Food Chemistry 173 (2015) 652–659

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Food Chemistry

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Evaluation of the in vitro antioxidant properties of a cod (Gadus morhua)protein hydrolysate and peptide fractions

http://dx.doi.org/10.1016/j.foodchem.2014.10.0790308-8146/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Human Nutritional Sciences,University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Tel.: +1 (204) 474 9555;fax: +1 (204) 474 7593.

E-mail address: rotimi.aluko@umanitoba.ca (R.E. Aluko).

Abraham T. Girgih a, Rong He a,b, Fida M. Hasan c, Chibuike C. Udenigwe d, Tom A. Gill c, Rotimi E. Aluko a,⇑a Department of Human Nutritional Sciences and The Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB R3T2N2, Canadab College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing, People’s Republic of Chinac Department of Process Engineering and Applied Science, Dalhousie University, Halifax, Nova Scotia, Canadad Department of Environmental Sciences, Dalhousie University, Agricultural Campus, Truro, Nova Scotia B2N 5E3, Canada

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 June 2014Received in revised form 25 September2014Accepted 15 October 2014Available online 22 October 2014

Keywords:CodProtein digestionHPLCAntioxidant peptidesMembrane ultrafiltration

Mechanically-deboned cod muscle proteins were sequentially hydrolysed using pepsin and a tryp-sin + chymotrypsin combination, which was followed by passing the digest through a 1 kDa equippedtangential flow filtration system; the permeate (<1 kDa peptides) was collected as the cod protein hydro-lysate (CPH). Reversed-phase high performance liquid chromatography (RP-HPLC) was used to separatethe CPH into four peptide fractions (CF1–CF4) and their in vitro antioxidant properties investigated.Results showed that most of the peptide fractions (CF2–CF4) displayed significantly higher (p < 0.05) oxy-gen radical absorbance capacity values (698–942 lM Trolox equivalents, TE/g) and 2,2-diphenyl-1-picrylhydrazyl scavenging activities (17–32%) than those of CPH (613 lM TE/g and 19%, respectively).However, the unfractionated CPH displayed improved capability to scavenge superoxide and hydroxylradicals as well as significantly higher (p < 0.05) ferric iron reduction and chelation of iron than theRP-HPLC peptides. The CPH and peptide fractions displayed a dose-dependent inhibition of linoleic acidoxidation.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Reactive oxygen species (ROS) and free radicals are generated inthe course of normal physiological activities especially during cel-lular respiration in humans and other aerobic organisms(Chalamaiah, Dinesh Kumar, Hemalatha, & Jyothirmayi, 2012).The ROS and free radicals play an important role in the etiologyof several diseases, such as neurodegenerative disorders, hyperten-sion, inflammation, cancer, diabetes, Alzheimer’s disease, Parkin-son’s disease and ageing problems (Bougatef et al., 2010; Ngo,Qian, Ryu, Park, & Kim, 2010). The ROS and free radicals containunpaired electrons and scavenge electrons from neighbouring bio-molecules, resulting in oxidative damage/injury to cells or tissues.In addition to the physiological production of ROS and free radicals,oxidation of fats and oils in food products during processing, trans-portation and storage leads to production of undesirable secondarylipid peroxidation products, which cause rancidity in foods(Sarmadi & Ismail, 2010). Consumption of oxidised foods may

result in potential injury to the cells leading to the progression ofvarious chronic diseases. In order to prevent lipid peroxidation infoods, many synthetic antioxidants, such as butylated hydroxy tol-uene (BHT), butylated hydroxy anisole (BHA), tertiary butyl hydroquinone (TBHQ) and propyl gallate (PG), have been used. However,due to the potential health risks associated with long term use ofthese synthetic antioxidants (Chalamaiah et al., 2012; Kim &Wijesekara, 2010; Sabeena Farvin et al., 2014), the search for safeas well as cheaper natural antioxidants is critical and timely. Nat-ural antioxidants are substances in foods which considerably delayor inhibit the oxidation of other substances when consumed. Anti-oxidant substances in foods play a significant role as healthenhancing agents by acting to protect the body from excessive pro-duction of ROS and free radicals that cause tissue damaging oxida-tive stress.

