ARTICLE IN PRESS

Journal of Cereal Science 44 (2006) 203211

www.elsevier.com/locate/yjcrs

The effects of malting and mashing on barley protein extractability

Inge Celus, Kristof Brijs, Jan A. Delcour
Laboratory of Food Chemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium
Received 24 March 2006; received in revised form 31 May 2006; accepted 1 June 2006

Abstract
Proteins in unmalted and malted barley and in brewers spent grain (BSG) obtained after mashing were fractionated on the basis of
their differential extractability in different media and characterised by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and highperformance liquid chromatography (HPLC). Albumins and globulins were rst extracted with 5.0% NaCl and hordeins (barley
prolamins) were extracted with 55.0% 1-propanol in the presence, or absence, of 1.0% DTT. Glutelins were then extracted with 2.0%
SDS/6.0 M urea/1.0% DTT or with 55.0% 1-propanol/6.0 M urea/1.0% DTT/0.036 M Tris-HCl (pH 8.4). Under non-reducing
conditions, monomeric C hordeins and some B hordeins were extracted from unmalted barley, whereas most if not all B, C and D
hordeins were extracted under reducing conditions. During malting, disulde bonds are reduced and B and D hordeins are broken down
by proteolysis. No D hordeins were extracted from malt and nearly the same levels of malt B hordeins were extracted both under nonreducing and reducing conditions. B hordeins present in BSG proteins were only extractable under reducing conditions. Whereas most of
the C hordeins were extracted from BSG under non-reducing conditions, more C hordeins were extracted under reducing conditions.
Mashing probably induced disulde bond formation resulting in aggregation. Although earlier literature suggested the formation of an
aggregate composed of B and D hordein (and glutelin) during mashing, the present work suggests the formation of an aggregate
composed of B hordeins in which C hordeins are entrapped.
r 2006 Elsevier Ltd. All rights reserved.
Keywords: Protein extractability; Barley; Malt; Brewers spent grain

1. Introduction
Barley (Hordeum vulgare L.) is a major food and animal
feed crop. It ranks fourth in area of cultivation of cereal crops
in the world. An important application of barley is as the raw
material for malting and subsequently production of beer.
Malting includes the controlled germination of barley in
which hydrolytic enzymes are synthesized and the cell walls,
proteins and starch of the endosperm are largely digested,
making the grain more friable (Bamforth and Barclay, 1993;
Abbreviations: ACN, acetonitrile; AX, arabinoxylan; BSG, brewers
spent grain; db, dry basis; DTT, dithiothreitol; Hcl, hordein crosslinking;
HMW, high molecular weight; HPLC, high-performance liquid chromatography; LMW, low molecular weight; MW, molecular weight; RP,
reversed phase; RT, room temperature; SDS, sodium dodecyl sulfate;
SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SE, size exclusion;
TFA, triuoroacetic acid; Tris: tris(hydroxymethyl)aminomethane.
Corresponding author. Tel.: +32 16321634; fax: +32 16321997.
E-mail address: inge.celus@biw.kuleuven.be (I. Celus).
0733-5210/$ - see front matter r 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.jcs.2006.06.003

Enari and Sopanen, 1986). In brewing, after malt mashing

and separation of the wort, brewers spent grain (BSG) is
obtained as the barley malt residue. The composition of BSG
varies with barley variety, time of harvest, adjuncts added and
brewing technology (Santos et al., 2003). To date, BSG has
been mainly used as an animal feed (Mussatto et al., 2006).
Barley contains 1012% proteins [dry basis (db);
MacGregor and Fincher, 1993], which can be classied
by their sequential extractabilities by the procedure
introduced by Osborne (1924). The hordein fraction,
extracted with alcoholic media in the presence of a
reducing agent, comprises 3555% of the total barley
grain proteins and is the main barley storage protein
(Shewry, 1993). Barley hordeins are divided into ve
groups based on their electrophoretic mobilities and amino
acid compositions: the B and C hordeins (7080% and
1020% of the hordein fraction, respectively) and the D
and g hordeins (less than 5% of the total hordein fraction)
(Shewry, 1993; Tatham and Shewry, 1995). The A

