lable at ScienceDirect

Journal of Cereal Science 54 (2011) 20e28

Contents lists avai

Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Effects of enzymatic hydrolysis on molecular structure and antioxidant activityof barley hordein

Fatemeh Bamdad, Jianping Wu, Lingyun Chen*

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada T6G 2P5

a r t i c l e i n f o

Article history:Received 16 November 2010Received in revised form24 December 2010Accepted 12 January 2011

Keywords:Barley hordeinEnzymatic hydrolysisMolecular weightAntioxidant activity

Abbreviations: AH, alcalase hydrolysates; ANS,phonic acid; BHA, butylated hydroxyanisole; BHT, budegree of hydrolysis; DPPH, 1,1-diphenyl-2-picryl hydinfrared spectroscopy; FH, flavourzyme hydrolysateTNBS, tri-nitro benzene sulfonic acid.* Corresponding author. Tel.: þ1 780 492 0038; fax

E-mail address: lingyun.chen@ualberta.ca (L. Chen

0733-5210/$ e see front matter � 2011 Elsevier Ltd.doi:10.1016/j.jcs.2011.01.006

a b s t r a c t

Hordein, the major storage protein of barley (Hordeum vulgare L.), was hydrolysed by three selectedproteases, including alcalase, flavourzyme and pepsin. The effects of protease type and hydrolysis timeon hordein molecular weight, surface hydrophobicity, secondary structure and antioxidant activity wereinvestigated. Flavourzyme hydrolysis of hordein was relatively more extensive and rapid, resulting in theformation of medium- and small-sized peptides with a broad distribution within 30 min. Alcalase andpepsin more gradually and less extensively hydrolysed hordein into medium- and larger-sized peptides,respectively. Protein surface hydrophobicity decreased with an increasing degree of hydrolysis. Theflavourzyme and alcalase hydrolysates had superior DPPH (1,1-diphenyl-2-picryl hydrazyl) free radicalscavenging activity (44e70 and 48e58%, respectively, at 0.5 mg/mL), Fe2þ-chelating ability (21e64% and39e73%, respectively, at 1 mg/mL), and superoxide radical scavenging capacity. It is proposed that thelarge- and medium-size hydrolysate fractions were most likely responsible for the antioxidant activitiesof hordein hydrolysates, and could be used as antioxidant peptides in food and nutraceuticalapplications.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

The oxidation of food ingredients, such as lipids, proteins,pigments, and aroma compounds, is triggered by light, oxygen andmetal ions. Oxidation is amajor cause of deleterious quality changesin food products, influencing flavour, colour and texture, and alsoleading to loss of nutritive value or food spoilage. In addition,oxidative stress in human tissues is associated with numerousdegenerative ageing diseases, such as cancer and arteriosclerosis(Halliwell, 2002). Intrinsic antioxidant systems in human metabo-lism can modulate the antioxidant/pro-oxidant balance to providea favourable environment. Such a favourable balance is more diffi-cult to maintain in food systems because processing operationsincrease the oxidative load by introducing oxygen, removing ordestroying natural antioxidants, and promoting pro-oxidativefactors (Rajapakse et al., 2005). Endogenous or added antioxidants

8-anilino-1-naphthalene sul-tylated hydroxytoluene; DH,razyl; FTIR, Fourier transforms; PH, pepsin hydrolysates;

: þ1 780 492 4265.).

All rights reserved.

can potentially protect food components or body tissues againstoxidative damage. Synthetic food antioxidant additives such as BHT(butylated hydroxytoluene), BHA (butylated hydroxylanisole), andpropyl gallate are under increasing regulatory scrutiny (Halliwell,2002); hence, recent attention has focused on antioxidants fromnatural resources. Vitamin E, phenolic compounds, glutathione,ascorbic acid, beta-carotenes and selenium are the major “natural”antioxidants known to prevent oxidation (Zhu et al., 2008).

Recently, food protein hydrolysates have attracted interest asnatural antioxidants (Marambe et al., 2008). Protein hydrolysatescan inactivate reactive oxygen species, scavenge free radicals,chelate pro-oxidative transition metals, reduce hydroperoxides,enzymatically eliminate specific oxidants, as well as form physicalbarriers to separate reactive species from food ingredients (Kongand Xiong, 2006; Zhu et al., 2008). Food protein hydrolysates areunique compared to other food antioxidants since they maypotentially act as multifunctional antioxidants to inhibit a variety offood ingredient oxidation pathways. Furthermore, protein additionin food products confers positive nutritional and functional prop-erties (Moure et al., 2006). Anti-oxidative peptides can be releasedfrom their protein precursors by in vitro proteolytic processes or bygastrointestinal digestion. During the past decade, the antioxidantactivity of protein hydrolysates has been reported from both plantand animal sources; soy protein (Moure et al., 2006), fish protein(Klompong et al., 2007; You et al., 2010), zein (Li et al., 2008), rice

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e28 21

endosperm protein (Zhang et al., 2009) and flaxseed protein(Marambe et al., 2008). The protein source, the type of protease andthe hydrolysis process determine the size, amino acid composition,hydrolysate peptide sequence, and subsequently their antioxidantactivity.

Barley (Hordeum vulgare L.) is the fourth most widely cultivatedcereal in the world after wheat (Triticum aestivum L.), rice (Oryzasativa L.) and corn (Zea mays L.) (Yalçın et al., 2008). Canada is thesecond largest barley producer in the world with an annualproduction of 12 million metric tons (Statistics Canada, 2006) withthe major growing areas located in the Prairie Provinces. In Canada,approximately 80% of the barley crop is used as livestock feed, 15%for malting and less than 5% for direct human consumption (Yalçınet al., 2008). The recent renewed interest in barley as a foodingredient is due to the growing public awareness of the ability ofbarley b-glucan to reduce blood cholesterol and glucose levels(Wood, 2002). Techniques have been developed to isolate b-glucanfrom barley grains as a health ingredient in food products(Vasanthan and Temelli, 2008). The by-products commerciallyavailable from this b-glucan isolation process are good sources ofprotein. Additionally, barley storage proteins normally precipitatewith the spent grains and are regarded as contaminants by thebrewing industry (Jadhav et al., 1998). The low lysine content inthese proteins limits their wide application as nutritional ingre-dient in foods, so they are normally sold cheaply as animal feed.