Several in vitro studies have shown that hydrolysates producedfrom fish proteins possess a myriad of bioactivities, such as actingas antioxidant, antihypertensive, antidiabetic and antimicrobialagents (Bougatef et al., 2008; Girgih, Udenigwe, Hasan, Gill, &Aluko, 2013; Raghavan & Kristinsson, 2008; Theodore, Raghavan,& Kristinsson, 2008). Thus, fish-based protein hydrolysates couldserve as suitable ingredients for the production of health-promot-ing foods and could limit oxidative deterioration of foods. In recent

A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659 653

years, fish protein sources have gained much interest as potentialantioxidative peptide sources, particularly due to the availabilityof large quantities of processing wastes (fins, heads, tails, visceraand frames) and underutilized species. Many studies have alsoindicated that peptides derived from fish proteins have shownantioxidative properties in different oxidative systems (Girgihet al., 2013; Klompong, Benjakul, Kantachote, Hayes, & Shahidi,2008; Klompong, Benjakul, Kantachote, & Shahidi, 2007; Yang,Ho, Chu, & Chow, 2008). The antioxidant activity of peptides canbe attributed to their specific ability to scavenge free radicalsformed during peroxidation, in addition to scavenging of oxygen-containing compounds, and metal-chelation. Therefore, productionof fish protein hydrolysates with antioxidant properties will enableincreased value-added utilisation of seafoods and processing by-products.

To the best of our knowledge, there are only a few studies in theliterature involving cod protein-derived peptides: these studieshave mainly evaluated the physico-chemical and functional prop-erties of cod hydrolysates (Petursson, Decker, & McClements,2004; Šlizyte, Daukšas, Falch, Storrø, & Rustad, 2005; Šlizyteet al., 2009). A recent report evaluated the in vitro antioxidantproperties of cod peptides including inhibition of lipid peroxida-tion in a liposome model system (Sabeena Farvin et al., 2014).Due to the paucity of information on the antioxidant propertiesof cod protein-derived peptides, the present study was designedto investigate the effect of simulated gastrointestinal digestion ofcod proteins followed by the removal of large peptides and non-digested proteins using a 1 kDa preparative scale ultrafiltrationmembrane to produce a cod protein hydrolysate (CPH). In addition,CPH was separated by RP-HPLC into fractions of different hydro-phobicity, followed by comparison of in vitro antioxidant proper-ties using seven standard antioxidant evaluation systems.

2. Materials and methods

2.1. Materials

DPPH (2,2-diphenyl-1-picrylhydrazyl), Triton X-100, potassiumferricyanide, reduced L-glutathione (GSH), pyrogallol (1,2,3-trihy-droxybenzene), ammonium thiocyanate, linoleic acid, 1,10-phe-nanthroline, porcine pepsin, and 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-40,400-disulphide acid sodium salt (ferrozine) were pur-chased from Sigma Chemicals (St. Louis, MO, USA). Methanol (HPLCgrade), trifluoroacetic acid (TFA), trichloroacetic acid (TCA), 0.45and 0.2 micron syringe filters, Trolox, Fluorescein, AAPH (2,20-azo-bis(2-amidino-propane)-dihydrochloride), rutin trihydrate, ethyl-enediaminetetraacetic acid (EDTA) and other analytical gradechemical reagents were obtained from Fisher Scientific (Oakville,ON, Canada). Enzeco Trypsin-Chymotrypsin� 1:1 mixture wasobtained from Enzyme Development Corporation, New York, USA.

2.2. Preparation of cod protein hydrolysate (CPH)

Approximately 68 kg of frozen cod (Gadus morhua) frames werereceived in insulated containers from Cooke Aquaculture (St. John,NB, Canada). Shipments were thawed at 4 �C overnight andmechanically deboned using a Bibun SDX meat-bone separatorequipped with a perforated drum with a 5 mm pore size (Bibun,Fukuyama, Hiroshima, Japan). The minced cod within the drum(�29 kg) was collected, packed into polyethylene bags and storedat �30 �C until required for further analysis. Fifty grammes of thethawed cod mince was weighed into a 1 l beaker, suspended in200 ml distilled water, and homogenised in a standard WaringBlender for about 2 min at high speed, then stirred in the cold roomovernight. The sample was then centrifuged at 7000g for 20 min at

4 �C to obtain a supernatant that was discarded while the pelletwas collected and dispersed in 200 ml of distilled water. The pro-tein dispersion was adjusted to pH 2.0 using 2 M HCl followed byaddition of pepsin (600–1800 units/mg protein) at 10,000 units/g fish protein. The enzyme-substrate mixture was incubated at37 �C for 12 h with continuous stirring and the digest adjusted topH 7.8 with 2 M NaOH to irreversibly deactivate pepsin (Bamdad,Wu, & Chen, 2011). A mixture of trypsin and chymotrypsin (EnzecoTrypsin-Chymotrypsin� 1:1) was then added at 1000 units per mgfish protein for both components. The trypsin/chymotrypsin diges-tion was carried out at 37 �C with stirring for 4 h, the digest wasthen boiled for 10 min to inactivate the enzymes, cooled to roomtemperature and centrifuged (5200g for 30 min at 4 �C); the super-natant was filtered using Whatman #2 qualitative filter paper. Thefiltrate was passed through a Prep/Scale Tangential Flow Filtration2.5 ft2 cartridge membrane ultrafiltration setup with 1 kDa exclu-sion limit (Millipore Corporation, Bedford, MA, USA). The permeatefraction with an average molecular weight of <1 kDa was collectedand lyophilized; samples were kept at �20 �C until required forfurther analysis.