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hordeins, the smallest polypeptides, average molecular

weight (MW) 15 k, may be alcohol-soluble albumins or
globulins or breakdown products of larger hordeins rather
than true hordeins (Baxter, 1981). The B hordeins can be
subdivided into B1, B2 and B3 subtypes (Skerritt and
Janes, 1992). Furthermore, a distinction is made between
the sulfur-rich (B and g hordeins), the sulfur-poor (C
hordeins) and high molecular weight (HMW) prolamins (D
hordeins) (Shewry, 1993; Shewry and Tatham, 1990;
Shewry et al., 1994; Tatham and Shewry, 1995). C, as well
as some B hordeins, appear as monomers, while most B
and D hordeins are linked by interchain disulde bridges.
Most g hordeins are monomers with intrachain disulde
bonds, but polymeric types may also occur (Sheehan and
Skerritt, 1997; Shewry, 1993; Shewry and Tatham, 1990;
Shewry et al., 1994; Tatham and Shewry, 1995). Barley gel
protein is a polymer containing HMW and low molecular
weight (LMW) subunits, the D and B hordeins, respectively (Smith and Lister, 1983; Smith and Simpson, 1983).
It has been hypothesized by Moonen et al. (1987) that in
barley, HMW subunits form a backbone, which binds
LMW subunits through disulde bridges to form a gel-like
aggregate. Hordein extraction with increasing concentration of sulphydryl reducing agents revealed that D hordeins
are extracted only at the highest 2-mercaptoethanol
concentration, suggesting that they form the gel protein
backbone (Skerritt and Janes, 1992).
Although barley glutelins are traditionally extracted with
dilute acid or alkali, it is now more common to use a
detergent [e.g. sodium dodecyl sulfate (SDS)] and/or
a chaotropic agent (e.g. urea), often in the presence of a
reducing agent. It has not been possible to prepare an
undenatured glutelin fraction totally free of contaminating
hordein. The non-hordein components tend to contain
HMW and some LMW components (Shewry, 1993). Few,
if any of the barley glutelins, have been characterized in
detail.
During malting, barley proteins are in part degraded to
amino acids and small peptides by a range of proteolytic
enzymes (Baxter, 1981; Enari and Sopanen, 1986; Jones,
2005). Well-modied malt contains less than half the
amount of hordeins present in the original barley (Baxter,
1981). The D hordeins are degraded more rapidly than
their B-type counterparts, and the latter are more rapidly
degraded than the C hordeins (Marchylo et al., 1986;
Tatham and Shewry, 1995; Wallace and Lance, 1988).
BSG, the main by-product of the brewing industry, is
rich in proteins and dietary ber (Mussatto et al., 2006).
BSG solids rich in protein have been extracted with
alkaline (Ernster, 1986; Frieden et al., 1957) or SDS (Chen
et al., 1993) solutions and the extracted proteins precipitated to yield a highly puried protein solid. Detergent
solutions solubilized more than 80% of the total nitrogen
of BSG. The low solubility of the nitrogenous constituents
in conventional protein solvents may be due to association
between cellulosic material and protein (Crowe et al.,
1985). During mashing, a complex is formed between

residual gel protein in the malt and the glutelins. As a

result, an impenetrable layer is formed on the spent grains
(Moonen et al., 1987). Disulde bridges between hordeins
can be formed causing mash separation difculties (Baxter,
1981). The oxidation of free protein thiol groups to
disulde bridges during mashing appears to be a good
indicator of the formation of a gel protein aggregate (Poyri
et al., 2002).
There is little information about the residual barley
proteins present in BSG and their interactions with other
BSG polymers. Moonen et al. (1987) suggested that during
malting, disulde bonds in the gel protein were reduced
and the subunits were proteolytically degraded. Poyri et al.
(2002) demonstrated the formation of a gel protein
aggregate by oxidation of free thiol groups of proteins
to disulde bridges during mashing. More research is
required to understand how the gel protein is disaggregated
during malting and how the aggregates form during
mashing.
To understand the effect of malting and mashing on
barley protein extractabilities we determined the protein
composition of barley, its corresponding malt and the
resulting BSG. The protein fractions were characterised
using size exclusion-high performance liquid chromatography (SE-HPLC) in combination with SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) and reversed phase-high
performance liquid chromatography (RP-HPLC). The
results will be used as a basis for the development of
further uses for BSG proteins. This may allow the selection
of enzymes with potential to generate BSG protein
hydrolysates with improved functional (health promoting
and technological) properties.

