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LWT 41 (2008) 868877

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Antioxidative activity of (1-3), (1-6)-b-D-glucan from

Saccharomyces cerevisiae grown on different media
Silke C. Jaehriga, Sascha Rohnb, Lother W. Krohb, Franz X. Wildenauerc,
Fred Lisdatc, Lutz-Guenther Fleischera, Tomas Kurza,
a
Department of Food Process Engineering, Institute of Food Technology and Food Chemistry, Technische Universitaet Berlin,
Amrumer Strasse 32, D-13353 Berlin, Germany
b
Department of Food Analysis, Institute of Food Technology and Food Chemistry, Technische Universitaet Berlin,
Gustav Meyer Allee 25, D-13355 Berlin, Germany
c
Department of Biosystems Technology/Bioinformatics, University of Applied Sciences Wildau, Wildau, Germany
Received 19 December 2006; received in revised form 12 June 2007; accepted 12 June 2007

Abstract

In many microorganisms, fungi and algae (1-3), (1-6)-b-D-glucan can be found as cell wall polysaccharide. Various publications
describe the strong positive inuence of these glucans on the immune system comprising antibacterial, wound-healing and antitumour
activities. Recently, it was found that (1-3)-b-D-glucan from the yeast Saccharomyces cerevisiae also exhibits antioxidative
capabilities.
In the present study, the antioxidative activity of glucan from the cell walls of S. cerevisiae grown on different media (wort,
yeastpeptoneglucose, yeastpeptonegalactose) was investigated.
Fermentation of the yeast took place on three different media. After fermentation the yeast was harvested and disrupted in a high-
pressure homogeniser. Subsequently, glucan was extracted from the yeast cell walls by a procedure including a combination of hot water
and enzymatic treatment. The level of (1-3), (1-6)-b-D-glucan in the cell walls was analysed enzymatically. The antioxidant activity
was determined by electron paramagnetic resonance (EPR) spectrometry, TEAC assay (Trolox equivalent antioxidant capacity) and an
amperometric biosensor.
The results show signicant differences in the glucan content of the cell walls and in their antioxidative activities depending on growth
medium. However, glucan itself seems to have a low antioxidative activity in contrast to other cell wall fractions e.g. proteins.
r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: b-Glucans; Bakers yeasts; Antioxidative activity; Media; Electron paramagnetic resonance

1. Introduction wall polymer (1-3), (1-6)-b-D-glucan possess a bio-

Spent brewers yeast (Saccharomyces cerevisiae) is a by-
product of the brewing process and makes up 23% of
total beer production (Bock & Oechsle, 1999; UNIDO,
2000). With a world beer production of about 1575 million
hl/year (Plato Logic, GB) 31,50047,250 million hl of yeast
waste accumulate every year. Most of this yeast is sold as
animal feed to a low price or has to be disposed with costs.
However, certain cell components e.g. the structural cell
Corresponding author. Tel.: +49 30 31427589; fax: +49 30 31427518.
E-mail address: tomas.kurz@tu-berlin.de (T. Kurz).
activity and thus a potential physiological value. Hence,
producing a high-value-added product e.g. functional food
from yeast glucan could benet breweries and the yeast
industry by receiving an additional source of income and
eliminating the costs of waste disposal.
Yeast cells are enclosed by a cell wall containing 2964%
b-glucans, 31% mannans, 13% proteins, 9% lipids and
12% chitin (Bacon, Farmer, Jones, & Taylor, 1969; Fleet
& Manners, 1976; Kath & Kulicke, 1999).
The cell wall glucans are composed of a 1,3-glycosidic
backbone and 1,6-glycosidic intermediate chains. Together
they form a three-dimensional network. Glucan can be