2.3. RP-HPLC separation of CPH

The CPH was fractionated by preparative RP-HPLC with a Varian940-LC system using the method described by Pownall, Udenigwe,and Aluko (2010). Freeze-dried CPH was dissolved (100 mg/ml) indouble-distilled water that contained 0.1 ml/100 ml TFA (solventA), and an aliquot of 4 ml (filtered sequentially through 0.45 lmand 0.2 lm membrane disks) was injected onto a 21 � 250 mmJupiter C-12 column with 5 lm particle size (Phenomenex, Tor-rance, CA). Fractions were eluted from the column at a flow rateof 10 ml/min using a linear gradient (0–100%) of methanol thatcontained 0.1% TFA (solvent B) for 60 min. Peptide elution wasmonitored at 220 nm and the eluatant was collected and pooledinto four fractions (�10 min intervals) using an automated fractioncollector. Pooled fractions were freeze-dried after rotary evapora-tion of solvent and stored at �20 �C until needed. The protein con-tents of CPH and HPLC peptide fractions were determined usingthe modified Lowry method (Markwell, Haas, Bieber, & Tolbert,1978).

2.4. Amino acid composition analysis

CPH and HPLC peptide fractions were hydrolysed for 24 h with6 M HCl followed by analytical HPLC detection and quantificationof amino acids according to the method of Bidlingmeyer, Cohen,and Tarvin (1984). To determine cysteine and methionine contents,performic acid oxidation was used instead of HCl (Gehrke, Wall,Absheer, Kaiser, & Zumwalt, 1985) while tryptophan was deter-mined after alkaline hydrolysis (Landry & Delhaye, 1992).

2.5. Radical scavenging activities

The ability of CPH and HPLC peptide fractions to scavenge theDPPH radical was determined using the method reported byAluko and Monu (2003), which was modified as follows for a 96-well plate. Cod peptide samples were dissolved in 0.1 M phosphatebuffer, pH 7.0 containing 1% (w/v) Triton X-100 while the DPPHradical was dissolved in methanol to a final concentration of100 lM. A 100 ll aliquot of the peptide sample was then mixedwith 100 ll of the DPPH solution in the 96-well plate to obtain afinal assay concentration of 1 mg peptide/ml followed by incuba-tion in the dark at room temperature for 30 min. The blank reac-tion consisted of 100 ll aliquot of buffer mixed with 100 ll ofthe DPPH solution while GSH (1 mg/ml) was used as the positivecontrol. The percentage DPPH radical scavenging activity (DRSA)

654 A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659

of each sample was determined using the following equation:DPPH radical scavenging activity (%) = [(Ab � As)/Ab] � 100; whereAb is absorbance of the blank and As is absorbance of the sample.

Oxygen radical antioxidant capacity (ORAC) of the CPH andHPLC peptide fractions was determined using the method of You,Udenigwe, Aluko, and Wu (2010), with the following modifica-tions. The cod peptide samples were dissolved in 75 mM phos-phate buffer, pH 7.4 and then mixed (final peptide concentrationof 1 mg/ml) with 300 nM fluorescein in a 96-well microplate fol-lowed by incubation of the mixture in the dark at 37 �C for15 min. After incubation, a 50-lL aliquot of AAPH (80 mM) wasadded to the mixture and the change in fluorescence due toAAPH-induced oxidation of fluorescein was measured at 1 minintervals for 90 min at excitation and emission wavelengths of485 and 528 nm, respectively, using a fluorescence microplatereader. A standard curve was prepared using different Trolox con-centrations (5–80 lM) and used to calculate the cod peptide ORACvalues, which was expressed as lM Trolox equivalent (TE)/g ofsample.

Hydroxyl radical scavenging activity (HRSA) was determinedusing the method described by de Avelar et al. (2004) with modi-fications. Peptide samples and 3 mM 1,10-phenanthroline wereseparately dissolved in 0.1 M phosphate buffer (pH 7.4) while3 mM FeSO4 and 0.01% (w/v) hydrogen peroxide were each sepa-rately dissolved in distilled water. The peptide sample or GSH(50 ll aliquots each) was first added to a 96-well plate followedby 50 ll each of the 1,10-phenanthroline and FeSO4. To initiatethe Fenton reaction in the wells, 50 ll of hydrogen peroxide wasadded to the mixture to give a final sample concentration of1 mg/ml and the covered plates incubated at 37 �C for 1 h withconstant shaking. The blank consisted of 50 ll phosphate bufferinstead of the sample. Absorbance of the coloured reaction mix-tures were measured at 10 min intervals for 1 h in a spectropho-tometer at a wavelength of 536 nm. The reaction rate (DA/min)was then used to calculate the HRSA value as follows: {[(DA/min)b

� (DA/min)s]/(DA/min)b} � 100, where b and s represent blank andsample, respectively.