2. Materials and methods

2.1. Materials
Barley (cv. Scarlett) and its corresponding malt were
from Cargill Malt (Herent, Belgium). An all-malt mash
was made in a pilot-scale brewery. The malt (82 kg) was
milled with a DLFU disc mill (setting 1.2 mm; BuhlerMiag, Braunschweig, Germany) and mashed in 500 l of
water at 59 1C. The mash pH was adjusted to 5.5 with
1.0 M lactic acid. During brewing, the following temperaturetime prole was followed: 20 min at 58 1C, increase
to 62 1C (1 1C/min); 45 min at 62 1C, increase to 72 1C
(1 1C/min); 30 min at 72 1C, increase to 78 1C (1 1C/min)
and was then typically held at this temperature for 10 min.
The wort was separated from the insoluble BSG in a
traditional lauter tun. The BSG was oven-dried (60 1C;
18 h) (Santos et al., 2003).
MW markers for gel electrophoresis were from GE
Healthcare (Uppsala, Sweden). All other chemicals were
from Sigma-Aldrich (Bornem, Belgium) and were of
analytical grade or better.

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2.2. Grinding of barley, malt and BSG

Barley and malt were ground with a Cyclotec 1093
sample mill (Tecator, Hoganas, Sweden). After drying,
BSG was ground in a universal mill (IKA Labortechnik,
Staufen, Germany) and sieved (250 mm).
2.3. Analysis of barley, malt and BSG
Moisture and ash contents were determined according to
AACC methods 44-15A and 08-12 (AACC, 2000),
respectively. Protein was determined using the Dumas
combustion method, an adaptation of the AOAC Ofcial
Method (AOAC, 1995) to an automated Dumas protein
analysis system (EAS, varioMax N/CN, Elt, Gouda, The
Netherlands), using 6.25 as the conversion factor. Monosaccharide compositions of barley, malt and BSG were
determined by the method of Englyst and Cummings
(1984). Arabinoxylan (AX) content was calculated as 0.88
times the sum of xylose and arabinose after correction of
the arabinose content for the presence of arabinogalactanpeptide based on an arabinose to galactose ratio of 0.7
and assuming that all the arabinose of arabinogalactan
peptide is present in the aqueous extract (Loosveld et al.,
1997). (1-3;1-4)-b-Glucan and total starch contents
were determined by the AACC methods 32-23 and 76-13
(AACC, 2000), respectively. (1-3;1-4)-b-Glucan and
total starch contents in malt and BSG were determined
after pretreatment with aqueous ethanol to remove glucose
and maltosaccharides as by AACC methods 32-23 and 7613, respectively.
2.4. Modified Osborne fractionation of barley, malt and
BSG proteins
Barley, malt and BSG [100 mg protein (db)] were
extracted twice with 3.0 ml of solvent A (Table 1) at room
temperature (RT). The residues were washed with 3.0 ml
deionized water. The supernatants and washing water
containing the water-soluble albumins and salt-soluble
globulins were combined. Hordeins in the residues were
extracted three times with 3.0 ml of solvent H1 or H2
(Table 1) at 60 1C. Glutelins in the residues after hordein
extraction under non-reducing or reducing conditions were
Table 1
Solvents used in the modied Osborne protein extraction of barley, malt
and brewers spent grain

