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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
1981). The hordeins have been further subdivided into
Most of the worlds barley is grown in Europe (60% A, B, C, D and c protein types based on amino acid
in 2005), and world production of the grain ranks composition and electrophoretic mobility (Shewry,
fourth behind maize, wheat and rice (Newman & New- 1993).
man, 2008). Although it has been an important dietary During the brewing process, barley polysaccharides
component for early societies, some cultures, mainly in and proteins are enzymatically hydrolysed, producing
North Africa and Asia, still depend on barley as a liqueed wort and an easily separable residue (30% by
food source. Presently, it is used as animal feed in the weight of the original barley) termed brewers spent
form of meal (Mussatto et al., 2006a) and in the bak- grain or brewers spent grain (BSG). BSG consists
ing industry but is principally associated with malting mainly of the leftover barley grain coverings, viz.,
and brewing (Mussatto et al., 2006b). Structurally, the pericarp, seed coat, husk and aleurone layer of the
barley consists of polysaccharides (starch, b-glucan, original grain (Mussatto et al., 2006a). Most of the
cellulose and hemicellulose), protein, lignin, lipid and phenolics of barley are covalently bound in these lay-
phenolics and to a lesser extent minerals and vitamins. ers, which contribute to the high phenolic acid content
Based on sequential extraction and solubility proper- and antioxidant activity of spent grains (Liyana-Path-
ties, barley proteins have been classied as water- and irana & Shahidi, 2006). BSG contains approximately
salt-soluble albumins and globulins, respectively, stor- 1526% protein, which is similar to that of the origi-
age proteins called hordeins (approximately 60% of nal barley grain.
total protein) and the cell wall structural proteins Interest in protein extraction and isolation stems
termed glutelins (Osborne, 1924). Hordeins belong to from the numerous food applications of this biochemi-
the cereal protein group prolamins, so-called because cal component present in BSG. Although proteins
from animal sources (e.g. milk proteins) have tradi-
*Correspondent: Fax: +353 (0) 61 331490; tionally been used in the food industry, recent aware-
e-mail: dick.tzgerald@ul.ie 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
Brewers spent grain A. Connolly et al. 1671