The cod peptides were analysed for superoxide radical scaveng-ing activity (SRSA) according to a previously described method(Xie, Huang, Xu, & Jin, 2008). Samples were prepared in 50 mMTris–HCl buffer containing 1 mM EDTA, pH 8.3. An 80 ll aliquotof sample was mixed with 80 ll of the buffer in a clear bottomed96-well plate in the dark; the blank reaction consisted of 160 llof buffer with no sample. Then, 40 ll of 1.5 mM pyrogallol dis-solved in 10 mM HCl was added to each well. The reaction rate(DA/min) was measured immediately at 420 nm for 4 min at roomtemperature. The SRSA was calculated using the followingequation:

Superoxide scavenging activity ¼ f½ðDA=minÞb � ðDA=minÞs�=ðDA=minÞbg � 100

where b and s represent blank and sample, respectively.

2.6. Metal chelation and ferric reducing activities

The metal (iron) chelating activity (MCA) was determinedaccording to the method of Xie et al. (2008), which was modifiedas follows. A 1 ml aliquot of the sample solution or buffer (blank)was mixed with 0.05 ml of 2 mM FeCl2 and 1.85 ml double distilledwater in a reaction tube to give a 1 mg/ml peptide concentration.This was followed by the addition of 0.1 ml of 5 mM Ferrozine,thorough mixing of the reaction mixture and incubation at roomtemperature for 10 min. After incubation, a 200 ll aliquot of thereaction mixture was transferred into a 96-well plate and absor-

bance values of both the blank (Ab) and samples (As) were mea-sured at 562 nm. The MCA (%) = [(Ab � As)/Ab] � 100.

The ferric reducing antioxidant power (FRAP) of samples wasdetermined according to the method reported by Zhang, Wang,and Xu (2008) with modifications. Peptide sample or GSH was pre-pared in 0.2 M phosphate buffer, pH 6.6. A 250 ll aliquot of thepeptide sample or GSH was mixed with 250 ll of the phosphatebuffer and 250 ll of 1 g/100 ml potassium ferricyanide solutionto give a final 1 mg/ml peptide concentration in the reaction mix-ture. The sample was omitted from the blank reaction, which con-tained 500 ll of the phosphate buffer mixed with 250 ll of thepotassium ferricyanide solution. All the reaction mixtures werethen incubated at 50 �C for 20 min followed by the addition of250 ll 10 g/100 ml aqueous TCA. After the incubation period,250 ll of TCA mixture was added to 50 ll of 0.1 g/100 ml ferricchloride and 200 ll of double distilled water, followed by incuba-tion at room temperature for 10 min. The solution was then centri-fuged at 1000g for 15 min and 200 ll of the clear supernatanttransferred into a 96-well plate for absorbance determination at700 nm.

2.7. Inhibition of linoleic acid oxidation

The method reported by Li, Jiang, Zhang, Mu, and Liu (2008) wasused to determine inhibition of linoleic acid oxidation by the pep-tide samples. A 1.5 ml 0.1 M phosphate buffer (pH 7.0) aliquot ofsample (or 1.5 ml of buffer as the blank) was added to 1 ml of50 mM linoleic acid (dissolved in ethanol) to give a final 0.1, 0.5,or 1 mg/ml peptide concentration. The mixtures were incubatedat 60 �C in the dark for 7 days during which 100 ll of the assaysolution was removed at 24 h intervals. The assay solution wasthen mixed with 4.7 ml of 75 g/100 ml aqueous ethanol, 0.1 ml ofammonium thiocyanate (30 g/100 ml) and 0.1 ml of 20 mM ferrouschloride that was dissolved in 1 M HCl. A 200 ll aliquot of theresultant mixture was added to a clear bottomed 96-well plateand incubated for 3 min at room temperature; absorbance wasthen measured at 500 nm. The degree of linoleic acid oxidation isdirectly proportional to the absorbance values.

2.8. Statistical analysis

The antioxidant assays were each conducted in triplicate andresults analysed by one-way analysis of variance (ANOVA). Datawere reported as mean ± standard deviation. Statistical signifi-cance of differences between mean values were evaluated by Dun-can’s multiple range test (p < 0.05) using the Statistical AnalysisSystems Software Version 9.2 (SAS, Cary, NC, USA).

