1670 International Journal of Food Science and Technology 2013, 48, 16701681

Original article
Characterisation of protein-rich isolates and antioxidative
phenolic extracts from pale and black brewers spent grain

Alan Connolly, Charles O. Piggott & Richard J. FitzGerald*

Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland

(Received 14 October 2012; Accepted in revised form 18 February 2013)

Summary Protein-enriched isolates and co-product fractions were obtained from sheared, pale and black brewers
spent grain (BSG) using sequential aqueous and alkaline (110 mM NaOH) extraction, followed by isoelec-
tric precipitation at pH 3.8. A recovery of 59% of the original pale BSG protein and 15% of the black
BSG protein was obtained for the nal isolates. Gel permeation HPLC (GP-HPLC) revealed that 59% of
the extracted pale BSG protein and only 6% of black BSG protein had a molecular mass >10 kDa.
Glutamine/glutamate and proline were the most abundant amino acids present in both isolates. Analysis
of four co-product fractions obtained during fractionation from both pale and black BSG revealed the
presence of phenolics, with higher concentrations in the black BSG extracts. These fractions possessed
antioxidant and free radical scavenging activity when tested using the ferric reducing ability of plasma
(0.16
0.01 to 4.33
0.11 mg Trolox equivalents g1 BSG dry weight) and diphenylpicrylhydrazyl
(12.85
1.16% to 59.50
3.47% DPPH sc) assays, respectively. The protein-enriched isolates and the
phenolic-rich extracts may nd use as value-added ingredients for incorporation into conventional and
functional foods.
Keywords Antioxidant activity, barley, functional food, pale and black brewers spent grain, phenolics, protein.

of their high glutamine and proline content (Baxter,

Introduction
Most of the worlds barley is grown in Europe (60%
in 2005), and world production of the grain ranks
fourth behind maize, wheat and rice (Newman & New-
man, 2008). Although it has been an important dietary
component for early societies, some cultures, mainly in
North Africa and Asia, still depend on barley as a
food source. Presently, it is used as animal feed in the
form of meal (Mussatto et al., 2006a) and in the bak-
ing industry but is principally associated with malting
and brewing (Mussatto et al., 2006b). Structurally,
barley consists of polysaccharides (starch, b-glucan,
cellulose and hemicellulose), protein, lignin, lipid and
phenolics and to a lesser extent minerals and vitamins.
Based on sequential extraction and solubility proper-
ties, barley proteins have been classied as water- and
salt-soluble albumins and globulins, respectively, stor-
age proteins called hordeins (approximately 60% of
total protein) and the cell wall structural proteins
termed glutelins (Osborne, 1924). Hordeins belong to
the cereal protein group prolamins, so-called because
*Correspondent: Fax: +353 (0) 61 331490;
e-mail: dick.tzgerald@ul.ie
1981). The hordeins have been further subdivided into
A, B, C, D and c protein types based on amino acid
composition and electrophoretic mobility (Shewry,
1993).
During the brewing process, barley polysaccharides
and proteins are enzymatically hydrolysed, producing
liqueed wort and an easily separable residue (30% by
weight of the original barley) termed brewers spent
grain or brewers spent grain (BSG). BSG consists
mainly of the leftover barley grain coverings, viz.,
the pericarp, seed coat, husk and aleurone layer of the
original grain (Mussatto et al., 2006a). Most of the
phenolics of barley are covalently bound in these lay-
ers, which contribute to the high phenolic acid content
and antioxidant activity of spent grains (Liyana-Path-
irana & Shahidi, 2006). BSG contains approximately
1526% protein, which is similar to that of the origi-
nal barley grain.
Interest in protein extraction and isolation stems
from the numerous food applications of this biochemi-
cal component present in BSG. Although proteins
from animal sources (e.g. milk proteins) have tradi-
tionally been used in the food industry, recent aware-
ness of the link between food supply, expanding global

doi:10.1111/ijfs.12137
2013 The Authors. International Journal of Food Science and Technology 2013 Institute of Food Science and Technology

