the role of lager beer yeast in oxidative stability of model beer

ORIGINAL ARTICLE

The role of lager beer yeast in oxidative stability of modelbeerT.S. Berner and N. Arneborg

Department of Food Science, Food Microbiology, University of Copenhagen, Frederiksberg, Denmark

Introduction

During industrial beer fermentation, the brewers yeast

will upregulate genes encoding antioxidants, thus indicat-

ing that it has been subjected to oxidative stress during

fermentation (Higgins et al. 2003; James et al. 2003). Oxi-

dative stress typically leads to the formation of reactive

oxygen species (ROS), which cause damage to the lipids,

proteins and DNA of yeast cells (Halliwell and Aruoma

1991; Temple et al. 2005). The exact cause of this oxida-

tive stress during the fermentation process has, as yet, not

been found, but it seems to be related more to starvation

than to the presence of oxygen during the initial stages of

the fermentation (Gibson et al. 2008). To prevent intra-

cellular oxidative damage, the yeast cell possesses an

oxidative stress response, consisting of both enzymatic

and nonenzymatic defence mechanisms to remove or

detoxify ROS. Enzymatic defence mechanisms include

peroxidases, catalases and superoxide dismutases, and the

nonenzymatic defence mechanisms include antioxidants

such as glutaredoxin, glutathion and thioredoxin (Grant

et al. 1998; Jamieson 1998). The expression of thioredoxin

may be induced by chemicals that cause oxidative stress,

such as H2O2, paraquat and diamide (Delaunay et al.

2000; Garrido and Grant 2002; Braconi et al. 2010). It is

well known that the tolerance of yeast to various other

stress conditions, such as ethanol, is strain dependent

(Kubota et al. 2004). So far, however, the oxidative stress

tolerance of different strains of brewers yeast has not been

studied.

In the past decade, the role of oxidation processes in

the formation of beer off-flavour compounds has received

considerable interest, because these compounds most

likely are oxidation products of components present in

the wort and ⁄ or the beer (Bamforth and Lentini 2009).

The oxidation of flavour compounds in wort and ⁄ or beer

Keywords

iron, lager beer, oxidative stability,

Saccharomyces pastorianus, thioredoxin,

yeast.

Correspondence

Torben S. Berner, Department of Food

Science, Food Microbiology, University of

Copenhagen, Rolighedsvej 30, Frederiksberg

1958, Denmark.

E-mail: [email protected]

2011 ⁄ 1145: received 9 July 2011, revised 16

November 2011 and accepted 7 December

2011

doi:10.1111/j.1472-765X.2011.03195.x

Abstract

Aims: In this study, we investigated the relationship between the ability of lager

brewing yeast strains to tolerate oxidative stress and their ability to produce

oxidative stable model beer.

Methods and Results: Screening of 21 lager brewing yeast strains against dia-

mide and paraquat showed that the oxidative stress resistance was strain

dependent. Fermentation of model wort in European Brewing Convention

tubes using three yeast strains with varying oxidative stress resistances resulted

in three model beers with different rates of radical formation as measured by

electron spin resonance in forced ageing experiments. Interestingly, the strain

with the lowest oxidative stress resistance and lowest secretion of thioredoxin,

as measured by Western blotting, resulted in the highest uptake of iron, as

measured by inductively coupled plasma-mass spectrometry, and the slowest

formation of radicals in the model beers.

Conclusions: A more oxidative stable beer is not obtained by a more-oxida-

tive-stress-tolerant lager brewing yeast strain, exhibiting a higher secretion of

thioredoxin, but rather by a less-oxidative-stress-tolerant strain, exhibiting a

higher iron uptake.

Significance and Impact of the Study: To obtain lager beers with enhanced

oxidative stability, yeast strains should be screened for their low oxidative stress

tolerance and ⁄ or high ability to take up iron rather than for their high oxida-

tive stress tolerance and ⁄ or high ability to secrete thioredoxin.