Hordein, a barley prolamin, is the major protein in barley by-products, and is themain storage barley protein (Shewry,1993). It isenriched with Glu, Pro, Leu, Val, Phe and Tyr, most of which havebeen reported to be related to antioxidant activity in their free formsor as residues in proteins and peptides (Marambe et al., 2008; Zhuet al., 2008). Additionally, a barley hordein subunit, C-hordein, isable to more effectively inhibit the oxidation of unsaturated fattyacids in its powder formcompared toa/b gliadin anda-zein (Kawaseet al., 1998). Thus, hydrolysing barley hordein to produce peptideswith antioxidant activity, may offer new opportunities for value-added food applications. Although limited recent data indicates thatthe antioxidant activity of barley hordein can be enhanced afterenzymatic hydrolysis (Chanput et al., 2009), there is little informa-tion regarding the impacts of the type of protease and the hydrolysisprocess on the peptide structures and their antioxidant activity.

The objective of this research was to perform the barley hordeinenzymatic hydrolysis to produce antioxidant peptides with a focuson protease type and hydrolysis time on subsequent peptidestructure (molecular weight, hydrophobicity and secondary struc-ture). The antioxidant activity of the peptide fractions of the proteinhydrolysates was investigated and their potential as commercialantioxidants discussed.

2. Experimental

2.1. Samples and reagents

Regular barley grains (cv. Falcon) were kindly provided byDr. James Helm, Alberta Agricultural and Rural Development,Lacombe, Alberta, Canada. Protein content was 13.2% (w/w) asdetermined by combustion with a nitrogen analyser (Leco Corpo-ration, St. Joseph, MI, USA) calibrated with analytical reagent gradeEDTA. A factor of 5.83 was used to convert the nitrogen to protein.Barley hordeinwas extracted according to our previouswork (Wanget al., 2010). The protein content of isolated hordeinwas 91.6% (w/w)determined by the nitrogen analyser. Alcalase 3.0T was purchasedfrom Novo Nordisk (3 AU/g solid, Novo Nordisk, Bagsvaerd,Denmark). Flavourzyme (�500 U/g), pepsin (800e2500 U/mgprotein), sodium dodecyl sulphate (SDS), ascorbic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH), Tri-nitro benzene sulfonic acid

(TNBS), the fluorescent dye 8-anilino-1-naphthalene sulphonic acid(ANS), 3-(2-pyridyl)-5,6-bis(4-phenyl-sulphonic acid)-1,2,4-triazine (Ferrozine) and standard molecular markers for HPLCanalysis (thyroglobulin, 670 kDa; ferritin, 440 kDa; BSA, 67 kDa;ovalbumin, 43 kDa; cytochrome C, 13.6 kDa; aprotinin, 6.5 kDa andvitamin B12, 1.4 kDa) were obtained from SigmaeAldrich CanadaLtd. (Oakville, ON, Canada). All other reagentswere of reagent grade.

2.2. Enzymatic hydrolysis of hordein

Hordein was hydrolysed by alcalase, flavourzyme and pepsin atoptimum reaction conditions for each of the enzymes used. Theenzyme/substrate ratio was optimised according to a preliminaryexperiment for each enzyme. Hordein (2% w/v) was first dispersedin deionised water using a homogeniser (PowerGen-1000, FisherScientific, Fairlawn, NJ, USA). After pH and temperature adjustment,proteases were added to the protein suspensions to initiatehydrolysis. Alcalase hydrolysis was conducted at the enzyme/substrate ratio of 0.24 AU/g protein, at pH 8.0 and 50 �C. Fla-vourzyme hydrolysis was performed at the enzyme/substrate ratioof 100 LAPU/g protein, at pH 7.0 and 50 �C, and pepsin at theenzyme/substrate ratio of 902 Units/mg protein, at pH 2.0 and37 �C. The pH value of the hydrolysis mixture was readjusted andoptimised every 15 min during hydrolysis.

Hydrolysis was carried out for 0.5, 1, 1.5, 2, 3 and 4 h. At the endof the hydrolysis period, the pH of the pepsin hydrolysate solutionwas adjusted to 7.0. All the hydrolysate solutions were heated at95 �C for 5 min to inactivate the enzyme and centrifuged at 5000 gat 23 �C for 10 min (Beckman Coulter Avanti J-E Centrifuge System,CA, USA) to separate the soluble hydrolysates from the non-solublesubstances. The supernatants were freeze dried to obtain thesoluble peptide powders which were stored in sealed glasscontainers at �18 �C till used.

2.3. Degree of hydrolysis (DH)

DH was calculated by reacting with TNBS to determine the freeamino groups (Adler-Nissen, 1979). Total number of amino groupswas determined in a sample completely hydrolysed with 6N HCl at110 �C for 24 h. The DH was calculated by the following equation:

DH ¼ ðh=htotÞ � 100

Where h (hydrolysis equivalents) is the amount of peptidebonds cleaved during hydrolysis, which is expressed as millimoleequivalents per gram of protein (mmol/g of protein); htot is the totalamount of peptide bonds in the protein substrate, which can bedetermined from the amino acid composition. For hordein,htot ¼ 7.52 mmol/g of protein. L-Leucine (0e4.0 mM) was used togenerate a standard curve. The average peptide chain length (PCL)in the hydrolysates was calculated according to the followingequation (Marambe et al., 2008).

PCL ¼ 100=DH

2.4. FTIR measurement

Infrared spectra was recorded at room temperature usinga Nicolet 6700 spectrometer (Thermo Scientific, Madison, WI, USA).The spectrometer was continuously supplied with nitrogen. Hor-dein hydrolysates (5%, w/v) were dissolved in D2O solution. Toensure complete H/D exchange, samples were prepared 2 daysbefore and kept at 4 �C prior to infrared measurements. Sampleswere placed between two CaF2 windows separated by 25 mmpolyethylene terephthalate film spacer for FTIR measurement. To

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e2822

study the amide I region of the protein, Fourier self-deconvolutionswere performed using the software provided with the spectrom-eter. Band narrowing was achieved with a full width at halfmaximum of 20e25 cm�1 and with a resolution enhancementfactor of 2.0e2.5 cm�1.

2.5. Surface hydrophobicity (Ho) measurement

Protein surface hydrophobicity was determined using the apolarfluorescent dye, ANS (Tang et al., 2008). Samples were prepared to1% w/v protein solution followed by five dilutions in phosphatebuffer to obtain a final concentration ranging from 0.0025 to0.0375% (w/v). Twenty mL ANS solution (8.0 mM in 0.l M phosphatebuffer, pH 7.4) was added to 4 mL sample. Fluorescence intensity(FI) was determined using a Fluorescence spectrophotometer (JascoFP-6300 spectrofluorometer, Tokyo, Japan) set at 390 and 470 nm asexcitation and emission wavelengths, respectively, with a constantexcitation and emission slit of 5 nm. The initial slope of the FI versusprotein concentration plot was calculated by linear regressionanalysis and used as an index of Ho.