population, health and global warming has led to
increased research interest in plant proteins. Using the
techniques of milling and sieving, a protein-rich
brous foodstu has also been made from BSG, which
is considered to improve the symptoms of ulcerative
colitis (Kanauchi & Agata, 1997). Extractants com-
monly used to obtain protein fractions from plants
include dilute acid or alkali, salt solutions, organic
solvents, surfactants and reducing agents (Shewry,
1993; Celus et al., 2007).
It has been observed that during the brewery mash-
ing process, type B and type D hordeins and glutelin
tend to aggregate into an impenetrable complex stabi-
lised by intra- and intermolecular disulphide bonding
(Moonen et al., 1987). These aggregates, also referred
to as gel protein, form part of the upper oberteig
layer of BSG and contribute to the protein comple-
ment of the residual spent grain. Use of pretreatment
or vigorous disruption procedures may therefore be
required to aid recovery of proteins from BSG.
Approximately 20 kg of BSG is generated for every
100 L of beer produced (Mussatto et al., 2006a). As a
result of increased cost of transportation coupled with
EU regulations on waste disposal, alternative uses for
BSG are being studied. A good source of protein,
BSG is currently primarily being used as feed for cattle
and other animals. In the present work, BSG obtained
during the standard brewing process is referred to as
pale BSG. A minor type of spent grain was also
included in the study. This is obtained by heating bar-
ley grains to over 200 C and extracting with water to
produce a dark wort, which is used to impart colour
to alcoholic beers. After ltering the wort, the leftover
solid residue is referred to as black BSG. The objective
of the present study was to develop strategies for the
recovery of both pale and black BSG protein-enriched
isolates and to characterise the proteins with respect to
amino acid composition and molecular mass distribu-
tion using SDS-PAGE and Gel permeation HPLC
(GP-HPLC). The various treatments and extraction
conditions investigated include mechanical shearing to
reduce particle size, addition of reducing agents to
disrupt disulphide bonds, as well as determining
optimal sodium hydroxide concentrations, weight/vol-
ume ratios and temperature eects. The co-product
fractions obtained in parallel with protein extraction
were also characterised with respect to their potential
value-added antioxidative components.
Experimental
Materials
The BSG samples used in this study were obtained from
a single batch which was brewed in November 2009,
and were collected from the brewery, vacuum-packed
2013 The Authors
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
Brewers spent grain A. Connolly et al. 1671
and stored in polypropylene bags at 20 C until use.
The kit (K-TSTA) used for starch analysis was
purchased from Megazyme (Bray, Ireland). All other
reagents and SDS-PAGE wide-range molecular mass
markers were from Sigma-Aldrich (Poole, UK).
Milling of brewers spent grain and extract preparation
Brewers spent grain samples were removed
from 20 C, thawed and oven-dried at 60 C for
18 h to a constant weight. The dried spent grains were
then ne-milled using a Micromark Minigrinder,
pulsed at 22 000 rpm for 1-min duration. To obtain
BSG extracts of the wet starting material or of dry-
milled samples, slurries were prepared by allowing the
BSG to extract while gently stirring for a given time
and temperature. Following centrifugation at 2700 g
for 20 min at 10 C (Hettich Zentrifugen Universal
320R centrifuge, Andreas Heittich GmbH & Co.,
Tuttlingen, Germany), the supernatant was carefully
poured o and retained at 20 C prior to analysis.
Slurries that required shearing were obtained
using an Ultra Turrax T25 high-performance
disperser (IKA Werke GmbH & Co. KG, Staufen,
Germany).
Quantification of protein
Protein content of raw BSG and precipitated protein
samples was quantied using a modication of the
macro-Kjeldahl procedure (IDF, 1993). Sample (350 mg
in triplicate) was digested in 50 mL concentrated sulphu-
ric acid with two Kjeldahl catalyst tablets. Initial diges-
tion was for 30 min at 220 C followed by a further 3-h
digestion at 420 C. After cooling, samples were analysed
using the Auto-Kjeldahl System K-370 (BUCHI Labor-
technik AG, Flawil, Switzerland). A value of 6.25 was
used as a protein conversion factor (Jones, 1941).
Protein concentration in extracts and ltrates was
determined in triplicate using the method of Lowry
et al. (1951) as modied by Bensadoun & Weinstein
(1976), using bovine serum albumen as standard.
Isoelectric precipitation of brewers spent grain proteins
A protein extract was prepared using 15 g of BSG dry
weight (dw) with 110 mM NaOH using a weight/vol-
ume ratio of 1:20. Two 1-h extractions were carried
out and resulting extracts were combined. Fourteen
10 mL aliquots were adjusted in triplicate to predeter-
mined pH values between pH 1.5 and 12.0 using HCl
and NaOH where applicable. The solutions were
allowed to equilibrate for 30 min under constant stir-
ring, and 2 mL aliquots were then centrifuged at
21 250 g for 15 min at 10 C. The resulting superna-
tants were then analysed in triplicate, by the method
International Journal of Food Science and Technology 2013
1672 Brewers spent grain A. Connolly et al.
of Lowry et al. (1951) as modied by Bensadoun &
Weinstein (1976) to determine protein content.
SDS-Polyacrylamide gel electrophoresis
SDS-polyacrylamide gel electrophoresis (PAGE) was
carried out as described by Laemmli (1970). Protein
samples from alkaline extracts were mixed with 109
buer [containing 3.3 mL 10% SDS, 0.8 mL 2-
mercaptoethanol, 2 mL 0.5 M TrisHCl buer pH 6.8,
1.6 mL glycerol, 0.05% (w/v) bromophenol blue and
0.4 mL ddH2O] and ddH2O resulting in a nal buer
concentration of 19. The samples were mixed, agitated
for half an hour and boiled for ten min at 100 C.
Samples were then separated on 12.5% gels at room
temperature with a constant voltage of 100 mV. Gels
were stained with 0.025% Coomassie Brilliant Blue in
40% methanol containing 7% acetic acid and
destained with a solution containing distilled water/
methanol/acetic acid (50:40:10).
Gel permeation chromatography
Gel permeation HPLC (GP-HPLC) was carried out on
samples of protein (0.80% w/v in dH2O) as previously
described (Spellman et al., 2005). The void volume
(Vo) and the column volume (Vt) were estimated using
thyroglobulin (660 000 Da) and L-tyrosine-HCl
(181 Da), respectively.
Amino acid analysis
Protein-rich isolates were subjected in duplicate to
23-h hydrolysis in 6 M HCl at 110 C. After hydroly-
sis, samples were deproteinised by mixing with equal
volumes of 24% trichloroacetic acid (TCA). These
were allowed to stand for 10 min before centrifuging
at 14 400 9 g (Microcentaur, MSE, London, UK) for
10 min. Amino acids in the supernatants were quanti-
ed using a Joel JLC-500/V amino acid analyser [Joel
(UK) Ltd., Herts, UK] tted with a Joel Na+ high-
performance cation-exchange column. Norleucine was
used as an internal standard.
Lipid content
Lipid content of oven-dried milled BSG samples was
determined gravimetrically following Soxhlet extrac-
tion. Dried-milled BSG (4 g) was added to a thimble
of known weight and covered with a known weight of
glass wool. The lipid fraction was extracted with
70 mL analytical grade chloroform for 20 h. The chlo-
roform-extracted lipid was rotary evaporated and
dried in an oven at 100 C until successive sample
weights did not dier by more than 0.005 g. Determi-
nations were carried out in triplicate.
International Journal of Food Science and Technology 2013
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
Total starch
Starch analysis was carried out using the AOAC Ocial
Method 996.11 with a Megazyme kit (K-TSTA). Briey,
dried-milled BSG samples, after dispersal in 80% etha-
nol, were incubated at 100 C (pH 7.0 for 12 min) with
a thermostable a-amylase. Samples were then equili-
brated to 50 C and incubated with amyloglucosidase
(pH 4.5) for 30 min. Final volumes were adjusted to
10 mL, centrifuged at 21 250 g for 6 min at 10 C, and
glucose concentration was measured with the GOD-
POD reagent using a 96-well Synergy HT Multi-Mode
Microplate Reader (BioTek, Mason Technology, Dub-
lin, Ireland). Starch concentration, reported as mg
starch g1 BSG dw, was then calculated based on the
amount of glucose liberated. A conversion factor of 0.9
was employed, as 1.0 mg D-glucose released corre-
sponds to 0.9 mg starch (McCleary et al., 1997), and
determinations were carried out in duplicate.
Klason lignin determination
Lignin measurements were carried out by modifying
the procedure of Treimo et al. (2009). Dried-milled
BSG (300 mg) was dispersed in 3.0 mL 12 M (72%)
H2SO4 and incubated with gentle stirring for 3 h at
room temperature. Samples were then diluted to 1 M
H2SO4 with dH2O and further incubated in sealed
glass tubes at 100 C for 2.5 h. Each sample was l-
tered under suction (using a porcelain ltering cruci-
ble), and the residues were exhaustively washed with
dH2O until a pH of 7.0 was reached. Crucibles were
then kept overnight in a 105 C oven and the lignin
was determined gravimetrically. Determinations were
carried out in duplicate.
Ash content
Dried-milled spent grain samples (1 g) that had been
desiccated for 1 h at 20 C were weighed into porcelain
crucibles. The ash content was determined by weight
dierence before and after incineration at 800 C for
2 h using triplicate samples.
Total phenolic content
Total phenolic content of dried BSG samples was
measured after alkali hydrolysis using a modication
of the procedure of Santos et al. (2003). Milled oven-
dried spent grain (100 mg) was incubated with 5.5 mL
1 N NaOH for 16 h at room temperature in the dark
under N2. Acetic acid (1.81 mL 3N solution) was then
added to neutralise the mixture, which was centrifuged
(Hettich Zentrifugen Universal 320R centrifuge,
Andreas Heittich GmbH & Co., Tuttlingen, Germany)
at 21 250 g for 15 min at 10 C. The total phenolic
2013 The Authors
 Brewers spent grain A. Connolly et al. 1673

