Full text of DOI:10.1002/j.2050-0416.2002.tb00550.x

VvsꢀBƒ†iˆwƒ‚ꢀi‚rꢀD“r†ƒ€“‡w‡ꢀƒtꢀ8i†€s“ꢀIi€ˆꢀ
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S. Pöyri,1,4 M. Mikola,2,3 T. Sontag-Strohm,2 A. Kaukovirta-Norja,1 and S. Home1
effect on the amount of gel-protein in spent grains and on
the wort separation rate.
Through the malting and mashing processes barley pro-
teins are also hydrolysed by proteolytic enzymes. During
J. Inst. Brew. 108(2), 261–267, 2002
The effect of oxidation and proteolysis on the amount of gel-
protein aggregate was investigated both in vivo during mashing
germination of barley the hordeins are partially degraded
and in vitro. The oxidation of the free thiol groups of proteins to
with D hordein being attacked first and then B and C hor-
disulphide bridges during mashing appeared to be a good indica-
deins32. The D hordein is also degraded more rapidly than
tor of the formation of gel-protein aggregate. The pH optimum
B and C hordeins when subjected to hydrolysis in vitro by
a purified 30 kDa cysteine proteinase23. Less proteolysis
occurs during mashing than in the malting process but still
slightly less than a quarter of the soluble nitrogen contain-
ing substances of wort are formed at this point of the pro-
cess by the endoproteinases13.
The aim of this work was to study the net results of for-
mation and hydrolysis of gel-protein during mashing and
possibilities for preventing its formation.
The effects of oxidation level, temperature and pH on
the amount of gel-protein were studied. In addition an at-
tempt was made to characterize the malt endoproteinases
that may be responsible for gel-protein hydrolysis during
mashing.
of the oxidation varied according to the isothermal mashing tem-
perature. The results suggested that the oxidation of the thiol
groups maybe a result of some kind of enzymatic activity. In
vitro experiments showed that the proteolysis of the gel-protein
aggregate was strongest at pH 5.0 and temperature denaturation
occurred only at temperatures over 80•C. Mashing experiments
on the other hand suggested that the proteolysis of the monomer
subunits of gel-protein (i.e. B- and D-hordein) had a stronger
effect on the final amount of the gel-protein aggregate than the
hydrolysis of the aggregate.
Key words: Gel-protein, hordein, hydrolysis, mashing, oxida-
tion, proteolysis.
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The barley gel-protein is a polymer that is formed by
polymerisation of high molecular weight (HMW; ca. 95
kDa) and low molecular weight (LMW; ca. 40 kDa) sub-
units which are considered to be barley storage proteins,
called D hordein and B hordein respectively26,27,30. Moonen
et al.19 have hypothesized that in barley HMW subunits
form a backbone, which binds LMW subunits with di-
sulphide bridges under oxidative conditions to form a gel-
like aggregate. The reducing conditions cause disruption
of disulphide bridges diminishing the amount of gel-protein
aggregate during malting but in mashing oxygen is present
and the aggregate is reformed20. The gel-protein of malt
represents a major part of the so called Oberteig-layer that
is formed on top of the spent grains during lautering and is
known to retard wort separation21. Moreover, wort separa-
tion can be accelerated by adding reducing substrates at
mashing that disrupt the disulphide bridges29. Recent re-
sults24 have shown that oxidation during mashing has an
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Commercial Kymppi malt harvested in 1996 was used
for the experiments. High-gravity mashings for preparing
high gravity worts were carried out according to a mash-
ing procedure (HGM) mimicking brewery practice (malt:
water ratio 1:4, temperature programme 48•C 30 min
/63•C 30 min/ 72•C 30 min / 80•C 10 min). The tempera-
ture increase rate was about 1.5-2•C/min25. The pH was
adjusted with 0.5 M H2SO4 (0-18 mL/L) to give mash pHs
of 6.0 to 4.6 (measured at 20•C). At mashing temperatures
the pH is somewhat lower than in the cooled wort12. After
mashing wort was separated in a Büchner-funnel and spent
grains were collected and freeze-dried.