205

extracted three times with 3.0 ml of solvent G1 or G2 at RT

(Table 1).
Each extraction step started by vortex mixing for 2 min
at RT and subsequent shaking for 10 min at RT (A, G1 and
G2 extracts) or 60 1C (H1 and H2 extracts). The suspensions were centrifuged (1500 g; 10 min; RT), the supernatants combined and diluted to 10.0 ml with the respective
extraction solvents.
Glutelins extracted with solvent G1 after hordein
extraction with solvent H1 or H2 are referred to as
G1(H1) and G1(H2) extracts, respectively. The G2(H1)
and G2(H2) extracts refer to glutelins extracted with
solvent G2 after hordein extraction with solvent H1 or
H2, respectively.
2.5. Protein determination of the protein fractions
The protein content of fractions from barley, malt and
BSG was determined as described in Section 2.3 and the
yield of extracted protein as (%) dry weight of the starting
material. The yields of protein in the G1 and G2 extracts,
which contained urea and/or Tris, were determined by SEHPLC. Their extraction yields were calculated from their
SE-HPLC peak areas (Section 2.6) based on the relationship between the SE-HPLC peak areas and the yields in the
respective H2 extracts. Hordein crosslinking (Hcl) (%) was
calculated from the ratio of DH, the difference in extraction
yield between the H2 and H1 extracts, to the extraction
yield of the H2 extract.
2.6. Size exclusion-high-performance liquid chromatography
SE-HPLC was conducted using a LC-2010 system
(Shimadzu, Kyoto, Japan) with automatic injection.
Aliquots of barley, malt and BSG protein fractions were
ltered through a 0.45 mm membrane (regenerated cellulose; Alltech Associates, Inc., Deereld, Massachusetts,
USA) before loading (60 ml) on a Biosep-SEC-S4000
column (Phenomenex, Torrance, CA, USA). The hordeins
extracted with solvents H1 and H2 were diluted (1:10)
before loading on SE-HPLC. The elution solvent was (1:1,
v/v) acetonitrile (ACN)/water containing 0.05% (v/v)
triuoroacetic acid (TFA). Chromatography conditions
were: ow rate 1.0 ml/min, temperature 30 1C (Veraverbeke
et al., 2000) with protein detection at 214 nm. SE-HPLC
peak fractions of the H1 and H2 extracts from barley were
collected and freeze-dried.
2.7. Reversed phase-high-performance liquid
chromatography

Solvent
A
H1
H2
G1
G2

5.0% (w/v) NaCl

55.0% (v/v) 1-propanol
55.0% (v/v) 1-propanol/1.0% (w/v) DTT
2.0% (w/v) SDS/6.0 M urea/1.0% (w/v) DTT
55.0% (v/v) 1-propanol/6.0 M urea/1.0% (w/v) DTT/0.036 M
Tris-HCl (pH 8.4)

Aliquots of the H1, H2 and G2 extracts of barley, malt

and BSG were ltered through a 0.45 mm membrane
(regenerated cellulose; Alltech Associates, Inc.) and loaded
(80 ml) on a Nucleosil 300-5 C8 column (Machery-Nagel,
Duren, Germany). The elution system consisted of
deionized water containing 0.1% (v/v) TFA (solvent 1)

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206

and ACN containing 0.1% TFA (v/v) (solvent 2). Proteins

were eluted with a linear gradient from 24.0% to 56.0%
solvent 2 in 50 min and detected at 214 nm (Wieser et al.,
1998).

20
18

H
16

2.8. SDS-polyacrylamide gel electrophoresis

3. Results and discussion

3.1. Partial compositions of barley, malt and BSG
The partial compositions of barley, malt and sieved BSG
are shown in Table 2. The sieved BSG fraction contained
the same proteins as the starting material (results not
shown), but at higher levels [26.7% (db)]. BSG contained
much less starch than the barley and malt and its protein,
AX and ash contents were much higher. Approximately
65% of the malt proteins were retained in the BSG while
ca. 35% were present in the wort.
3.2. Yields of extracted protein fractions of barley, malt and
BSG
The yields for the different Osborne protein fractions of
barley, malt and BSG expressed as the percentage of the
initial total dry material are shown in Fig. 1. Higher
amounts of albumins and globulins were extracted from
malt than from barley, whereas, as expected, only low
Table 2
Partial compositions [% (db)] of barley, malt and brewers spent grain
(BSG) (passed through a 250 mm sieve)
Component [% (db)]

Barley

Malt

BSG (o250 mm fraction)