0023-6438/$34.00 r 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2007.06.004
 ARTICLE IN PRESS
S.C. Jaehrig et al. / LWT 41 (2008) 868877
further subdivided into an alkali insoluble (8085% of the
entire content) and an alkali soluble part (Kath & Kulicke,
1999).
The exact structure and composition of the yeast cell
wall depends strongly on the cultivation conditions.
Aguilar-Uscanga and Francois (2003) found that the dry
mass and polysaccharides content of the cell wall could
vary by more than 50% with the nature of the carbon
source, nitrogen limitation, pH, temperature and aeration,
and with the mode of cell cultivation.
(1-3), (1-6)-b-D-Glucan from S. cerevisiae is a well-
known immunomodulator with a strong positive inuence
on the human and animal immune system (Fitzpatrick,
Haynes, Silver, & Dicarlo, 1964; Bohn & BeMiller, 1995;
Li, Li, Xing, Cheng, & Lai, 2006). Recently, Kogan et al.
(2005) found that carboxymethylated (1-3)-b-D-glucan
from S. cerevisiae cell walls exhibits antioxidative activity
in terms of free radical scavenging.
However, S. cerevisiae not only contains antioxidants in
the cell wall but also in the cytoplasm which holds several
endogenous antioxidants (Demasi, Pcrcira, & Netto, 2001;
Pinheiro, Belo, & Mota, 2002).
The aim of the present work was to evaluate glucan
content and antioxidative activity of (1-3), (1-6)-b-D-
glucans from S. cerevisiae grown on three different media.
Since the cell wall composition depends on growth medium
two model media containing glucose and galactose as
carbon source, respectively, were used. Additionally, a
medium used in traditional brewery fermentationwort
was applied. The comparison of the different media should
take into consideration whether yeasts grown on a
comparatively impure medium as wort show the same
bioactivity of the cell wall glucan as yeasts grown on the
model media.
In contrast to previous works (Krizkova et al., 2003;
Kogan et al., 2005) underivatised glucan was investigated.
The reason is that drastic conditions in the chemical
extraction and derivatisation can degrade the glucan
structure severely (Mueller, 1993; Mueller et al., 1997).
Thus, the present study uses a mild enzymatic method for
the isolation of glucan (Freimund, Sauter, Kaeppeli, &
Dutler, 2003). Furthermore, (1-3), (1-6)-b-D-glucan
from the yeast cell wall of bakers yeast is classied as
GRAS by the FDA (FDA Specications 184.1983).
However, the yeast cell wall contains more potentially
antioxidant constituents than just glucan (e.g. mannans
and proteins) (Krizkova, Durackova, Sandula, Sasinkova,
& Krajcovic, 2001). Therefore, the contribution of the
other cell wall components to the antioxidative activity was
investigated, too.
The antioxidative activity was detected by electron
paramagnetic resonance (EPR) measurements, Trolox
equivalent antioxidant capacity (TEAC) assay and with
an electrochemical method called amperometric biosensor
(Ignatov, Shishniashvili, Ge, Scheller, & Lisdat, 2002). In
this study, the applicability of the new and fast biosensor
for the evaluation of the antioxidative activity of (1-3),
869
(1-6)-b-D-glucan from yeast cell walls was investigated
and compared with the two standard methods.
2. Materials and methods
2.1. Biomass
2.1.1. Micro-organism and culture conditions
The yeast strain used in this study, S. cerevisiae W34/70,
was obtained from Hefebank Weihenstephan (85350
Freising, Germany). Originally, the strain was isolated
from industrial beer fermentation. The yeast strain was
maintained on wort agar slants (Merck, Darmstadt,
Germany) which were incubated at 30 1C for 48 h and
then stored at 4 1C.
2.1.2. Fermentation media
For fermentation three different media with different
carbon sources were used. The composition of the media
was as follows. The wort medium consisted of 158 g/l wheat
wort extract, 8.3 g/l ammonium sulphate, 1 mg/l zinc and
vitamins. The main carbon source was maltose. The yeast
peptone glucose medium (YPD) consisted of 25 g/l yeast
extract, 50 g/l peptone and 100 g/l glucose. The carbon
source was glucose. The yeast peptone galactose medium
(YP-Gal) consisted of 25 g/l yeast extract, 50 g/l peptone
and 100 g/l galactose. The carbon source was galactose.
The wort extract Bavarian Hefeweizen was obtained
from Weyermann (Bamberg, Germany), yeast extract from
Serva Electrophoresis GmbH (Heidelberg, Germany),
peptone from Sigma-Aldrich Chemie GmbH (Deisenhofen,
Germany), glucose from VWR (Darmstadt, Germany) and
galactose from Serva.
2.1.3. Preparation of inocula for fermentation
To initiate yeast growth, inocula from the slants were
transferred to ve test-tubes each containing 5 ml of the
medium. The test-tubes were incubated at 30 1C for 72 h.
Subsequently, inocula (5 ml) from the test-tubes were
transferred to ve 500 ml Erlenmeyer asks each contain-
ing 200 ml medium. The asks were incubated at 30 1C for
24 h on a rotary shaker at 200 rpm. Inoculum of 1 l from
such a liquid culture was used to inoculate the fermentation
medium.
2.1.4. Equipment and experimental procedure
A 30 l Biostat UD fermenter (B. Braun, Melsungen,
Germany) with an operating volume of 20 l, equipped with
an agitator, pH, and temperature control systems was used
as the batch fermenter in this study. Fermentations were
performed on 20 l medium. The fermenter (containing the
medium) was steam sterilised at 1.5 bar for 15 min. Then 1 l
inoculum was transferred to the fermenter. The tempera-
ture of fermentation was maintained at 25 1C. The pH of
the fermentation broth was regulated at pHX4. The
fermentations were carried out at atmospheric pressure.
An aeration rate of 1 vvm (volume of gas per volume of
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870 S.C. Jaehrig et al. / LWT 41 (2008) 868877