Isothermal mashings were done at temperatures of 40,
50, 60, 70, 80 and 90•C for 60 min. After mashing, sam-
1 VTT Biotechnology, PO Box 1500, FIN-02044 VTT, Finland
2 University of Helsinki, Department of Food Technology, PO Box
27, FIN-00014 University of Helsinki, Finland
TABLE I. The effect of added H2SO4 on the achieved pH of the cold
worts after isothermal mashings at 40-90 C.
Addition of H2SO4 (mL/L)
Range of achieved pH values
3 Current address, AVENA oy, Kansakoulunkatu 1 B, FIN-00100
Helsinki, Finland
0
1.5
8
11
15
18
5.9-5.7
5.8-5.6
5.4-5.1
5.2-4.9
4.9-4.7
4.7-4.6
4 Corresponding author. E-mail: saara.poyri@vtt.fi

ples were immediately cooled (<5•C) in ice-water and cen-
trifuged (8 000g, 10 min, +4•C). Unwashed spent grains
were freeze-dried.
measured with NTSB (2-nitro-5-thiosulphobenzoate)6,28
.
The amount of 200 ml of diluted wort (1:10 with water)
was incubated for 30 min with 1 mL of reaction buffer
consisting of 1mM NTSB, 0.1M sodium sulphite, 0.2 M
Tris-HCl, pH 9.5, 1% SDS and 3mM EDTA. NTSB was
synthesized from DTNB in the presence of sodium sul-
phite and O2 as described by Thannhauser28. The absor-
bance of the solution was read at 412 nm. A blank sample
of wort and buffer without added NTSB was used as a
reference. The method was calibrated with cysteine hydro-
chloride monohydrate (Merck).
E‡ƒ€iˆwƒ‚ꢀƒtꢀus€ꢁ„†ƒˆsw‚ꢀ
Gel-protein was isolated from freeze-dried spent grains
or barley. The material was milled through a 0.5 mm sieve
(Fritsch GmbH). The flour (4g/100 mL) was extracted
with petroleum ether (B.P. 40-60) for an h to remove ex-
cess of lipids. Gel-protein was isolated by extracting flour
with 63 mL of cold 1.5% SDS (sodium dodecyl sulphate)
for 10 min and centrifuging the material in a Beckman
Ultracentrifuge (70 000g) for 40 min11. Gel-protein is in-
soluble in SDS and it forms a gel-like material on the top
of the flour pellet. The gel was carefully scraped off the
pellet, solubilised in 8M urea with 5% 2-mercaptoethanol
and the amount of the soluble protein was measured using
the Bradford method5.
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[SHfree]*100
DoO(%) 100
2*[S – S] [SHfree
]
[SHfree] = free thiol group content
[S – S] = the amount of disulphide bridges (total
sulphydryl group content – free thiol group content)
In order to measure the degree of oxidation during
mashing the results were compared with the blank malt
extract. Malt was extracted with deaerated water (1:4
malt: water ratio) for 10 min at room temperature under a
nitrogen blanket to minimize oxidation.
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Degree of oxidation (DoO) was measured as a ratio of
free thiol groups and disulphide bridges in wort during
mashing.
B†ssꢀˆvwƒ€ꢀu†ƒ‰„ꢀqƒ‚ˆs‚ˆꢀ
(DoO DoO0)*100
Relative DoO(%)
The amount of free thiol groups of the wort was mea-
sured with the colorimetric method first described by Ell-
man9,10. Wort (200 mL) was incubated for 30 min with 1
mL of reaction buffer containing 0.4 mM 5,5’-dithiobis 2-
nitrobenzoic acid (DTNB; Sigma), 0.2 M Tris-HCl, pH
8.0, 1% SDS and 3mM EDTA. The absorbance of the so-
lution was read at 412 nm. Blank samples of wort with
buffer without added DTNB were used as a reference. The
method was calibrated with cysteine hydrochloride mono-
hydrate (Merck).
100 DoO0
DoO = Degree of oxidation of a sample
DoO0 = Degree of oxidation of a blank malt extract
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The endoproteinases were extracted from barley malt
according to Zhang and Jones33. One gram of malt was
homogenized with 2 mL of 50 mM Na-acetate buffer (pH
5.0) including 2 mM cysteine. Extraction was carried out
under agitation at + 4•C for one h followed by centrifu-
gation (10,000g, 4•C, 15 min) after which the supernatant
was frozen in aliquots and used for hydrolysis experi-
ments.
