Eur Food Res Technol (2004) 219:2026DOI 10.1007/s00217-004-0882-9

O R I G I NA L PA P ER

Shin-Young Jun Pyo-Jam Park Won-Kyo Jung Se-Kwon Kim

Purification and characterization of an antioxidative peptidefrom enzymatic hydrolysate of yellowfin sole (Limanda aspera)frame proteinReceived: 10 November 2003 / Revised: 29 December 2003 / Published online: 31 March 2004 Springer-Verlag 2004

Abstract In order to utilize yellowfin sole (Limandaaspera) frame protein (YFP), which is normally discardedas industrial waste in the process of fish manufacture,yellowfin sole frame protein hydrolysates (YFPHs) werefractionated using an ultrafiltration (UF) membrane sys-tem following hydrolysis with pepsin and mackerel in-testines crude enzyme (MICE). The YFPHs were sepa-rated into five major types, YFPH-I (3010 kDa), YFPH-II (105 kDa), YFPH-III (53 kDa), YFPH-IV (31 kDa),and YFPH-V (below 1 kDa) by using UF membranes withmolecular weight cut-offs of 30, 10, 5, 3, and 1 kDa,respectively. The antioxidative activity of the YFPHs wasinvestigated and compared with that of a natural antiox-idant, a-tocopherol, used as a reference. Furthermore, thefraction showing strong antioxidative activity was iso-lated from the YFPHs using consecutive chromatographicmethods on an SP-Sephadex C-25 column, on a SephadexG-75 column, and high-performance liquid chromatog-raphy (HPLC) on an octadecylsilane column. The mo-lecular mass of the antioxidant was identified as 13 kDausing HPLC on a gel permeation chromatography (GPC)column, and the antioxidative peptide was composed of10 N-terminal amino acid residues, RPDFDLEPPY.

Keywords Antioxidative peptide Hydrolysate Yellowfin sole Characterization

Introduction

An antioxidant is defined as any substance that signifi-cantly delays or inhibits oxidation of a substrate whenpresent at low concentrations compared to that of anoxidizable substrate. Antioxidants can act at different

levels in an oxidative sequence. This can be illustrated byconsidering one of the many mechanisms by which oxi-dative stress can cause damage by stimulating the freeradical chain reaction of lipid peroxidation. Free radicalchain reactions within a material can be inhibited eitherby adding chemicals that retard the formation of freeradicals or by introducing substances that compete withthe existing radicals and remove them from the reactionmedium.

Lipid oxidation is of great concern to the food industryand consumers, since it leads to the development of un-desirable off-flavors and potentially toxic reaction prod-ucts [1]. Antioxidants are used to preserve food productsby retarding discoloration and deterioration as a result ofoxidation. Therefore, antioxidants are increasingly usedas a means of enhancing shelf-life and to improve thestability of lipid and lipid-containing foods. Syntheticantioxidants such as butylated hydroxyanisole (BHA),butylated hydroxytoluene (BHT), tertiary butylhydro-quinone, and propyl gallate are added to food products toretard lipid oxidation [2]. However, use of synthetic an-tioxidants in food products is under strict regulation, be-cause of their potential health hazards [3]. In addition,free radical-mediated modification of DNA, proteins, li-pids, and small cellular molecules is associated with anumber of pathological processes, including atheroscle-rosis, arthritis, diabetes, cataractogenesis, muscular dys-trophy, pulmonary dysfunction, inflammatory disorders,ischemia-reperfusion tissue damage, and neurological dis-orders such as Alzheimers disease [4]. Therefore, thesearch for natural antioxidants as alternatives to syntheticones is of great interest among researchers. Severalstudies have described the antioxidative activity of pro-teins such as milk casein [5], soy protein [6], bovine se-rum albumin [7], oil seed protein [8], wheat gliadin [9],beach pea [10], evening primrose [11], maize zein [12],egg yolk protein [13], Pollack skin gelatin [14], chitosan[15], tomato products [16], and pork protein [17]. Aminoacids have also been reported to exhibit antioxidantproperties against linoleic acid oxidation in the freeze-dried emulsion condition [18]. However, little is known

S.-Y. Jun P.-J. Park W.-K. Jung S.-K. Kim ())Department of Chemistry,Pukyong National University,Busan 608737, Koreae-mail: sknkim@mail.pknu.ac.krTel.: +82-51-620 6375Fax: +82-51-628 8147

about the structure of antioxidative peptides from variousfood proteins.