population, health and global warming has led to and stored in polypropylene bags at 20 C until use.
increased research interest in plant proteins. Using the The kit (K-TSTA) used for starch analysis was
techniques of milling and sieving, a protein-rich purchased from Megazyme (Bray, Ireland). All other
brous foodstu has also been made from BSG, which reagents and SDS-PAGE wide-range molecular mass
is considered to improve the symptoms of ulcerative markers were from Sigma-Aldrich (Poole, UK).
colitis (Kanauchi & Agata, 1997). Extractants com-
monly used to obtain protein fractions from plants
Milling of brewers spent grain and extract preparation
include dilute acid or alkali, salt solutions, organic
solvents, surfactants and reducing agents (Shewry, Brewers spent grain samples were removed
1993; Celus et al., 2007). from 20 C, thawed and oven-dried at 60 C for
It has been observed that during the brewery mash- 18 h to a constant weight. The dried spent grains were
ing process, type B and type D hordeins and glutelin then ne-milled using a Micromark Minigrinder,
tend to aggregate into an impenetrable complex stabi- pulsed at 22 000 rpm for 1-min duration. To obtain
lised by intra- and intermolecular disulphide bonding BSG extracts of the wet starting material or of dry-
(Moonen et al., 1987). These aggregates, also referred milled samples, slurries were prepared by allowing the
to as gel protein, form part of the upper oberteig BSG to extract while gently stirring for a given time
layer of BSG and contribute to the protein comple- and temperature. Following centrifugation at 2700 g
ment of the residual spent grain. Use of pretreatment for 20 min at 10 C (Hettich Zentrifugen Universal
or vigorous disruption procedures may therefore be 320R centrifuge, Andreas Heittich GmbH & Co.,
required to aid recovery of proteins from BSG. Tuttlingen, Germany), the supernatant was carefully
Approximately 20 kg of BSG is generated for every poured o and retained at 20 C prior to analysis.
100 L of beer produced (Mussatto et al., 2006a). As a Slurries that required shearing were obtained
result of increased cost of transportation coupled with using an Ultra Turrax T25 high-performance
EU regulations on waste disposal, alternative uses for disperser (IKA Werke GmbH & Co. KG, Staufen,
BSG are being studied. A good source of protein, Germany).
BSG is currently primarily being used as feed for cattle
and other animals. In the present work, BSG obtained
Quantification of protein
during the standard brewing process is referred to as
pale BSG. A minor type of spent grain was also Protein content of raw BSG and precipitated protein
included in the study. This is obtained by heating bar- samples was quantied using a modication of the
ley grains to over 200 C and extracting with water to macro-Kjeldahl procedure (IDF, 1993). Sample (350 mg
produce a dark wort, which is used to impart colour in triplicate) was digested in 50 mL concentrated sulphu-
to alcoholic beers. After ltering the wort, the leftover ric acid with two Kjeldahl catalyst tablets. Initial diges-
solid residue is referred to as black BSG. The objective tion was for 30 min at 220 C followed by a further 3-h
of the present study was to develop strategies for the digestion at 420 C. After cooling, samples were analysed
recovery of both pale and black BSG protein-enriched using the Auto-Kjeldahl System K-370 (BUCHI Labor-
isolates and to characterise the proteins with respect to technik AG, Flawil, Switzerland). A value of 6.25 was
amino acid composition and molecular mass distribu- used as a protein conversion factor (Jones, 1941).
tion using SDS-PAGE and Gel permeation HPLC Protein concentration in extracts and ltrates was
(GP-HPLC). The various treatments and extraction determined in triplicate using the method of Lowry
conditions investigated include mechanical shearing to et al. (1951) as modied by Bensadoun & Weinstein
reduce particle size, addition of reducing agents to (1976), using bovine serum albumen as standard.
disrupt disulphide bonds, as well as determining
optimal sodium hydroxide concentrations, weight/vol-
Isoelectric precipitation of brewers spent grain proteins
ume ratios and temperature eects. The co-product
fractions obtained in parallel with protein extraction A protein extract was prepared using 15 g of BSG dry
were also characterised with respect to their potential weight (dw) with 110 mM NaOH using a weight/vol-
value-added antioxidative components. 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-
Experimental
mined pH values between pH 1.5 and 12.0 using HCl
and NaOH where applicable. The solutions were
Materials
allowed to equilibrate for 30 min under constant stir-
The BSG samples used in this study were obtained from ring, and 2 mL aliquots were then centrifuged at
a single batch which was brewed in November 2009, 21 250 g for 15 min at 10 C. The resulting superna-
and were collected from the brewery, vacuum-packed tants were then analysed in triplicate, by the method

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
1672 Brewers spent grain A. Connolly et al.