Letters in Applied Microbiology ISSN 0266-8254

ª 2011 The Authors

Letters in Applied Microbiology 54, 225–232 ª 2011 The Society for Applied Microbiology 225

is initiated by the reaction of oxygen with transition met-

als, such as iron and copper, thus generating the superox-

ide anion (O�2 ), which may be further reduced and

protonated to hydrogen peroxide (H2O2). The superoxide

anion and hydrogen peroxide can then undergo the

Haber–Weiss and Fenton reactions, respectively, with iron

and ⁄ or copper ions to produce the very reactive hydroxyl

radical (OH·) (Kaneda et al. 1988). The generated ROS

species may react with various wort and beer compounds,

resulting in flavour changes in the beer (Vanderhaegen

et al. 2006).

One of the strategies to prevent oxidation in beer is

to capture ROS and free radicals by antioxidants (Van-

derhaegen et al. 2006). Sulphite is the most abundant

antioxidant in beer, produced and secreted by the brew-

ers yeast during fermentation (Kaneda et al. 1994, 1996;

Andersen et al. 2000). Thioredoxin, a cytosol-localized

antioxidant, has been reported to be secreted out of the

cell during sake and beer production (Swan et al. 2003;

Inoue et al. 2007). Furthermore, a proteomic study of

11 lager beers identified thioredoxin as one out of only

four proteins originating from yeast (Iimure et al. 2010).

Thus, besides sulphite, thioredoxin may also be a poten-

tial candidate for protecting the beer against oxidation

of flavour compounds. Moreover, it may be postulated

that more-oxidative-stress-tolerant yeast strains may pro-

duce and secrete more thioredoxin to the beer during

fermentation, thereby resulting in a beer with enhanced

oxidative stability.

In this study, we identify lager beer yeast strains with

different tolerances towards thiol-depleting and superox-

ide-generating oxidative stress. We find that model beers

fermented with the most-oxidative-stress-tolerant strain

contain the highest amount of thioredoxin as compared

with beers fermented with two less-oxidative-stress-toler-

ant strains. However, the oxidative stability of the beer, as

measured by electron spin resonance (ESR), is not gov-

erned by the ability of the yeast to secrete thioredoxin.

Rather, it seems to depend on the ability of the yeast to

take up iron.

Materials and Methods

Yeast strains and media

The yeast strains (n = 21) used in this study were all lager

brewing strains, belonging to the species Saccharomyces

pastorianus, obtained from White Labs (WL, San Diego,

CA, USA) and our own collection (KVL) at the Depart-

ment of Food Science, Food Microbiology, University of

Copenhagen (see Table 1). Yeast strains were grown in

synthetic beer media (SBM), composed of 4% maltose;

1Æ5% sucrose; 1% dextrin; 1% glucose; 0Æ5% fructose;

2Æ94% balanced peptone; and 1Æ7% yeast nitrogen base

w ⁄ o aa adjusted to pH 5Æ5.

Screening for oxidative stress tolerance

Yeast was grown to exponential phase in 10 ml of SBM at

11�C without shaking and diluted to OD600 0Æ1, 0Æ01 and

0Æ001. Cells were spotted on SBM plates containing either

diamide (1Æ5; 1Æ75 or 2 mmol l)1) or paraquat (0Æ5 or

0Æ75 mmol l)1). Plates were incubated at 11�C for 6 days.

For each strain, growth was scored against colony size

without any additives annotated as (+++) no effect of

additive, (++) minor effect – smaller colonies, (+) large

effect – tiny colonies and ()) no growth.