liquid per minute) was introduced into the medium whilst
stirring at 250 rpm.
After 26 h, the yeast cells were harvested, concentrated
by cross ow ltration (Sartocon II, Sartorius, Goettingen,
Germany) and washed three times. Fermentations were
performed in duplicate for each medium.
2.2. Processing of the yeast cell walls
Glucan was enriched in the yeast cell walls by a
procedure of Freimund et al. (2003). The schematic process
for the fractionation is shown in Fig. 1. After cell
disruption in a high-pressure homogeniser (fraction F1)
mannoproteins and water-soluble glucans were extracted
from the cell walls by a hot water treatment (F2).
Afterwards, the remaining cell wall proteins were removed
by the commercially available protease SavinaseTM (F4).
After protease treatment the sample was treated with a
commercially available lipase to remove the cell wall lipids
(F5). For the purpose of analysis a sample was taken after
Cell disruption with high
pressure homogeniser
(F1)
every step of the extraction procedure. Glucan rich
sediment was separated by centrifugation from a super-
natant which contained the cell components extracted in
the previous step. Thus, different fractions of glucan
enriched yeast cell walls (sediments) and supernatants
containing cell wall components were gained.
The sediments were lyophilised and ground to powder,
the supernatants were ltered through a membrane lter
with 0.2 mm pore width to remove solids.
2.3. Preparation of the cell walls
2.3.1. Yeast cell disruption
The separation of the cell walls was carried out by
mechanical disruption in a high-pressure homogeniser
(type Panda, 1000 bar, Niro Soavi, Parma, Italy). There-
fore, the dry mass of the yeast suspension was adjusted to
7.5% (w/w) with water. Three homogenisation cycles at
700 bar were needed. The yeast cell walls were separated in
a Beckman J2-HS lab centrifuge yielding a sediment
consisting of disrupted cell walls (fraction F1) and a
supernatant containing the cell content. Subsequently, the
sediment was washed three times with water. For analytical
purposes, an aliquot of the sediment was lyophilised in an
Alpha 24 freeze dryer (Christ, Osterode, Germany). The
supernatant was stored at 20 1C.