Vƒˆi€ꢀ‡‰€„v“r†“€ꢀu†ƒ‰„ꢀqƒ‚ˆs‚ˆꢀ
The amount of disulphide groups was calculated as the
difference between sulphydryl groups before and after
reduction of disulphide bridges with sodium sulphite.
The amount of free thiol groups after reduction was
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Temperature program
Relative degree of oxidation
Gel-protein
FIG. 2. The effect of mashing pH on the amount of gel-proteins
in spent grains compared to the HGM procedure with a pH of
5.6. Results are from series of mashings made by using statisti-
cal design.
FIG. 1. Changes in the degree of oxidation and the amount of
gel-proteins in spent grains during mashing. Results are repre-
sentative data from series of experiments (n>30).

For the model hydrolysis experiments 0.25 mL of 1%
(w/v) of freeze dried gel-protein preparation was mixed
with 0.5 mL of appropriate buffer including possible addi-
tives and then the reaction was started by adding 0.25 mL
of endoproteinase extract. Hydrolysis was carried out at
40•C and the reaction was stopped by adding reducing
SDS sample buffer and incubating the mixture for 5 min
in a boiling water bath.
SDS-PAGE on 12% homogeneous gels was performed
and the buffers were prepared according to the manufac-
turers instructions (MiniProteanII, BioRad Laboratories,
Hercules, CA). After the separation of proteins the gels
were stained using 0.06% Coomassie brilliant blue R-250
in 6% trichloracetic acid overnight. The gels were de-
stained in water until the backgrounds were clear and
photographed.
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Oxidation-reduction reactions are known to be influ-
enced by pH. In order to find out what effect a mash pH
has on the formation of gel-protein a series of high-gravity
mashings were conducted under different pH conditions.
The HGM procedure at pH 5.6 was used as a reference.
The amount of gel-protein in spent grains decreased ap-
proximately linearly with mash pH-value (Fig. 2). At pH
4.8 a decrease of 60% in gel-protein content of spent
grains was measured compared to the reference mashing.
This decrease in gel-protein did not accelerate the wort
separation rate (results not shown). Probably the starch
that remains unhydrolysed at low pH conditions has a
stronger adverse effect on wort separation. The decrease
of gel-protein at low pH indicates that endoproteinase ac-
tivity may have an impact on the formation of gel. Cys-
teine endoproteinases (the major class of proteinases in
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In the present study the formation of aggregate (gel-
protein) in the high gravity mashing (HGM) was approx-
imately parallel to the relative degree of oxidation mea-
sured as the oxidation of free thiol groups (Fig. 1). The
formation of gel-protein was quite slow and the oxidation
minimal at the temperature of the first rest (48•C) but dur-
ing and after the saccharification rest (63•C) the mash was
oxidized vigorously and the amount of gel-protein in spent
grains rose rapidly. At the temperatures over 72•C the
oxidation ceased but the amount of gel-proteins kept in-
creasing.
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FIG. 3. The effect of pH on the degree of oxidation in isothermal
mashings at temperatures of 40-70°C (A) and 80-90°C (B). Re-
sults are averages of triple measurements.
Fig. 4. The amount of gel-proteins in spent grains after isother-
mal mashings at different temperatures with addition of 1.5 (A),
11 (B), and 18 (C) mL/L of sulphuric acid.

malt) have their pH optima near 3.8-4.533. Thiol-oxidation
is also pH dependant but as can be seen from Figures 4 A-
C the difference in oxidation with different pH values is
much smaller than the difference in formation of gel-
proteins (Fig. 2).
Moreover, the effect of pH and temperature together on
the oxidation of mash and on the formation of gel-protein
was studied in isothermal mashings at six different tem-
peratures (40-90•C) and with addition of six different
amounts of 0.5 M H2SO4 (0, 1.5, 8, 11, 15 and 18 mL/L).