content (TPC) of the supernatant was determined
using the FolinCiocalteu procedure of Singleton &
Rossi (1965). To 100 lL of extract or gallic acid stan-
dard solutions were added in succession 50 lL of
FolinCiocalteu reagent, 200 lL of 20% Na2CO3 and
dH2O to a nal volume of 1000 lL. After 40 min at
37 C, the absorbance change was measured at
765 nm relative to a dH2O blank and the phenolic
content, expressed as gallic acid equivalents (GAE),
was determined using gallic acid as standard polyphe-
nol. Samples were prepared and analysed in triplicate
and the results expressed as mg gallic acid equivalents
(mg GAE)/g BSGdw, where dw represents dry weight.
The total phenolic content of BSG supernatant
extracts obtained during the protein extraction process
was also estimated in triplicate, using the Folin
Ciocalteu procedure.
radical scavenging activity. The percentage of DPPH
that was scavenged (%DPPHsc) was calculated using
the following equation:
%DPPH:sc Acont  Asamp  100=Acont
where Acont is the absorbance of the control and Asamp
the absorbance of the sample.
Production of protein-rich isolates and co-product
fractions from pale and black brewers spent grain
The method used to obtain protein-rich isolates and
co-product fractions (P1/B1 P4/B4) from wet BSG is
schematically outlined in Fig. 1. The three-step proto-
col employed is based on results from preliminary
extraction studies described in the Results and Discus-
sion section.

Ferric reducing antioxidant power assay
The antioxidant activity of test solutions and standards
was estimated according to the procedure of Grin &
Bhagooli (2004). Briey, a 150-mL sample of ferric
reducing antioxidant power (FRAP) reagent, prepared
freshly and warmed at 37 C was added to each well in
a 96-well microtitre plate. A time-zero blank reading
was taken at 595 nm using a BioTek Synergy HT
(Winooski, VT, USA) microtitre plate reader. Test
sample (20 lL) was then added to wells containing the
FRAP reagent, and a second reading was performed at
595 nm after 8 min. The FRAP reading for each sam-
ple was obtained by subtracting the time-zero blank
reading from the corresponding 8-min reading.
The FRAP reagent contained 1.0 mL of a 10 mM
2,4,6-tripyridyl-5-triazine (TPTZ) solution in 40 mM
HCl, 1.0 mL of 20 mM FeCL36H2O and 10 mL of
300 mM sodium acetate buer, pH 3.6. Methanolic
solutions of known Trolox (6-hydroxy-2,5,7,8-tetrame-
Step 1
A 1:20 (dw/v) slurry was prepared with distilled water
by shearing wet BSG for 60 s at 24 000 rpm as a pre-
treatment homogenisation step. The slurry was then
subjected to extraction under continuous stirring at
room temperature for 1 h and centrifuged at 2700 g
for 20 min at 10 C (all further centrifugation steps
were carried out under these same conditions). The
sediment was set aside at 4 C, and the decanted
supernatant represents the rst co-product fraction, P1
(B1).
Step 2
The sediment from Step 1 was subjected to two
sequential 1-h extractions in 110 mM NaOH (1:20,
original dw/v) at 50 C. Both protein-rich extracts
BSG
Shear with dH2O
Ultra Turrax , 24,000 rpm,
2 min

thylchroman-2-carboxylic acid) concentrations in the Centrifuge

Sn* P1 (B1)
range of 0.0120.125 mg mL1 were used for calibra- Extract (x2) pH 7.0
tion. Samples were tested in triplicate, and results were With 110mM NaOH

expressed as mg Trolox equivalents (TE) g1 BSG dw. Centrifuge Sediment:

Extract with 1N NaOH
Protein extract: Centrifuge
acid ppt. at pH 3.8
DPPH free radical scavenging assay Sn P2 (B2)
Centrifuge pH 7.0
Sn P4 (B4)
The antioxidant activity of test extracts was evaluated pH 7.0
by the DPPH free radical scavenging method described Sediment:
Sediment:
Extract with dH2O
by Gizdavic-Nikolaidis et al. (2004). In its radical Suspend in dH2O and
adjust to pH 7.0
Centrifuge
form, DPPH absorbs at 516 nm, but upon reduction Sn P3 (B3)
by an antioxidant, its absorption decreases. Test Freeze-Dry pH 7.0
extracts (100 lL) were added to 1.5 mL of a 72 mM a, Protein-Rich Extract
Fibre-rich sediment

a,-diphenyl-b-picrylhydrazyl (DPPH) methanolic Sn*: Supernatant

preparation, vortexed and left stand at room tempera-
ture, in the dark for 30 min. The absorbance of tripli- Figure 1 Flowchart for production of protein-rich isolates and
cate samples was then measured at 516 nm relative to co-product fractions from pale and black brewers spent grain
a dH2O blank. Lower absorbance indicates higher free (BSG).