sediment: native cell walls 2.3.2. Hot water extraction

supernatant: cytoplasm After cell disruption the washed sediment (fraction F1)
was diluted with water to 13% dry mass (w/w) and
adjusted to pH 7 with NaOH (30% w/w), according to
Freimund et al. (2003). The suspension was heated to
Hot water 125 1C in a VARIOKLAV steam autoclave (H+P Labor-
extraction (F2) technik AG, Oberschleissheim, Germany) for 5 h. After-
wards, the suspension was cooled down to room
temperature and was diluted with 72.5 ml water per 100 g
sed.: glucans, proteins, lipids
suspension. The sediment was separated by centrifugation
sup.: mannoproteins, soluble glucans
and washed twice with water. An aliquot of the sediment
(fraction F2) was lyophilised and the supernatant was
stored at 20 1C.
Savinase Lipase
treatment (F3) 2.3.3. Protease treatment
treatment (F4)
Following hot water extraction the washed sediment
sed.: glucans, (fraction F2) was diluted with water to 4% (w/w) dry mass.
sed.: glucans, lipids proteins After heating to 45 1C and adjustment of the pH to 10.5
sup.: peptides sup.: glycerol, with NaOH (30% w/w), the protease SavinaseTM 16.0 L
fatty acids type EX (Novozymes, Bagsvaerd, Denmark) was added
under continuous stirring three times (t 0; 1.5 and 3 h).
Each time 0.075 ml SavinaseTM per 100 ml suspension was
Lipase needed. After 5 h, the sediment was separated by centrifu-
treatment (F5) gation and washed twice with water. An aliquot of the
sediment (fraction F4) was lyophilised and the supernatant
sed.: glucans was stored at 20 1C.
sup.: glycerol,
fatty acids
2.3.4. Lipase treatment
Fig. 1. Schematic process for the fractionation of yeast cell walls. Subsequent to either hot water extraction (fraction F2)
sed. sediment; sup. supernatant. or protease treatment (fraction F4) the washed sediment
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S.C. Jaehrig et al. / LWT 41 (2008) 868877
was diluted with water to 4% (w/w) dry mass and further
treated with LipolaseTM 100 L EX (Novozymes, Bags-
vaerd, Denmark) at 45 1C and pH 10.5 with stirring for 3 h
(1 g LipolaseTM per 100 ml suspension). The glucan
particles (fractions F3, F5) were harvested by centrifuga-
tion, washed twice and were lyophilised. The supernatant
was stored at 20 1C.
2.4. Analytical procedure
2.4.1. Determination of (1-3), (1-6)-b-D-glucan content
The amount of (1-3), (1-6)-b-D-glucan in the cell wall
fractions was determined enzymatically by the commercial
assay YEAST BETA GLUCAN ASSAY KIT (Mega-
zyme Int., Bray, Ireland). For the analysis of the
lyophilised, ground sediments of each fraction were used.
The measurements were carried out with a Novaspec II
spectrophotometer (Pharmacia LKB, Sweden). The ab-
sorption of all solutions was measured at 510 nm against a
blank and the b-glucan content was calculated.
2.4.2. Determination of mannoprotein content
Determination of the mannoprotein content of the
native cell walls was carried out by ethanol precipitation.
After hot water extraction (F2) 9 g of the supernatant was
added to ethanol under stirring until the water content
reached 30%. The mixture was stored overnight at 4 1C.
The precipitate was centrifuged, washed with ethanol/H2O
(70% w/w) and dried at 105 1C in an incubator until no
residual moisture was left. The dried precipitate was
weighed and mannoprotein content was calculated in
respect to cell wall dry mass (dry mass of F1).
2.4.3. Determination of protein content
Protein contents of the cell wall fractions were deter-
mined by a Kjeldahl method (Kjeldahtherm-Turbosog-
Vapodest 33, C. Gerhardt GmbH & Co. KG, Bonn,
Germany).
2.5. Determination of antioxidative activity
Three methods were applied to measure the antioxida-
tive activity: EPR spectrometry, TEAC assay and an
amperometric biosensor.
2.5.1. EPR spectrometry
The ability of the samples to donate a hydrogen atom or
electron to a free radical was monitored by EPR spectro-
metry (Rohn & Kroh, 2005) using a synthetic stabilised
radical.
Both, sediments and supernatants of the cell wall
fractions were analysed with a Miniscope MS 100
(Magnettech GmbH, Berlin, Germany). For the sediments,
the lipophilic radical TEMPO (2,2,6,6-tetramethyl-piper-
idine-1-oxyl) was chosen since the sediments were dissolved
in DMSO. For the aqueous supernatants the hydrophilic
Fremys salt (potassium nitrosodisulfonate) was used as a
871
radical, according to Roesch, Bergmann, Knorr, and Kroh
(2003).
A 10-fold dilution of the supernatants was added to an
equal volume of Fremys salt radical solution (1 mM in
phosphate buffered saline, pH 7.4). The spectrum of the
low-eld resonance of Fremys salt was recorded from
90 s after mixing the sample with the radical for a du-
ration of 25 min by EPR. Signal intensity was obtained by
double integration and the concentration calculated by
comparison with a control reaction (phosphate buffer
instead of supernatant). Spectra were obtained at 20 1C.
The antioxidative activity (mM Fremys salt per l super-
natant) was expressed as the percentage of Fremys
salt radicals reduced by the samples and was calculated
by Eq. (1).
reduction of Fremys salt % initial amount of Fremys salt mM
AA .
100  initial amount of sample undiluted l
(1)
Hundred ml of dissolved sediment (50 mg lyophilised,
ground sediment per 5 ml dimethyl sulfoxide) was added to
an equal volume of TEMPO radical solution (1 mM in
DMSO). The spectrum of the low-eld resonance of
TEMPO was recorded from 90 s after mixing of the sample
with the radical for a duration of 25 min by EPR. Signal
intensity was obtained by double integration and the
concentration calculated by comparison with a control
reaction with DMSO instead of the dissolved sediment.
Spectra were obtained at 20 1C. The antioxidative activity
(mM TEMPO per g sediment) was expressed as the
percentage of TEMPO radicals reduced by the samples
and was calculated by Eq. (2).
reduction of TEMPO %  initial amount of TEMPO mM
AA .
100  initial amount of sample g
(2)
2.5.2. TEAC
As a second method, a modied TEAC assay (Rohn,
Rawel, & Kroll, 2004) was used to measure the antioxidant
activity of the supernatants. Originally described by Miller,
Rice-Evans, Davies, Gopinathan, and Milner (1993),
the TEAC assay is based on scavenging of the long-living
2,2-azinobis-3-ethylbenzthiazolin-6-sulfonic acid (ABTS)
radical anions. The radicals are generated by potassium
persulfate and can easily be detected with a spectro-
photometer at 734 nm (Re et al., 1999). Present antiox-
idants reduce the absorption according to their
concentration. As reference substance Trolox (a water
soluble analogue of vitamin E, Sigma-Aldrich Chemie
GmbH, Deisenhofen, Germany) was used. Absorbance
was measured exactly 6 min after the reaction was
started by addition of persulfate to a mixture of ABTS
and the supernatants (10-fold diluted with PBS buffer,
pH 7.27.4).
The antioxidative activity of the sample was quantied
by comparison with Trolox (constructing a calibration