The achieved pH values of the cold worts varied depend-
ing on mashing temperature (Table I).
tures (Fig. 3A). Oxidation in isothermal mashings (60
min) at temperatures of 40•C and 50•C occurred at a me-
dium rate and was enhanced at low pH levels. At tempera-
tures of 60•C and 70•C oxidation was more rapid than at
lower temperatures but the degree of oxidation was lower
when pH decreased. At a temperature of 80•C the DoO
was small when the pH was low (Fig. 3B). At 90•C the
conditions caused reducing of disulphide bridges at every
pH value. In mashing at 90•C with acid addition of 18
mL/L (pH under 4.6) the separation of wort from solid
particles was so difficult that no analysis could be carried
out. This was probably due to negligible starch hydrolysis.
Present results suggest that the formation of gel-protein
is, at least partly, related to oxidative enzyme activity dur-
The relative degree of oxidation of mash could be di-
vided into three categories according to mashing tempera-
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FIG. 5. The effect of pH and class specific proteinase inhibitors on model digestion of barley gel-proteins by malt endoproteinases.
Hydrolysis products analysed by 12% homogenous SDS-PAGE. A, lane 1, gel-protein preparation, reduced; lane 2, gel-protein prepa-
ration, nonreduced; lane 3, molecular weight standard. B, lane 1, pH 3.8, 0 h; lane 2, pH 3.8, 24 h; lane 3, pH 5.0, 0 h; lane 4, pH 5.0,
24 h; lane 5, pH 6.2, 0 h; lane 6, pH 6.2, 24 h; lanes 7-13, pH 5.0; lane 7, as lane 3; lane 8, as lane 4; lane 9, as lane 8, methanol added;
lane 10, as lane 8, o-phen added; lane 11, as lane 8, pep A added; lane 12, as lane 8, E-64 added; lane 13, as lane 8, PMSF added; lane
14, empty; lane 15, molecular weight standard.
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FIG. 6. The effect of a narrow pH range on model digestion of barley gel-proteins by malt endoproteinases. The conditions were as
described in Materials and Methods and the hydrolysis products were analysed by 12% homogenous SDS-PAGE. Lanes 1-6, pH 4.5;
lanes 7-12, pH 5.0; and lanes 14-19, pH 5.5. Lane 1, 0 h; lane 2, 1 h; lane 3, 2 h; lane 4, 3 h; lane 5, 4 h; lane 6, 24 h; lane 7, 0 h; lane
8, 1 h; lane 9, 2 h; lane 10, 3 h; lane 11, 4 h; lane 12, 24 h; lane 13, molecular weight standard; lane 14, 0 h; lane 15, 1 h; lane 16, 2 h;
lane 17, 3 h; lane 18, 4 h; lane 19, 24 h; lane 20, molecular weight standard.

ing mashing. Most oxygen-scavenging enzymes are able
to catalyse the formation of disulphide bridges. The most
common oxygen scavenging enzymes that are active in
barley malt are peroxidase, catalase, superoxidedismutase
and lipoxygenase.
Catalase and superoxidedismutase are known to be in-
activated rapidly during mashing at 65•C1,7. Peroxidase
can retain its activity even at 80•C3,7. Peroxidases are con-
sidered to act mostly on the oxidation of polyphenols. In
the present experiments the mashing pH and temperature
did not have the same impact on oxidation of polyphenols
as on oxidation of disulphide bridges (results not shown).
It seems that the formation of disulphide bridges during
mashing is mostly related to some other enzyme.
mashings after 1 h at 50•C the degree of oxidation was
over 65% but the amount of aggregate was still 1 mg/g. It
seems that the proteases cleaved either gel-protein sub-
units or the aggregate. The same phenomena could be seen
with the HGM procedure at 63•C and the isothermal mash-
ing at 60•C. At these temperatures degradation of gel-
protein could be enhanced by lowering the pH (Fig. 4 A,
B, C). In isothermal mashings there was a dramatic in-
crease in the amount of aggregate when the temperature
was raised from 60 to 70•C (Fig. 4A,B, C). At the mash-
ing temperatures of 72•C in HGM and 70•C in isothermal
mashings the relative degree of oxidation was ca. 80% and
the amount of gel-protein was near its maximum at 8-9
mg/g. No effect of proteolysis could be seen. At tempera-
tures over 80•C there was less oxidation and the formation
of gel-protein aggregate was minor. Although proteolysis
at such a high temperature is negligible, denaturation and
precipitation of proteins also diminishes the amount of
aggregate.