3. Results and discussion

3.1. RP-HPLC fractionation of CPH

Based on hydrophobicity, the CPH was fractionated using RP-HPLC and pooled into four peptide fractions (CF1–CF4) as shownin Fig. 1. The protein content of the lyophilized low molecularweight peptide mixture and its constituent HPLC fractions wasdetermined by the Lowry method and this may or may not in allcases represent the true levels in peptide mixtures. The Lowry pro-teins were 55%, 40%, 98%, 91% and 97% for CPH, CF1, CF2, CF3, andCF4, respectively. The CPH had lower protein content than the 67%reported for a similar salmon 1 kDa permeate (Girgih et al., 2013).Peptides in CF1 started to elute from the column after the first5 min, which indicates least net hydrophobicity, and hence wereweakly bound to the column when compared to the peptides inCF2–CF4.

U

Fig. 1. Chromatogram showing four pooled fractions (CF1–CF4) of RP-HPLCseparated CPH which were lyophilized and used for various analyticaldeterminations.

A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659 655

3.2. Amino acid composition

Table 1 shows the amino acid profile of the <1 kDa ultrafiltra-tion membrane CPH permeate peptides when compared to theRP-HPLC fractionated peptides. Apart from the CF1 (23.2%), allthe other peptide fractions (CF2–CF4) contained higher hydropho-bic amino acid (HAA) contents (41.4–50.3%) than the CPH (39.6%),which represents a 1.3-fold increase as a result of fractionation.Specifically, the increases in the individual HAA (Ala, Val, Leu,Cys, Trp, Pro & Ile) ranged from 1.2 to 2.2-fold, which is supportedby previous reports (Girgih et al., 2013; He, Girgih, Malomo, Ju, &Aluko, 2013). The presence of high HAA contents in the RP-HPLCcod peptide fractions offers structural properties that can enhanceinteractions with lipid foods. The high hydrophobic nature of

Table 1Amino acid composition of <1 kDa cod protein hydrolysate (CPH) and its RP-HPLCderived peptide fractions (CF1–CF4).

Amino acid CPH (%) CF1 (%) CF2 (%) CF3 (%) CF4 (%)

ASX 9.46 9.74 9.54 8.98 12.2THR 4.62 4.41 4.46 4.56 4.16SER 5.10 5.21 5.03 5.02 4.49GLX 14.1 16.6 14.0 12.4 16.0PRO 4.36 2.73 4.38 5.27 8.59GLY 7.54 7.94 7.50 6.84 5.74ALA 6.48 7.53 5.69 5.71 3.82CYS 0.38 0.54 0.34 0.58 0.18VAL 4.72 3.83 4.85 5.33 6.25MET 2.37 1.58 2.63 3.76 1.89ILE 3.84 1.76 4.33 5.15 8.50LEU 8.01 2.63 9.07 11.1 7.05TYR 3.88 1.80 5.04 4.48 1.26PHE 4.55 0.52 4.62 7.41 4.27HIS 2.72 3.37 2.61 2.58 3.21LYS 8.29 15.8 7.52 5.04 2.14ARG 8.83 13.9 7.94 4.77 10.2TRP 0.96 0.20 0.49 1.45 0.11HAA 39.6 23.2 41.4 50.3 50.0AAA 9.40 2.57 10.2 13.3 5.64PCAA 19.8 33.1 18.1 12.4 15.6NCAA 23.6 26.4 23.5 21.4 28.2EAA 40.10 34.30 41.00 46.23 38.15

ASX = aspartic acid + asparagine; GLX = glutamic acid + glutamine; combined totalof hydrophobic amino acids (HAA) = alanine, valine, isoleucine, leucine, tyrosine,phenylalanine, tryptophan, proline, methionine and cysteine; aromatic amino acids(AAA) = phenylalanine, tryptophan and tyrosine; positively charged amino acids(PCAA) = arginine, histidine, lysine; negatively charged amino acids(NCAA) = aspartic + asparagine, glutamic + glutamine, threonine, serine; essentialamino acids (EAA) = histidine, isoleucine, leucine, lysine, methionine, phenylala-nine, threonine, tryptophan and valine.

CF2–CF4 can also contribute to enhanced peptide entry into targetorgans through hydrophobic interactions with membrane lipidbilayers; this mechanism is believed to enhance in vivo antioxidanteffects (Sarmadi & Ismail, 2010). Fractionation of the CPH by RP-HPLC increased the aromatic amino acid (Phe, Tyr & Trp) contentmainly in CF2 and CF3 by 9% and 42%, respectively. Aromaticamino acids (AAA) possess the ability to scavenge free radicalsand, therefore, inhibit the propagation of oxidised lipid by-prod-ucts. Tryptophan and tyrosine have been reported to be responsi-ble for the antioxidative properties of AAA, particularly inpreventing blindness resulting from cataract formation (Rathore& Gupta, 2010). As expected, the positively charged amino acid lev-els (His, Arg & Lys) are significantly (p < 0.05) higher in the mosthydrophilic fraction (CF1). Histidine in particular is credited withstrong radical scavenging activity due to the presence of an imid-azole ring (Samaranayaka & Li-Chan, 2011). The content of nega-tively charged acidic amino acids (NCAA) was similar in all thepeptide samples though CF3 had the lowest. Previous reports (Heet al., 2013; Udenigwe & Aluko, 2011) have shown that the NCAA(ASX-aspartic and asparagine; GLX- glutamic and glutamine) havestrong antioxidant effects due to the presence of excess electrons,which can easily be donated to quench free radicals.