Protein
Arabinoxylan
(1-3;1-4)-b-Glucan
Total starch
Ash

10.1
6.5
4.2
53.5
1.9

10.4
7.0
0.1
46.5
1.9

26.7
22.5
0.3
1.0
3.3

14
Extraction yield (%)

The protein fractions (100 ml) and freeze-dried peak

fractions were mixed with sample buffer (20 and 100 ml,
respectively) containing 17.7 mM Tris-HCl (pH 6.8), 1.0%
(w/v) SDS, 20.0% (w/v) sucrose, 5.0% (v/v) b-mercaptoethanol and 0.01% (w/v) Bromophenol Blue. Samples were
shaken overnight (4 1C), held in boiling water for 5 min and
centrifuged (11, 000 g; 5 min; RT).
Samples (40 ml) were subjected to SDS-PAGE in a SE
600 Series gel electrophoresis unit (Hoefer, GE Healthcare). The slab gels (14.0  16.0  1.5 cm) consisted of
stacking (3.88% T and 1.33% C) and running gels (17.57%
T and 0.455% C). Gels were run at 18 1C and 30 mA.
Separation was stopped when the tracking dye ran off the
gel. Gels were stained with 0.025% Coomassie Brilliant
Blue R250 in 40.0% methanol containing 7.0% acetic acid
and destained with a solution containing 5.0% methanol
and 7.0% acetic acid.

G1(H2)

H1
A

12
10
8
6
4
2
0
Barley

Malt

BSG

Fig. 1. Yields (%) of dry material of sequentially extracted barley, malt

and brewers spent grain (BSG) protein fractions. The solvents used to
extract the albumins and globulins (A extract), the H1 hordeins and the
G1 glutelins are listed in Table 1. DH represents the difference in
extraction yield between the H2 and H1 extract (see text).

amounts were present in BSG. For barley, the amount of

hordeins extracted under reducing conditions was higher
than that extracted under non-reducing conditions, in
agreement with the presence of disulde linkages in barley
hordeins. Under reducing conditions, more barley hordeins
were extracted at 60 1C than at RT (results not shown).
Malt apparently contained less hordeins than barley, and
the amount was not inuenced by the presence of reducing
agents. This suggests that the apparently higher amounts of
albumins and globulins in malt compared to barley may be
due not only to de novo synthesis of a number of proteins
during malting, but also to the formation of hordein
fragments which are extractable in dilute salt solution
(Section 3.3). The yield of hordeins from BSG (ca. 11.5%)
exceeded that of barley (ca. 4.5%) and malt (ca. 2.5%). The
apparent hordein cross-linking (Hcl) is lower in malt (ca.
15%) than in barley (ca. 39%) which indicates degradation
and/or reduction of disulde-linked hordeins during
malting. Hordeins accounted for ca. 43% of both the
barley and BSG proteins when extracted under reducing
conditions. Under such conditions, more hordeins were
extracted from BSG than under non-reducing conditions
which may indicate the presence of disulde bonds in BSG
proteins. Furthermore, the higher Hcl of BSG (ca. 58%)
than for malt (ca. 15%) suggests that disulde linkages in
BSG hordeins are formed during mashing.
As expected, more glutelin was extracted after H1 than
after H2 extraction (results not shown). Furthermore, more
glutelin was extracted with solvent G1 than with solvent
G2 (results not shown). While comparable levels of glutelin
were extracted from barley and malt, more glutelin was

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extracted from BSG. When expressed as a percentage of

protein, the levels of hordeins extracted from barley and
BSG under reducing conditions were comparable, while
more glutelin was subsequently extracted from BSG (ca.
22%) than from barley (ca. 14%). In general, more
proteins remained unextracted in BSG and malt than in
barley. Whereas ca. 72% of the proteins of BSG were
extracted with solvents A, H2 and G1, extraction with
solvent G2 in combination with solvents A and H2 yielded
only ca. 54% of the proteins. Solvent G1 extracted more
proteins than solvent G2, probably due to the presence of
SDS in the former.
3.3. SDS-PAGE of the protein fractions of barley, malt and
BSG
3.3.1. Hordein fraction
Hordeins extracted with solvents H1 and H2 were
analyzed with SDS-PAGE (Fig. 2). Protein bands were
assigned to various subfractions based on published
classications (Bilgi and C
- elik, 2004; Howard et al., 1996;
Shewry et al., 1978). The B hordeins have been subdivided
into B1, B2 and B3 hordeins (Janes and Skerritt, 1993). The
g hordeins are classied as B hordeins as both types
migrated similarly in SDS-PAGE (Howard et al., 1996;
Shewry, 1993). Whereas the C (between 55 and 80 k) and A
hordeins (smaller than 20 k) were completely extracted
from barley with 55.0% 1-propanol (Fig. 2; lane b), B
hordeins (between 35 and 50 k) were almost completely