of Lowry et al. (1951) as modied by Bensadoun &


Total starch
Weinstein (1976) to determine protein content.
Starch analysis was carried out using the AOAC Ocial
Method 996.11 with a Megazyme kit (K-TSTA). Briey,
SDS-Polyacrylamide gel electrophoresis
dried-milled BSG samples, after dispersal in 80% etha-
SDS-polyacrylamide gel electrophoresis (PAGE) was nol, were incubated at 100 C (pH 7.0 for 12 min) with
carried out as described by Laemmli (1970). Protein a thermostable a-amylase. Samples were then equili-
samples from alkaline extracts were mixed with 109 brated to 50 C and incubated with amyloglucosidase
buer [containing 3.3 mL 10% SDS, 0.8 mL 2- (pH 4.5) for 30 min. Final volumes were adjusted to
mercaptoethanol, 2 mL 0.5 M TrisHCl buer pH 6.8, 10 mL, centrifuged at 21 250 g for 6 min at 10 C, and
1.6 mL glycerol, 0.05% (w/v) bromophenol blue and glucose concentration was measured with the GOD-
0.4 mL ddH2O] and ddH2O resulting in a nal buer POD reagent using a 96-well Synergy HT Multi-Mode
concentration of 19. The samples were mixed, agitated Microplate Reader (BioTek, Mason Technology, Dub-
for half an hour and boiled for ten min at 100 C. lin, Ireland). Starch concentration, reported as mg
Samples were then separated on 12.5% gels at room starch g1 BSG dw, was then calculated based on the
temperature with a constant voltage of 100 mV. Gels amount of glucose liberated. A conversion factor of 0.9
were stained with 0.025% Coomassie Brilliant Blue in was employed, as 1.0 mg D-glucose released corre-
40% methanol containing 7% acetic acid and sponds to 0.9 mg starch (McCleary et al., 1997), and
destained with a solution containing distilled water/ determinations were carried out in duplicate.
methanol/acetic acid (50:40:10).
Klason lignin determination
Gel permeation chromatography
Lignin measurements were carried out by modifying
Gel permeation HPLC (GP-HPLC) was carried out on the procedure of Treimo et al. (2009). Dried-milled
samples of protein (0.80% w/v in dH2O) as previously BSG (300 mg) was dispersed in 3.0 mL 12 M (72%)
described (Spellman et al., 2005). The void volume H2SO4 and incubated with gentle stirring for 3 h at
(Vo) and the column volume (Vt) were estimated using room temperature. Samples were then diluted to 1 M
thyroglobulin (660 000 Da) and L-tyrosine-HCl H2SO4 with dH2O and further incubated in sealed
(181 Da), respectively. 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
Amino acid analysis
dH2O until a pH of 7.0 was reached. Crucibles were
Protein-rich isolates were subjected in duplicate to then kept overnight in a 105 C oven and the lignin
23-h hydrolysis in 6 M HCl at 110 C. After hydroly- was determined gravimetrically. Determinations were
sis, samples were deproteinised by mixing with equal carried out in duplicate.
volumes of 24% trichloroacetic acid (TCA). These
were allowed to stand for 10 min before centrifuging
Ash content
at 14 400 9 g (Microcentaur, MSE, London, UK) for
10 min. Amino acids in the supernatants were quanti- Dried-milled spent grain samples (1 g) that had been
ed using a Joel JLC-500/V amino acid analyser [Joel desiccated for 1 h at 20 C were weighed into porcelain
(UK) Ltd., Herts, UK] tted with a Joel Na+ high- crucibles. The ash content was determined by weight
performance cation-exchange column. Norleucine was dierence before and after incineration at 800 C for
used as an internal standard. 2 h using triplicate samples.

Lipid content Total phenolic content


Lipid content of oven-dried milled BSG samples was Total phenolic content of dried BSG samples was
determined gravimetrically following Soxhlet extrac- measured after alkali hydrolysis using a modication
tion. Dried-milled BSG (4 g) was added to a thimble of the procedure of Santos et al. (2003). Milled oven-
of known weight and covered with a known weight of dried spent grain (100 mg) was incubated with 5.5 mL
glass wool. The lipid fraction was extracted with 1 N NaOH for 16 h at room temperature in the dark
70 mL analytical grade chloroform for 20 h. The chlo- under N2. Acetic acid (1.81 mL 3N solution) was then
roform-extracted lipid was rotary evaporated and added to neutralise the mixture, which was centrifuged
dried in an oven at 100 C until successive sample (Hettich Zentrifugen Universal 320R centrifuge,
weights did not dier by more than 0.005 g. Determi- Andreas Heittich GmbH & Co., Tuttlingen, Germany)
nations were carried out in triplicate. at 21 250 g for 15 min at 10 C. The total phenolic

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
Brewers spent grain A. Connolly et al. 1673