2.6. Size Exclusion High Performance Liquid Chromatography(SE-HPLC)

The average molecular weight (Mw) of the hordein hydrolysateswas determined by SE-HPLC using an Agilent 1100 series HPLCsystem equipped with a BiosuiteTM 125/5 mm HR-SEC column(7.8 � 300 mm, Waters Corp., Mass., USA). Phosphate buffer(100 mM) containing 300 mM NaCl was used as mobile phase ata flow rate of 0.5 mL/min at 25 � 0.5 �C. Sample solution (20 mL)was injected into the HPLC system and the protein was monitoredat the UV wavelength of 220 nm. Standard molecular markers wereused to calculateMw of the hydrolysed hordeins. A calibration curvewas made from the log Mw of the markers and their respectiveelution times (R2 ¼ 0.99).

2.7. Antioxidant properties

Since antioxidants may have various mechanisms in trappingand/or intercepting free radicals, the evaluation of antioxidantactivity in multiple assays may lead to a better understanding ofthese mechanisms (Frankel and Meyer, 2000).

2.7.1. Radical scavenging activity2.7.1.1. DPPH free radical scavenging activity. Scavenging activity ofhordein hydrolysates on DPPH free radical was assessed accordingto the method of Tang et al. (2010) with slight modifications.Aliquots of samples (0.5 mg/mL) were mixed 1:1 (v/v) with 0.1 mMDPPH in anhydrous ethanol. The mixture was shaken vigorouslyand incubated at 25 �C for 30 min under light protection. BHT andascorbic acid at concentrations of 0.01 and 0.1 mg/mL were used aspositive controls. The reduction of DPPH free radicals was deter-mined by measuring the absorbance at 517 nm with a UVevisiblespectrophotometer (model V-530, Jasco, CA, USA). The ability of thehydrolysates to scavenge the DPPH free radicals was calculatedaccording to the following equation:

%DPPH free radical scavenging ¼ 1� ðAs=AcÞ � 100

where As and Ac represent the absorbencies of the sample and thecontrol (deionised water instead of hydrolysates), respectively.

2.7.1.2. Superoxide radical (O $�2 ) scavenging assay. In this assay,

O $�2 was generated from autoxidation reaction of pyrogallol

(Marklund and Marklund, 1974). Eighty mL of hordein hydrolysates

at 0.5 mg/mL was mixed with 80 mL of 50 mM TriseHCl buffer (pH8.3) in a 96-well microplate followed by the addition of 40 mL of1.5 mM pyrogallol in 10 mM HCl. The rate of O $�

2 -induced poly-merisation of pyrogallol (DAs/min) was measured as increase inabsorbance at 320 nm for 5 min at 23 �C. BHT at concentrations of0.01 and 0.1 mg/mL was applied as positive control and TriseHClbuffer was used instead of hydrolysates in blank experiments (DAc/min). The O $�

2 scavenging activity of hydrolysates was calculatedusing the following equation:

The O�2 scavenging activity ¼ ½ðDAc=minÞ � ðDAs=minÞ�=

�ðDAc=minÞ � 100

2.7.2. Ferrous ion chelating activityFerrous ion chelating activity was determined according to the

method of Kong and Xiong (2006). 1 mL of 20 mM FeCl2 was addedto 0.5 mL of hordein hydrolysates (1 mg/mL) and the reaction wasthen initiated by the addition of 1 mL ferrozine (0.5 mM). Themixture was vortexed and left standing at 23 �C for 10 min. Fer-rozine-Fe2þ is a pink chromophore that absorbs strongly at 562 nm.EDTA (0.1 mg/mL), a strong metal chelator, was used as a positivecontrol. The ferrous ion chelating ability was calculated by thefollowing equation:

% Ferrous ion chelating ability ¼ 1� ðBs=BcÞ � 100

Where Bs and Bc represent the absorbance of the sample and thecontrol (deionised water instead of hydrolysates), respectively.

2.7.3. Reducing powerThe reducing power of hordein hydrolysates was measured

according to the method of Zhang et al. (2009). One mL of hordeinhydrolysates (1 mg/mL) was added to a solution containing 2.5 mLof 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassiumferricyanide. The mixture was incubated at 50 �C for 20 min.Subsequently, 2.5 mL of 10% trichloroacetic acid (TCA) was added tostop the reaction. After centrifugation at 5000 g,10min at 23 �C, thesupernatant was collected and 2.5 mL was diluted with 2.5 mLdeionised water and 0.5 mL of 0.1% FeCl3 in a test tube. Aftera 10 min reaction, the absorbance of the resulting solution wasmeasured at 700 nm. The blank contained everything except thesample. An increased absorbance of the reaction mixture indicatedthe increased reducing power.

2.8. Statistical analysis

All experiments were performed at least in three independenttrials and the results were reported as mean � standard deviation.Results were subjected to the analysis of variance using the SAS(SAS Institute, Inc., Cary, NC) and statistical significance of differ-ences (p < 0.05) was evaluated by the least significant difference(LSD) procedure.

3. Results and discussion

3.1. Enzymatic hydrolysis of barley hordein

Fig. 1 shows the degree of hydrolysis of hordein determined atvarious incubation times by three selected proteases: alcalase, fla-vourzyme and pepsin. The hydrolysis proceeded at a low rateinitially with degree of hydrolysis reaching only 1.5e2% after 1 hincubation, but thehydrolysis rate increased rapidly forflavourzymeand alcalase afterwards. Analogous research for other proteinsubstrates, such as porcine plasma protein (Liu et al., 2010), yellow

Fig. 1. Degree of hydrolysis of hordein with different proteases.

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e28 23

stripe trevally (Klompong et al., 2007) and soy proteins (Radha andPrakash, 2009), demonstrated a more rapid hydrolysis rate duringthe initial 0.5e1h of the incubation. This slower initial hydrolysis forhordein can be attributed to its greater content of inter-chaindisulfide bonds making it more resistant to proteases (Shewry,1993). Additionally, abundant glutamine residues (32%) in certainhordein molecular chains could form intermolecular hydrogenbonds to further stabilise protein structures (Wieser, 2007).

