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LWT - Food Science and Technology 60 (2015) 452e461

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LWT - Food Science and Technology

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Isolation, purification and identification of three novel antioxidativepeptides from patin (Pangasius sutchi) myofibrillar proteinhydrolysates

Leila Najafian*, Abdul Salam BabjiSchool of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

a r t i c l e i n f o

Article history:Received 25 December 2013Received in revised form26 July 2014Accepted 30 July 2014Available online 15 August 2014

Keywords:Patin (P. sutchi)Antioxidant peptidesMyofibrillar protein hydrolysatePurificationMass spectrometry

* Corresponding author. Tel.: þ60 3 89215988; fax:E-mail address: najafian_5828@yahoo.com (L. Naj

http://dx.doi.org/10.1016/j.lwt.2014.07.0460023-6438/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Myofibrillar protein from patin was hydrolysed using papain, alcalase and flavourzyme with differingdegrees of hydrolysis (DH) to obtain antioxidative peptides. The protein solubility and peptide content ofthe myofibrillar protein hydrolysates (MPHs) were observed. The antioxidant activity of the hydrolysateswas evaluated. The results showed that the highest DH (89.17%) of MPH was produced by the 120-minpapain treatment. When the DH of MPHs increased, protein solubility and peptide content increased.Papain-MPHs exhibited the highest antioxidant activity. The papain hydrolysate was purified using ionexchange chromatography, gel filtration chromatography and RP-HPLC. The potent fraction (MI 4) ob-tained from RP-HPLC had DPPH radical scavenging activity that was 2.97 times higher than MPH. Threeantioxidative peptide sequences were identified as VPKNYFHDIV, LVMFLDNQHRVIRH, and FVNQPYL-LYSVHMK according to HPLC and connected to the electrospray ionization-time-of-flight mass spec-trometer (ESI-TOF MS/MS). The FVNQPYLLYSVHMK peptide exhibited the highest antioxidant activity.The presence of hydrophobic amino acids (Leu, Val and Phe), hydrophilic and basic amino acids, (His, Proand Lys), and aromatic amino acids (Phe and Tyr) in the peptide sequences is believed to contribute tothe high antioxidant activity of MPHs. These results suggest that MPHs from patin have potential as anatural antioxidants ingredient in foods.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Antioxidants have been proven to be benefit human healthbecause they may protect the body against molecules known asreactive oxygen species (ROS), which can attack membrane lipids,protein and DNA. The formation of ROS has been associated withmany human diseases, such as heart disease, stroke, arterioscle-rosis, diabetes, cancer and Alzheimer's disease. Antioxidants arewidely used in dietary supplements to boost health and to reducethe risk of diseases such as cancer and coronary heart disease. Manysynthetic antioxidants such as butylatedhydroxytoluene (BHT),butylatedhydroxyanisole (BHA), tert-butylhydroquinone (TBHQ),and propyl gallate (PG), are used in the food and pharmaceuticalindustries to retard lipid oxidation (Bernardini et al. 2011). How-ever, the use of these synthetic antioxidants must be strictlycontrolled because of the potential health issues (Hettiarachch,Gnanasanbanda & Johnson, 1996). There is growing interest in

þ60 3 89213232.afian).

the replacing synthetic antioxidants with natural antioxidants fromfood sources to capture their potential health benefits with few orno side effects (Sarmadi & Ismail, 2010). Protein hydrolysates fromseveral fish species, such as round scad (Thiansilakul, Benjakul, &Shahidi, 2007), mackerel (Wu, Chen, & Shiau, 2003), sardinelle(Bougatef, Nedjar-Arroume & Manni, 2010), yellow stripe trevally(Klompong, Benjakul, Kantachote, & Shahidi, 2007), loach (You,Zhao, Regenstein, & Ren, 2010) and tilapia (Raghavan, Kristinsson& Leeuwenburgh, 2008) have been reported to demonstrate anti-oxidant activity.

Antioxidative peptides generally contain 2e20 amino acid units.The amino acid composition and sequences can affect the activity ofbiopeptides. Some bioactive peptides contain hydrophobic aminoacid residues such as Val or Leu at the N-terminus of the peptides,and Pro, His, Tyr, Trp, Met, and Lys in their sequences (Elias et al.,2006). Some contained mostly the acidic amino acid residues(Glu, Asp) (Saiga, Tanabe, & Nishimura, 2003). However, moreresearch is required to describe the relationship between thestructure and function of antioxidative peptides.

Pangasius sutchi is a popular freshwater fish used as food inMalaysia. This fish species is found in abundance in the Amazon

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461 453

River, in parts of Russia and in other places around the world underdifferent names. There is increasing demand for protein hydroly-sates with antioxidative properties in the pharmaceutical andhealth food industries as well as the food processing and preser-vation industries (Alasalvar, Taylor, & Shahidi, 2002; Hagen &Sandnes, 2004). Thus, the production of fish protein hydrolysates(FPHs) with antioxidative capacity from patin muscle proteins andthe development of FPH-based ingredients for food and pharma-ceutical applications would be an ideal approach for utilizing thisfreshwater fish caught in the waters of Malaysia.

In the present study, the antioxidant activity (DPPH and ABTSradical scavenging activities, reducing power and metal chelatingactivity) of myofibrillar protein hydrolysates (MPHs) with variouscommercial enzymes was investigated. The degree of hydrolysis(DH) solubility and the peptide content of the MPHs weremeasured. Antioxidant peptides from MPHs were separated usingion exchange chromatography, gel filtration chromatography andhigh-performance liquid chromatography (RP-HPLC). The antioxi-dant activity of each fraction collected was evaluated using DPPHradical scavenging activity. The sequence of the purified anti-oxidative peptide was identified by HPLC connected to electrosprayionization (ESI) quadropole-time of flight mass spectrometer (ESIQ-TOF-MS/MS).

