Food Biochemistry and Food Processing (2 edition)

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33 Biochemistry of Beer Fermentation 637

and elevated wort aeration or wort lipid concentration (Ver-

strepen et al. 2003b). Enhancing ester production is slightly more

complicated.

If it is possible, overpressure or wort aeration can be reduced.

Otherwise, worts rich in glucose and nitrogen combined with

higher fermentation temperatures and lower pitching rates or

application of the drauflassen technique may prove helpful.

Biosynthesis of Organic Acids

Over hundred different organic acids have been reported in

beer (Meilgaard 1975b). Important organic acids detected in

beer include pyruvate, acetate, lactate, succinate, pyroglutamate,

malate, citrate,α-ketoglutarate, andα-hydroxyglutarate; and the

medium-chain length fatty acids, caproic (C6), caprylic (C8),

and capric (C10) acid (Coote and Kirsop 1974, Meilgaard 1975b,

Klopper et al. 1986). They influence flavor directly when present

above their taste threshold 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,

which are excreted by yeast cells, are synthesized via amino

acid biosynthesis pathways and carbohydrate metabolism. Es-

pecially, they are overflow products of the incomplete Krebs

cycle during beer fermentation. Excretion of organic acids is

influenced by yeast strain and fermentation vigor. Sluggish fer-

mentations lead to lower levels of excretion. Pyruvate excretion

follows the yeast growth: maximal concentration is reached just

before the maximal yeast growth and is next taken up by the

yeast and converted to acetate. Acetate is synthesized quickly

during early fermentation and is later partially reused by the

yeast during yeast growth. At the end of the fermentation, ac-

etate is accumulated. The reduction of pyruvate results in the

production ofd-lactate ofl-lactate (most yeast strains produce

preferentiallyd-lactate). The highest amount of lactate is pro-

duced during the most active fermentation period.

The change in organic acid productivity by disruption 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) disrup-

tant, 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 productivi-

ties were isolated by gene disruption (Arikawa et al. 1999). Sake

fermented using the aconitase gene (ACO1) disruptant contained

a twofold higher concentration of malate and a twofold 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 theα-ketoglutarate dehydrogenase gene (KGD1)and

fumarase gene (FUM1) disruptants gave lower succinate con-

centrations. InS. cerevisiae, there are two isoenzymes of fu-

marate reductase (FRDS1 and FDRS2), encoded by theFRDS

andOSM1genes, respectively (Arikawa et al. 1998). Recent

results suggest that these isoenzymes are required for the reox-

idation of intracellular NADH under anaerobic conditions, but

not under aerobic conditions (Enomoto et al. 2002).

Succinate dehydrogenase is an enzyme of the TCA cycle

and thus essential for respiration. InS. cerevisiae, this enzyme

is composed of four nonidentical subunits, that is, the flavo-

protein, the iron–sulfur protein, the cytochromeb 560 , and the

ubiquinone reduction protein encoded by theSDH1,SDH2,

SDH3,andSDH4genes, respectively (Lombardo et al. 1990,

Chapman et al. 1992, Bullis and Lamire 1994, Daignan-Fournier

et al. 1994). Sdh1p and Sdh2p comprise the catalytic domain in-

volved 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 (com-

plex II) of the electron transport chain. Single or double disrup-

tants of theSDH1,SDH1b(which is a homologue of theSDH1

gene),SDH2,SDH3,andSDH4genes have been constructed and

shown that the succinate dehydrogenase activity was retained in

theSDH2disruptant and that double disruption ofSDH1and

SDH2orSDH1bgenes is necessary to cause deficiency of suc-

cinate dehydrogenase activity in sake yeast (Kubo et al. 2000).

The role of each subunit in succinate dehydrogenase activity and

the effect of succinate dehydrogenase on succinate production

using strains that were deficient in succinate dehydrogenase,

have also been determined. The results suggested that succinate

dehydrogenase activity contributes to succinate production un-

der shaking conditions, but not under static and sake brewing

conditions.

The medium-chain fatty acids account for 85–90% of the

fatty acids in beer and impart an undesirable goaty, sweaty, and

yeasty flavor (Chen 1980). These fatty acids are produced de

novo by yeast during anaerobic fermentation and are not the

result ofβ-oxydation of wort or yeast long-chain fatty acids. As

a result, any change in fermentation conditions that promote the

extent of yeast growth also favor increased levels of medium-

chain fatty acids in beer. Higher temperature, increased wort

oxygenation, and possibly elevated pitching rates are all effective

in this respect (Boulton and Quain 2006). The presence of these

medium-chain fatty acids in beer is also related to yeast autolysis

(Masschelein 1981). Yeast autolytic off-flavors are stimulated

at high temperatures, high yeast concentrations, and prolonged

contact times at the end of primary fermentation and during

secondary fermentation.

Biosynthesis of Vicinal Diketones

Vicinal diketones are ketones with two adjacent carbonyl groups.

During fermentations, these flavor-active compounds are pro-

duced as by-products of the synthesis pathway of isoleucine,

leucine, and valine (ILV pathway) (see Fig. 33.3) and 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 com-

pounds are important in beer, that is, diacetyl (2,3-butanedione)

and 2,3-pentanedione. Diacetyl is quantitatively more impor-

tant than 2,3-pentanedione. It has a taste threshold in lager

beer from 17μg/L (Saison et al. 2009) to 150μg/L (Meilgaard