872
curve) and was given in Trolox equivalents (mM Trolox
per l).
2.5.3. Amperometric biosensor
As a third method for the detection of antioxidants an
electrochemical method has been applied, which is based
on a radical measurement with a cytochrome c modied
gold electrode and on the measurement of steady-state
superoxide levels. Superoxide radicals based on xanthine
oxidase were produced by a controlled enzymatic system.
The addition of antioxidants facilitates the decomposition
of the radical and thus reduces the superoxide level. This
results in a decreased current level at the electrode.
Antioxidant activity can be quantied from the response
of the sensor electrode by the percentage of the signal
decrease. The concentration which induces a 50% inhibi-
tion of the superoxide level (IC50) was calculated and
compared with the IC50 of ascorbic acid as a standard
substance (Ignatov et al., 2002).
3. Results and discussion
3.1. Content of (1-3), (1-6)-b-D-glucan
Fig. 2 illustrates the content of (1-3), (1-6)-b-D-
glucan in the different cell wall fractions.
The fractions with the lowest amount of glucan are the
F1 fractions, which represent the native yeast cell walls.
The highest content of (1-3), (1-6)-b-D-glucan (47/
100 mg sediment dry mass) is shown by the native cell walls
grown on YPD (D-F1) with glucose as carbon source. YP-
Gal (G-F1) only yields a glucan content of 42.6/100 mg
sediment dry mass. The lowest glucan content (30/100 mg
sediment dry mass) is exhibited by the cell walls grown on
wort (W-F1) where maltose is the main carbon source.
90
Wort
80
g -Glucan/ 100 g sediment dry mass
70
60
50
40
30
20
10
0
W-F1 W-F2 W-F3 W-F4 W-F5 D-F1 D-F2 D-F3 D-F4 D-F5 G-F1 G-F2 G-F3 G-F4 G-F5
Fig. 2. (1-3), (1-6)-b-D-Glucan content of the cell wall fractions (sediments). Key to fraction numbers: W wort medium; D yeast peptone dextrose
medium (YPD); G yeast peptone galactose medium (YP-Gal); F1 after cell disruption; F2 after hot water treatment; F3 after lipase treatment;
F4 after Savinase treatment; F5 after Savinase and lipase treatment.
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Thus, medium and especially the carbon source seem to
inuence the cell wall composition greatly.
The fractions with the highest amount of glucan are the
F5 fractions. The other cell wall components as mannans,
proteins and lipids have been extracted in the previous
steps.
For the hot water extracted cell wall fractions F2 the
glucan content of cells grown wort (46.9/100 mg sediment
dry mass) is lower than that of cells grown on model media
(60/100 mg sediment dry mass). The fractions F2 are
devoid of mannoprotein but still contain all other proteins
and lipids.
The fractions F4 are products of protease treatment of
the fractions F2. The protease used in this study was a
commercial preparation (SavinaseTM) whose major enzy-
matic activity is that of subtilisin (EC 3.4.21.62), hydro-
lysing peptide bonds with low specicity (Bond, 1989). The
protease treated fractions of cells grown on wort (W-F4)
and on YPD (D-F4) show similar contents of b-glucan
(76/100 mg sediment dry mass). Cells grown on YP-Gal
only reach a glucan content of 70.3/100 mg sediment dry
mass.
The increase in glucan content from fractions F2 to F4 is
due to the protein content of the cell wall. However, the
increase between fractions F1 and F2 accounts for
mannoproteins as conrmed by ethanol precipitation (data
not shown). The protein content of the mannoproteins is
3.4 mg protein/100 mg sediment dry mass for fraction W-F2,
2.3 mg protein/100 mg sediment dry mass for fraction D-F2
and 1.9 mg protein/100 mg sediment dry mass for fraction
G-F2. Thus, total protein content of the native cell wall is
32.2 mg protein/100 mg sediment dry mass for cells grown
on wort, 14 mg protein/100 mg sediment dry mass for cells
grown on YPD and 12.3 mg protein/100 mg sediment dry
mass for cells grown on YP-Gal.
YPD YP-Gal
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F3 and F5 are the fractions gained after enzymatic lipid
extraction of the fractions F2 and F4, respectively.
Actually, the differences in glucan content between F2
and F3 and F4 and F5, respectively, should be similar and
represent the lipid content of the native cell walls.
However, there are signicant differences between the lipid
contents calculated from F2 and F3 and F4 and F5,
respectively. Lipid contents gained from the difference
between F5 and F4 are 5.7 mg lipid/100 mg sediment dry
mass for cells grown on wort, 3.5 mg lipid/100 mg sediment
dry mass for cells grown on YPD and 6.5 mg lipid/100 mg
sediment dry mass for cells grown on YP-Gal. Lipid
contents gained from the difference between F3 and F2 are
much lower (between 0.9 and 3 mg lipid/100 mg sediment
dry mass). An explanation for these observations could be
found in the structure of the cell wall and in the way the
lipase works. The removal of the mannoprotein complex
on the outside of the cell wall (F2) reveals the glucan
network, which is still entangled with fat and proteins.
Maybe the lipase applied to fraction F2 is not able to
remove all the present lipids since they might be still
covered with protein. On the other hand, there is strong
evidence that a signicant amount of proteins is removed in
this extraction step (see 3.3 TEAC assay). This might be
due to a proteolytic coactivity of the lipase. When the
lipase is applied to fraction F4 it might be easier to remove
the lipids since proteins have been removed in the previous
step. However, some residual proteins could be removed by
proteolytic coactivity, as well.
Finally, glucan content is lowest in cell walls grown on
wort but protein and lipid contents show the highest values
compared to the other media. Apparently, it is easier for
yeast cells to build up glucan from glucose than from
maltose and galactose, which are more difcult to
metabolise. The elevated protein and lipid contents of cells
grown on wort might account for the presence of proteins
and lipids in the medium. This would correspond with the
6
Wort
5
Antioxidative activitity [mM/l]
0
W-F1 W-F2 W-F3 W-F4 W-F5 D-F1 D-F2 D-F3 D-F4 D-F5 G-F1 G-F2 G-F3 G-F4 G-F5
Fig. 3. Antioxidative activity (EPR) of the supernatants. Fraction numbers refer to Fig. 2.
873
ndings of other authors who barely found lipids in the
pure yeast cell wall and quote that the presence of lipids is
mainly due to membrane contaminant (Klis, 1994; Dallies,
Francois, & Paquet, 1998).
3.2. EPR measurements
According to the methods of Roesch et al. (2003), both,
sediments and supernatants of the cell wall fractions were
analysed with EPR to determine the antioxidative activity.
The antioxidative activity of the supernatants deter-
mined by EPR measurements is shown in Fig. 3. The
highest antioxidative activities are observed for the
cytoplasm obtained after cell disruption (F1), which
contains several endogenous antioxidative substances, e.g.
superoxide dismutase and ubiquinone (Santiago & Mori,
1993). The strongest antioxidant is W-F1 (wort medium)
followed by D-F1 (YPD medium) and G-F1 (YP-Gal
medium). The antioxidative activity of W-F1 is approx.
two times higher than that of D-F1. An explanation could
be found in the composition of the media. All media
contain different carbon sources. This probably accounts
for the difference between D-F1 and G-F1. In contrast to
the model media YPD and YP-Gal the wort medium
contains secondary plant metabolites, e.g. polyphenols
which are known to be good antioxidants (Floridi,
Montanari, Marconi, & Fantozzi, 2003). Yeasts are able
to adsorb (Rizzo, Ventrice, Varone, Sidari, & Caridi, 2006)
and absorb (Leibnitz, Behrens, & Seifert, 1961; Schanderl,
1962) these phenolic compounds. Thus, the cells grown on
wort medium could have taken up polyphenols from the
medium leading to an increased antioxidative activity of
the cell cytoplasm. As illustrated in Fig. 3, all wort
fractions show higher antioxidative activities than YPD
or YP-Gal fractions. This could account for adsorbed
phenols which could have been released into the super-
natants during the extraction procedure.
YPD YP-Gal
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874 S.C. Jaehrig et al. / LWT 41 (2008) 868877