2. Materials and methods

2.1. Materials and chemicals

Patin (Pangasius sutchi) was taken from a nearby farm inMalaysia. The meat (without the skin, head, tail, bones and blood)was collected and ground. The ground meat was stored at �18 �C.Amino acid standards were purchased from Pierce (Rockford, IL,USA) 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) dia-mmonium salt (ABTS), 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonicacid)-1,2,4-triazine (ferrozine), 1,1-diphenyl-2-picrylhydrazyl(DPPH), ferric chloride, ammonium thiocyanate and trichloro-acetic acid (TCA) were purchased from SigmaeAldrich (St. Louis,MO, USA). Flavourzyme® 500 L (a protease from Aspergillus oryzae)

DH ð%Þ ¼ ðsoluble nitrogen in the 10% TCA sample=total nitrogen in the sampleÞ � 100

was obtained fromNovo Nordisk. Alcalase® 2.4 L, an endopeptidasefrom Bacillus licheniformis and papain from papaya were purchasedfrom SigmaeAldrich. Other analytical grade chemicals were alsopurchased from SigmaeAldrich.

2.2. Extraction of myofibrillar proteins

Myofibrillar proteins were extracted using the alkali solubili-sation method descibed by Hultin and Kelleher (2000). The mincedmuscle was mixed and homogenised with nine parts cold (6 �C)deionized water for each part of muscle using a BioHomogenizer ata high speed for 1 min. In the alkaline solubilization step, the pH ofthe homogenized sample was adjusted to 11.0 using 2 M NaOH andincubated at 4 �C for 30 min. After centrifuging the mixture at10,000 � g for 20 min at 4 �C, the supernatant containing the sol-uble proteins was separated and filtered through a double layer ofcheese cloth and adjusted to pH 5.5 using 1MHCl to precipitate theproteins. Themixturewas then centrifuged at 10,000� g for 20minat 4 �C to recover the myofibrillar proteins in the pelleted material.

This separated protein was freeze-dried and used as a substrate forthe enzymatic hydrolysis.

2.3. Preparation of patin myofibrillar protein hydrolysate

The freeze-dried proteins obtained from Pangasius sutchi weremixed with distilled water at a ratio of 1:100 (w/v), and incubatedwith papain (pH 7; 50 �C), flavourzyme (pH 7; 50 �C) or alcalase (pH8; 60 �C) for 30, 60, 90 and 120 min. The ratio of the myofibrillarprotein fraction to each enzyme was 100:1 (w/w). The pH of eachsolutionwas adjusted using 2M HCl or 2 MNaOH. After incubation,the enzymatic hydrolysis was stopped by boiling the solution in awater bath for 5 min or heating at 98 �C for 10 min. The hydrolysatewas centrifuged at 3000 � g for 20 min and then lyophilized.

2.4. Determination of amino acid composition

Amino acid compositions were determined as described byAlaiz, Navarro, Gir�on, and Vioque (1992), with slight modifications.Samples were hydrolysed with 6 M hydrochloric acid at 110 ± 1 �Cfor 24 h. The amino acids were detected using a C18 AccQ-Tag col-umn (150 � 3.9 mm, Waters, USA), with the temperature set at37 �C and a flow rate of 1 ml/min. UV absorbance was measured at248 nm (for peak identification), and the associated fluorescencedetector was operated at excitation and emission wavelengths of250 nm and 395 nm (for amino acid quantification), respectively.Alkaline hydrolysis was also performed to quantify the level oftryptophan (Rutherfurd & Gilani, 2009).

2.5. Determination of the degree of hydrolysis

The degree of hydrolysis (DH) was measured according usingthe method of Hoyle and Merritt (1994). A 20-ml sample of proteinhydrolysate was added to 20 ml of 20% (w/v) TCA to produce asoluble material consisting of 10% TCA. Each mixture was left atroom temperature for 30 min and then centrifuged at 7800 � g for15 min. The degree of hydrolysis (DH) was calculated using thefollowing equation:

2.6. Solubility

The soluble content of myofibrillar protein was determinedusing the Folin-Lowry method (Gerhardt, Murray, Wood, & Krieg,1994). An aliquot of 0.5 ml of the sample was mixed with 0.7 mlof an alkaline-copper reagent and incubated for 20 min at roomtemperature. The mixture was added to 0.1 ml of Folin-Ciocalteu’sphenol reagent at 2 times dilutionwith deionized water and left for30 min or longer at room temperature. The absorbance at 750 nmwas measured with a spectrophotometer (Model UV-160A, Shi-madzu, Japan). The soluble protein content was quantified usingbovine serum albumin as the standard.

2.7. Peptide content

The peptide content of the hydrolysates was determined withslight modifications to the method described by Church,Swaisgood, Porter, and Catignani (1983) using an o-phthaldialde-hyde (OPA) spectrophotometric assay. The fresh OPA reagent was

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461454

prepared by mixing 25 ml of 100 mM sodium tetra hydroborate,2.5 ml of 20% (w/w) sodium dodecyl sulfate, 40 ml of OPA sodium(dissolved in 1 ml of methanol) and 100 ml of bemercaptoethanoland then adjusting the volume to 50 ml with deionized water. Fiftymicroliters of each hydrolysate, containing 5e100 mg proteins weremixed with 2ml of OPA reagent and incubated for 2 min at ambienttemperature. The absorbance at 340 nm was measured withspectrophotometer. Casein (tryptone) in a phosphate buffer (pH7.4) was used as the standard to quantify the peptide content.