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

Step 1
Ferric reducing antioxidant power assay A 1:20 (dw/v) slurry was prepared with distilled water
The antioxidant activity of test solutions and standards by shearing wet BSG for 60 s at 24 000 rpm as a pre-
was estimated according to the procedure of Grin & treatment homogenisation step. The slurry was then
Bhagooli (2004). Briey, a 150-mL sample of ferric subjected to extraction under continuous stirring at
reducing antioxidant power (FRAP) reagent, prepared room temperature for 1 h and centrifuged at 2700 g
freshly and warmed at 37 C was added to each well in for 20 min at 10 C (all further centrifugation steps
a 96-well microtitre plate. A time-zero blank reading were carried out under these same conditions). The
was taken at 595 nm using a BioTek Synergy HT sediment was set aside at 4 C, and the decanted
(Winooski, VT, USA) microtitre plate reader. Test supernatant represents the rst co-product fraction, P1
sample (20 lL) was then added to wells containing the (B1).
FRAP reagent, and a second reading was performed at
595 nm after 8 min. The FRAP reading for each sam- Step 2
ple was obtained by subtracting the time-zero blank The sediment from Step 1 was subjected to two
reading from the corresponding 8-min reading. sequential 1-h extractions in 110 mM NaOH (1:20,
The FRAP reagent contained 1.0 mL of a 10 mM original dw/v) at 50 C. Both protein-rich extracts
2,4,6-tripyridyl-5-triazine (TPTZ) solution in 40 mM
HCl, 1.0 mL of 20 mM FeCL36H2O and 10 mL of BSG
300 mM sodium acetate buer, pH 3.6. Methanolic Shear with dH2O
Ultra Turrax , 24,000 rpm,
solutions of known Trolox (6-hydroxy-2,5,7,8-tetrame- 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 in black BSG than in pale BSG, while the level of
supernatant (AS) was set aside for further treatment. lipid, starch and ash were, in comparison, lower in
The leftover sediments were combined and extracted in black than in pale BSG. It was not possible to deter-
the dark with gentle stirring using 1 N NaOH (1:14, mine the lignin or carbohydrate content of black BSG,
original dw/v) at room temperature for 16 h (Kroon this may be due to component interaction during
et al., 1997). The supernatant obtained after centrifu- production of this BSG type. The values obtained for
gation was designated co-product P2 (B2). The sedi- most components in pale BSG in the present study are
ment was further extracted in the dark for 1 h with in general agreement with previously published results
dH2O (1:14, original dw/v) by gently stirring at room where total carbohydrate ranges from 45.0% to 47.2%
temperature and the resultant supernatant obtained and protein from 14.0% to 26.7% have been reported
following centrifugation was designated co-product (Celus et al., 2006; Forssell et al., 2008; Xiros et al.,
fraction, P3 (B3). 2008). There is little information in the literature
regarding the level of alkali-extractable phenolics in
Step 3 BSG. Two reports for pale BSG give values of 1.87%
The combined protein-rich supernatants from Step 2 (Santos et al., 2003) and 1.16% (Forssell et al., 2008).
were adjusted to pH 3.8 using 2 N HCl and stirred In the present study, the value obtained for pale BSG
gently for 15 min at room temperature. Isoelectrically (1.70%) lies within this range, while the value for