Model beer fermentation

Aerobic propagation of yeast was started from a single

colony in 10 ml of SBM – in duplicates. After incubation

at 20�C for 24 h, the suspensions were transferred to

100 ml SBM in 250-ml Erlenmeyer flasks with a magnetic

stirrer at 200 rev min)1. Yeast suspensions were trans-

ferred after 2 days at 20�C to 400 ml SBM and incubated

Table 1 Screening for pro-oxidant effect on lager yeast*

Yeast stain

Synthetic beer media

Paraquat

(mmol l)1) DIAMIDE (mmol l)1)

0.5 0.75 1.5 1.75 2

KVL001 ++ ++ ++ ++ ++

KVL002 ++ ++ +++ +++ +++

KVL003 + + ++ + )KVL004 ) ) ) ) )KVL005 + + ++ + )KVL006 ++ ++ + + )KVL007 + + + ) )KVL008 ++ + + ) )KVL009 + + + + )KVL010 ++ + ++ + )KVL016 ++ ++ + ) )KVL017 +++ ++ ) ) )KVL018 ) ) ) ) )KVL019 ++ ++ ++ + )KVL020 ++ + + + +

WLP800 ) ) + + )WLP810 + + +++ +++ +++

WLP830 ++ ++ ++ ) )WLP833 +++ ++ +++ ++ +

WLP838 + ) + + )WLP840 +++ +++ ++ ++ +

(+++), no effect of additive; (++), minor effect – smaller colonies; (+),

large effect – smal colonies; ()), no growth.

*Yeast strains were spotted in 10-fold dilutions on SBM. Colony sizes

were determined after 6 days at 11�C.

The role of lager beer yeast in oxidative stability T. Berner and N. Arneborg

226 Letters in Applied Microbiology 54, 225–232 ª 2011 The Society for Applied Microbiology

ª 2011 The Authors

for 24 h at 20�C. Yeast cells were harvested (3000 g,

10 min, 20�C) and pitched at 7 · 106 cells ml)1 in 2 l of

SBM saturated with air. Fermentations were carried out

in 2Æ5-litres European Brewing Convention (EBC) tubes

at 11�C for 12 days. Samples of culture broth were col-

lected aseptically with a syringe from the top of the EBC-

tubes at days 0, 1, 2, 3, 6, 9 and 12. Cell density was

determined by measuring the optical density at 600 nm

(UV-1800; Shimadzu Scientific Instruments, Columbia,

MD), and pH was determined using a pH-meter

(pHM220; Radiometer Analytical SAS, Villewbanne

Cedex, France) before further treatment of samples.

Protein analysis

Immediately after sampling, proteins were precipitated

from a 15-ml sterile filtered sample with trichloroacetic

acid (TCA; finale concentration 12Æ5%) at )20�C O ⁄ N.

Proteins were pelleted (15 000 g, 4�C, 30 min), washed

twice in 1 ⁄ 3 volume ice-cold acetone (15 000 g for

15 min at 4�C) and re-suspended in buffer (400 ll of

10 mmol l)1 MES pH 6Æ0, 1 mmol l)1 EDTA).

2D Quant (80-6483-56; GE Healthcare Bio-Sciences,

Hillerød, Denmark) was used to determine the protein

concentration according to the manufacturer’s protocol,

with BSA as standard. 60 lg of protein was separated on a

20% RunBlue minigel using an Xcell II electrophoresis sys-

tem (Life Technologies Europe BV, Naerum, Denmark).

Western blotting

Separated proteins were blotted on polyvinylidene diflou-

ride membranes. Bound anti-yeast thioredoxin (a kind

gift from Professor Chris M. Grant) was visualized by

alkaline phosphatase after incubation with anti-rabbit

immunoglobulin-alkaline phosphatase conjugate. ImageJ

64 (Rasband, W.S., ImageJ; US National Institutes of

Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/,

1997–2009) was used to quantify band intensity of the

developed blots by estimating the peak intensity. Thiore-

doxin concentrations were determined from a dilution

row of yeast-purified thioredoxin (Genway, San Diego,

CA, USA) on the same blot.