2.8. Determination of the antioxidant activity of SPH

2.8.1. DPPH radical scavenging activityDPPH radical scavenging activity was measured using the

method described by Wu et al. (2003) with some modifications. A1.5-ml samplewas added to 1.5ml of 0.15mMDPPH in 95% ethanol.The solution was then allowed to stand for 30 min at room tem-perature in the dark after vigorous mixing. The absorbance wasmeasured at 517 nm. A control solution using distilled waterinstead of samples was prepared in the same manner. The scav-enging effect was quantified according to the following equation:

DPPH radical scavenging activity ð%Þ ¼ ½ðB� AÞ=B� � 100

Where A represents the absorbance of the sample and B representsthe absorbance of the control.

2.8.2. Reducing powerThe reducing power of the SPHs was measured according to the

method described by Oyaizu (1988) with slight modifications.Briefly, 1 ml of the sample was mixed with 2 ml of 0.2 M phosphatebuffer (pH 6.6) and 1 ml of a 1% potassium ferricyanide solution.After incubation at 50 �C for 20 min, 1 ml of 10% TCA was added tothe reaction mixture, and the reaction mixture was then centri-fuged at 1500 � g for 10 min. Finally, 1 ml of the supernatant so-lution was subsequently mixed with 1 ml of distilled water and200 ml of a 0.1% ferric-chloride solution. After standing for 10 min atroom temperature, the absorbance was measured at 700 nm.

2.8.3. Metal-chelating activityThe ability of MPHs to chelate pro-oxidative Feþ2 was measured

according to the method of Decker and Welch (1990). For eachsample, 1 ml of solution was mixed with 3.7 ml of distilled water.This mixture was then allowed to react with a solution containing0.1 ml of 2 mM FeCl2 and 0.2 ml of 5 mM ferrozine. The absorbanceof this reaction mixture was monitored at 562 nm after 20 min. Acontrol solution using 1 ml of distilled water instead of the samplewas prepared in the same manner. The chelating activity wascalculated according to the following equation:

Chelating activity ð%Þ ¼ ½1� ðA=BÞ� � 100

Where A represents the absorbance of the sample and B representsthe absorbance of the control.

2.8.4. ABTS radical-scavenging activityThe ABTS radical-scavenging activities of the MPHs were

determined according to the method described by Re et al. (1999)with some modifications. A stock solution containing of 7 mMABTS in 2.45 mM potassium persulphate, was prepared and used asthe ABTS solution. This mixture was left for 12e16 h at roomtemperature in the dark. An aliquot of the stock solution wasdiluted with distilled water until it indicated an absorbance of0.70 ± 0.02 at 734 nm. A 50-ml aliquot of the sample or distilledwater (in the case of the control experiment) was added to 950 mL

of the diluted ABTSþ solution. The mixture was shaken vigorouslyfor 30 s and allowed to stand in a dark environment for 10 min. Theabsorbance values were then measured at 734 nm. A control so-lution using distilled water instead of the sample was prepared inthe same manner. The scavenging effect was quantified accordingto the following equation:

ABTS radical scavenging activity ð%Þ ¼ ½ðB� AÞ=B� � 100

Where A represents the absorbance of the sample and B representsthe absorbance of the control.

2.8.5. Thiobarbituric acid value assayTBA value was assessed using the method of Buege and Aust

(1978). A 0.5 g sample was homogenated and reacted with 2.5 mlof TBA reagent containing 0.375% TBA solution, 15% TCA and 0.25 Nof hydrochloric acid (HCl). The mixture was heated for 10 min atboiling temperature (100 �C) to formation of pink colour. Aftercooling the solution was centrifuged at 2540 � g for 25 min. Theabsorption of supernatant was read by using a spectrophotometer(Varian Cary 50) at wavelength of 532 nm. The TBAvalue calculatedbased on the following formula:

TBA Value ¼ Absorbance at 532 nm� 2:77

The extent of oxidation is described as the TBA value and isexpressed as milligrams of malonaldehyde (MA) equivalents perkilogram sample.

2.9. Purification of antioxidant peptides from SPHs

2.9.1. Ion exchange chromatographyThe dried fractions with the highest antioxidant activity that

obtained from myfibrillar protein hydrolysate using papain, werere-dissolved in 50 mM acetate buffer (pH 4.0) to obtain a proteinconcentration of 25 mg/ml and loaded onto an ion exchange col-umnHiTrap CM FF (GEHealthcare, UK), whichwas equilibratedwith50 mM sodium acetate buffer (pH 4.0) and eluted with a lineargradient of NaCl (0e1.0 M) in the same buffer at a flow rate of 3 ml/min. Fractions were collected andmonitored at 280 nm at a volumeof 6 ml to isolate the peptide fractions. The antioxidant activity offractions was measured using a DPPH radical scavenging activityassay. The highest antioxidative fraction was lyophilized.

2.9.2. Gel filtration chromatographyAfter ion exchange chromatography separation, the fraction that

showed the highest DPPH radical scavenging activity was furtherpurified using gel filtration chromatography. Samples of 250 mgwere dissolved in 2 ml of 10 mM sodium phosphate buffer (pH 7.2),loaded onto a Hiprep 26/60 sephacryl S-100HR column,(26 � 600 mm, GE Healthcare, UK), and eluted with 10 mM sodiumphosphate buffer (pH 7.2) at a flow rate of 1 ml/min. The elutedfractions (5 ml) were pooled into several fractions based on theabsorbance peaks observed at 280 nm and freeze dried. The anti-oxidant activity of the fractions was determined using the DPPHradical scavenging activity assay.