After the initial hydrolysis, hordein likely unfolds and becomessusceptible to the action of proteases, leading to an increased

Fig. 2. SEC chromatogram of hordein hydrolysates after 30 min (A) and 3 h of hydrolysis (B)with flavourzyme (FH), alcalase (AH) and pepsin (PH) after 3 h of digestion. Means with di

hydrolysis rate. Flavourzyme hydrolysis proceeded at the highestrate, with a degree of hydrolysis of 9.0 and 16.4% at 3 and 4 h ofincubation, respectively. Alcalase hydrolysis proceeded at anintermediate rate and reached a degree of hydrolysis of 5.3% after3 h of hydrolysis. Pepsin hydrolysis proceeded very slowly, witha degree of hydrolysis of 2.53% even after 4 h of hydrolysis. Fla-vourzyme is a fungus-origin enzyme containing a mixture ofexopeptidases and endoproteases. This enzyme has been used toprepare short chain peptides through extensive hydrolysis of foodproteins (Marambe et al., 2008). Alcalase is an endoprotease char-acterised by a very broad specificity in peptide cleavage, and hasbeen widely used to hydrolyse food proteins in the preparation ofbioactive peptides (Zhang et al., 2009). The efficient hydrolysisobserved for flavourzyme and alcalase demonstrates their highproteolytic activity towards hordein. The low degree of hydrolysisof pepsin hydrolysates can be attributed to pepsin’s inability tohydrolyse the proline peptide bond efficiently. Major barley storageproteins are sulphur-rich, and contain proline/glutamine repeateddomains at their N-terminus (Jadhav et al., 1998; Shewry, 1993).Some proteases and peptidases such as pepsin are unable tohydrolyse proline residues (Hausch et al., 2002; Simpson, 2001).The three dimensional conformation of hordein, combined with itshigh proline content, makes it a challenging endoproteasesubstrate (Simpson, 2001). Similar resistance to pepsin hydrolysiswas reported for genetically related prolamins such as wheatgliadin and rye secalin (Sollid, 2000).

3.2. FTIR measurement

The FTIR spectrum, especially in the range of 1600e1700 cm�1, isa reliable indicator of secondary structures of proteins and peptides(Surewicz et al., 1993). The amide I region of the FTIR spectra forhordein hydrolysates is shown in supplementary Fig.1. After 30min

and the relative area (%) of the peptide peaks in hordein hydrolysate fractions preparedfferent letters differ significantly (p < 0.05).

Fig. 2. (continued).

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e2824

hydrolysis, the pepsin hydrolysate (PH) showed several bands in theamide I region,whichwere assigned toprotein secondary structuresaccording to previous reports (Haris andSevercan,1999; Lefevre andSubirade, 1999): b-sheets (1630 cm�1), b-turn (1677 and1692 cm�1), and random coils (1643 cm�1). The band at 1660 cm�1

was assigned to the carbonyl stretching of the glutamine side chain.Such a spectrum indicated that most of the secondary structureswere intact after short periods of pepsin hydrolysis. After 3 h ofpepsin hydrolysis, the intensity of bands at 1692, 1677, 1660 and1643 decreased significantly, and two major peaks at 1617 and1610 cm�1 appeared that correspond to intermolecular b-sheet andamino acid side chain residues. This means that by 3 h of pepsinhydrolysis, hordein polypeptide chains were undergoing denatur-ation with marked changes in secondary structure. Significant andmore rapid secondary structure changes and polypeptide denatur-ation were also observed in alcalase hydrolysate (AH) and fla-vourzyme hydrolysate (FH) samples within 30 min of hydrolysis.Deconvoluted FTIR spectra of hordein hydrolysates confirmed thatinitial hydrolysis could unfold hordein peptides to facilitate furtherhydrolysis, and some secondary structures still remained even afterextensive hordein hydrolysis.

3.3. Molecular mass distribution

The SEC-HPLC chromatograms of the hordein hydrolysates byflavourzyme, alcalase and pepsin exhibited different elutionprofiles. At 30 min, the FH chromatogramwas characterised by one

sharp major peak (atMw of 1.2 kDa) and several peaks of almost thesame height ranging from 17 to 1.7 kDa (Fig. 2). After 3 h, four mainpeaks were observed at 6.7, 3.7, 2.2 and 1.2 kDa. Hordein appearedto be hydrolysed extensively by flavourzyme in the first 30 min.This extensive hydrolysis by flavourzyme resulted in peptides witha broad size distribution. For both AH and PH chromatograms after30 min of hydrolysis, one major peak was identified representingaMw of around 7.6 kDa. This peak shifted to a Mw of approximately2.3 kDa after 3 h of alcalase hydrolysis, whereas no analogous shiftwas observed for the similar peptides in the PH chromatogram. Inthe PH chromatogram, the highMw (21 kDa) peptides instead werereduced while the three peaks atMw of 3.7, 2.0 and the 1.2 kDa peakwere slightly enhanced. The SEC-HPLC profiles were divided intothree fractions based on their apparent Mw, calculated from thecalibration curve. Fraction I corresponds to large-sized peptidefragments with Mw exceeding 5 kDa, fraction II to medium-sizedpeptide fragments withMw between 2 and 5 kDa, and fraction III tosmall-sized peptide fragments withMw lower than 2 kDa. Fig. 2 alsodisplays the three fractions and summarises their quantitativechanges during hydrolysis time, represented by the area of eachfraction relative to the total area of the SE-HPLC chromatogram.Fractions II (40e48%) and III (30e35%) are the major ones for FH,and increasing incubation time did not result in obvious changes inlevels of protein fragments of each fraction. For AH, the levels ofprotein fragments in fraction I decreased, whereas those in fractionII increased with an increasing degree of hydrolysis, indicatinga gradual degradation of large polypeptides to medium-sized

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e28 25

peptides (p < 0.05). Fraction II dominated the chromatograms after2 h of hydrolysis. However in PH, the major peptides were largerthan 5 kDa. With increasing hydrolysis time, the level of fraction Idecreased significantly within 30 min, then slightly decreasedduring the following hours. This was accompanied by a slightincrease of fraction II and III levels.

The above results demonstrated that the type of protease used isa key factor in determining hordein hydrolysis. With flavourzymehydrolysis, a fast and extensive degradation occurred within 30 minof incubation, resulting in the formation ofmedium- and small-sizedpeptides with a broad distribution with major peaks correspondingto 4.7, 1.2 and 0.1 kDa. Alcalase, however, more gradually hydrolysedhordein large peptides intomedium-sized oneswith amajor peak at2.3 kDa. Pepsin could hydrolyse hordein into large peptideswith onemajor peak at 10 kDa, however, further degradation of large peptidesinto medium- and small-sized peptides was slow.