Sulphite analysis

Enzymatic determination of total sulphite was performed

using a Sulphite UV determination kit according to

the manufacturer’s protocol with some modifications

(10725854035; Roche Diagonostics A ⁄ S, Hvidovre, Den-

mark). In brief, reaction size was scaled down 10 times for

measurement in microtitre plates. Samples were sterile fil-

tered through a 0Æ22-lm filter to separate yeast cells from

sample and diluted threefold in modified reaction buffer

(700 mmol l)1 triethanolamine, 0Æ4 g l)1 NADH, pH 8Æ0).

0Æ01 U NADH peroxidase was added, and the start absor-

bance was recorded at 340 nm followed by the addition of

0Æ1225 U sulphite oxidase. The reduction in NADH was

measured at 340 nm, and sodium sulphite was used as stan-

dard. Values were determined as a minimum in triplicates.

Sugar and ethanol determination

Samples were filtrated through a 0Æ22-lm sterile filter and

kept at )20�C until analysis. Sugar and ethanol concen-

trations were determined using a HPLC (HP series 1100;

Hewlett-Packard ApS, Allerød, Denmark) with a Micro-

Guard cation H cartridge followed by an Aminex HPX-

87H column (Bio-Rad Laboratories, Hercules, CA, USA)

connected to a RI detector (HP1047A; Hewlett-Packard).

The column was eluted with a degassed mobile phase

containing 2Æ5 mmol l)1 H2SO4, pH 2Æ75, at 50�C and at

a flow rate of 0Æ6 ml min)1.

ESR experiments

The lag phase experiments were performed by heating

approximately 1 ml of sterile filtered samples containing

30 mmol l)1 of the spin trap phenyl N-tert-butylnitrone

(PBN) in 3Æ6-ml screw-cap tubes in a 60�C water bath. The

first sample (50 ll) was withdrawn after 10 min followed

by intervals of 20 min. ESR spectra of the samples were

recorded with a Miniscope MS 200 X-band spectrometer

(Magnetteech Gmbh, Berlin, Germany) using 50-ll micro-

pipettes as sample cells. The settings used were as follows:

microwave power, 10 mW; sweep width, 60 G; modulation

frequency, 2000 mG; receiver gain, 900; and sweep time

30 s. All spectra consisted of single scans and were recorded

at room temperature. The amplitudes of the spectra were

measured and are reported as the height of the central dou-

blet. The lag phases of the ESR spectra, that is, the antioxi-

dant capacity, were determined according to Uchida and

Ono (2000), and the relative radical formation rates, that

is, the oxidative stability, were determined by linear regres-

sion of the straight line and related to the slowest forma-

tion. All samples were measured in duplicates.

Metal ion determination

To determine the concentrations of iron and copper, sam-

ples were filtered through a 20-lm sterile filter, condensed

and acid digested in 45% HNO3 and 10% H2O2

(Wyrzykowska et al. 2001). Ion analysis of samples were

analysed using inductively coupled plasma-mass spectrom-

etry (ICP-MS) (Agilent 7500c; Agilent Technologies

Denmark ApS, Hørsholm, Denmark). Settings for the

T. Berner and N. Arneborg The role of lager beer yeast in oxidative stability

ª 2011 The Authors


ICP-MS were as described in Hansen et al. (2009). All

samples were measured in duplicates.

Statistical analysis

All results represent the mean values ± standard error of

the mean (SEM) from two independent fermentations

with at least duplicate measurements. Statistical analysis

was performed by one-way analysis of variance (anova)

and Tukey’s post hoc using StatPlus software (Analyst-

Soft, Inc., Vancover, British Columbia, Canada). Proba-

bilities <0Æ05 were considered significant.

Results

Screening of brewers yeast against pro-oxidants

Initially, a collection of 21 lager brewing yeast strains

was screened for their ability to withstand oxidative

stress. Two pro-oxidants, that is, diamide – a thiol-

depleting agent and paraquat – a superoxide-generating

agent, were used to induce oxidative stress by different

pathways (Bus and Gibson 1984; Kosower and Kosower

1995). Yeast was grown on agar plates in aerobic condi-

tions in tenfold dilutions against near lethal concentra-

tions of the selected pro-oxidants. The screening results

show that some yeast strains were clearly more tolerant

to both pro-oxidants than others (Table 1). Moreover,

paraquat seemed to be more reactive than diamide

(Table 1).