precipitated protein was retrieved by centrifugation, black BSG (2.61%) indicates that the latter BSG type
and the supernatant was designated co-product P4 is a richer source of polyphenols. No previous litera-
(B4). The precipitated protein obtained was re-suspe- ture reports appear to exist on black BSG phenolic
nded in, pH 3.8, dH2O and neutralised to pH 7.0 using content. BSG phenolics, of which ferulic and p-couma-
1 N NaOH. The resulting protein-rich isolate was ric are the most abundant, are mainly in the bound
freeze-dried and stored at 20 C, and the supernatant form (Liyana-Pathirana & Shahidi, 2006), requiring
co-product fractions were adjusted to pH 7.0 using strong alkali or hydrolytic enzymes for their solubilisa-
2 N NaOH or 2 N HCl as required and stored at tion.
20 C until further analysis.
Effect of sodium hydroxide concentration, weight/volume
Results and discussion ratio and isoelectric precipitation on the extraction of
protein from pale and black brewers spent grain
Composition of brewers spent grain
Components of interest may be extracted from BSG
Pale and black spent grains were removed from using either predried grains (Celus et al., 2007) or wet
20 C, thawed and oven-dried at 60 C for 18 h. grains, as starting material. The present study was under-
Pale and black BSG contained 73.85  0.09 and taken with a view to developing a BSG protein extraction
74.24  0.35% moisture, respectively. Dried samples protocol, which could be transferred to semi-pilot scale.
were then milled and used for compositional analysis. Because of the potentially high energy requirement for
The results, expressed on a dry weight basis, are sum- scaled-up production of oven-dried spent grains, it was
marised in Table 1. decided to proceed with the investigation of optimal con-
Pale BSG consists mainly of protein, lignin and car- ditions for protein extraction using wet BSG as starting
bohydrate, with lesser amounts of phenolics, lipid and material. BSG samples were collected on the day of pro-
starch. The levels of protein and phenolics were higher 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.
Table 1 Composition (w/w, %) of brewers spent grain samples Furthermore, the BSG was found to be microbiologically
stable over a period of 1-week storage at 4 C. Various
Component Pale BSG (g 100 g1 dw) Black BSG
parameters, such as alkali concentration, isoelectric pre-
Protein 23.10  0.09 26.93  0.69 cipitation pH and weight/volume ratio during extraction
Klason lignin 23.39  0.56 n.d. were investigated. Alkaline solutions are widely recogni-
Polyphenols 1.70  0.02 2.61  0.07 sed as the most eective GRAS solvents for extraction of
Lipid 13.51  0.78 9.96  0.09 proteins from plants and cereals (Lusas & Riaz, 1995;
Starch 1.48  0.01 0.85  0.02 Celus et al., 2007), and many seed storage proteins such
Ash 3.29  0.06 2.07  0.03
as barley glutelins are solubilised by weak alkali.
Carbohydrate (nonstarch) 34a n.d.
Preliminary alkaline extractions from pale BSG gave
Values are the mean of two or more determinations. values of 94  3% and 24  1% for protein recovery
BSG, brewers spent grain; n.d., not determined. using KOH and Na2CO3, respectively, where the
a
Measured by difference. recovery using 110 mM NaOH was assigned a value of