In this study, we purified an antioxidative peptidederived from enzymatic hydrolysate of yellowfin soleframe protein (YFP), which is normally discarded as in-dustrial waste in the process of fish manufacture, anddetermined the amino acid sequence.

Materials and methods

Materials

Fresh samples of yellowfin sole frame (moisture, 79%) were do-nated by Daerim Co. (Busan, Korea), and stored at 20 C untiluse. Mackerel intestine was obtained from a local fish market andstored at 20 C until use. Alcalase 0.6 L (0.6 AU/g) and Neutrase0.5 L (0.5 AU/g) were acquired from Novo Co. (Novo Nordisk,Bagsvaerd, Denmark), and papain from papaya latex (type IV),pepsin from porcine stomach mucosa, trypsin from bovine pancreas(type II), a-chymotrypsin from bovine pancreas (type II), pronase Efrom Streptomyces griseus (type XIV), 2-thiobarbituric acid (TBA),ammonium thiocyanate, linoleic acid, a-tocopherol. SP-SephadexC-25, and Sephadex G-75 were purchased from Sigma ChemicalCo. (St. Louis, MO., USA). The ultrafiltration membrane (UF)reactor (Minitan) system and membranes for the fractionations ofyellowfin sole hydrolysate, based on molecular weights, were fromMillipore Co. (Bedford, USA). All other reagents were of thehighest grade available commercially.

Extraction of mackerel intestine crude enzyme (MICE)

The crude proteinase from mackerel intestine was extracted ac-cording to the method of Kim et al. [19]. Briefly, the minced in-testine was added to two volumes of 20 mM Tris-HCl buffer (pH7.0) containing 5 mM CaCl2, and homogenized twice at 12,000 rpmfor 2 min using an homogenizer (Ace homogenizer, Nissei AM-7,Nihonseiki Kaisha, Tokyo, Japan). The homogenate was incubatedat 37 C for 2 h, and centrifuged at 9,500g for 20 min. Afteradjusting the supernatant to a 50% saturated solution with coldacetone (v/v), it was centrifuged again as described above. To re-move insoluble protein from the precipitated protein, the samevolume of distilled water was added, and the mixture was cen-trifuged at 9,500g for 10 min. The supernatant was lyophilizedand stored at 20 C until use.

Preparation of yellowfin sole frame protein hydrolysates (YFPH)

Eight proteases (Alcalase, a-chymotrypsin, MICE, Neutrase, pa-pain, pepsin, pronase E, trypsin) were used for the digestion ofYFP. YFP was hydrolyzed with each protease for 6 h in a batchreactor under optimal conditions and then heated at 98 C for10 min to inactivate the proteases. The resulting hydrolysates werelyophilized, and assayed for antioxidative activity. To purify theantioxidative peptides, YFP was hydrolyzed with MICE (enzyme tosubstrate ratio, 1:50) for 3 h at pH 10.0 and 50 C, and subsequentlyprepared with pepsin (enzyme to substrate, 1:50) for 3 h at pH 2.0and 37 C, after the pH of the solution had been adjusted to 2.0with conc. HCl. Finally, the resultant hydrolysate was fractionatedthrough five different UF membranes having a range of molecularweight cut-offs (MWCO), i.e., 30, 10, 5, 3, and 1 kDa. ThoseYFPHs which passed through the 30 kDa membrane but notthrough the 10 kDa membrane were categorized as YFPH-I. Thosewhich passed through the 10 kDa membrane but not passed throughthe 5 kDa membrane were YFPH-II. Those that passed throughthe 5 kDa membrane but not through the 3 kDa membrane wereYFPH-III. Those which passed through the 3 kDa membrane butnot through the 1 kDa membrane were YFPH-IV, and YFPH-V

were those which passed through the 1 kDa membrane. All theYFPHs recovered were lyophilized on a freeze-drier for 5 days.