The fractions F2 and F4 show a much lower antiox- antioxidative activity probably accounts for a slight proteo-
idative activity than the fractions F1. The F2 fractions are lytic coactivity of the lipase preparation cleaving proteins and
obtained after cell wall disruption and hot water treatment. thus releasing peptides (Jaehrig et al., 2007).
They consist mainly of mannoproteins. The F4 fractions Fig. 4 illustrates the antioxidative activities of the native
result from protease treatment of sediment F2 causing a yeast cell walls (selected sediments of fraction F1). The
release of many peptides which may act as antioxidants highest antioxidative activity is observed for the YP-Gal
(Jaehrig, Rohn, Kroh, Fleischer, & Kurz, 2007). medium (G-F1). For YPD (D-F1) and wort medium
The lowest antioxidative activities exhibit the supernatants (W-F1) the antioxidative activities are signicantly lower
after lipase treatment (F3, F5). The lipase treatment than for YP-Gal. Apparently, the medium has a strong
hydrolyses fat into mono- and diglycerides, glycerol and free inuence on the cell wall composition whereas the carbon
fatty acids (Jensen et al., 1990). All these do not have any source seems to be more important than other compounds
groups which could act as antioxidants. The residual of the medium as indicated by the great difference between
the carbon sources galactose (YP-Gal) and glucose (YPD).
0.02 The results of other sediment fractions are not shown since
YP-Gal the signals of the EPR measurements were too low to be
0.018 evaluated. Although F1 is the most impure fraction (native
cell wall) it shows the highest antioxidative activity. The
0.016 other fractions are gradually puried residues of this cell
wall with an increasing content of (1-3), (1-6)-b-D-
YPD
glucan (F1 to F5). Thus, a higher glucan content does not
Antioxidative activity [mM/g]