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
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
Brewers spent grain A. Connolly et al. 1677

were considered. Hydrochloric acid 12% (v/v) and pale BSG (incorporating alkaline extraction steps at 50
acetic acid 1% (v/v) and the oxidising agent hydrogen or 20 C) produced a protein-rich isolate, which
peroxide 0.3% (v/v) were included in the initial BSG consisted of 46  4% and 45.74  0.66% protein for
aqueous extraction step. In these experiments, dilute the 50 and 20 C pale BSG extractions, respectively.
acid treatment did not aect protein recovery levels This represented 59  5% (at 50 C) and
(results not shown). Hydrogen peroxide treatment, 28.55  1.03% (at 20 C) recovery of the original
when included in the aqueous medium for 1 h at spent grain protein for pale BSG. Scaled-up extrac-
20 C, increased protein yield by 27%. This treatment tions for black BSG (300 mL) resulted in protein-rich
released the protein bound to BSG insoluble structures isolate with lower protein content and lower protein
during the extraction process. However, it was decided recovery than those obtained for pale BSG. For black
not to include this step due to possible oxidising eects BSG extraction, protein-rich isolates consisting of
on the extracted protein. 17.72  0.05% protein at 50 C and 19.42  0.72%
Shearing, a physical pretreatment similar to extru- protein were obtained at 20 C. This corresponded to
sion, proved benecial during spent grain protein a recovery of 15.26  0.33% protein at 50 C and
extraction. Shearing experiments were carried out on 11.04  1.03% protein at 20 C of original black BSG
aqueous BSG suspensions, prior to alkaline (110 mM protein. The protein-rich extract and the co-product
NaOH) extraction. The results obtained indicate that fractions P1P4 and B1B4 were subjected to further
shear speed and duration aect protein yield (Fig. 4). study. For protein isolation aided by NAC, which has
For pale BSG, shearing for both 20 and 60 sec at been shown to increase protein yield, the reducing
11 000 rpm resulted in a 3-fold increase in extracted agent may be incorporated in the 110 mM NaOH
protein. Shearing was also benecial for extraction extraction steps. This oers the possibility of using an
from black BSG, where a 1.4-fold increase was alternative isolation procedure. A disadvantage of the
obtained when shearing (11 000 rpm) for both 20 and latter protocol is that it would require further steps
60 s. Little change was observed in the amount of (for example, by washing the protein precipitate with
extracted protein obtained for both BSG types, when dH2O at pH 3.8) for the removal of NAC due to its
shearing speed was increased from 11 000 to possible interference with subsequent bioactive assays
24 000 rpm. Pretreatment of lignocellulosic material is of individual isolates.
often used to aid the enrichment of individual compo-
nents. For example, disruption of the bre matrix of
SDS-PAGE profile of pale and black brewers spent grain
BSG was achieved using a milling pretreatment (Niemi
alkaline extracts
et al., 2012).
Pale and black BSG alkaline protein extracts (AS
See Materials and Methods: Production of protein-rich
Protein-rich isolate and co-product fractions from pale
isolates and co-product fractions from pale and black
and black brewers spent grain
BSG) were subjected to SDS-PAGE. At least nine
The optimised extraction scheme (Fig. 1), devised for polypeptides ranging in molecular mass from 71 to
isolation of protein-rich isolates from BSG, involves 24 kDa along with a range of low molecular mass
shearing, alkaline extraction, heat and isoelectric peptide bands were observed on the pale BSG gel
precipitation. The alkaline extraction steps may be (data not shown). The barley proteins present in the
performed at 50 or 20 C, with an increased protein alkaline extracts of BSG (in the above scheme) consist
yield being obtained when extracting at the higher tem- mainly of hordeins and some glutelins, because any
perature. Scaled-up (5000 mL) laboratory extractions of 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)
140 Pale 20s Pale 60s Black 20s Black 60s based on amino acid composition and electrophoretic
Protein (mg g1 BSG dw)

120 mobility and the B proteins are further subdivided into


100 three subgroups (Shewry, 1993). The B and C hordeins
80 comprise 80% and 15% of these storage proteins,
60 respectively, while D and c hordeins make up the
40 remaining 5%. Celus et al. (2006) reported that barley
20 (pale) BSG proteins corresponded with type B and C
0 hordeins as described in the Shewry classication. In
Control 11000 16000 24000 the present study, the main polypeptides of SDS-
Speed (rpm)
PAGE-analysed pale BSG proteins (4271 kDa) were
Figure 4 Eect of shearing on alkaline extraction of protein from in the same molecular weight range of type B and C
brewers spent grain (n = 2). hordeins as described by Celus et al. (2006). Black

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
1678 Brewers spent grain A. Connolly et al.