207

extracted from barley under non-reducing conditions. The

addition of 1.0% dithiothreitol (DTT) resulted in the
extraction of all B (between 35 and 50 k) and D hordeins
(96 k) (Fig. 2; lane e). The C hordeins were present as six
bands and were divided into C hordeins with LMW and C
hordeins with HMW, while the B hordeins appeared as a
broad band of B3 hordeins and two smaller bands of B2
and B1 hordeins with lower MW (Fig. 2; lane b). Malt
hordeins extracted with solvent H1 had reduced amounts
of C hordeins of higher MW, lower amounts of B hordeins
(in particular, no B1 hordeins were present), and lower
amounts of A hordeins than barley (Fig. 2; lane c). During
malting, D hordeins were degraded (Fig. 2; lane f). In BSG
(Fig. 2; lanes d and g), two bands of C hordeins with higher
MW were present, as several bands of C hordeins with
lower MW were visible. Lower amounts of B hordeins,
particularly B1 and B2 hordeins, and no D hordeins were
extracted from BSG. More B3 hordeins were extracted
from BSG under reducing than under non-reducing
conditions.
3.3.2. Glutelin fraction
SDS-PAGE patterns of barley glutelins extracted with
solvents G1 and G2 after H1 extraction, showed the
presence of D and B hordeins (Fig. 3; lanes b and f). Only
low proportions of D and B hordeins were present in the
malt glutelin extracts (Fig. 3; lanes c and g), whereas no D
hordeins and more B hordeins were present in the BSG
extracts (Fig. 3; lanes d and h). Barley glutelins extracted
after previous hordein extraction under reducing conditions showed some residual D, C and B hordeins and also

k
D
94

67
43

94

B3

67

B2
B1

43

30
30

20.1
20.1

A
14.4
a

14.4

h
a

Fig. 2. SDS-PAGE of hordein fractions of barley (lanes b and e), malt

(lanes c and f) and brewers spent grain (lanes d and g). Lanes bd
represent hordeins extracted with solvent H1, while lanes eg represent
hordeins extracted with solvent H2 (Table 1). Molecular weight markers
are shown in lanes a and h and the position of A, B (and subfractions B1,
B2 and B3), C and D hordeins are indicated.

Fig. 3. SDS-PAGE of glutelin fractions of barley (lanes b, f and i), malt

(lanes c, g and j) and brewers spent grain (lanes d, h and k). Lanes bd
represent G1(H1) extracts, lanes fh represents G2(H1) extracts and lanes
ik represent G1(H2) extracts (Table 1). Molecular weight markers are
shown in lanes a, e and l.

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208

some other protein bands (Fig. 3; lane i), while those

extracted from malt and BSG showed no clear protein
bands (Fig. 3; lanes j and k).

k
D

94

3.4. HPLC of barley protein fractions

3.4.1. Hordein fraction
The protein peak of barley hordeins extracted with 55%
1-propanol under reducing conditions was more prominent
than that resulting from extraction under non-reducing
conditions (Fig. 4a). SDS-PAGE of the peak fractions of
the SE-HPLC proles showed that the main peak (89 min)
in the SE-HPLC prole (Fig. 4a) contained B and C
hordeins (Fig. 5; lanes b and d), while a small peak of D
hordeins eluting at 78 min was observed only when the
extracts were made under reducing conditions (Fig. 5; lane
c). The main peak (89 min) from the H2 extract contained
all B and C hordeins (Fig. 5; lane d), and the H1 extract
had the same amount of C hordeins but less B hordeins
(Fig. 5, lane b). The apparent average MW of proteins in
the SE-HPLC prole of the H2 extract (Fig. 4a) was
somewhat lower than that of the H1 extract because more
B hordeins and the same amount of C hordeins were

Absorbance at 214 nm (AU)