Measurement of antioxidative activity

The antioxidative activity of the YFPHs was measured in a linoleicacid model system according to the methods of Osawa et al. [20]. Asample (1.3 mg) was dissolved in10 ml of 50 mM phosphate buffer(pH 7.0) and added to a solution of 0.13 ml of linoleic acid and10 ml of 99.5% ethanol. Then the total volume was adjusted to25 ml with distilled water. The solution was incubated in a conicalflask with a screw cap at 401 C in a dark room, and the degree ofoxidation was evaluated by measuring the TBA and ferric thiocy-anate values. The TBA value was measured using a modifiedversion of the method of Ohkawa et al. [21]. The reaction mixture(50 ml) was added to a mixture of 0.8 ml distilled water, 0.2 ml of8.1% sodium dodecyl sulfate, and 1.5 ml of 0.8% TBA solution.The mixture was incubated at 5 C for 1 h, and then heated at 95 Cfor 1 h in the dark. The TBA value was measured by reading theabsorbance at 532 nm. The ferric thiocyanate value was measuredaccording to the method of Mitsuda et al. [22]. The reaction solu-tion (100 ml) incubated in the linoleic acid model system describedabove [20] was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 30%ammonium thiocyanate, and 0.1 ml of 210-2 M ferrous chloridesolution in 3.5% HCl. After 3 min, the PV was measured by readingthe absorbance at 500 nm following color development with FeCl2and thiocyanate at different intervals during the incubation periodat 401 C . All analyses were run in triplicate and averaged.

Molecular weight distribution profile

Molecular weight distributions of the hydrolysates were determinedby gel permeation chromatography (GPC) using a high-perfor-mance liquid chromatography (HPLC) system (Hewlett-Packard,Palo Alto, CA). Two GPC columns, Zorbax PSM 300 and 60(Hewlett-Packard), with exclusion limits of 3103 to 3105 Da(6.2 mm, 254.6 cm) and 110103 Da (6.2 mm, 254.6 cm), wereconnected in series, and the hydrolysates were chromatographedand monitored at 230 nm at room temperature.

Purification of the antioxidative peptide and determinationof amino acid sequence

The lyophilized YFPH-I was dissolved in 20 mM sodium acetatebuffer (pH 4.0) and fractionated by ion-exchange chromatographyon a SP-Sephadex C-25 column (440 cm). The column wasequilibrated with the same buffer and fractions eluted with a lineargradient of NaCl concentration (01.0 M). Fractions of 5 ml werecollected at a flow rate of 60 ml/h. The fractions showing antiox-idative activity were pooled and lyophilized. The lyophilized frac-tion was dissolved in 50 mM sodium phosphate buffer (pH 7.0) andloaded onto a Sephadex G-75 gel filtration column (2.590 cm)which had previously been equilibrated with the same buffer. Thecolumn was then eluted with the same buffer, and 5-ml fractionswere collected at a flow rate of 60 ml/h. The fractions exhibitingantioxidative activity were pooled and lyophilized. The antioxida-tive fraction was dissolved in distilled water and separated usingreversed-phase HPLC on a Primesphere 10 C18-HC 120 (10 mm,1.025 cm; Phenomenex, Macclesfield, UK) column using a lineargradient of acetonitrile (050% in 40 min) in 0.1% trifluoroaceticacid (TFA) at a flow rate of 2.0 ml/min. The elution peaks weremonitored at 215 nm, and their antioxidative activities were mea-sured using the same method. The active peaks were concentratedusing a centrifugal evaporator. The peaks representing the anti-oxidative activity were rechromatographed on the same columnusing a linear gradient of acetonitrile (030% in 40 min) in 0.1%TFA at a flow rate of 2.0 ml/min. The sequence of antioxidativepeptide was determined by automated Edman degradation with aPerkinElmer 491 protein sequencer (Branchburg, NJ., USA).