2.9.3. Reverse-phase high performance liquid chromatography (RP-HPLC)

Based on the gel filtration chromatography, 5-mg samples offractions that showed the highest DPPH radical scavenging activitywere dissolved in sodium phosphate buffer and filtered with filter a0.22-mm filter; 200 ml of the sample were loaded onto an XBridgeBEH130 Prep C18 (10 � 250 mm, 5 mm, Waters, USA) column. Sol-vent A was 0.1% (v/v) trifluoroacetic acid (TFA) in deionized water,

Table 1The effect of time on the degree of hydrolysis and the soluble protein and peptidecontent of myofibrillar proteins.

Samples Times ofincubation(min)

DH (%) Proteinsolubility(mg/g)

Peptidecontent(mg/g)

MyofibrillarProtein

e 82.83 ± 2.16l 85.4 ± 1.47k

30 46.16 ± 1.41g 342.3 ± 1.47i 245.2 ± 0.98d

Papain-MPH 60 65.83 ± 1.28d 455.8 ± 1.69f 261.4 ± 1.14c

90 88.53 ± 0.25a 496.4 ± 1.47d 279.2 ± 1.47b

120 89.17 ± 0.31a 559.8 ± 2.33a 299.1 ± 1.71a

30 40.60 ± 0.43h 325.4 ± 1.96j 206.0 ± 1.22h

Alcalase-MPH 60 60.33 ± 0.50e 441.1 ± 1.71g 224.0 ± 1.55f

90 82.18 ± 1.37b 482.3 ± 1.10e 240.0 ± 1.55e

120 83.60 ± 0.50b 536.0 ± 2.04b 263.8 ± 1.47c

30 36.53 ± 0.70i 310.5 ± 1.31k 188.0 ± 1.02j

Flavourzyme-MPH 60 52.80 ± 1.36f 424.4 ± 1.18h 196.8 ± 1.22i

90 72.80 ± 0.86c 456.6 ± 1.75f 210.2 ± 0.98g

120 74.16 ± 0.53c 502.1 ± 1.22c 224.2 ± 1.47f

aec Mean % values in the same column without a common superscript letter aresignificantly different (p < 0.05).e Values shown are the mean ± standard deviation of three replicates.

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461 455

and solvent B was 0.1% (v/v) TFA in 100% (v/v) acetonitrile solution.Peptide separation was performed with a linear gradient of 100%eluent A for 5 min and with the following increasing eluent B:0e5 min, 0% eluent B; 5e10 min, 0e50% eluent B; 10e30 min,50e65% eluent B; 30e35 min, 65e100% eluent B; 35e40 min, 100%eluent B; 40e45 min, 100-0% eluent B. The flow rate was 4.73 mg/min and the UV absorbance of the eluents was monitored at214 nm. All the peaks were collected separately and then concen-trated using a centrifugal concentrator at 130 rpm for 3 h. Theconcentrated samplewas used for DPPH radical scavenging activity.

2.10. Identification of antioxidant peptides using LC-MS-TOF

The desirable fraction with the highest antioxidant activity afterRP-HPLC purificationwas dissolved in 0.1% formic acid inwater to aconcentration of 20 mg/ml and loaded onto an Agilent 1200 HPLC-chip MS interface. An aliquot of 1 ml of the test sample was loadedonto the large capacity chip, 300 A, C18, 160 nl enrichment columnand 75 mm � 150 mm analytical column (Agilent part no: G4240-62010). The mobile phase A was 0.1% formic acid (FA) in water,and B was 90% acetonitrile (ACN) inwater and 0.1% formic acid. Theflow gradient with the Agilent 1200 Series nanoflow LC pump wasas follows: baseline, 3% B; 0e30min, 3e50% B; 30e32min, 50e95%B; 32e37 min, 95% B; 37e38 min, 95-3% B; 38e47 min, a flow rateof 0.3 ml/min. Mass detection was performed with an Agilent 6520Accurate-Mass Q-TOF LC/MS operating in positive ion mode.Spectrawere acquired in theMS/MSmode, and the 6520 Q-TOFwasoperated in 2 GHz Extended Dynamic Range modewith an MS scanrange of 110e3000 m/z and an MS/MS scan range of 50e3000 m/z.Precursor selection I and II included a threshold 200 counts abs. and2, 3, >3 in the precursor charge state selection and preferencesrespectively. The Agilent ESI Q-TOF was used for data acquisitionand processing. Agilent Spectrum Mill MS Proteomics Workbenchsoftware was used to perform a database search usingSwissProt.MAR.2013.fasta.

2.11. Peptide synthesis

Sample for commercial synthesis of peptide sequence was sentto First BASE Laboratories Sdn Bhd, Malaysia who then sent forsynthesizing to Mimotopes Pty Ltd ABN, The Peptide Company inAustralia. The synthesized peptides were then purified by RP-HPLCon a Monitor C18, Column Engineering (150 � 4.6 mm). Solvent Awas 0.1% trifluoroacetic acid in 100% water, and solvent B was 0.1%trifluoroacetic acid in 90% acetonitrile with linear gradient 10% B for1.0 min; 10e66.6% B over 15 min; then column reequilibration(flow rate, 1.5 ml/min; monitoring 214 nm). The molecular massesof the isolated peptide were determined by mass spectrometry.

2.12. Statistical analyses

A one-way analysis of variance (ANOVA)was performed, and themeans were compared using Duncan's multiple range test. All an-alyses were conducted in triplicate. The statistical analyses wereperformed using SPSS (SPSS 18.0 for Windows, SPSS Inc.). Differ-ences were considered significant at p < 0.05.