3.4. Surface hydrophobicity (Ho)

The importance of surface hydrophobicity to stability, confor-mation, and function of proteins is well recognised. Surfacehydrophobicity of hordein hydrolysates is displayed in Fig. 3. Allhydrolysates showed reduction in hydrophobicity as the hydrolysisproceeded. FH had the highest Ho value at 30 min of hydrolysis(Ho ¼ 1040) and its hydrophobicity decreased significantly(p < 0.05) to 480 after 4 h. AH also exhibited the same trend (Ho

decreased from 460 to 200) while pepsin hydrolysis resulted ina mild reduction of the Ho value from 890 to 620. Liu et al. (2010)indicated that proteolysis may result in a gain or loss of hydro-phobicity, depending on protein substrate nature, specificity ofproteolytic enzyme and molecular weight of the producedpeptides. Hordein is known to be hydrophobic, and according to theGoldmaneEngelmaneSteitz (GES) scale (Engelman et al., 1986) itshydrophobic amino acid content (33.12%) is almost one third of thetotal amino acid content, with the highest level corresponding toLeu, Val, Phe and Tyr (Table 1, supplementary items). Phenylalanineand tyrosine, although hydrophobic in nature, are not alwaysburied inside the protein molecule due to their bulky structure(Akita and Nakai, 1990). Some aromatic amino acids may be situ-ated at the surface where they contribute to surface hydrophobicityof the molecule. Considering the hydrophobicity changes of hor-dein hydrolysates and its amino acid composition, hordein may

Fig. 3. Changes in surface hydrophobicity of hordein hydrolysates during hydrolysis.Means with different letters differ significantly (p < 0.05).

have a unique tertiary structure: a hydrophilic core covered bya hydrophobic surface. In enzymatic hydrolysis of proteins, initialconformational changes occur due to cleavages in the polypeptidechain, unfolding buried sections and, as in case of hordein, exposinghydrophilic sections. Further hydrolysis results in shorter peptideswith a higher charge density (Radha and Prakash, 2009). Incontrast, many globular proteins, such as those from soybean(Radha and Prakash, 2009), possess hydrophobic residues buriedinside and thus a hydrophobic molecular core with relatively morehydrophilic groups outside (Krause et al., 1996). In the case of theseglobular proteins, hydrolysis can result in conformational changesthat lead to increasing surface hydrophobicity by exposing previ-ously buried and susceptible peptide bonds (Radha and Prakash,2009). The highest hordein surface hydrophobicity observed inthe PH fractions may be due to buried hydrophilic patches insidethe large peptides. The higher surface hydrophobicity of FH thanAH may be attributed to the different specificity of these twoenzymes to particular regions in the hordein sequence.

3.5. Antioxidant activity of hydrolysates

The antioxidant activity of the hordein hydrolysates by fla-vourzyme, alcalase and pepsin at different incubation times wasdetermined by the DPPH free radical scavenging assay, superoxide(O $�

2 ) free radical scavenging assay, the reducing power activityassay and the ferrous ion chelating activity assay. Based onpreliminary data, the peptide concentrations used in each assaywere optimised and the lowest effective concentrations (0.5 or1 mg/mL) were selected for each assay. Ascorbic acid and BHT wereused as positive controls representing a natural and a syntheticantioxidant, respectively, and EDTA was used as a positive controlfor ferrous ion chelating ability. The antioxidant activity of unhy-drolysed hordein was not tested because it produced a turbidsolution due to low solubility.

3.5.1. Free radical scavenging activity3.5.1.1. DPPH free radical scavenging activity. The relatively stableDPPH free radical in ethanol has beenwidely used to test the abilityof some compounds to act as free radical scavengers or hydrogendonors (Zhang et al., 2009). These antioxidants donate hydrogen tofree radicals, leading to non-toxic species and therefore protectingbiomolecules from further damage. As shown in Fig. 4-A, allhydrolysates could scavenge DPPH free radicals. FH showed a sharpdecrease in scavenging ability during the first 90 min of hydrolysis(26.8% decrease) which thereafter remained almost unchanged(p < 0.05). AH, however, displayed a maximum DPPH free radicalscavenging activity at 1.5e2 h incubation. This suggests a criticalpeptide size is necessary to manifest the optimal scavenging ability.The DPPH free radical scavenging ability of PH was significantlylower than FH and AH, and did not improve significantly througha 4 h digestion period (p < 0.05). This suggests that medium-sizedpeptides may play an important role in antioxidant activity. Thehigh level of DPPH free radical scavenging activity of proteinhydrolysates is associated with a high amount of hydrophobicamino acids or peptide hydrophobicity (Rajapakse et al., 2005).Although PH exhibited the highest surface hydrophobicity, it ismainly composed of large peptides. A low diffusivity in the reactionsolution due to a low degree of hydrolysis may limit the DPPH freeradical scavenging activity. Medium-sized peptides in FH and AHmay possess an optimal balance of diffusivity and hydrophobicityto better manifest scavenging capacity. However, further peptidebond cleavages can result in an accumulation of shorter hydrophilicpeptides inaccessible to DPPH free radicals (You et al., 2010),resulting in a decreased DPPH free radical scavenging activity.The dependence of DPPH free radical scavenging activity of AH on

Fig. 4. Free radical scavenging activity of hordein hydrolysates at 0.5 mg/mL with threeproteases. (A, DPPH free radical and B, superoxide radical scavenging activity). Meanswith different letters differ significantly (p < 0.05).

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e2826

the degree of hydrolysis was in accordance with that of loachprotein hydrolysates which showed an increase in scavengingactivity within a limited range of degree of hydrolysis (18e23%) andthen a slight decrease in activity with a greater degree of hydrolysis(You et al., 2009). In case of FH, the maximum peak may have beenachieved before 30 min of hydrolysis due to fast and extensivehydrolysis by flavourzyme. This could explain the decreasing trendin DPPH radical scavenging ability of FHs.