0.014
mean higher antioxidative activity. An explanation for this
0.012 behaviour could be that (1-3), (1-6)-b-D-glucan is
Wort hardly responsible for the antioxidative activity of the
0.01 yeast cell wall (Jaehrig et al., 2007). Instead, the interaction
of the different cell wall components, as present in the
0.008 native cell wall, could account for the antioxidative
potential. For this, composition and structure of the cell
0.006 wall components which are strongly inuenced by growth
conditions (e.g. medium and carbon source) seem to be of
0.004 great importance.

0.002 3.3. TEAC assay

0 The results of the TEAC assay for the supernatants are

W-F1 D-F1 G-F1
shown in Fig. 5. Cytoplasm (F1) and peptides (F4) exhibit
Fig. 4. Antioxidative activity (EPR) of the F1 fractions (sediments). high antioxidative activities. The antioxidative activities of
Fraction numbers refer to Fig. 2. the F2 fractions (mannoproteins) are a bit lower. The

0.3
Wort YPD YP-Gal

0.25
Equivalent trolox [mM/l]

0.2

0.15

0.1

0.05

0
W-F1W-F2W-F3W-F4W-F5 D-F1 D-F2 D-F3 D-F4 D-F5 G-F1 G-F2 G-F3 G-F4 G-F5

Fig. 5. Total antioxidative activity (TEAC) of the supernatants. Fraction numbers refer to Fig. 2.
 ARTICLE IN PRESS
S.C. Jaehrig et al. / LWT 41 (2008) 868877 875

supernatants after lipid extraction (F3 and F5) only show electrode. Since cytochrome c can act as an oxidant of
little antioxidative activities. As in the EPR measurements superoxide the superoxide level in solution can be detected
the highest antioxidative activities are gained on wort as an oxidation current at the sensor electrode due to
medium, the lowest on YP-Gal medium. These ndings electron transfer from the radical via cytochrome c to the
correlate with the results of the EPR measurements. electrode. After the addition of an antioxidant the current
However, the TEAC assay is much more sensitive to level decreases since the antioxidative substances facilitate
peptides than EPR. Apparently, the ABTS radical reacts the decomposition of the radical in addition to sponta-
stronger with peptides than Fremys salt. neous dismutation. This can be detected with the electrode
and antioxidant activity can be quantied of the current
3.4. Amperometric biosensor decrease (Ignatov et al., 2002).
The results of the biosensor were compared with the two
This method of antioxidant detection uses the redox standard methods EPR and TEAC assay. As illustrated in
transformation of cytochrome c at a modied gold Fig. 6(A) the cytoplasm fractions (F1) are the strongest

