biochemistry of Beer Fermentation 633shown that ergosterol and unsaturated fatty acids increase both
in concentration as long as oxygen is present in the wort (e.g.,
Haukeli and Lie 1979). A maximum concentration is obtained in
5–6 hours after pitching, but the formation rate is dependent upon
the pitching rate and the temperature. Unsaturated fatty acids can
also be taken up from the wort, but all malt wort does not contain
sufficient unsaturated lipids to support a normal growth rate of
yeast. Adding lipids to the wort, especially unsaturated fatty
acids might be an interesting alternative (Moonjai et al. 2000a,
2000b).
The oxygen required for lipid biosynthesis can also be in-
troduced by oxygenation of the separated yeast cells. The use
of the preoxygenation technique resulted in more controllable
and consistent fermentations, and as a consequence, in a more
balanced beer flavor profile (Jakobsen 1982,Ohno and Taka-
hashi 1986a, 1986b, Boulton et al. 1991, Devuyst et al. 1991,
Masschelein et al. 1995, Depraetere et al. 2003, Depraetere
2007). Recently, the impact of yeast preoxygenation on yeast
metabolism has been assessed (Verbelen et al. 2009). Therefore,
expression analysis was performed of genes that are of impor-
tance in oxygen-dependent pathways, oxidative stress response
and general stress response during 8 hours of preoxygenation.
The gene expressions of both the important transcription fac-
tors Hap1 and Rox1, involved in oxygen sensing, were mainly
increased in the first 3 hours, whileYAP1expression, which is
involved in the oxidative stress response, increased drastically
only in the first 45 minutes. The results also show that stress-
responsive genes (HSP12,SSA3,PAU5,SOD1,SOD2,CTA1,
andCTT1) were induced during the process, together with the
accumulation of trehalose. The accumulation of ergosterol and
unsaturated fatty acids was accompanied by the expression of
ERG1,ERG11, andOLE1. Genes involved in respiration (QCR9,
COX15,CYC1,andCYC7) also increased during preoxygena-
tion. Yeast viability did not decrease during the process, and
the fermentation performance of the yeast reached a maximum
after 5 hours of preoxygenation. These results suggest that yeast
cells acquire a stress response along the preoxygenation period,
which makes them more resistant against the stressful conditions
of the preoxygenation process and the subsequent fermentation.
Different devices are used to aerate the cold wort: ceramic or
sintered metal candles, aeration plants employing venturi pipes,
two component jets, static mixers, or centrifugal mixers (Kunze
1999). The principle of these devices is that very small air (oxy-
gen) bubbles are produced and quickly dissolve during turbulent
mixing.
As a result of this aeration step, carbohydrates are degraded
aerobically during the first few hours of the “fermentation”
process. The aerobic carbohydrate catabolism takes typically
12 hours for a lager fermentation.
During the first hours of the fermentation process, oxidative
degradation of carbohydrates occurs through the glycolysis and
Krebs (TCA) cycle. The energy efficiency of glucose oxidation
is derived from the large number of NADH 2 +produced for each
mole of glucose oxidized to CO 2. The actual wort fermentation
gives alcohol and carbon dioxide via the Embden-Meyerhof-
Parnas (glycolytic) pathway. The reductive pathway from pyru-
vate to ethanol is important since it regenerates NAD+.Energyis obtained solely from ATP-producing steps of the Embden-
Meyerhof-Parnas pathway. During fermentation, the activity
of the TCA cycle is greatly reduced, although it still serves
as a source of intermediates for biosynthesis (Lievense and
Lim 1982).
Lagunas (1979) observed that during aerobic growth ofS.
cerevisiae, respiration accounts for less than 10% of glucose
catabolism, the remainder being fermented. Increasing sugar
concentrations resulting in a decreased oxidative metabolism is
known as the Crabtree effect. This was traditionally explained as
an inhibition of the oxidative system by high concentrations of
glucose. Nowadays, it is generally accepted that the formation
of ethanol at aerobic conditions is a consequence of a bottleneck
in the oxidation of pyruvate, for example, in the respiratory
system (Petrik et al. 1983, Rieger et al. 1983, Kappeli et al. ̈
1985, Fraleigh et al. 1989, Alexander and Jeffries 1990).
A reduction of ethanol production can be achieved by
metabolic engineering of the carbon flux in yeast resulting in an
increased formation of other fermentation product. A shift of the
carbon flux towards glycerol at the expense of ethanol formation
in yeast was achieved by simply increasing the level of glycerol-
3-phosphate dehydrogenase (Michnick et al. 1997, Nevoigt and
Stahl 1997, Remize et al. 1999, Dequin 2001). TheGDP1gene,
which encodes glycerol-3-phosphate dehydrogenase, has been
overexpressed in an industrial lager brewing yeast to reduce
the ethanol content in beer (Nevoigt et al. 2002). The amount
of glycerol produced by theGDP1-overexpressing yeast in fer-
mentation experiments—simulating brewing conditions—was
increased 5.6 times and ethanol was decreased by 18% com-
pared to the wild-type strain. Overexpression did not affect the
consumption of wort sugars and only minor changes in the
concentration of higher alcohols, esters and fatty acids could
be observed. However, the concentrations of several other by-
products, particularly acetoin, diacetyl, and acetaldehyde, were
considerably increased.
Mutants ofS. cerevisiaestrains deficient in tricarboxylic acid
cycle genes have been reported as suitable strains for the produc-
tion of nonalcoholic beer (Navratil et al. 2002). Strains deficient
in fumarase andα-ketoglutarate dehydrogenase made nonal-
coholic beers with an alcohol content lower than 0.5% (v/v)
(Selecky et al. 2008). The low ethanol content was compensated
by the considerable increase of organic acids (citrate, succinate,
fumarate, and malate). Some of the mutants released high levels
of lactic acid, which protects beers against contamination and
masks an unacceptable worty off-flavor.METABOLISM OF BIOFLAVORING
BY-PRODUCTSYeast is an important contributor to flavor development in fer-
mented beverages. The compounds, which are produced dur-
ing fermentation, are many and varied, depending on both the
raw materials and the microorganisms used. The interrelation
between yeast metabolism and the production of bioflavoring
by-products is illustrated in Figure 33.2. The important flavor
compounds produced by yeast can be classified into five cat-
egories: alcohols, esters, organic acids, carbonyl compounds