2013 The Authors International Journal of Food Science and Technology 2013
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
1674 Brewers spent grain A. Connolly et al.
were centrifuged and mixed, and the resulting alkaline
supernatant (AS) was set aside for further treatment.
The leftover sediments were combined and extracted in
the dark with gentle stirring using 1 N NaOH (1:14,
original dw/v) at room temperature for 16 h (Kroon
et al., 1997). The supernatant obtained after centrifu-
gation was designated co-product P2 (B2). The sedi-
ment was further extracted in the dark for 1 h with
dH2O (1:14, original dw/v) by gently stirring at room
temperature and the resultant supernatant obtained
following centrifugation was designated co-product
fraction, P3 (B3).
Step 3
The combined protein-rich supernatants from Step 2
were adjusted to pH 3.8 using 2 N HCl and stirred
gently for 15 min at room temperature. Isoelectrically
precipitated protein was retrieved by centrifugation,
and the supernatant was designated co-product P4
(B4). The precipitated protein obtained was re-suspe-
nded in, pH 3.8, dH2O and neutralised to pH 7.0 using
1 N NaOH. The resulting protein-rich isolate was
freeze-dried and stored at 20 C, and the supernatant
co-product fractions were adjusted to pH 7.0 using
2 N NaOH or 2 N HCl as required and stored at
20 C until further analysis.
Results and discussion
Composition of brewers spent grain
Pale and black spent grains were removed from
20 C, thawed and oven-dried at 60 C for 18 h.
Pale and black BSG contained 73.85
0.09 and
74.24
0.35% moisture, respectively. Dried samples
were then milled and used for compositional analysis.
The results, expressed on a dry weight basis, are sum-
marised in Table 1.
Pale BSG consists mainly of protein, lignin and car-
bohydrate, with lesser amounts of phenolics, lipid and
starch. The levels of protein and phenolics were higher
Table 1 Composition (w/w, %) of brewers spent grain samples
Component Pale BSG (g 100 g1 dw)
Protein 23.10
0.09
Klason lignin 23.39
0.56
Polyphenols 1.70
0.02
Lipid 13.51
0.78
Starch 1.48
0.01
Ash 3.29
0.06
Carbohydrate (nonstarch) 34a
Values are the mean of two or more determinations.
BSG, brewers spent grain; n.d., not determined.
a
Measured by difference.
International Journal of Food Science and Technology 2013
in black BSG than in pale BSG, while the level of
lipid, starch and ash were, in comparison, lower in
black than in pale BSG. It was not possible to deter-
mine the lignin or carbohydrate content of black BSG,
this may be due to component interaction during
production of this BSG type. The values obtained for
most components in pale BSG in the present study are
in general agreement with previously published results
where total carbohydrate ranges from 45.0% to 47.2%
and protein from 14.0% to 26.7% have been reported
(Celus et al., 2006; Forssell et al., 2008; Xiros et al.,
2008). There is little information in the literature
regarding the level of alkali-extractable phenolics in
BSG. Two reports for pale BSG give values of 1.87%
(Santos et al., 2003) and 1.16% (Forssell et al., 2008).
In the present study, the value obtained for pale BSG
(1.70%) lies within this range, while the value for
black BSG (2.61%) indicates that the latter BSG type
is a richer source of polyphenols. No previous litera-
ture reports appear to exist on black BSG phenolic
content. BSG phenolics, of which ferulic and p-couma-
ric are the most abundant, are mainly in the bound
form (Liyana-Pathirana & Shahidi, 2006), requiring
strong alkali or hydrolytic enzymes for their solubilisa-
tion.
Effect of sodium hydroxide concentration, weight/volume
ratio and isoelectric precipitation on the extraction of
protein from pale and black brewers spent grain
Components of interest may be extracted from BSG
using either predried grains (Celus et al., 2007) or wet
grains, as starting material. The present study was under-
taken with a view to developing a BSG protein extraction
protocol, which could be transferred to semi-pilot scale.
Because of the potentially high energy requirement for
scaled-up production of oven-dried spent grains, it was
decided to proceed with the investigation of optimal con-
ditions for protein extraction using wet BSG as starting
material. BSG samples were collected on the day of pro-
duction, and no bacterial growth was detected in the
samples. Samples were immediately stored at 20 C in
sealed, vacuum-packed polypropylene bags until use.
Furthermore, the BSG was found to be microbiologically
stable over a period of 1-week storage at 4 C. Various
Black BSG
parameters, such as alkali concentration, isoelectric pre-
26.93
0.69 cipitation pH and weight/volume ratio during extraction
n.d. were investigated. Alkaline solutions are widely recogni-
2.61
0.07 sed as the most eective GRAS solvents for extraction of
9.96
0.09 proteins from plants and cereals (Lusas & Riaz, 1995;
0.85
0.02 Celus et al., 2007), and many seed storage proteins such
2.07
0.03
as barley glutelins are solubilised by weak alkali.
n.d.
Preliminary alkaline extractions from pale BSG gave
values of 94
3% and 24
1% for protein recovery
using KOH and Na2CO3, respectively, where the
recovery using 110 mM NaOH was assigned a value of
2013 The Authors
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
 Brewers spent grain A. Connolly et al. 1675