35
Wort YPD YP-Gal
25

15

5
Ascorbic Acid Equivalent

2.5

1.5

0.5

0
W-F1 W-F2 W-F3 W-F4 W-F5 D-F1 D-F2 D-F3 D-F4 D-F5 G-F1 G-F2 G-F3 G-F4 G-F5

35 8 0.30
Biosensor:Ascorbicacid equivalent

EPR: Antioxidative activity [mM/l]

Biosensor
TEAC:Equivalent Trolox [mM/l]

30 EPR 0.25
TEAC 6
25
0.20
20
4 0.15
15
0.10
10
2
0.05
5

0 0 0.00
W-F1 W-F2 W-F3 W-F4 W-F5

Fig. 6. (A) Superoxide scavenging activity of supernatants (related to ascorbic acid) measured with amperometric biosensor. (B) Comparison of EPR K,
TEAC assay m and biosensor (wort medium). Fraction numbers refer to Fig. 2.
 ARTICLE IN PRESS
876 S.C. Jaehrig et al. / LWT 41 (2008) 868877
antioxidants whereas the antioxidative activity is highest
for wort medium (W-F1) and lowest for YP-Gal (G-F1).
The superoxide anion seems to react extremely well with
components of the cytoplasm, much less with mannopro-
teins and peptides (F2, F4) and hardly with hydrolysis
products of lipids (F3, F5). The amperometric biosensor
shows similar results like EPR and TEAC assay. The
qualitative consistency between the three methods is
demonstrated in Fig. 6(B).
4. Conclusions
Our results suggest an inuence of growth medium and
particularly carbon source on the composition of the yeast
cell wall. These ndings correlate with those of Aguilar-
Uscanga and Francois (2003). The growth medium also
affects the antioxidative activity of the yeast cells and their
cell wall components. The highest antioxidative activities
are reached by the supernatants of cells grown on wort.
Especially the cell cytoplasm gained from the wort grown
cells (W-F1) acts highly antioxidative caused by well-
known endogeneous antioxidants (Santiago & Mori, 1993).
However, the cytoplasm of the wort grown cells shows a
signicantly higher antioxidative activity as the cytoplasm
of cells grown on the model media. This might be due to
absorbed polyphenols present in the wort medium (Floridi
et al., 2003; Leibnitz et al., 1961). Consequently, yeast from
the brewing process is more suitable for applications as
antioxidant than yeast grown on expensive model media.
The sediments, in contrast, exhibit very low antioxida-
tive activities whereas the highest values are reached by
cells grown on YP-Gal (galactose as carbon source).
Obviously, composition and structure of the cell wall
components are positively inuenced by the galactose
medium with regard to antioxidative potential.
Nevertheless, the sediments hold some antioxidative
potential, too. Apart from the cytoplasm (F1) the super-
natant fractions after hot water extraction (F2) and after
protease treatment (F4) exhibit non-negligible antioxida-
tive activities. These supernatants are gained from the
sediments F1 (native cell wall) and F2 (sediment after hot
water extraction). From these sediments the incorporated
mannoproteins and proteins were hydrolysed and extracted
resulting in antioxidative supernatants. This supports the
idea of Jaehrig et al. (2007) that the antioxidative potential
of yeast cell walls is less determined by (1-3), (1-6)-b-D-
glucan than by cell wall proteins (especially aromatic side
chains and thiol groups of the hydrolysed peptides in the
supernatants).
Thus, yeast cells and cell walls become valuable products
after a few inexpensive and easy extraction steps (disrup-
tion, hot water extraction, proteolytic treatment). This
could be of great interest for breweries and the yeast
processing industries.
Additionally, three methods for the determination of
antioxidative activity were compared and evaluated. The
methods applied were the standard methods EPR and
TEAC assay and a new and fast electrochemical method
called amperometric biosensor. It was found that all three
methods show comparable results. Thus, the amperometric
biosensor is applicable as a rapid and inexpensive method
for quality control.
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