3.3. Radical scavenging activities

DPPH is a relatively stable non-physiologically relevant freeradical that accepts an electron or hydrogen from natural bioactivecompounds to become a less reactive, more stable molecule(Šlizyte et al., 2009). Fig. 2A shows that fractionation of CPH byRP-HPLC led to the increased DRSA of the cod peptide fractionsfrom 19% (unfractionated CPH) to 32% (CF4). The DRSA was directlyproportional (p < 0.05) to the elution time and content of HAA,which possibly suggests that the more hydrophobic peptides inter-act with the hydrophobic radical species. However, GSH had a sig-nificantly (p < 0.05) higher DRSA value than the CPH and peptidefractions, which suggests a superior radical scavenging ability forGSH in vitro. Results of the DRSA tests are similar to those reportedfor RP-HPLC separated salmon peptide fractions (9–33%), whichwere also evaluated at 1 mg peptide/ml (Girgih et al., 2013). Com-pared to the DRSA tests on chickpea peptide fractions prepared byLi et al. (2008) and fractionated using gel permeation chromatogra-phy, the cod peptides would appear to be somewhat less effective.

Fig. 2B shows that the ORAC values of CPH were only marginallyimproved after the RP-HPLC peptide fractionation because only theCF3 fraction had a significantly (p < 0.05) higher value. In fact, bothCF1 and CF4 had significantly (p < 0.05) lower ORAC values whencompared to the unfractionated CPH. As expected the GSH hadan ORAC value (1552 lM TE/g) that was significantly (p < 0.05)higher than those of the CPH and peptide fractions. The highestORAC value (940 lM TE/g) reported in this study for CPH andCF3 is lower than the 1541 and 1690 lM TE/g values previouslyreported for salmon (Girgih et al., 2013) and ovotransferrin(Huang, Majumder, & Wu, 2010), respectively. However, the CPHhad a higher ORAC value (613 lM TE/g) when compared to the269 and 434 lM TE/g reported for alcalase and trypsin oat proteinhydrolysates, respectively (Tsopmo, Cooper, & Jodayree, 2010).

In contrast to the results obtained for DRSA, the HRSA was sig-nificantly (p < 0.05) higher for the unfractionated CPH when com-pared to the fractionated peptides and the values are inverselyrelated to elution time of the peptides (Fig. 2C). The results suggestthat RP-HPLC fractionation of CPH to its component fractions led toa reduction of potency, probably due to loss of synergistic effects ofthe individual peptides. Thus, it seems likely that the higher thelevel of hydrophobic amino acids, the lower the HRSA of cod pep-tides. In contrast, an unfractionated pea protein hydrolysateshowed no detectable HRSA but the peptide fractions (F1–F5)

GSH CPH CF1 CF2 CF3 CF40

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Fig. 2. Radical scavenging activities of cod protein hydrolysate (CPH) and RP-HPLC separated peptide fractions (CF1–CF4) compared to reduced glutathione (GSH): (A) DPPH;(B) oxygen radical absorbance capacity; (C) hydroxyl radicals; (D) superoxide. Bars with different letters have significantly different (p < 0.05) mean values.

656 A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659

obtained after RP-HPLC fractionation had detectable HRSA levelsthat increased with higher HAA contents (Pownall et al., 2010).The present results also differ from those obtained for RP-HPLCfractionation of salmon peptides, which showed lack of HRSAdependency on content of HAA (Girgih et al., 2013).

The superoxide radical is an in vivo free radical that could pro-mote oxidative reactions because of its ability to reduce transitionmetals and react with the hydroxyl radical, causing damage to vitalcellular components, such as DNA, cell membrane lipids, proteinsand enzymes (Winczura, Zdzalik, & Tudek, 2012). Therefore, thebody’s ability to scavenge superoxide anion radicals is criticallyimportant in order to reduce oxidative stress-related destructionof cell biomolecules. Our results show that unfractionated CPHhad significantly (p < 0.05) higher SRSA (45%) than the RP-HPLCpeptide fractions, whose scavenging abilities ranged from 15% to40% (Fig. 2D). However, the CPH and peptide fractions had signifi-cantly (p < 0.05) lower SRSA values when compared to GSH (64%).The effect of the level of hydrophobic amino acids was also ambig-uous because for fractions CF1–CF3, the SRSA levels appeared todecline as hydrophobicity increased; whereas for CF4 (with highestrelative hydrophobicity, the SRSA level seemed to increase. Onecan, therefore conclude that relative hydrophobicity is perhapsnot the only factor involved in determining SRSA activity. How-ever, the presence of higher levels of electron-donating aminoacids seemed to contribute to better SRSA as evident in the resultsobtained for CPH and CF4 with �26% and 28% contents of nega-tively charged amino acids (NCAAs), respectively (Table 1). Theresults suggest that ability to donate electrons is an important con-tributing structural feature for improved scavenging of superoxide