3.5.1.2. Superoxide radical scavenging activity. The superoxideradical is produced in vivo by autoxidation (or metal-catalysedoxidation) of several food constituents (e.g. flavonoids), andphotochemical reactions of O2. Superoxide radicals react withseveral enzymes that possess ironesulphur clusters at their activesites, including mammalian aconitase. These reactions can lead totransition metal ion release, which could conceivably facilitatehydroxyl radical formation (Halliwell, 2002). Superoxide radicalscan kill cells, inactivate enzymes, and degrade DNA, cellmembranes, and polysaccharides. These radicals may also play animportant role in the peroxidation of unsaturated fatty acids andpossibly other susceptible substances. Pyrogallol can autoxidize

rapidly, especially in alkaline solutions to produce O $�2 , H2O2 and

possibly OH� (Udenigwe and Aluko, 2010). This reaction has beenemployed for scavenging activity evaluation of potential antioxi-dants. The pattern of superoxide free radical scavenging activity ofhordein hydrolysates at different incubation times is presented inFig. 4-B. FH possesed siginficantly higher scavenging capacity(higher than 30% at 0.5 mg/mL) than hydrolysates by alcalase andpepsin (p < 0.05). AH and PH showed similar ranges of scavengingcapacity, with that of AH slightly higher than PH. With hydrolysistime increasing, all hydrolysates exhibited only a subtle decrease inO $�2 scavenging ability. The lack of relationship between scav-

enging ability and hydrolysis time suggests that the specificcleavage of protein chains to release peptides of certain sequencesrather than the hydrolysis process may be the main factor todetermine hordein hydrolysates’ superoxide free radicals scav-enging activity. Flavourzyme efficiently created barley hordeinpeptides with O $�

2 scavenging ability. Although the scavengingactivity of hordein hydrolysates against O $�

2 was lower than that ofDPPH free radicals, especially for AH and FH, these values arecomparable to and even higher than those of many other proteinhydrolysates. Zein and rice endosperm protein hydrolysatesexhibited an O $�

2 scavenging ability of 11.5% at 10 mg/mL and<20%at 0.5mg/mL, respectively (Tang et al., 2010; Zhang et al., 2009). Theremarkable O $�

2 scavenging ability of barley hordein hydrolysatesmay be related to the unique structure of hordein. Saito et al. (2003)indicated that His, Pro, Tyr, and Trp are themost important residuesin radical scavenging activity of antioxidant peptides. These aminoacids comprise more than 26% of the total residues in hordein.Hordein hydrolysates, especially those hydrolysed by flavourzyme,can efficiently scavenge O $�

2 .

3.5.2. Ferrous ion chelating activityTransition metal ions such as iron and copper can catalyse

reactive radicals that trigger oxidation chain reactions in biologicaland food systems (Halliwell, 2002). Metal chelation of proteins andpeptides may be accomplished either electrostatically, throughinteraction with charged amino acid residues, or structurally,through peptide transition metal ion entrapment (Zhang et al.,2009). The structureefunction relationship for metal bindingpeptides has been well-studied. For example, a “cage structure” inmetallothionein excludes surrounding water and allows segmentsof the thiol protein to bind more Cu2þ than a loose structure (Zhuet al., 2008). As shown in Fig. 5, the chelating ability of AH andPH increased significantly in the first hour of hydrolysis and thendecreased sharply, whereas FH possessed a decrease after 90min ofhydrolysis and then increased thereafter. The maximum chelatingactivity of FH was reached within 30 min of hydrolysis. Thissuggests that the relatively highermolecular weight peptides of themedium-sized group may be responsible for ferrous ion chelatingof hordein hydrolysates. A “cage” structure presumably exists inhordein, and limited hordein hydrolysis converts it to moreamphoteric and structurally flexible polypeptides, capable ofentrapping ions. The sharp decrease in chelation activity of allhydrolysates after 1 h of hydrolysis, may be due to the loss of a cagestructure caused bymore extensive cleavages in peptide bonds. Theincrease in chelating capacity of FH at 3 h incubation can beattributed to extensive hydrolysis of hordein by flavourzymecomposed of both endo- and exopeptidases. This means that aminoacids such as histidine, having awell knownmetal binding capacityare exposed, leading to a significant increase in charged groupscapable of forming complexes with iron via electrostatic interac-tions. Such a high activity of FH at 3 h incubationmay also be due topresence of metal binding free amino acids (e.g. histidine, cysteine)as flavourzyme can produce both amino acids and peptides(Klompong et al., 2008). The ferrous ion chelating evaluation

Fig. 5. Ferrus ion chelating activity of hordein hydrolysates at 1 mg/mL concentration.Means with different letters differ significantly (p < 0.05).

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e28 27

revealed that the hordein hydrolysates, specially AH and FH, areefficient Fe2þ-chelators. These chelators may decrease risks ofhypertension and cancers potentially associated with ferrous ioncatalysed lipid oxidation. Corn zein hydrolysed with alcalaseshowed very poor Fe2þ-chelating ability, even at 30 mg/mLconcentration (Kong and Xiong, 2006). However, Chang et al.(2007) reported a chelating ability ranging from 8 to 63% forhydrolysates derived from porcine haemoglobin by one and twostages of hydrolysis, at 5 mg/mL assay concentration.

3.5.3. Reducing powerTo determine the reducing power of hordein hydrolysates, we

used the ferric reducing antioxidant assay, based on the ability of anantioxidant to reduce Fe3þ to Fe2þ in a redox-linked colourimetricreaction. As shown in Fig. 6, the reducing power of AH increasedsignificantly (p<0.05) in thefirst 1.5 h, and then stabilised,whereas,a significant increase in reducing power was only observed for PHafter 2 h of hydrolysis. On the contrary, FH showed a decreasingreducing power during the first 1.5 h of hydrolysis. After a mild

Fig. 6. Reducing power of hordein hydrolysates at 1 mg/mL. Means with differentletters differ significantly (p < 0.05).

increase during 1.5e2 h, no significant change was observed duringthe following hydrolysis period. A decrease in reducing power withincreasing degree of hydrolysis was observed for fish proteinhydrolysate and the authors indicated that low-Mw fractions, such ashighly hydrolysed hydrolysates, had lower reducing power (Ahnet al., 2010). Barley hordein hydrolysates also were dependent ontheMw of the peptide fractions, with the relatively highermolecularweight peptides of the medium-sized range group displayinggreater activity. The increase in reducing power for AH and PH canbe attributed to the exposure of electron-dense amino acid sidechain groups, such as polar or charged moieties. An increasedavailability of free amino acids during flavourzyme hydrolysis mayprovide an additional source of protons and electrons to maintaina high redox potential (Zhu et al., 2008). The delay in increase of thereducing power for PH can be explained by the slow hydrolysis rateof pepsin with hordein, causing a delay in the release of peptideswith appropriateMw and structure. The highest reducing power forFH thatwas observed at 0.5 h of hydrolysis can be attributed to quickand extensive hordein hydrolysis by flavourzyme producingmedium-sized peptides. Further hydrolysis led to the formation ofmore low-Mw fractions, resulting in a reduced reducing poweractivity. Hordein hydrolysates are electron donors and can reactwith free radicals to convert them to more stable products andterminate radical chain reactions (Chang et al., 2007).