3. Results and discussion

3.1. Degree of hydrolysis and the soluble protein and peptidecontent

The hydrolysis of proteins with certain enzymes can increasethe antioxidant activity of hydrolysates. The protein substrate, theenzymes used for the proteolysis, the hydrolysis conditions and the

degree of hydrolysis (DH) greatly influence the amino acid com-positions, the molecular weights of the protein hydrolysates, andthe resulting biological activities (Balti et al. 2011). The effect oftime on DH and the soluble protein and peptide content ofextracted myofibrillar proteins from Pangasius sutchi hydrolysedwith papain (papain-MPH), alcalase (alcalase-MPH) and fla-vourzyme (flavourzyme-MPH) are shown in Table 1. The degree ofhydrolysis depended on the type of enzyme (papain, alcalase andflavourzyme) and the reaction time. The results indicated that DHincreased with increasing hydrolysis time for all the enzymes(Table 1). For a given hydrolysis time, the DH associated withpapain digestion was significantly higher than that associated withthe alcalase and flavourzyme treatments (p < 0.05). The highest DH(89.17%) for the hydrolysis of the myofibrillar proteinwas producedwith a papain treatment for 120 min. This result showed that theconcentration of peptide bonds available for hydrolysis by papain ishigher than other enzymes used. Papain action is broad in natureand able to hydrolyse more of peptide bonds of the myofibrillarprotein. This also commonly associated with its tenderizing func-tion, which is actually hydrolysis on most of the peptide bond ofamino acids. Therefore, the higher (p < 0.05) levels of DH observedin association with the papain treatment may have occurredbecause the papain enzyme is more effective than alcalase andflavourzyme in preparing the patin protein hydrolysates.

Solubility is primarily dependent on the distribution of hydro-phobic and hydrophilic amino acids on the surface of a protein andon the thermodynamics of protein-water interactions (Kristinsson& Rasco, 2000). The soluble protein content of myofibrillar pro-tein hydrolysates with proteases increased as the time of hydrolysisincreased from 82.83 to 559.8 mg/g (Table 1). The results showedthat when the DH of the MPHs increased, protein solubilityincreased. These results are similar to the results obtained withhydrolysates from capelin muscle (Shahidi, Han, & Synowiecki,1995), shark muscle (Diniz & Martin, 1997), yellow stripe trevallymuscle (Klompong et al., 2007) and silver carp muscle (Dong et al.,2008). The increased solubility of intact fish myofibrillar proteinswith partial enzymatic hydrolysis is well documented and attrib-utable to the smaller size of peptides and to newly exposed aminoand carboxyl groups that enable more interaction with water andincrease the hydrophilic nature of the hydrolysates (Kristinsson &Rasco, 2000). The peptide content of myofibrillar proteins andtheir hydrolysates increased from 85.4 to 299.1 mg/g (Table 1).Papain myofibrillar proteins had the highest peptide content(299.1 mg/g), with a DH of 89.17%. The results showed that as the

Table 2Antioxidant activities of MPH prepared with papain, alcalase and flavourzyme.a

Sample IC50 (mg/ml) Reducing power Thiobarbituricacid value(mg malonaldehyde/kg)

DPPH radical scavenging activity ABTS radical scavenging activity Metal chelating activity

Papain-MPH 1.12 ± 0.09 0.840 ± 0.08 80.46 ± 0.49 0.315 ± 0.006 1.89 ± 0.01Alcalase-MPH 2.95 ± 0.22 0.893 ± 0.31 42.98 ± 0.37 0.282 ± 0.008 1.44 ± 0.02Flavourzyme-MPH 1.94 ± 0.15 1.490 ± 0.23 65.39 ± 0.24 0.224 ± 0.005 0.995 ± 0.04

e Values shown are the mean ± standard deviation of three replicates.a DPPH and ABTS radical scavenging activities, reducing power and thiobarbituric acid value of MPH after 60 min incubation and metal chelating activity of MPH after

120 min incubation were indicated.

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incubation time increased, the peptide content of protein hydro-lysates increased. A similar result, with peptide content rangingfrom 84.2 to 272.3 mg/g, was reported in freshwater clam(Corbicula fluminea, Muller) muscle protein hydrolysates (Tsai, Lin,Chen, & Pan, 2006). This number may vary, depending on the pu-rity, sequence and purification.

3.2. The antioxidant activity of MPHs

The antioxidant activity of the hydrolysates was determinedusing DPPH and ABTS radical scavenging activities, reducing power,metal chelating activity and thiobarbituric acid value assays. Asshown in Table 2, alcalase-MPH demonstrated chelating activity forthe Fe2þ ion with an IC50 value of 42.98 ± 0.37 mg/ml, which washigher than the values for flavourzyme-MPH and papain-MPH (IC50values of 65.39 ± 0.24 and 80.46 ± 0.49 mg/ml, respectively).Papain-MPH had the highest DPPH and ABTS radical scavengingactivity with IC50 values of 1.12 ± 0.09 and 0.840 ± 0.08 mg/ml,respectively (p < 0.05). Moreover, papain-MPH had greaterreducing power, with an absorbance of 0.315± 0.006, than alcalase-MPH and flavourzyme-MPH, with absorbance values of0.282 ± 0.005 and 0.224 ± 0.005, respectively. According to ourprevious research (Najafian, Jafarzade, Said, & Babji, 2013), DPPH

Fig. 1. Separation scheme for the radical-scavenging peptide obtained from patin protein hyelution profiles. e Different letters within the same parameter indicate significant differen

and ABTS radical scavenging activity, reducing power of MPHs after60 min incubation and metal chelating activity of MPHs after120 min were very high.