1
0.8
0.6
0.4
0.2
0
5

(a)

8
Time (min)
C

10

11

Absorbance at 214 nm (AU)

43

30

20.1
A
14.4

Fig. 5. SDS-PAGE of SE-HPLC peak fractions from the H1 extract (lane

b) and H2 extract (lanes c and d) from barley (Table 1). Lane c represents
the rst small peak (78 min), while lane d represents the second large peak
(89 min). Molecular weight markers are shown in lanes a and e and the
position of AD hordeins are indicated.

extracted under reducing conditions and the B hordeins

have a lower MW than the C hordeins (Fig. 2).
RP-HPLC separated the extracted hordeins into different groups (Fig. 4b). Extraction under reducing conditions
yielded three groups of fractions. Whereas under nonreducing conditions the fractions corresponding to the C
hordeins remained similar, lower amounts of B hordeins
and almost no D hordeins were extracted. The B hordein
peak extracted under reducing conditions appeared in six
fractions that fell into two groups and a smaller group of
fractions eluting late in the chromatogram, which possibly
contained some g hordeins (Janes and Skerritt, 1993)
(Fig. 4b). D hordeins were extracted under reducing
conditions at 60 1C, but not at RT (results not shown).

0.8
0.6
0.4
0.2
0
10

(b)

67

15

20

25
30
Time (min)

35

40

45

Fig. 4. SE-HPLC (a) and RP-HPLC (b) proles of barley hordeins

extracted with 55.0% 1-propanol (
) and with 55.0% 1-propanol in the
presence of 1% DTT (
). Elution times of molecular weights markers
with following molecular weights are given on SE-HPLC with crosses:
232, 158, 43 and 13.7 k. The D, C, B and g hordeins are indicated on RPHPLC. Absorbance expressed in arbitrary units (AU).

3.4.2. Glutelin fraction

After H1 extraction, glutelins were extracted with
solvents G1 or G2. The RP-HPLC prole of the G2(H1)
extract showed the presence of both D and B hordeins
(Fig. 6).
The results described in Sections 3.4.1 and 3.4.2 showed
that it is possible to fractionate the B, C and D hordeins by
RP-HPLC. After extraction of albumins and globulins, C
hordeins (and some B hordeins) can be extracted with 55%
1-propanol at RT. B hordeins are then extracted with 55%
1-propanol in the presence of 1% DTT at RT. In the next
step, the D hordeins are extracted with solvent G2 or with
55% 1-propanol in the presence of 1% DTT at 60 1C.

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D

disulde-linked B and D hordeins are degraded enzymically. SDS-PAGE showed that mainly the B1 (and B2)
hordeins were degraded (Fig. 2). This is in agreement with
the results of Marchylo et al. (1986) and Weiss et al. (1992)
that D hordeins are degraded most rapidly, followed by the
B and C hordeins.

1
Absorbance at 214 nm (AU)

209

0.8
0.6
0.4
0.2

3.6. HPLC of BSG protein fractions

0
10

15

20

25

30

35

40

45

Time (min)

Fig. 6. RP-HPLC prole of barley glutelins extracted with 55.0% (v/v) 1propanol/6.0 M urea/1.0% (w/v) DTT/0.036 M Tris-HCl (pH 8.4) after
hordeins have been extracted under non-reducing conditions. The D and B
hordeins are indicated on RP-HPLC. Absorbance expressed in arbitrary
units (AU).

Absorbance at 214 nm (AU)

1
0.8
0.6
0.4
0.2
0
10

15

20

25
30
Time (min)

35

40

45

Fig. 7. RP-HPLC prole of malt hordeins extracted with 55.0% 1propanol (

) and with 55.0% 1-propanol in the presence of 1% DTT
(
). The C, B and g hordeins are indicated on RP-HPLC. Absorbance
expressed in arbitrary units (AU).