3.6. Comparison with commercial antioxidants

Antioxidant activities of hordein hydrolysates prepared at themost effective hydrolysis time were then compared to the selectedpositive controls. As summarised in Table 2 in supplementary items,FH or AH at 0.5 mg/mL possessed a similar DPPH free radicalscavenging activity compared to BHTor ascorbic acid at 0.01mg/mL.The O $�

2 scavenging activities of alcalase and pepsin hydrolysates at0.5 mg/mL were weaker than that of BHT (46.6 and 32.0% scav-enging activity at 0.1 and 0.01mg/mL concentration), however FH at0.5 mg/mL showed the same activity as BHT at 0.01 mg/mL.Therefore, hordein hydrolysates can be considered as effective freeradical scavengers. EDTA, a standard metal ion chelator, displayed38.5% chelating ability at 0.01 mg/mL. In this test, all three types ofhydrolysates showed higher ferrous ion chelating activity at 1 mg/mL than EDTA at 0.01mg/mL. AH and FHdemonstrated a reasonablereducing power, but ascorbic acid exhibited significantly higherreducing power than hordein hydrolysates (p < 0.05). Althoughcommercial synthetic or natural antioxidants are effective at lowerconcentrations, hordein hydrolysates could be incorporated intofood or cosmetic formulations in much higher proportions withoutsignificantly impacting food sensory quality potentially.

4. Conclusion

The enzymatic degradation of barley hordein can producehydrolysates with antioxidant properties such as radical scav-enging, metal chelating and oxidative reducing power. The type ofprotease used is a key factor in determining the subsequent hordeinhydrolysate molecular weight, secondary structure, surfacehydrophobicity and ultimately its antioxidant activity. AH and FHcan effectively scavenge DPPH free radicals. The superoxide radicalscavenging ability of FH and AH, although lower than that of DPPHfree radicals, was still higher than many other plant proteins. AHpossessed significant Fe2þ-chelating and reducing power, espe-cially at the first stages of hydrolysis. The majority of previousantioxidant peptide research has shown that the lower molecularweight peptides are themost efficient antioxidants. In our research,however, the large- andmedium-sized peptide fractions weremoreeffective antioxidants in hordein hydrolysates. This could be due to

F. Bamdad et al. / Journal of Cereal Science 54 (2011) 20e2828

the specific structure of hordeinwith an abundance of hydrophobicclusters, since hydrophobic amino acids with bulky and aromaticside chains may act as hydrogen donors and as direct radicalscavengers (Farvin et al., 2010). Barley hydrolysates, particularlythose produced by flavourzyme and alcalase, show potential to beused as a natural antioxidant ingredient for food and pharmaceu-tical applications. Further investigation is required to elucidate themechanism of antioxidation and to identify the specific peptidesresponsible for each anti-oxidative activity.

Acknowledgements

The authors are grateful to the Natural Sciences and EngineeringResearch Council of Canada (NSERC), Alberta Crop Industry Devel-opment Fund Ltd. (ACIDF) and Alberta Barley Commission forfinancial support.

Appendix. Supplementary data

Supplementary data associated with the article can be found inonline version, at doi:10.1016/j.jcs.2011.01.006.

References

Adler-Nissen, J., 1979. Determination of the degree of hydrolysis of food proteinhydrolysates by trinitrobenzenesulfonic acid. Journal of the Agricultural andFood Chemistry 27 (6), 1256e1262.

Ahn, C.-B., Lee, K.-H., Je, J.-Y., 2010. Enzymatic production of bioactive proteinhydrolysates from tuna liver: effects of enzymes and molecular weight onbioactivity. International Journal of Food Science and Technology 45, 562e568.

Akita, E.M., Nakai, S., 1990. Lipophilization of b-lactoglobulin: effect on hydropho-bicity, conformation and surface functional properties. Journal of Food Science55 (3), 711e717.

Chang, C.-Y., Wu, K.-C., Chiang, S.-H., 2007. Antioxidant properties and proteincompositions of porcine haemoglobin hydrolysates. Food Chemistry 100,1537e1543.

Chanput, W., Theerakulkait, C., Nakai, S., 2009. Antioxidative properties of partiallypurified barley hordein, rice bran protein fractions and their hydrolysates.Journal of Cereal Science 49, 422e428.

Engelman, D.M., Steitz, T.A., Goldman, A., 1986. Identifying nonpolar transbilayerhelices in amino acid sequences of membrane proteins. Annual Review ofBiophysics and Biophysical Chemistry 15, 321e353.

Farvin, K.H.S., Baron, C.P., Nielsen, N.S., Otte, J., Jacobsen, C., 2010. Antioxidantactivity of yoghurt peptides: part 2 e characterization of peptide fractions. FoodChemistry 123, 1090e1097.

Frankel, E.N., Meyer, A.S., 2000. The problems of using one-dimensional methods toevaluate multifunctional food and biological antioxidants. Journal of theScience of Food and Agriculture 80, 1925e1941.

Halliwell, B., 2002. Food-derived antioxidants: how to evaluate their importance infood and in vivo. In: Cadenas, E., Packer, L. (Eds.), Handbook of Antioxidants.Marcel Dekker, Inc., New York, pp. 1e45.

Haris, P.I., Severcan, F., 1999. FTIR spectroscopic characterization of protein structurein aqueous and non-aqueous media. Journal of Molecular Catalysis B: Enzy-matic 7, 207e221.

Hausch, F., Shan, L., Santiago, N.A., Gray, G.M., Khosla, C., 2002. Intestinal digestiveresistance of immunodominant gliadin peptides. American Journal of Physi-ology-Gastrointestinal and Liver Physiology 283, G996eG1003.

Jadhav, S.J., Lutz, S.E., Ghorpade, V.M., Salunkhe, D.K., 1998. Barley: chemistry andvalue-added processing. Critical Reviews in Food Science and Nutrition 38,123e171.

Kawase, S.I., Matsumura, Y., Murakami, H., Mori, T., 1998. Comparison of anti-oxidative activity among three types of prolamin subunits. Journal of CerealScience 28, 33e41.