Model beer fermentations in EBC-tubes

Based on the screening results on SBM, a highly-oxida-

tive-stress-resistant strain, that is WLP833; and a low-

resistant strain, that is KVL018, were selected for model

beer fermentations. Besides, KVL001 was included as a

medium-resistant strain. Growth and metabolic profiles

00 2 4 6 8 10 12

0 2 4 6 8 10 12

0 2 4 6Days Days

8 10 12 0 2 4 6 8 10 12

0 2 4 6 8 10 12

0

4·8

5·0

5·2

5·4

5·6

4·8

5·0

5·2

5·4

5·6

4·8

5·0

5·2

5·4

5·6

2 4 6 8 10 120

1·0

2·0

3·0

4·0

10

20

30

40

g l–

1

pH

OD

600

0

1·0

2·0

3·0

4·0

OD

600

0

1·0

2·0

3·0

4·0

OD

600

pHpH

0

10

20

30

40

g l–

1

0

10

20

30

40

g l–

1

(a)

(b)

(c)

Figure 1 Fermentation profiles for yeast strains KVL001 (a), KVL018 (b) and WLP833 (c), respectively, grown in 2 l synthetic beer media in Euro-

pean Brewing Convention tubes, showing (s) glucose; (h) fructose; (m) maltose; (4) ethanol; (n) OD and (d) pH. Values are means of two bio-

logically independent fermentations, and error bars indicate standard deviations.



ª 2011 The Authors

for fermentations in 2 l SBM in EBC-tubes using yeast

strains KVL001, KVL018 and WLP833 are shown in

Fig. 1. The three fermentations were all similar and

resembled typical beer fermentation (Fig. 1).

Oxidative stability of model beers

Forced ageing of the fermentation samples followed by

ESR were performed to determine the oxidative stability

of the model beers after 12 days. ESR measures the level

of radicals in samples indirectly by detecting the forma-

tion of radicals trapped by the spin trap, PBN. The for-

mation of stable spin trap radical follows a two-phase

course; first a lag phase is observed and then the rate of

radical formation increases and signals from the spin trap

increase linearly with time (Fig. 2). The lag phase is pro-

posed to be the time where antioxidants quench the radi-

cals before reacting with a spin trap and may thus reflect

the antioxidant capacity (Uchida and Ono 1996, 2000).

The model beers showed similar lag phase (Table 2), indi-

cating that the amount of antioxidants produced by the

different yeast strains were not significantly different.

However, model beers fermented with KVL018 generated

radicals at a low rate, whereas those fermented with

KVL001 and WLP833 produced radicals at 3- and 1Æ8-

fold higher rates, respectively (Table 2).

Sulphite content of model beers

The concentration of sulphite were similar in all three

model beers after 12 days of fermentation, ranging from

3Æ75 mg l)1 in model beer fermented with KVL001 to

3Æ48 and 2Æ50 mg l)1 in model beers fermented with

WLP833 and KVL018, respectively (Table 2).

Thioredoxin content of model beers

Swan et al. (2003) have earlier reported that brewers yeast

are able to secrete thioredoxin to the beer during fermen-

tation. Our data support these findings. Western blotting

of protein samples during fermentation showed that the

level of thioredoxin in the model beers increased from

being nondetectable in the beginning of the fermentation

(data not shown) to 40–80 pg lg)1 total protein after

12 days of fermentation (Fig. 3). Here, KVL001 and

WLP833 had produced twofold more thioredoxin, that is

80 pg lg)1 total protein, than KVL018, that is,

40 pg lg)1 total protein (Fig. 3).