radicals. The SRSA data obtained for cod peptides are different fromthose previously reported for salmon (Girgih et al., 2013) and pea(Pownall et al., 2010), where RP-HPLC fractionation led toincreased scavenging of superoxide radicals at the same level ofpeptide concentration (1 mg/ml).

3.4. Metal (iron) chelation and reducing properties

The metal chelation assay evaluates the ability of peptides toform complexes with transition metals (such as Fe2+ and Cu2+) thatcatalyse and promote lipid peroxidation, making them unavailableto participate in reactions that lead to generation of ROS and freeradicals. Peptides have been reported to act as indirect antioxidantagents through their chelating activities with metal ions, whichultimately reduce the susceptibility of lipids to oxidative peroxida-tion and associated negative health effects. Fig. 3A shows that CPHexhibited the strongest (44%) metal chelating activity (MCA),which was significantly (p < 0.05) higher than those of the fractions(25–30% range) and GSH (26%). The observed stronger chelatingactivity of CPH suggests a synergistic effect of the component pep-tides on inhibition of metal catalysed lipid oxidation. This synergywas lost after HPLC separation of CPH as reflected in the lower MCAvalues in the peptide fractions. The results are consistent with ourprevious reports that have shown unfractionated protein hydroly-sates to be better metal ion chelators than their fractionated coun-terparts (Girgih, Udenigwe, & Aluko, 2011; Girgih et al., 2013;Pownall et al., 2010). In contrast, the African yam bean seed proteinhydrolysate exhibited similar metal chelating activities to theirseparated ultrafiltration membrane fractions (Ajibola, Fashakin,

GSH CPH CF1 CF2 CF3 CF40

10

20

30

40

50

e

a

b df

c

Sample (1 mg/mL)

AM

etal

(Iro

n) C

hela

ting

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ivity

(%)

GSH CPH CF1 CF2 CF3 CF40.0

0.5

1.0

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2.0

2.5

3.0

c

a

c cb c

Sample (1 mg/mL)

B

FRA

P (a

bsor

banc

e at

700

nm

)

Fig. 3. (A) Metal chelating activity and (B) ferric reducing antioxidant power (FRAP) of reduced glutathione (GSH), cod protein hydrolysate (CPH), and its RP-HPLC derivedfractions (CF1–CF4). Bars with different letters have significantly different (p < 0.05) mean values.

0 1 2 3 4 5 6 7 80.0

0.1

0.2

0.3

0.4

0.5

0.6BlankGSHCPHCF1CF2CF3CF4

A

Incubation period (days)

a

bAbs

orba

nce

at 5

00 n

m

0 1 2 3 4 5 6 7 80.0

0.1

0.2

0.3

0.4

0.5

0.6 BlankGSHCPHCF1CF2CF3CF4

B

a

b

c

Incubation period (days)

Abs

orba

nce

at 5

00 n

m

0 1 2 3 4 5 6 7 80.0

0.1

0.2

0.3

0.4

0.5

0.6BlankGSHCPHCF1CF2CF3CF4

C

a

b

c

Incubation period (days)

Abs

orba

nce

at 5

00 n

m

Fig. 4. Inhibition of linoleic acid oxidation by cod protein hydrolysate (CPH) and RP-HPLC separated peptide fractions (CF1–CF4) compared to reduced glutathione (GSH) atsample concentrations of: (A) 1 mg/ml; (B) 0.5 mg/ml; and (C) 0.1 mg/ml respectively. Lines with differing letters have significantly different (p < 0.05) mean values.

A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659 657

Fagbemi, & Aluko, 2011), whereas the membrane fractions of rape-seed protein hydrolysate showed superior metal chelating proper-ties than the unfractionated protein hydrolysate (He et al., 2013).