100%. As a result, NaOH was chosen as the protein chosen for further extraction experiments. Even though a
extraction solvent. The eect of varying NaOH higher amount of protein was extracted from black BSG
concentration on the amount of protein extracted at a higher weight/volume ratio, a weight/volume ratio
was measured (Fig. 2a). Over a concentration range of of 1:20 was also employed for black BSG extraction for
50150 mM NaOH, the amount of extracted protein direct comparison purposes. The pH required for isoelec-
increased 2- and 2.5-fold for pale and black BSG, tric precipitation of BSG proteins was investigated. The
respectively. Although protein extraction levels for results (Fig. 2c) indicate that precipitation occurs over
both pale and black BSG peaked at 200 mM NaOH, a the range pH 7.03.5. It can also be observed that the
lower value (110 mM) was chosen for subsequent black BSG proteins show greater solubility over the acid
extractions to minimise alkali utilisation while main- pH range. In further experiments, pH 3.8 was chosen as
taining good extraction eciencies. Bals et al. (2009) the precipitation pH for recovery of alkali-extracted pro-
found that it was possible to extract 22% of total pro- teins from both pale and black BSG. Previous workers
tein from corn distillers grains using 500 mM NaOH, have reported isoelectric precipitation values of pH 4.0
while other workers have reported the use of a lower (Celus et al., 2007) and pH 5.3 (Wu et al., 1979) for pale
alkali concentration (100 mM NaOH) to extract pro- BSG proteins and barley protein concentrate, respec-
tein-rich fractions from brewers spent grain (Celus tively. No previous data appear to be available for black
et al., 2007). BSG.
Weight/volume ratio is another important variable
in determining the eectiveness of the alkali extraction.
Effects of temperature on protein extraction from pale
The experimental conditions used for the weight/
and black brewers spent grain
volume ratio study were extraction with 110 mM
NaOH for 1 h at 20 C. The level of pale BSG protein The eect of temperature on alkaline extraction of pro-
extracted increased from 40.61
0.82 to 49.49
tein from BSG was also investigated over the range
1.69 mg g1 BSG dw over a range of weight/volume 2060 C. The amount of protein extracted from pale
ratios from 1:14 to 1:20 (Fig. 2b). For black BSG, the BSG with 110 mM sodium hydroxide gradually increased
extracted protein increased from 45.49
3.00 to from 37.17
0.05 to 88.20
2.87 mg g1 BSG dw over
53.87
0.39 mg g1 BSG dw over a weight/volume this range, while protein from black BSG increased from
ratio range of 1:14 to 1:40. With pale BSG, a decrease 37.02
2.12 to 64.32
4.30 mg g1 BSG dw (Fig. 3a).
in extracted protein was observed using weight/volume The dierences in extracted protein between pale and
ratios greater than 1:20, an eect that was not observed black BSG may reect dierences in the properties of
for black BSG. Typically, reported values of weight/vol- the protein constituents. Temperatures over 60 C were
ume ratios used during alkaline extraction of cereal pro- not investigated due to the possibility of protein dena-
teins range from 1:6 to 1:10 (Wu & Sexson, 1976; turation. Previous studies (Celus et al., 2007; Bals et al.,
Anderson & Guraya, 2001). As the maximum protein 2009) have shown good recovery of alkaline extractable
level was extracted from pale BSG when using a weight/ protein from BSG with increasing temperature, and
volume ratio of 1:20, this weight/volume ratio was positive temperature eects on protein extraction from

80 60
Protein (mg g1 BSG dw)

(a) (b)
Protein (mg g1 BSG dw)

70 55
60
50
50
45
40
40
30
Pale BSG Pale BSG
20 35
Black BSG Black BSG
10 30
40 80 120 160 200 1:15 1:17 1:20 1:27 1:40
NaOH (mM) Weight-volume Ratio

20 (c)
Total protein (mg)

15

Figure 2 The eect of (a) varying sodium 10

hydroxide concentrations (b) change in
5
weight/volume ratio on protein extraction Pale BSG
and (c) variation in pH on the precipitation Black BSG
0
of alkali-extracted proteins from pale and 0 1 2 3 4 5 6 7 8 9 10 11 12 13
pH
black brewers spent grain (BSG n = 3).

2013 The Authors International Journal of Food Science and Technology 2013
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
1676 Brewers spent grain A. Connolly et al.

100 100
(a) Pale BSG (b) Pale BSG
Protein (mg g1 BSG dw)

Protein (mg g1 BSG dw)

80 Black BSG 80 Black BSG

60 60

40 40

20 20

0 0
20 30 40 50 60
Temperature (oC)

100 (c) 100 (d)

Pale BSG
Protein (mg g1 BSG dw)

Protein (mg g1 BSG dw)

Pale BSG
80 Black BSG 80

60 60 Figure 3 Eect of (a) temperature, (b)

40 40 reducing agent, (c) combined temperature
and reducing agent (N-acetyl-L-cysteine) and
20 20
(d) varying concentrations of N-acetyl-L-
0 0 cysteine on the recovery of protein from
20 30 40 50 60 Control 0.1 0.2 0.3 0.4 0.5
brewers spent grain (BSG) during alkaline
Temperature (oC) N-acetyl-l-cysteine (% w/v)
extraction (n = 2).

other plant sources have also been observed (Wani (2060 C) and the reducing agent (NAC) was investi-
et al., 2008). Studies on rice proteins by Lim et al. gated. The reducing agent (0.5% w/v) was included in
(1999) have indicated that temperature eects observed the 110 mM NaOH extraction medium. Protein extracted
during protein extraction may be related to solubilisa- from pale BSG increased from 58.57
2.25 to 96.58

tion of swollen starch molecules bound to protein. 0.86 mg g1 BSG dw with increasing temperature
(Fig. 3c). A protein level of 88.20
2.87 mg g1 BSG
dw was obtained when no reducing agent was used.
Effect of reducing agents on alkaline extraction of
The combination of reducing agent (NAC) and heat
proteins from pale and black brewers spent grain
also increased the amount of black BSG protein
Three reducing agents, L-cysteine (0.5% w/v), b-merc- extracted from 26.65
2.01 to 49.03
2.65 mg g1
aptoethanol (0.5% v/v) and N-acetyl-L-cysteine (NAC: BSG dw over the temperature range 2060 C. This
0.5% w/v), were separately incorporated into the increase was similar to that obtained by application of
sodium hydroxide solutions (110 mM) used to extract heat without reducing agent. However, the gradual
protein from pale and black BSG. NAC had the great- increase in protein extracted over the temperature
est impact on protein extraction from pale BSG, giving range studied (Fig. 3a) indicates that application of
a value of 72.80
0.08 mg g1 BSG dw compared heat appears to counteract the negative eects of the
with a control (without NAC) of 45.40
2.06 mg g1 reducing agent on black BSG protein extraction.
BSG dw (Fig. 3b).
Of the three reducing agents, NAC is most preferable,
Effect of varying concentrations of N-acetyl-L-cysteine on
due to its food-grade GRAS status, while b-mercapto-
alkaline extraction of protein from pale brewers spent
ethanol is generally considered to be non-food-grade.
grain
When compared to the results obtained with pale spent
grain, the reducing agents used appeared to have a neg- The protein concentration obtained from alkaline
ative eect on protein extraction from black BSG. On extracts, when varying NAC concentration, was high-
the other hand, some of the pale BSG proteins, which est when using 0.4% (w/v) and lowest with 0.1% (w/v,
consist of polymeric structures known to be linked by Fig. 3d). A concentration of 1% (w/v) dithiothreitol
disulphide bonds, may be susceptible to disruption in has previously been used to extract disulphide-bonded
the presence of reducing agents. Celus et al. (2006) suc- proteins from BSG (Celus et al., 2006). Due to the
cessfully extracted hordein proteins from pale BSG with negative eect of NAC on extraction of protein from
the aid of 1% (w/v) dithiothreitol. black BSG, this spent grain type was not studied in
this experiment.
Effect of combined temperature and reducing agent on
alkaline extraction of protein from pale and black The effect of shearing as a pretreatment step for alkaline
brewers spent grain extraction of protein from brewers spent grain
In an eort to increase the amount of extractable protein In an eort to further improve yield of extractable
from BSG, the combined eects of temperature protein from the spent grains, various pretreatments