Papain-MPH showed the highest Thiobarbituric acid value with1.89 ± 0.01 mg malonaldehyde/kg after 60 min in compare toalcalase (1.44 ± 0.02)and flavourzyme (0.995 ± 0.04). Thio-barbituric acid (TBA) value measures secondary lipid oxidationproducts, which were also responsible for the rancid taste devel-oped during storage. TBA value was previously found to be from0.36 to 1.08 (Das, Anjaneyulu, & Biswas, 2006) in ground buffalomeat, which is comparable with the present study.

Totally, the results suggest that MPHs might contain peptidesthat act as hydrogen donors, terminating the radical chain reactionby scavenging with the free radicals to produce stable products andalso can reduce lipid peroxidation in food systems. The differencesin antioxidant activity between the myofibrillar protein hydroly-sates may be attributed to the differences in the exposed sidechains of the peptides that were generated by the different en-zymes for the several levels of the DH. As shown in Table 2, papain-MPH showed the highest antioxidant activities and Kim et al.(2009) showed papain hydrolysate from venison (family Cervi-dae) had the highest antioxidant activity. Furthermore, reportedthat papain is a protease that exhibits specific substrate preferences

drolysate. (A) ion exchange chromatography and (B) gel filtration chromatography (B)ces (p < 0.05).

Table 4Amino acid (AA) compositions of the patin myofibrillar protein and MPHs (%).

AA% UPMPd MAe MI

Asp 10.17 ± 0.16b 10.39 ± 0.18ab 10.57 ± 0.20a

Ser 4.18 ± 0.11a 3.29 ± 0.25b 2.20 ± 0.19c

Glu 15.99 ± 0.22b 16.33 ± 0.14ab 16.44 ± 0.16a

Gly 3.56 ± 0.14a 2.97 ± 0.21b 2.40 ± 0.16c

Hisi 2.37 ± 0.16b 2.89 ± 0.13a 3.04 ± 0.16a

Arg 4.88 ± 0.26a 3.30 ± 0.20b 2.20 ± 0.15c

Thri 6.26 ± 0.82a 5.98 ± 0.25a 6.60 ± 0.12a

Ala 5.36 ± 0.88a 5.41 ± 0.17a 5.11 ± 0.16a

Pro 2.99 ± 0.64a 3.16 ± 0.22a 3.22 ± 0.15a

Tyr 3.49 ± 0.28a 3.48 ± 0.08a 3.71 ± 0.16a

Vali 4.77 ± 0.02c 5.36 ± 0.16b 6.00 ± 0.19a

Meti 5.43 ± 0.57a 5.53 ± 0.19a 5.61 ± 0.16a

Lysi 9.34 ± 0.66a 10.02 ± 0.16a 10.11 ± 0.16a

Ilei 4.32 ± 0.42a 4.39 ± 0.14a 4.43 ± 0.14a

Leui 7.86 ± 0.27b 8.10 ± 0.12b 8.60 ± 0.13a

Phei 3.63 ± 0.29b 3.95 ± 0.15b 4.46 ± 0.13a

Cys 1.56 ± 0.15a 1.00 ± 0.16b 0.80 ± 0.16b

Trpi 3.84 ± 0.17b 4.50 ± 0.14a 4.67 ± 0.14a

TEAAf 47.82c 50.72b 53.52a

HAAg 37.85c 39.38b 41.14a

ARMh 13.33c 14.82b 15.88a

e Values are reported as the mean ± SD; experiments were performed in triplicate.e Different letters within the same parameter indicate significant differences(p < 0.05).

d UPMP: Unhydrolyzed Patin Myofibrillar Protein.e MA and MI: Fractions with the highest DPPH radical scavenging activity ob-

tained from ion exchange (MA) and gel filtration chromatography (MI).f Total essential amino acids.g Hydrophobic amino acids (Ala,Val, Met, Ile, Leu, Phe, Pro and Tyr).h Aromatic amino acids (Phe, His, Trp and Tyr).i Essential amino acid.

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primarily for bulky hydrophobic or aromatic residues at this sub-strate. Therefore, papain hydrolysates were selected for furtherstudy.

3.3. Purification of antioxidant peptides

3.3.1. Ion-exchange chromatographyPapain-MPH was dissolved in 50 mM acetate buffer (pH 4.0)

loaded onto an anion exchange column HiTrap CM FF (GE Health-care, UK) and fractionated into two proteins (Fig. 1A). The MAfraction was not bound to negatively charged groups on the solidsupporters in the column and was eluted. The MB fraction wasbound to negatively charged groups on the solid supporters andeluted later with increasing NaCl. Therefore, the fraction of MA hada neutral or acidic charged, while the fraction of MB had basiccharged in a solution of 50 mM sodium acetate buffer with a pH of4.0.

As shown in Table 3, the DPPH radical scavenging activity of MA(with an IC50 value of 0.610 mg/ml: 1.84 times higher than MPH)was higher than that of MB (with an IC50 value of 5.88 mg/ml)(p < 0.05) (Fig. 1A).

3.3.2. Gel filtration chromatographyGel filtration is a method that separates substances based on

differences in their molecular dimensions. It is also used to removesalt from the protein solutions and to separate proteins and othercolloids from low-molecular-weight substances (Gelotte, 1960).Fig. 1B shows the DPPH radical trapping activity (IC50) and the gelfiltration chromatography elution profile of the MA. The MA frac-tion of patin fish obtained from ion exchange chromatography wasfurther separated using gel filtration chromatography. Fractioninghas resulted in three fractions (MI, MII and MIII). The MI fractionpossessed the highest radical scavenging activity, with IC50 value of0.509 mg/ml (2.20-fold higher than MPH) (p < 0.05) (Table 3). TheMIII fraction had the lowest radical scavenging activity, with an IC50of 8.59 mg/ml (p < 0.05).