The FRAP assay is often used to evaluate the capacity of naturalantioxidants to donate hydrogen or transfer electrons to transitionmetals responsible for catalysing the formation of harmful ROS/free radicals. In fact, Tang et al. (2012) have shown a direct corre-lation between enhanced antioxidant activities (measured as

inhibition of ROS production) and the reducing power of naturalpeptides. In this assay, the ability of CPH and its RP-HPLC peptidefractions to reduce Fe3+ to Fe2+ was determined through changesin the absorbance at 700 nm of the supernatant of the reactionmixture; the higher the absorbance, the stronger the metal reduc-ing power of the sample. Among the peptides, the CPH (unfraction-ated hydrolysate) showed the strongest FRAP (absorbance of 0.18)while its RP-HPLC peptide fractions (CF1–CF4) exhibited lower

658 A.T. Girgih et al. / Food Chemistry 173 (2015) 652–659

FRAP activities with 0.056–0.112 absorbance values, which is anindication that fractionation did not improve this property. Thus,the higher FRAP potency of CPH may be attributed to synergisticeffect of the peptide components, which was lost upon RP-HPLCseparation into fractions. The FRAP values of the fractionated pep-tides were partially related to content of sulphur-containing aminoacids (SCAAs) with CF1, CF2, CF3 and CF4 containing 2.12%, 2.97%,4.34% and 2.07%, respectively. Despite the comparable level of SCA-As in CF1, the observed low FRAP value may be due to the high con-tent (33%) of positively charged amino acids (especially lysine),which could have reduced the intensity of interactions with thepositively charged ferric ions. The results are consistent with thereport of Udenigwe and Aluko (2011), which had previously sug-gested that SCAAs and lysine make positive and negative contribu-tions, respectively, to FRAP.

3.5. Inhibition of linoleic acid oxidation

Fig. 4 shows the abilities of different concentrations of CPH andits HPLC peptide fractions to inhibit linoleic acid oxidation during a7-day incubation period in comparison to a blank that lacked thepeptides. At 1 mg/ml peptide concentration (Fig. 4A) the CPH, HPLCfractions and GSH significantly (p < 0.05) inhibited linoleic acidoxidation when compared to the blank reaction mixture. Thestrong inhibitory effects of all of the peptide fractions is evidentby the almost linear clustering of the curves at the lower portionof the graph, indicating an immediate and almost complete pre-vention of linoleic acid oxidation by the added cod peptide inhibi-tors. In the blank control (with no added peptide fractions), therewas a rapid increase in the absorbance, which reached its maxi-mum value (0.516 nm) on the third day and began a steep declineas the incubation period progressed, possibly due to the formationand decomposition of linoleic oxidation products (Jayaprakasha,Singh, & Sakariah, 2001). As peptide concentration was decreasedfrom 1 mg/ml (Fig. 4A) to 0.5 (Fig. 4B) and 0.1 mg/ml (Fig. 4C),there was reduced potency against linoleic acid peroxidation asshown by the increases in absorbance values, which suggests adose-dependent peptide inhibition of linoleic acid oxidation. Theinhibition of linoleic acid peroxidation results in this study are sim-ilar to those obtained for salmon peptides (Girgih et al., 2013). TheCF2 peptides seem to exhibit weakest activity at 0.1 and 0.5 mg/mlconcentrations, though the factor responsible is not clear since thisfraction contains high HAA, AAA and NCAA levels. A possible rea-son for the low activity of CF2 could be a reduced ability to interactwith the lipid phase. This is because increased lipid solubility ofpeptides is believed to enhance inhibition of lipid peroxidationby facilitating better interaction with free radical species(Rajapakse, Mendis, Jung, Je, & Kim, 2005).

4. Conclusions

This study has shown that CPH and its HPLC fractions havestrong in vitro antioxidant properties as evident in their strong rad-ical scavenging and metal binding activities. In comparison to GSH,cod peptides displayed lower radical scavenging activities buthigher metal chelation and ferric reducing properties. Peptide frac-tionation on the basis of hydrophobicity had a negative effect byreducing the antioxidant synergy within the unfractionated CPHwith respect to SRSA, HRSA metal chelation and FRAP. In contrast,the most hydrophobic peptides displayed higher ORAC and DPPHvalues. Since the negative effect of fractionation was observed fornatural free radicals, the unfractionated CPH may provide a betteralternative as an ingredient to formulate antioxidant foods whencompared to the fractionated peptides. The small size (<1 kDa) ofthe peptides used in this work could contribute to their enhanced

absorption and potency as in vivo antioxidants. Therefore, futurework will concentrate on examining the ability of these peptidesto reduce oxidative stress in living tissues. However, the increasedpotency of the fractionated peptides against artificial radicals(ORAC and DPPH) suggests the fractions have a higher potentialthan unfractionated CPH as ingredients to formulate antioxidantingredients for increased food lipid stability and qualitypreservation.

Acknowledgements

The work was funded in part through a Strategic Grant awardedto author Gill by the Natural Sciences and Engineering ResearchCouncil (NSERC) of Canada. Author Aluko’s research program isfunded in part by an NSERC Discovery grant award.

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