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Results and discussion

Every year, about 100 million tons of fish are harvested;however, 30% of the total catch is transformed intofishmeal [23, 24]. Over 50% of the harvest is processingwaste which includes bone, skin, fins, internal organs,heads, and so on [25]. In Korea, the annual yellowfin soleharvest exceeds 13,828 tons, and 1,327 tons were pro-cessed in Korean fish plants in 2002. In particular, fishframes obtained after filleting include bones, heads andtails. The total solid mass of the frame consist of con-siderable amount of protein, which can be used as po-tential bioactive substances. Therefore, we investigatedthe antioxidative activity of enzymatic hydrolysate ofYFP, a by-product, which is normally transformed intofishmeal or discarded by fish processing plants.

Digestion of YFP with various enzymesand their antioxidative activity

In order to select suitable proteases for the hydrolysis ofYFP, the YFP was independently hydrolyzed with MICE,alcalase, a-chymotrypsin, papain, pepsin, pronase E,neutrase, and trypsin using a batch reactor. The degree ofhydrolysis of YFP by MICE was the highest (67%), andthe degree of hydrolysis of YFP by pepsin was the lowest(22%) compared to hydrolysis using commercial pro-teases (data not shown). In addition, the antioxidativeactivities of the resultant YFPHs were measured andcompared with that of a-tocopherol. As shown in Fig. 1,the oxidation of linoleic acid was markedly inhibited byYFPHs derived from YFP with various proteases. Amongthe hydrolysates resulting from various enzymes, thehighest antioxidative activity was observed in the pepsinhydrolysate, which exhibited about 70% inhibition of li-noleic acid peroxidation. Therefore, these results indicatethat the hydrolysates of YFP seemed to contain someantioxidative peptides.

Preparation and molecular weight profiles of YFPHs

The pepsin hydrolysate showed the highest antioxidativeactivity compared to those of other enzymatic hy-drolysates. However, the degree of hydrolysis of YFP bypepsin was the lowest. Therefore, we combined MICE,which showed the highest degree of hydrolysis of YFP,with pepsin, which exhibited the highest antioxidativeactivity. The YFPH was prepared with MICE for 3 h atpH 10.0 and 50C, and subsequently hydrolyzed withpepsin for 3 h at pH 2.0 and 37C after the pH of thesolution had been adjusted to 2.0 with HCl. The antiox-idative activity of the YFPH derived from the MICE andpepsin combination was similar to or slightly higher thanthat of pepsin hydrolysate (data not shown). To isolate anantioxidative peptide from YFP with the MICE andpepsin combination, five different kinds of YFPHs wereprepared by a batch system, and fractionated by passing

the protein through five UF membranes with molecularweight cut-offs (MWCO) of 30, 10, 5, 3, and 1 kDa. TheYFPHs were named as YFPH-I, which passed through the30 kDa membrane but not through the 10 kDa membrane;YFPH-II, which passed through the 10 kDa membranebut not through 5 kDa; YFPH-III, which passed throughthe 5 kDa membrane but not through 3 kDa; YFPH-IV,which passed through the 3 kDa membrane but notpassed through1 kDa, and YFPH-V, which passedthrough 1 kDa. The molecular weight distributions variedaccording to the MWCO size of the membrane used(Fig. 2). YFPH-I had a size distribution of 23 to 10 kDa,and the main peaks were located at 9 and 7 kDa. Themolecular weight distribution of YFPH-II was between 13and 6 kDa, and the major peaks were at 9 and 7 kDa.YFPH-III showed a major peak at 5 kDa. The molecular

Fig. 1 Antioxidative activities of hydrolysates from yellowfin soleframe protein (YFP) from various proteases in a linoleic acid au-toxidation system measured by the thiobarbituric acid (TBA)method (A), and by the ferric thiocyanate method (B). l,control;, a-tocopherol; t, mackerel intestine crude enzyme (MICE)hy-drolysate; 5, alcalse hydrolysate; n, a-chymotrypsin hydrolysate;o, papain hydrolysate; u, pepsin hydrolysate; }, pronase E hy-drolysate; s, neutrase hydrolysate; 4, trypsin hydrolysate. Allexperiments were carried out in triplicate, and the results wereexpressed as means

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weight distribution of YFPH-IV was between 3 and0.9 kDa, except for the appearance of peak at 0.8 kDa,and a major peak at 2.6 kDa. YFPH-V showed two majorpeaks at 0.8 and 0.5 kDa. The result of the molecularweight profiles of each hydrolysate showed a distinct

decrease of molecular weight according to the pore size ofthe membrane.