Kaukovirta-Norja14 found a correlation between lipoxy-
genase activity of malt and the wort separation. This effect
may also be related to oxidation of free thiol groups of
gel-protein17. The function of lipoxygenase (LOX) during
mashing is rather controversial2,16. Lipoxygenase is sug-
gested to be heat labile because only a minor part of the
activity present in green malt survives kilning. However,
this remaining part of LOX is known to be very stable
towards heating. According to Baxter4 almost 60% of the
LOX activity of malt extract survived for 1 h at a tempera-
ture of 67•C. Recent results also indicate that LOX may
survive temperatures as high as 95•C in spent grains31. In
addition, Doderer8 reported that lipoxygenase purified
from germinating barley has a pH optimum at 6.5. This is
in agreement with the present results where oxidation in-
creased as the pH increased from 4.5 to 6. The effect of
pH higher than 6 has not been studied in this experiment.
The enhancement of proteolysis by low pHs and its pos-
sible effect on the amount of gel-protein aggregate can be
seen by comparing Figs. 1 and 4 A, B and C. In the HGM
procedure after the first 30 min at 48•C the relative degree
of oxidation was ca 20% and the amount of gel-protein
aggregate was ca. 1 mg / g spent grains. In isothermal
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The final amount of the gel-protein aggregate after
mashing depends highly on the balance between formation
and hydrolysis of gel-protein. In order to find out the real
impact of protein hydrolysis on the amount of gel-protein
a series of model experiments was made with barley gel-
protein. Barley gel-protein was chosen for the substrate
because in the reducing conditions during malting the
amount of gel-material diminishes and purification of the
protein is quite difficult.
The gel-protein fraction of barley consisted mainly of
proteins with molecular weights of 98 kDa and 40-55 kDa
(Fig. 5A lane 1). According to Smith26,27 these are D and B
hordeins. When this fraction was electrophoresed under
non-reducing conditions none of the protein could pene-
trate into the gel indicating a polymeric nature (Fig. 5A
lane 2). The protein band between 28.3 kD and 34.6 kD
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
MW
104,000
81,000
47,700
34,600
28,300
19,200
FIG. 7. The effect of heat treatment on model digestion of barley gel-proteins at pH 5.0 by malt endoproteinases. Prior to the hydrolysis
reaction, the enzyme extracts were pre-incubated for 15 min at temperatures shown below. The reaction conditions were as described in
Materials and Methods and the hydrolysis products were analysed by 12% homogenous SDS-PAGE. Lane 1, no treatment, 0 h; lane 2, no
treatment, 24 h; lane 3, 60°C, 0 h; lane 4, 60°C, 24 h; lane 5, 65°C, 0 h; lane 6, 65°C, 24 h; lane 7, 70°C, 0 h; lane 8, 70°C, 24 h; lane 9,
80°C, 0 h; lane 10, 80°C, 24 h; lane 11, 90°C, 0 h; lane 12, 90°C, 24 h; lane 13, 100°C, 0 h; lane 14, 100°C, 24 h; lane 15, molecular
weight standards.

molecular weight markers (for example lane 1gel 5B)
which is visible in Figs. 5-7 is not derived from the sub-
strate gel-protein but from the enzyme extract. The gel-
protein preparation was incubated at 40•C for 24 h with
proteinase mixture extracted from malt. These hydrolysis
reactions were carried out at three different pH values (pH
3.8, 5.0 and 6.2). Different patterns of hydrolysis were
detected (Fig. 5). The D hordein was hydrolyzed at all of
the pH values (Fig. 5B lanes 1-6). At pH 3.8 some of the
B hordein was not hydrolysed (Fig. 5B lanes 1,2). This
was also the case at pH 6.2 although not as clearly as at
pH 3.8 (Fig. 5B lanes 5,6). At pH 5.0 no proteins after the
incubation were detectable (Fig. 5B lanes 3,4).
dent on the pH and the temperature of the mashing. The
different pH optima of the oxidation in different tempera-
tures implied that the oxidation of the thiol groups to di-
sulphide bridges maybe a result of enzymatic activity,
possibly lipoxygenase activity.