Metal contents of model beers

According to Zufall and Tyrell (2008), the iron and cop-

per concentrations in wort are in the range of 100–270

and 20–400 lg l)1, respectively. In the model wort used

00:30 1:00 1:30

Time (min)

Rel

ativ

e ra

dica

l sig

nal i

nten

sity

2:302:00

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

Figure 2 Formation of spin trap radicals measured by electron spin

resonance during forced ageing at 60�C of model beers fermented in

duplicate with (4) KVL001; (s) KVL018 and (h) WLP833.

Table 2 Sulphite concentration, antioxidante capacity and radical for-

mation rate in model beers after 12 days of fermentation*

Yeast

strain

Sulphite

(mg l)1)

ESR lag

phase

(min)

Relative radical

formation rate

compared to

KVL018 (fold)

KVLO01 3.75 ± 0.08 55 ± 8.1 3.2 ± 0.33A

KVL018 2.50 ± 0.06 53 ± 1.9 1 ± 0.22B

WLP833 3.48 ± 0.04 60.5 ± 2.3 1.8 ± 0.03B

ESR, electron spin resonance.

*Values represent the mean ± SEM from duplicate measurements

from two independent fermentations. Values with different capital let-

ters in superscripts, within a column, are significantly different

(P < 0.05).

y-TRX2

TRX2 standard

4·7

KVL001

B

KVL001

A

KVL018

A

KVL018

B

WLP

833

A

WLP

833

B

4·9 4·3 5·1 5·22·4 2·4 2·61·3 10·4 ng

Figure 3 Western blot detection of extracellular thioredoxin after

12 days of fermentation with KVL001, KVL018 and WLP833 in 2 l

synthetic beer media in European Brewing Convention tubes. 60 lg

of TCA precipitated proteins from two independent fermentations

(A ⁄ B) were separated on a 20% SDS-PAGE gel blotted on to a PVDF

membrane, detected with an anti-yeast-thioredoxin antibody, and

quantified from the dilution row of 1Æ3–10Æ4 ng yeast thioredoxin

(TRX2) standard.


ª 2011 The Authors


in this study, the iron concentration is higher (600–

815 lg l)1), yet the copper concentration is in the interval

reported by Zufall and Tyrell (25–30 lg l)1) (Table 3).

Both the copper and iron ion concentrations detected in

the model beer were comparable to those reported earlier

(Wyrzykowska et al. 2001; Pohl and Prusisz 2010; Sancho

et al. 2011).

The copper ion concentrations in the model beers were

virtually similar (9–12 lg l)1) after 12 days of fermenta-

tion (Table 3). The iron ion concentrations were, how-

ever, very different ranging from 57 ± 9 lg l)1 in the

model beer fermented with KLV018 to 396 ± 27 lg l)1 in

the one fermented with KVL001 and 160 ± 14 in the

WLP833 fermented model beer (Table 3).

Discussion

To the best of our knowledge, this is the first report com-

paring the ability of brewing yeast strains to endure oxi-

dative stress. We show here that the ability of lager

brewing yeast to tolerate the two different oxidative-

stress-inducing compounds, diamide and paraquat is

strain dependent. Because brewers yeast somehow seem

to be subject to oxidative stress during beer fermentation

(Higgins et al. 2003; James et al. 2003), and because

intracellularly produced antioxidants, such as thioredoxin,

have been reported to be secreted to the beer during fer-

mentation (Swan et al. 2003), we expected that strains

with increased resistance to pro-oxidants would be able

to circumvent this stress condition better by producing

and secreting more antioxidants to the beer. Interestingly,

this seems to be the case, that is, the two more-oxidative-

stress-tolerant strains (KVL001 and WLP833) produced

beer with higher amounts of thioredoxin than the low-

oxidative-stress-tolerant strain (KVL018).

In this study, the antioxidant capacity of the three

model lager beers, as represented by the lag phase in the

ESR experiments, are virtually similar. This may be

explained by the fact that the sulphite concentrations in

all three model beers are the same, and it is in accordance

with previous studies reporting that the ESR lag phase is

correlated with the amount of sulphite in beer (Uchida

et al. 1996; Andersen et al. 2000).