The RP-HPLC prole of the hordeins extracted from

BSG under non-reducing conditions showed only C
hordeins (Fig. 8). Most of the C hordeins were extracted
under non-reducing conditions, while more C hordeins
could be extracted under reducing conditions. Under
reducing conditions, B hordeins were also extracted, but
no D hordeins. As was the case for barley and malt, the B
hordeins appeared in two groups of fractions and in a small
more hydrophobic fraction, the g hordeins (Fig. 8). As for
malt hordeins, a peak eluting at 4142 min (Fig. 8) was
detected. Since no B hordeins were extracted under nonreducing conditions, this probably indicates that all B
hordeins present in BSG are in the form of a disuldelinked aggregate. SE-HPLC proles of the BSG glutelins
extracted after the H1 extract showed no peak corresponding to the D hordeins, a large peak containing C and B
hordeins and an additional peak of lower MW (results
not shown). The latter may well represent B hordeins that
are partly dissociated during malting but re-aggregate
during mashing. These aggregated proteins were not
removed in the wort but remained in the BSG fraction.
The RP-HPLC prole of the glutelins (Fig. 9) showed the
presence of B and some C hordeins as well as proteins coeluting with the C hordeins which were not present in the
H1 extract. During mashing, an aggregate of B hordeins
in which some C hordeins are entrapped may have been
formed.

3.5. HPLC of malt protein fractions

The RP-HPLC prole of malt hordeins extracted under

non-reducing conditions was very similar to that for barley
(Fig. 7). Under reducing conditions, the peak corresponding to the B hordeins appeared in two groups of fractions
and a small more hydrophobic fraction of the g hordeins.
In contrast to barley, extraction of malt under both
reducing and non-reducing conditions showed a distinct
peak at 4142 min (Fig. 7) corresponding to hydrophobic
proteins with MW lower than 40 kDa as revealed by SDSPAGE (results not shown). Although the amount of B
hordeins extracted under reducing conditions was almost
the same as that obtained by extraction under nonreducing conditions, no D hordeins were extracted under
reducing conditions. This implies that, during malting,
disulde bonds in B and D hordeins are reduced and/or

Absorbance at 214 nm (AU)

0.8
0.6
0.4
0.2
0
10

15

20

25
30
Time (min)

35

40

45

Fig. 8. RP-HPLC prole of brewers spent grain hordeins extracted with

55.0% 1-propanol (
) and with 55.0% 1-propanol in the presence of 1%
DTT (
). The C, B and g hordeins are indicated on RP-HPLC.
Absorbance expressed in arbitrary units (AU).

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I. Celus et al. / Journal of Cereal Science 44 (2006) 203211

210

Absorbance at 214 nm (AU)

0.8
0.6
0.4
0.2
0
10

15

20

25
30
Time (min)

35

40

45

Fig. 9. RP-HPLC prole of brewers spent grain glutelins extracted with

55.0% (v/v) 1-propanol/6.0 M urea/1.0% (w/v) DTT/0.036 M Tris-HCl
(pH 8.4) after hordeins have been extracted under non-reducing
conditions. The C and B hordeins are indicated on RP-HPLC.
Absorbance expressed in arbitrary units (AU).

4. Conclusions
In summary it appears that during malting, disulde
bonds are reduced and B and D hordeins are proteolytically degraded, since almost the same levels of B hordeins
were obtained after extraction either under non-reducing
or reducing conditions, and almost no D hordeins were
extracted from malt. The apparent increase in albumins
and globulins during malting is the result of the proteolytic
breakdown of the disulde-linked hordeins and the
secretion of enzymes in the endosperm. Taken together
with those of Moonen et al. (1987), the results presented
here provide good evidence for the reduction of disulphide
bonds and proteolytic degradation of B and D hordeins
during malting.
Although it was earlier suggested that an aggregate
composed of B and D hordein (and glutelin) is formed
during mashing (Moonen et al., 1987; Poyri et al., 2002),
the failure to extract B and all C hordeins from BSG under
non-reducing conditions may indicate that all B hordeins
present in BSG form a disulde-linked aggregate in which
some of the C hordeins are entrapped. It is speculated that
this disulde-linked aggregate is formed during mashing.
Acknowledgements
The authors acknowledge the Instituut voor de aanmoediging van Innovatie door Wetenschap en Technologie
in Vlaanderen (I.W.T., Brussels, Belgium) for their
nancial support. We thank Dr Filip Delvaux (Centre for
Malting and Brewing Science) for the brewing experiments.
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