Gel-protein hydrolysis was found to have a clear pH
optimum near pH 5.0 according to model experiments.
The same result was not observed in mashing experi-
ments. Instead the amount of gel-protein decreased further
when the pH was lowered under 5.0. Since the proteolysis
of gel-protein subunits (D- and B-hordeins) by cysteine
proteinases has a pH optima near 3.8-4.5 it seems that the
proteolysis of D- and B-hordeins has a stronger effect on
the hydrolysis-formation -balance of the gel-protein dur-
ing mashing than the proteolysis of gel-protein aggregate
(pH optimum at 5.0). Also the oxidizing conditions during
mashing may have an effect on the proteolytic activity of
cysteine endoproteinases and alter their characteristics.
There was also a difference between the thermal inacti-
vation of enzymes that hydrolyse gel-protein. In model
experiments thermal inactivation happened only at tem-
peratures over 80•C while in mashing experiments no pro-
teolysis could be observed after 70•C. It is possible that
the extraction of the malt enzymes in model experiments
may offer some protection against thermal inactivation.
As a conclusion the balance between formation and hy-
drolysis of gel-protein aggregate appears to be difficult to
control. It is not possible to adjust the pH value low
enough to accelerate gel-protein hydrolysis without caus-
ing inefficient starch hydrolysis. Lengthening the time of
the proteolysis rest is also not suitable because it causes
formation of excess FAN. Instead, the level of oxygen can
be adjusted without adverse effects. The amount of the
gel-protein aggregate can be diminished by preventing oxi-
dation and furthermore the wort separation is improved.
In order to find out the nature of the proteinases par-
ticipating in the hydrolysis of the gel-protein the class-
specific proteinase inhibitors were added into the reaction
mixtures at pH 5.0. Only minor effects were detected (Fig.
5B lanes 7-14). O-phenanthroline (metalloproteinase in-
hibitor, Fig. 5B, lane10), PMSF (serine proteinase inhibi-
tor, lane 13) and pepstatin A (aspartic proteinase inhibitor,
lane 11) had no effect on the hydrolysis products. How-
ever, E-64 a cysteine proteinase inhibitor partially inhib-
ited the hydrolysis (lane 12) suggesting a role of cysteine
proteinases in the reaction. This is in agreement with ear-
lier results showing that cysteine proteinases most prob-
ably are responsible for the bulk hydrolysis of the hordeins
during barley germination15,22,23
.
To narrow the area of the pH optimum further, tests
were then conducted using shorter incubation periods;
samples were taken at 1h intervals up to 4 h at pH 4.5, 5.0
and 5.5 (Fig. 6). The hydrolysis proceeded fastest at pH
5.0 (Fig. 6 lanes 7-12), slower at 4.5 (Fig. 6, lanes 1-6)
and most slowly at pH 5.5 (Fig. 6 lanes 14-19). Further
analyses were performed at pH 5.0 since it was the opti-
mum pH for the gel-protein hydrolysis and this is also
within the area of pH 4.9-5.1, which has been reported to
be the pH of germinating barley endosperm18.
To study the thermal stability of the proteinases the en-
zyme extract was pre-incubated for fifteen min at different
temperatures prior to hydrolysis reactions (Fig. 7). Heat-
ing at 50, 60, 65 or 70•C did not alter the extent of hy-
drolysis (Fig. 7 lanes 1-8). Pre-incubation at 80•C did af-
fect the hydrolysis, because all of the substrate-derived
protein bands were visible after the hydrolysis but the in-
tensity of the bands was lower than in the beginning (Fig.
7 lanes 9,10). Pre-incubation at 90 or 100•C inactivated
the proteinases totally so that the substrate polypeptides
were intact after the incubation (Fig. 7 lanes 11-14).
Addition of an oxidizing agent, hydrogen peroxide, par-
tially inhibited the hydrolysis (results not shown) which
would be logical since the catalytic activity of cysteine
proteinases requires that their active site cysteines are re-
duced.
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(Manuscript accepted for publication May 2002)