3.3.3. Amino acid composition of fractions from ion exchange andgel filtration chromatography

Table 4 shows the amino acid composition of myofibrillar pro-tein, the fraction of MA from ion exchange chromatography and theMI fraction from gel filtration chromatography. The major aminoacids in unhydrolyzed patin myofibrillar protein (UPMP) and in theMA and MI fractions are Glu, Asp, Lys and Leu. The percentages ofUPMPs and MA and MI fractions containing hydrophobic aminoacids are high: 37.85%, 39.38% and 41.14%, respectively. The MIfraction exhibited significantly more hydrophobic amino acidscompared with UPMP and MA (p < 0.05). It was found that thecontent of aromatic amino acids (Trp, Phe and Tyr) in the MI frac-tion increased to 15.88% compared with smaller increases in MPH-III (14.82%) and UPMP (13.33%) (p < 0.05). The MI fraction showed a

Table 3A summary of the antioxidant peptide fractionation from myofibrillar protein hy-drolysate (MPH).

Fraction DPPH radicalscavengingactivity (%)a

IC 50 (mg/ml) Fractionation rate

MPH 49.80 ± 1.43 1.120 ± 0.02Ion exchange (MA) 60.22 ± 1.05 0.610 ± 0.03 1.84Gel filtration (MI) 61.78 ± 1.45 0.509 ± 0.09 2.20RP-HPLC (MI 4) 64.40 ± 1.55 0.378 ± 0.10 2.97

e Values were reported as themean ± SD, experiments were performed in triplicate.a DPPH radical scavenging activity was measured at a hydrolysate peptide con-

centration of 1 mg/ml.

significant increase in essential amino acids (53.52%) comparedwith MA (50.72%) and UPMP (47.82%) (p < 0.05).

The amino acid composition of food protein hydrolysates has astrong influence on their antioxidant properties. For example, theamount of cysteine, histidine, methionine, proline, and aromaticamino acids have been reported to contribute to the antioxidantactivity of food peptides (Aluko, 2012, chap. 3). Hydrophobic aminoacids such as Val and Lue have been reported to have antioxidantproperties (Chen, Muramoto & Yamauchi, 1995; Uchida &Kawakishi, 1992).

3.3.4. Reverse- phase high performance liquid chromatography (RP-HPLC)

The fraction of MI obtained from gel filtration that showed thehighest radical scavenging activity was further separated usingreverse-phase high performance liquid chromatography (RP-HPLC)on an XBridge™ BEH130 Prep C18 (10 � 250 mm, 5 um, Waters,USA) column. The antioxidant peptide separation of the MI fractionof MPH with RP-HPLC was performed by taking advantage of themolecular polarity differences in the sample solution.

Fig. 2 shows the DPPH radical scavenging activity (IC50) and theelution profile of RP-HPLC for the MI fraction of MPH. For the MIfraction of the MPH, four fractions were separated using RP-HPLC,the fraction of MI 1-MI 4 (Fig 2). The MI 4 fraction, at an elutiontime of 12:63, showed the greatest amount of DPPH radical scav-enging and the lowest IC50 value of 0.378mg/ml (p < 0.05). Throughthree successive separation methods, the antioxidant peptidesderived from MPH has demonstrated antioxidant activity that was2.97-fold higher than that of the MPH sample without separation(Table 3). Given the large elution time in the hydrophobic chro-matography column, it has been suggested that antioxidant pep-tides from MPHs possess strong hydrophobic properties. Theseresults have been proven by their amino acid composition, shown

Fig. 2. DPPH radical scavenging activity (IC50) and RP-HPLC for the MI fraction obtained from MPH. e Values are reported as the mean ± SD, experiments were performed intriplicate. e Different letters within the same parameter indicate significant differences (p < 0.05).

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461458

in Table 4; the hydrophobic amino acid content of the fractionsseparated from MPHs is high. These results were supported byMegias et al. (2004) and You et al. (2010).

3.4. Identification of antioxidative peptides by LC-MS-TOF andpeptide synthesis

Fig. 3 shows the mass spectra (MS/MS) of the antioxidant pep-tide from myofibrillar protein hydrolysates (MPH). Three aminoacid sequences Val-Pro-Lys-Asn-Tyr-Phe-His-Asp-Ile-Val(VPKNYFHDIV), position 287-296 of the parent protein Glucan1,3-beta-glucosidase 1 OS¼Hansenula anomala (SwissProt acces-sion number O93939), Leu-Val-Met-Phe-Lue-Asp-Asn-Gln-His-Arg-Val-Ile-Arg-His (LVMFLDNQHRVIRH) with a molecular massof 1231 Da, position 124-137 of the parent protein UPF0758 proteinYE0063 OS¼Yersinia enterocolitica serotype O:8/biotype 1B (strain

8081) (SwissProt accession number A1JHX2) and Phe-Val-Asn-Gln-Pro-Tyr-Lue-Lue-Tye-Ser-Val-His-Met-Lys (FVNQPYLLYSVHMK)with a molecular mass of 1739 Da, position 247-260 of the parentprotein Guanylate cyclase soluble subunit alpha-3 OS ¼ Homo sa-piens (SwissProt accession Q02108) were identified using spectrummill MS proteomics workbench software packages. These peptidescontain 10-14 amino acid residues.

This result is comparable to the peptides isolated from proteinhydrolysates coger eei (928 Da) (Ranathunga, Rajapakse, & Kim,2006) and alaska Pollak (1000 Da) (Je, Kim, & Kim, 2005). Bioac-tive peptides usually contain 2-20 amino acid residues (Pihlanto-Leppala & Korhonen, 2003). This figure is comparable to the re-sults reported in this study, where the number of amino acid res-idues of antioxidant peptides of MPH is 10e14. Antioxidantpeptides are very much influenced by molecular weight and mo-lecular structure (Suetsuma, Ukeda, & Ochi, 2000). Many studies

Fig. 3. The mass spectra (MS/MS) of the antioxidant peptide from patin myofibrillar protein hydrolysate (MPH). (A): VPKNYFHDIV; (B): LVMFLDNGHRVIRH; (C): FVNQPYLLYSVHMK.