Our results, however, also demonstrate that the rates of

radical formation are different in the three model lager

beers. The rate of radical formation is expected to be slow

when the sample has either a high oxidant defence, which

hinders oxidative reactions, and ⁄ or a low level of pro-

oxidants favouring radical formation (Uchida et al. 1996).

As the most-oxidative-stress-tolerant strains secrete the

most thioredoxin, we expected that these strains would

also result in beers with an increased oxidative stability.

Surprisingly, our results show more or less the opposite,

that is, the yeast strain most prone to oxidative stress in

the screening experiment (KVL018) gives beer with the

slowest formation of radicals. Together, our data indicate

that the oxidative stability of lager model beers is not

related to yeast strains having a high oxidative stress tol-

erance, and thus a good ability to secrete thioredoxin.

Rather, it seems as if KVL018 is superior to take up

iron, which, in turn, may explain its low oxidative stress

tolerance. Perhaps its higher iron uptake may lead to

increased intracellular iron levels and thus higher ROS

levels as a result of Haber–Weiss and Fenton reactions

(Vanderhaegen et al. 2006). The good ability of KVL018

to take up iron, however, seems to decrease the rate of

radical formation outside the cells and may thus be bene-

ficial for the oxidative stability of the lager model beer.

Interestingly, the three strains do not differ in their cop-

per uptake. Further experiments are required to identify

the mechanisms underlying this phenomenon. Many

strategies have so far been proposed to inhibit metal-

induced, oxidative changes in beer (Vanderhaegen et al.

2006). Based on our results presented here, we suggest a

new strategy for lager beers: to use lager yeast strains that

are susceptible to oxidative stress and ⁄ or are highly capa-

ble of capturing iron ions.

It remains to be investigated if other yeast produced

antioxidants; for example, glutaredoxin and glutathione

have an influence on the oxidative stability of lager beer.

It should also be noted that although high levels of an-

tioxidants may be desired to obtain oxidative stable beer,

they may have a negative influence on foam stability of

beer, as reported for thioredoxin (Iimure et al. 2008).

In conclusion, we find that lager beer yeast resistant to

oxidative stress does not produce more oxidative stable

model beer. On the other hand, we see increased thiore-

doxin production and secretion to the beer correlating

with the tolerance towards pro-oxidants. We have identi-

fied a lager beer yeast strain that has a low oxidative

stress tolerance and is able to assimilate almost all the

Table 3 Transition metal concentration in wort*

Yeast strain

Fe (ppb) Cu (ppb)

Days of fermentation Days of fermentation

0 12 0 12

KVL001 831 ± 35 396 ± 27A 28 ± 3.0 12 ± 1.4

KVL018 665 ± 32 57 ± 9.6B 27 ± 0.8 9 ± 1.2

WLP833 624 ± 46 160 ± 14C 26 ± 1.9 9 ± 0.9

*Metal ion concentrations were determinated by ICPMS. Values repre-

sent the mean ± SEM from duplicate measurements from two inde-

pendent fermentations. Values with different capital letters in

superscripts, within a column, are significantly different (P < 0.05).



ª 2011 The Authors

iron present in model wort, thereby decreasing the rate of

radical formation in model beer. Thus, our results suggest

that, to obtain lager beers that are oxidatives, yeast strains

should be screened for their low oxidative stress tolerance

and ⁄ or their good ability to take up iron rather than for

their high oxidative stress tolerance and ⁄ or high secretion

of thioredoxin.

Acknowledgements

This project was financed by the Danish Ministry of

Food, Agriculture and Fisheries, project no. 3304-FVFP-

07. We thank Christopher White from White Labs, San

Diego, USA, for kindly providing yeast strains and are

grateful to Professor Chris Grant, University of Manches-

ter, UK, for donating the yeast thioredoxin antibody.

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