BSG alkali-extracted proteins were not eciently protein breakdown and degradation. Montavon et al.
resolved with SDS-PAGE, and therefore, proteins (2003) have reported degradation of green coee bean
from the latter type of BSG could not be analysed storage and nonstorage proteins during the roasting
using the same classication system. In this case, band- process.
ing was observed at the interface between the stacking
and separating gels as well as at the dye front. The
Amino acid composition
former possibly represents complexed proteins too
large to enter the gel and the latter representing low The amino acid composition of pale and black BSG
molecular weight proteins and peptides. 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
Gel permeation chromatography
amino acids with the exception of cysteine. Of these,
Samples of the alkaline protein extract (AS See glutamine/glutamate, proline and leucine were the
Materials and Methods: Production of protein-rich most abundant with values ranging from 24.73  1.31
isolates and co-product fractions from pale and black to 7.19  0.23 g 100 g1 protein. The sulphur-contain-
BSG) for both pale and black BSG at 50 and 20 C ing amino acids cysteine and methionine were present
were subjected to Gel permeation chromatography in lowest quantities. When compared to the pale 50 C
(GPC)-HPLC analysis (Fig. 5). For the 50 C pale protein-enriched isolate, pale 20 C protein contained
BSG extract, it can be observed that a large propor- less amino acids, 100 g1 protein, and glutamine/gluta-
tion of the proteins (58.8%) have molecular masses mate and proline showed highest levels. Phenylalanine,
>10 kDa, while 33.5% appear lower than 5 kDa. The asparagine/aspartate and leucine were the next most
20 C pale BSG extract had 72% of protein above abundant, while cysteine (1.56  0.00 g 100 g1 pro-
10 kDa and 20.6% below 5 kDa. On the other hand, tein) and methionine (1.07  0.04 g 100 g1 protein)
86.29% of the 50 C and 87.61% of the 20 C black were present in lowest levels mirroring results from the
BSG proteins elute below 5 kDa, suggesting a substan- pale 50 C protein-enriched isolate.
tial degree of degradation of proteins in the latter Compared to both pale isolates, the black 50 and
BSG type. The molecular mass distribution prole 20 C protein-enriched isolates contained lower levels
(using size-exclusion HPLC), obtained using a BSG of amino acid, 100 g1 protein. All amino acids pres-
protein concentrate, was similar to that obtained in ent in the pale BSG isolates were found in the black
the present study for pale BSG (Celus et al., 2007). protein isolates with the exception of serine and argi-
Literature data are not available for the molecular nine. Glutamine/glutamate, proline and leucine showed
mass distribution of black BSG proteins. highest levels in both black BSG protein-enriched
The dierences in the GPC proles exhibited for the isolates but were lower than levels in the pale isolates.
two BSG types may arise from dierences in their The black 50 C isolate had highest cysteine levels
mode of production. Pale BSG is a by-product of the (2.51  0.41 g 100 g1 protein) when compared to the
brewery mashing process, during which protein com- other isolates. Threonine and methionine were the
plexes may form through intermolecular disulphide least abundant amino acids found in both black BSG-
bonding, which may help to stabilise the protein extracted protein isolates. The overall loss in amino
(Celus et al., 2006). Black BSG on the other hand is acid content for black BSG proteins may possibly be
the product of a barley grain roasting procedure, where ascribed to the roasting of the barley grain at high
high temperatures (200 C) may result in signicant temperatures (200 C). At these high temperatures,
components in barley may be involved in Maillard
reactions (Quail, 1996).
10 kDa 5 kDa Barley hordein fractions analysed by Shewry (1993)
0.6
Pale 20C
showed that glutamine/glutamate and proline were the
Detector response @214nm

0.5 Pale 50C


Black 20C prominent amino acids present, and germinated barley
0.4 Black 50C
foodstu (GBF), a protein- and bre-rich fraction
0.3 extracted (by milling and sieving) from BSG (Kanau-
0.2 chi & Agata, 1997), also showed high glutamine and
glutamate (22.66 g 100 g1 protein). The GBF amino
0.1
prole exhibited good correlation with values obtained
0
10 20 30 40 50 60
for amino acids shown in Table 2 for pale BSG (50 C
Retention time (min) extracted) protein-rich isolate. Treimo et al. (2008) also
found that the amino acid composition of both pale
Figure 5 Gel permeation HPLC proles of pale and black brewers BSG and enzymatically solubilised protein from the
spent grain protein extracts obtained at 20 and 50 C. same source had high levels of glutamine/glutamate,

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
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.

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2013 The Authors International Journal of Food Science and Technology 2013
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