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461 459

Table 5Antioxidant activities of the synthesized peptides from myofibrillar protein hydro-lysate (MPH).

Synthesized peptides DPPH radicalscavengingactivity, IC50 (mg/ml)

Thiobarbituric acidvalue (mg malonaldehyde/kg)

VPKNYFHDIV 0.354 ± 0.02b 1.92 ± 0.08a

FVNQPYLLYSVHMK 0.268 ± 0.06a 2.00 ± 0.03a

LVMFLDNQHRVIRH 0.443 ± 0.12c 1.75 ± 0.04b

e Values were reported as themean ± SD, experiments were performed in triplicate.

L. Najafian, A.S. Babji / LWT - Food Science and Technology 60 (2015) 452e461460

have reported that peptides with low molecular weight have highantioxidant activity (Rajapakse, Mendis, Byun, & Kim, 2005).

On the basis of the sequence of isolated three peptides, peptideswere synthesized, and the antioxidant activity of these peptideswas determined (Table 5). All synthesized peptides had antioxidantactivity, however FVNQPYLLYSVHMK possessed the highest DPPHradical scavenging activity and thiobarbituric acid value with IC50value of 0.268 mg/ml and 2 mg malonaldehyde/kg, respectively.

The characterization of the amino acid composition of thepeptides is also important for antioxidant properties. TheVPKNYFHDIV and FVNQPYLLYSVHMK peptides from MPH hadproline (P) and lysine (K) amino acids, and the FVNQPYLLYSVHMKand LVMFLDNQHRVIRH peptides had methionine (M) and lucine(L) amino acids. Valine (V), histidine (H), asparagine (N) andphenylalanine (F) amino acids were identified in all of the anti-oxidative peptides fromMPH. The antioxidative peptides fromMPHhad a high content of hydrophobic amino acids, including leucine(L), valine (V) and phenylalanine (F), and hydrophilic amino acids,histidine (H) and proline (P). Aromatic amino acids such as Phe (F)and Tyr (Y) were identified from MPH. Elias, Kellerby, and Decker(2008) and Najafian and Babji (2014) reported that antioxidativepeptides often include hydrophobic amino acid residues such as Valor Leu at the N-terminus of the peptides and Pro, His, Tyr, Trp, Met,and Cys in their sequences. In addition, Sarmadi and Ismail (2010)reported that aromatic amino acids (Phe, Tyr, His, and Trp) couldconvert radicals to stable molecules by donating electrons. Theseamino acid sequences are believed to contribute to the high anti-oxidant activity of peptides from MPH. The presence of hydro-phobic amino acid like Phe at the N-terminal and Basic amino acidlike Lys at C-terminal of FVNQPYLLYSVHMK peptide can possesshigh antioxidant activity.

4. Conclusions

Patin myofibrillar protein was hydrolysed using various en-zymes with differing degrees of hydrolysis (DH) to obtain anti-oxidative peptides. The highest DH (89.17%) for the hydrolysis of themyofibrillar protein was produced with a papain treatment for120 min. The results showed that when the DH of MPHs increased,protein solubility and peptide content increased. Papain-MPH hadthe highest protein solubility and peptide content (559.8 ± 2.33 and299.1 ± 1.71 mg/g, respectively). Myofibrillar protein hydrolysate(MPH) exhibited scavenging activities for DPPH and ABTS radicals,metal chelating, thiobarbituric acid value and reducing power.Papain-MPH had the highest antioxidant activity. Papain-MPH waspurified using consecutive chromatographic methods, includingion exchange chromatography, gel filtration chromatography andreverse-phased high performance liquid chromatography. The ionexchange and gel filtrated fractions with the highest antioxidantactivity that were separated from papain myofibrillar, were testedfor amino acid composition. The results showed that the fractionshad higher amounts of hydrophobic and aromatic amino acids thanunhydrolyzed patin myofibrillar protein (UPMP). The potent

fractions obtained using RP-HPLC of myofibrillar (MI 4 fraction)hydrolysates presented DPPH radical scavenging activity that was2.97 times greater higher than that of MPH. By comparisonwith theSwissProt database, the amino acid sequence of antioxidant peptideof the MPH exhibited the three peptides (VPKNYFHDIV,LVMFLDNQHRVIRH, and FVNQPYLLYSVHMK) Antioxidant peptidesfromMPH contained hydrophobic amino acids: Leu (L), Val (V) andPhe (F). In addition, the presence of basic and hydrophilic aminoacids, Lys (K), His (H) and Pro (P), and aromatic amino acids such asPhe (F) and Tyr (Y) in the peptide sequences is believed tocontribute to the high antioxidant activity of MPH. Among of thesepeptides, FVNQPYLLYSVHMK peptide showed the highest antioxi-dant activity. Therefore, patin myofibrillar protein hydrolysate canbe used as source of novel peptides for natural antioxidants inpreventing oxidation reactions in food processing and in enhancingthe antioxidant properties of functional foods.

Acknowledgements

This study was financially supported by Government ofMalaysia, Grant ERGS/1/2012/STWNO3/UKM/01/1 as well as STGL-004-2013.We also acknowledged Agilent Technologies for assistingin the analysis of peptide sequences and First BASE LaboratoriesSdn Bhd, for synthesizing the peptide sequences.

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