Klompong, V., Benjakul, S., Kantachote, D., Shahidi, F., 2007. Antioxidative activityand functional properties of protein hydrolysate of yellow stripe trevally(Selaroides leptolepis) as influenced by the degree of hydrolysis and enzymetype. Food Chemistry 102, 1317e1327.

Klompong, V., Benjakul, S., Kantachote, D., Hayes, K.D., Shahidi, F., 2008. Compar-ative study on antioxidative activity of yellow stripe trevally protein hydroly-sate produced from alcalase and flavourzyme. International Journal of FoodScience & Technology 43, 1019e1026.

Kong, B., Xiong, Y.L., 2006. Antioxidant activity of zein hydrolysates in a liposomesystem and the possible mode of action. Journal of Agricultural and FoodChemistry 54, 6059e6068.

Krause, J.-P., Mothes, R., Schwenke, K.D., 1996. Some physicochemical and interfacialproperties of native and acetylated legumin from faba beans (Vicia faba L).Journal of Agricultural and Food Chemistry 44, 429e437.

Lefevre, T., Subirade, M., 1999. Structural and interaction properties of b-lacto-globulin as studied by FTIR spectroscopy. International Journal of Food Scienceand Technology 34, 419e428.

Li, X.-X., Han, L.-J., Chen, L.-J., 2008. In vitro antioxidant activity of protein hydro-lysates prepared from corn gluten meal. Journal of the Science of Food andAgriculture 88, 1660e1666.

Liu, Q., Kong, B., Xiong, Y.L., Xia, X., 2010. Antioxidant activity and functionalproperties of porcine plasma protein hydrolysate as influenced by the degree ofhydrolysis. Food Chemistry 118, 403e410.

Marambe, P.W.M.L.H.K., Shand, P.J., Wanasundara, J.P.D., 2008. An in-vitro investi-gation of selected biological activities of hydrolysed flaxseed (Linum usitatissi-mum L.) proteins. Journal of American Oil Chemists Society 85, 1155e1164.

Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in theautoxidation of pyrogallol and a convenient assay for superoxide dismutase.European Journal of Biochemistry 47, 469e474.

Moure, A., Domínguez, H., Parajo, J.C., 2006. Antioxidant properties of ultrafiltra-tion-recovered soy protein fractions from industrial effluents and their hydro-lysates. Process Biochemistry 41, 447e456.

Radha, C., Prakash, V., 2009. Structural and functional properties of heat-processedsoybean flour: effect of proteolytic modification. Food Science and TechnologyInternational 15, 453e463.

Rajapakse, N., Mendis, E., Byun, H.-G., Kim, S.-K., 2005. Purification and in vitroantioxidative effects of giant squid muscle peptides on free radical-mediatedoxidative systems. Journal of Nutritional Biochemistry 16, 562e569.

Saito, K., Jin, D.-H., Ogawa, T., Muramoto, K., Hatakeyama, E., Yasuhara, T.,Nokihara, K., 2003. Antioxidative properties of tripeptide libraries prepared bythe combinatorial chemistry. Journal of Agricultural and Food Chemistry 51,3668e3674.

Shewry, P.R., 1993. Barley seed proteins. In: MacGregor, A.W., Bhatty, R.S. (Eds.),Barley: Chemistry and Technology. American Association of Cereal ChemistsInc., St. Paul, Minnesota, USA, pp. 131e197.

Simpson, D.J., 2001. Proteolytic degradation of cereal prolaminsethe problem withproline. Plant Science 161, 825e838.

Sollid, L.M., 2000. Molecular basis of celiac disease. Annual Review of Immunology18, 53e81.

Statistics Canada, 2006. Estimated production of field crops. Available at. http://www.statcan.ca/daily/English/061005/d061005a.htm.

Surewicz, W.K., Mantsch, H.H., Chapman, D., 1993. Determination of proteinsecondary structure by fourier transform infrared spectroscopy: a criticalassessment. Biochemistry 32 (2), 389e394.

Tang, C.-H., Sun, X., Yin, S.-W., Ma, C.-Y., 2008. Transglutaminase-induced cross-linking of vicillin-rich kidney protein isolate: influence on functional propertiesand in vitro digestion. Food Research International 41, 941e947.

Tang, X., He, Z., Dai, Z., Xiong, Y.L., Xie, M., Chen, J., 2010. Peptide fractionation andfree radical scavenging activity of zein hydrolysate. Journal of Agricultural andFood Chemistry 58, 587e593.

Udenigwe, C.C., Aluko, R.E., 2010. Antioxidant and angiotensin converting enzyme-inhibitory properties of a flaxseed protein-derived high fischer ratio peptidemixture. Journal of Agricultural and Food Chemistry 58, 4762e4768.

Vasanthan, T., Temelli, F., 2008. Grain fractionation technologies for cereal beta-glucan concentration. Food Research International 41, 876e881.

Wang, C., Tian, Z., Chen, L., Temelli, F., Liu, H., Wang, Y., 2010. Functionality of barleyproteins extracted and fractionated by alkaline and alcohol methods. CerealChemistry 87 (6), 597e606.

Wieser, H., 2007. Chemistry of gluten proteins. Food Microbiology 24, 115e119.Wood, P.J., 2002. Relationships between solution properties of cereal b-glucans and

physiological effects e a review. Trends in Food Science and Technology 15,313e320.

Yalçın, E., Çelik, S., _Ibano�glu, E., 2008. Foaming properties of barley protein isolatesand hydrolysates. European Food Research and Technology 226, 967e974.

You, L., Zhao, M., Cui, C., Zhao, H., Yang, B., 2009. Effect of degree of hydrolysis onthe antioxidant activity of loach (Misgurnus anguillicaudatus) protein hydroly-sates. Innovative Food Science and Emerging Technologies 10, 235e240.

You, L., Zhao, M., Regenstein, J.M., Ren, J., 2010. Changes in the antioxidant activityof loach (Misgurnus anguillicaudatus) protein hydrolysates during a simulatedgastrointestinal digestion. Food Chemistry 120, 810e816.

Zhang, J., Zhang, H., Wang, L., Guo, X., Wang, X., Yao, H., 2009. Antioxidant activitiesof the rice endosperm protein hydrolysate: identification of the active peptide.European Food Research and Technology 229, 709e719.

Zhu, L., Chen, J., Tang, T., Xiong, Y.L., 2008. Reducing, radical scavenging, andchelation properties of in vitro digests of alcalase-treated zein hydrolysate.Journal of Agricultural and Food Chemistry 56, 2714e2721.