International Journal of Food Science and Technology 2013 2013 The Authors
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology

were considered. Hydrochloric acid 12% (v/v) and
acetic acid 1% (v/v) and the oxidising agent hydrogen
peroxide 0.3% (v/v) were included in the initial BSG
aqueous extraction step. In these experiments, dilute
acid treatment did not aect protein recovery levels
(results not shown). Hydrogen peroxide treatment,
when included in the aqueous medium for 1 h at
20 C, increased protein yield by 27%. This treatment
released the protein bound to BSG insoluble structures
during the extraction process. However, it was decided
not to include this step due to possible oxidising eects
on the extracted protein.
Shearing, a physical pretreatment similar to extru-
sion, proved benecial during spent grain protein
extraction. Shearing experiments were carried out on
aqueous BSG suspensions, prior to alkaline (110 mM
NaOH) extraction. The results obtained indicate that
shear speed and duration aect protein yield (Fig. 4).
For pale BSG, shearing for both 20 and 60 sec at
11 000 rpm resulted in a 3-fold increase in extracted
protein. Shearing was also benecial for extraction
from black BSG, where a 1.4-fold increase was
obtained when shearing (11 000 rpm) for both 20 and
60 s. Little change was observed in the amount of
extracted protein obtained for both BSG types, when
shearing speed was increased from 11 000 to
24 000 rpm. Pretreatment of lignocellulosic material is
often used to aid the enrichment of individual compo-
nents. For example, disruption of the bre matrix of
BSG was achieved using a milling pretreatment (Niemi
et al., 2012).
Protein-rich isolate and co-product fractions from pale
and black brewers spent grain
The optimised extraction scheme (Fig. 1), devised for
isolation of protein-rich isolates from BSG, involves
shearing, alkaline extraction, heat and isoelectric
precipitation. The alkaline extraction steps may be
performed at 50 or 20 C, with an increased protein
yield being obtained when extracting at the higher tem-
perature. Scaled-up (5000 mL) laboratory extractions of
140 Pale 20s
Protein (mg g1 BSG dw)
120
100
80
60
40
20
0 hordeins as described in the Shewry classication. In
Control
Figure 4 Eect of shearing on alkaline extraction of protein from
brewers spent grain (n = 2).
2013 The Authors
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
Brewers spent grain A. Connolly et al. 1677
pale BSG (incorporating alkaline extraction steps at 50
or 20 C) produced a protein-rich isolate, which
consisted of 46
4% and 45.74
0.66% protein for
the 50 and 20 C pale BSG extractions, respectively.
This represented 59
5% (at 50 C) and
28.55
1.03% (at 20 C) recovery of the original
spent grain protein for pale BSG. Scaled-up extrac-
tions for black BSG (300 mL) resulted in protein-rich
isolate with lower protein content and lower protein
recovery than those obtained for pale BSG. For black
BSG extraction, protein-rich isolates consisting of
17.72
0.05% protein at 50 C and 19.42
0.72%
protein were obtained at 20 C. This corresponded to
a recovery of 15.26
0.33% protein at 50 C and
11.04
1.03% protein at 20 C of original black BSG
protein. The protein-rich extract and the co-product
fractions P1P4 and B1B4 were subjected to further
study. For protein isolation aided by NAC, which has
been shown to increase protein yield, the reducing
agent may be incorporated in the 110 mM NaOH
extraction steps. This oers the possibility of using an
alternative isolation procedure. A disadvantage of the
latter protocol is that it would require further steps
(for example, by washing the protein precipitate with
dH2O at pH 3.8) for the removal of NAC due to its
possible interference with subsequent bioactive assays
of individual isolates.
SDS-PAGE profile of pale and black brewers spent grain
alkaline extracts
Pale and black BSG alkaline protein extracts (AS
See Materials and Methods: Production of protein-rich
isolates and co-product fractions from pale and black
BSG) were subjected to SDS-PAGE. At least nine
polypeptides ranging in molecular mass from 71 to
24 kDa along with a range of low molecular mass
peptide bands were observed on the pale BSG gel
(data not shown). The barley proteins present in the
alkaline extracts of BSG (in the above scheme) consist
mainly of hordeins and some glutelins, because any
residual water-soluble albumens would be removed by
the initial aqueous extraction step in P1. Barley horde-
ins are classied into ve groups (A, B, C, D and c)
Pale 60s Black 20s Black 60s based on amino acid composition and electrophoretic
mobility and the B proteins are further subdivided into
three subgroups (Shewry, 1993). The B and C hordeins
comprise 80% and 15% of these storage proteins,
respectively, while D and c hordeins make up the
remaining 5%. Celus et al. (2006) reported that barley
(pale) BSG proteins corresponded with type B and C
11000 16000 24000 the present study, the main polypeptides of SDS-
Speed (rpm)
PAGE-analysed pale BSG proteins (4271 kDa) were
in the same molecular weight range of type B and C
hordeins as described by Celus et al. (2006). Black
International Journal of Food Science and Technology 2013
1678 Brewers spent grain A. Connolly et al.
BSG alkali-extracted proteins were not eciently
resolved with SDS-PAGE, and therefore, proteins
from the latter type of BSG could not be analysed
using the same classication system. In this case, band-
ing was observed at the interface between the stacking
and separating gels as well as at the dye front. The
former possibly represents complexed proteins too
large to enter the gel and the latter representing low
molecular weight proteins and peptides.
Gel permeation chromatography
Samples of the alkaline protein extract (AS See
Materials and Methods: Production of protein-rich
isolates and co-product fractions from pale and black
BSG) for both pale and black BSG at 50 and 20 C
were subjected to Gel permeation chromatography
(GPC)-HPLC analysis (Fig. 5). For the 50 C pale
BSG extract, it can be observed that a large propor-
tion of the proteins (58.8%) have molecular masses
>10 kDa, while 33.5% appear lower than 5 kDa. The
20 C pale BSG extract had 72% of protein above
10 kDa and 20.6% below 5 kDa. On the other hand,
86.29% of the 50 C and 87.61% of the 20 C black
BSG proteins elute below 5 kDa, suggesting a substan-
tial degree of degradation of proteins in the latter
BSG type. The molecular mass distribution prole
(using size-exclusion HPLC), obtained using a BSG
protein concentrate, was similar to that obtained in
the present study for pale BSG (Celus et al., 2007).
Literature data are not available for the molecular
mass distribution of black BSG proteins.
The dierences in the GPC proles exhibited for the
two BSG types may arise from dierences in their
mode of production. Pale BSG is a by-product of the
brewery mashing process, during which protein com-
plexes may form through intermolecular disulphide
bonding, which may help to stabilise the protein
(Celus et al., 2006). Black BSG on the other hand is
the product of a barley grain roasting procedure, where
high temperatures (200 C) may result in signicant
10 kDa 5 kDa
0.6
Detector response @214nm
0.5
0.4
0.3
0.2
0.1
0
10 20 30 40
Retention time (min)
Figure 5 Gel permeation HPLC proles of pale and black brewers
spent grain protein extracts obtained at 20 and 50 C.
International Journal of Food Science and Technology 2013
protein breakdown and degradation. Montavon et al.
(2003) have reported degradation of green coee bean
storage and nonstorage proteins during the roasting
process.
Amino acid composition
The amino acid composition of pale and black BSG
protein-enriched isolates (alkali extracted at 50 and
20 C) are shown in Table 2. The pale 50 C protein-
enriched isolate contained the highest levels for all
amino acids with the exception of cysteine. Of these,
glutamine/glutamate, proline and leucine were the
most abundant with values ranging from 24.73
1.31
to 7.19
0.23 g 100 g1 protein. The sulphur-contain-
ing amino acids cysteine and methionine were present
in lowest quantities. When compared to the pale 50 C
protein-enriched isolate, pale 20 C protein contained
less amino acids, 100 g1 protein, and glutamine/gluta-
mate and proline showed highest levels. Phenylalanine,
asparagine/aspartate and leucine were the next most
abundant, while cysteine (1.56
0.00 g 100 g1 pro-
tein) and methionine (1.07
0.04 g 100 g1 protein)
were present in lowest levels mirroring results from the
pale 50 C protein-enriched isolate.
Compared to both pale isolates, the black 50 and
20 C protein-enriched isolates contained lower levels
of amino acid, 100 g1 protein. All amino acids pres-
ent in the pale BSG isolates were found in the black
protein isolates with the exception of serine and argi-
nine. Glutamine/glutamate, proline and leucine showed
highest levels in both black BSG protein-enriched
isolates but were lower than levels in the pale isolates.
The black 50 C isolate had highest cysteine levels
(2.51
0.41 g 100 g1 protein) when compared to the
other isolates. Threonine and methionine were the
least abundant amino acids found in both black BSG-
extracted protein isolates. The overall loss in amino
acid content for black BSG proteins may possibly be
ascribed to the roasting of the barley grain at high
temperatures (200 C). At these high temperatures,
components in barley may be involved in Maillard
reactions (Quail, 1996).
Barley hordein fractions analysed by Shewry (1993)
Pale 20C
showed that glutamine/glutamate and proline were the
Pale 50C
Black 20C prominent amino acids present, and germinated barley
Black 50C
foodstu (GBF), a protein- and bre-rich fraction
extracted (by milling and sieving) from BSG (Kanau-
chi & Agata, 1997), also showed high glutamine and
glutamate (22.66 g 100 g1 protein). The GBF amino
prole exhibited good correlation with values obtained
50 60
for amino acids shown in Table 2 for pale BSG (50 C
extracted) protein-rich isolate. Treimo et al. (2008) also
found that the amino acid composition of both pale
BSG and enzymatically solubilised protein from the
same source had high levels of glutamine/glutamate,
2013 The Authors
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
 Brewers spent grain A. Connolly et al. 1679

Table 2 Amino acid composition of pale and black brewers spent obtained using 1 N NaOH treatment, which aids the
grain protein-rich isolates release of bound polyphenols (Kroon et al., 1997). The
outer layers of cereal grains possess a high percentage
Amino acid content (g 100 g1 protein)a
of phenolics as bound cell wall forms (Liyana-Pathir-
Pale BSG Black BSG ana & Shahidi, 2006), which require chemical
treatment or specic enzyme digestion to enable disso-
Amino acid 50 C 20 C 50 C 20 C
ciation. The TPC levels in P4 and B4 fractions are also
Cysteine 1.37
0.24 1.56
0.00 2.51
0.41 1.51
1.48 high (4.59
0.11 and 4.35
0.08 mg GAE g1 BSG
Methionine 1.36
0.08 1.07
0.04 0.46
0.00 0.43
0.05 dw, respectively), indicative of possible release of
Asparagineb 6.57
0.32 5.98
0.02 2.50
0.00 2.24
0.25 phenolics during the 50 C alkaline extraction step. P1
Threonine 3.16
0.14 2.75
0.01 0.24
0.29 0.13
0.19 and B1 display lowest values, because these correspond
Serine 4.09
0.22 3.44
0.03 n.d. n.d. to residual water-extractable components, most of
Glutaminec 24.73
1.31 22.33
0.01 14.56
0.33 14.33
1.92 which would have been extracted or ltered o during
Glycine 3.75
0.15 3.31
0.04 2.48
0.17 2.22
0.38
the spent grain production process.
Alanine 4.29
0.08 3.76
0.11 3.06
0.08 2.88
0.25
All the co-product extracts possessed antioxidant
Valine 5.97
0.70 4.91
0.04 4.40
0.21 3.87
0.75
Isoleucine 4.19
0.32 3.56
0.10 2.53
0.07 2.37
0.13
activity (using both FRAP and DPPH assays), and
Leucine 7.19
0.23 5.84
0.07 4.91
0.13 4.57
0.53 there was good correlation between antioxidant levels
Tyrosine 3.49
0.30 3.21
0.11 2.43
0.01 2.12
0.06 and TPC values. This is indicative of the contribution
Phenylalanine 6.23
0.31 6.09
0.05 4.26
0.25 4.01
0.35 of polyphenolics to the observed in vitro antioxidant
Hisitidine 3.63
0.25 2.70
0.03 2.00
0.11 2.35
0.33 activity. On a comparative basis, fractions B1, B2 and
Lysine 3.15
0.06 2.92
0.06 0.90
0.05 0.78
0.08 B3 had higher TPC values and antioxidant activity
Arginine 5.95
0.73 4.67
0.01 n.d. n.d. than the corresponding pale co-product fractions (P1,
Proline 9.71
0.77 9.50
0.54 5.59
0.90 5.61
0.83 P2 and P3), and overall highest antioxidant activity
n.d., not detectable.
was observed in fractions P2, B2, P4 and B4. All black
a
Isolates were analysed in duplicate (values are mean
SD). BSG co-product fractions possess a darker colour than
b
Includes asparagine and aspartate. the corresponding pale BSG fractions. Data obtained
c
Includes glutamine and glutamate. in the present study (Table 1) show that the black
BSG starting material has higher TPC than pale BSG
proline, leucine and asparagine/aspartate. It has been starting material (26.15
0.69 and 17.04
0.19 mg
reported that pale BSG had an amino acid balance GAE g1 BSG dw, respectively). It is thought that
equivalent to that of barley grain, both showing high nonenzymatically produced dark coloured components
levels of glutamine/glutamate, proline and leucine (the result of Maillard reactions) present in dietary
(Wu, 1986). No previous amino acid data appear to be components such as cocoa, coee and malt may
available for black BSG. contribute to this type of increased phenolic level and
antioxidant activity (Tagliazucchi et al., 2010). The
TPC of the combined pale co-product fractions
Total phenolic content and antioxidant activity of
(9.58 mg GAE g1 BSG dw) represents a recovery of
co-product fractions
A previous study (McCarthy et al., 2012) showed that Table 3 Total phenolic content, antioxidant activity using ferric
BSG co-product fractions (obtained during 20 C reducing ability of plasma (FRAP) and diphenylpicrylhydrazyl
alkaline protein extraction) had the ability to protect (DPPH) assays of co-product fractions obtained during 50 C alka-
human lymphocytic U937 cells against genotoxic line extraction of pale and black brewers spent grain (BSG)
eects of oxidants such as hydrogen peroxide (H2O2)
Total phenolic content FRAP assay (mg
and 3-morpholinosydnonimine hydrochloride (SIN-1).
Fraction (mg GAE g1 BSG dw) TE g1 BSG dw) DPPH sc (%)
The protective eects, which may relate to iron chela-
tion, also correlated with the phenolic acid content P1 0.25
0.01 0.16
0.01 n.d.
and antioxidant activity of the extracts. P2 3.75
0.12 1.68
0.07 52.91
1.08
In the present study, samples of co-product fractions P3 0.99
0.03 0.47
0.01 12.85
1.16
P1P4 and B1B4 (obtained during protein extraction P4 4.59
0.11 4.33
0.11 53.34
3.22




using 110 mM aqueous NaOH at 50 C) were analysed B1 1.48 0.03 0.80 0.04 14.79 0.32
B2 5.29
0.05 2.41
0.10 59.50
3.47
for TPC and antioxidant activity (FRAP and DPPH
B3 1.38
0.03 0.73
0.03 31.84
0.22
assays). The TPC values obtained for co-product frac- B4 4.35
0.08 2.72
0.08 42.41
1.64
tions (P2 + P3) and (B2 + B3) correspond to the high-
est levels (4.74 and 6.67 mg GAE g1 BSG dw, Values are mean
SD, (n = 3).
respectively) of phenolics extracted (Table 3). This GAE, gallic acid equivalents; TE, Trolox equivalents; n.d., not detect-
result was not unexpected, as these fractions were able.

2013 The Authors International Journal of Food Science and Technology 2013
International Journal of Food Science and Technology 2013 Institute of Food Science and Technology
1680 Brewers spent grain A. Connolly et al.
56% of the phenolics from pale BSG starting material,
while 12.50 mg GAE g1 BSG dw (48% phenolic
recovery) was obtained for the black BSG.
Further studies are in progress to investigate the
potential application of the protein isolates and
co-product fractions as value-added food ingredients.
Conclusions
This study showed that protein-enriched isolates can
be obtained from wet pale and black BSG using an
alkaline extraction process. Amino acid analysis of the
protein-rich isolate indicated that glutamine/glutamate
and proline were the most prominent amino acids
present. These results and SDS-PAGE analysis indi-
cate the presence of hordein protein, a cereal grain
prolamin (Baxter, 1981), in the pale BSG protein-
enriched isolate. GP-HPLC analysis showed that most
of the black BSG protein isolate was degraded to
components of molecular weight below 5 kDa. Four
co-product fractions obtained during the protein
extraction process from both pale and black BSG were
shown to have antioxidant activity and black BSG
possessed higher phenolic content and antioxidant
activity than pale BSG. This study appears to be the
rst to investigate extracts of black BSG and the high
antioxidant activity therein indicates that these extracts
could act as a source of value-added functional food
ingredients. The protein and phenolics of BSG repre-
sent low-cost products and BSG is regularly available
in large quantities. Most other BSG research groups
have worked with dried-milled BSG, while the present
study used wet BSG, further ensuring a potential low-
cost approach to obtaining protein-rich isolates. Addi-
tional studies on the properties and bioactivity of the
protein isolates and phenolic extracts are in progress.
The data presented here furthermore provide useful
information, which may be applied in robust protocols
for industrial-scale production of protein-enriched iso-
lates from brewers spent grain by the food industry.
Acknowledgments
Funding for this research was provided under the
National Development Plan 20072013, through the
Food Institutional Research Measure (FIRM), admin-
istered by the Department of Agriculture, Food and
Marine, Ireland under grant issue 08RDUL669.
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