Antioxidative activity of YFPHs

There are only a few reports on the antioxidative efficacyof amino acids. Tryptophan and histidine showed highantioxidant activity whereas glycine and alanine showedonly weak activity, and methionine and cysteine had anantioxidative effect in Soybean oil [30]. However, allamino acids have exhibited antioxidant activity in somesystems, which probably reflects the antioxidant nature ofthe NH3R group [31]. The use of a protein or a hydrol-ysate for the improvement of the antioxidative activity infunctional foods might be more practical than the useof amino acids, because proteins and hydrolysates haveother desired functional properties. The antioxidative ac-tivity of soybean protein hydrolysates has been docu-mented [32]. In a recent paper, the antioxidative effect ofpeptides derived from the enzymatic hydrolysates of fish

Fig. 2 Molecular weight distribution profiles of YFPH-I and II (A),and YFPH-III, IV and V (B) on high-performance liquid chroma-tography (HPLC) with a gel permeation chromatography column.HPLC was carried out with deionized water as the mobile phase ata flow rate of 1.0 mL/min. A, albumin (MW 66,000 Da); B, car-bonic anhydrase (MW 29,000 Da); C, cytochrome C (MW12,327 Da); D, aprotinin (MW 6,000 Da); E, pentaphenylalane(MW 753.9 Da)

Fig. 3 Antioxidative activities of hydrolysates by MICEpepsincombination from YFP in the linoleic acid autoxidation systemmeasured by the TBA method (A), and by the ferric thiocyanatemethod. l,control; , a-tocopherol; t, YFPH-I; n, YFPH-II; o,YFPH-III; u, YFPH-IV; }, YFPH-V

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skin gelatin was described [27]. In this study, the an-tioxidative activity of five hydrolysates fractionated fromYFP was investigated and compared with that of a-to-copherol, a widely used natural antioxidant. YFPH-I ex-hibited the highest activity (Fig. 3). In addition, the syn-ergistic antioxidative effects of the YFPH with the non-peptidic antioxidant a-tocopherol were studied. All hy-drolysates of YFP from various enzymes exhibited syn-ergistic effects with a-tocopherol (Fig. 4). The synergisticeffects of nonpeptidic antioxidants on antioxidative ac-tivity have previously been demonstrated with the hy-drolysates of a vegetable protein, yeast protein, AlaskaPollack skin gelatin hydrolysates, and bovine serum al-bumin [26, 27, 28]. Chen et al. [29] reported that thehydrolysates of soybean protein showed a strong syner-gistic effect with nonpeptidic antioxidants although some

hydrolysates had very low antioxidative activity. In thisstudy, the hydrolysates of YFP had both antioxidativeactivity and a synergistic effect with a-tocopherol usingthe linoleic acid in water/alcohol system. Therefore, wefocused on the isolation and structural characterization ofpotent antioxidative peptides from the YFPH.

Purification of an antioxidative peptide and determinationof amino acid sequence

During the process of identifying the antioxidative pep-tide derived from YFP, the protein was hydrolyzed withthe MICE and pepsin combination, and five hydrolysates(YFPH-I, II, III, IV, and V) were obtained using UFmembranes with 30, 10, 5, 3, and 1 kDa MWCO. YFPH-Iwas then separated using ion-exchange chromatographyon an SP-Sephadex C-25 column and fractionated intothree portions. When these fractions were tested for an-tioxidative activity, fraction III was found to possess astrong activity and was then lyophilized (Fig. 5A). Thelyophilized fraction III was subjected to size exclusionchromatography on Sephadex G-75 and fractionated intoa major portion. Fraction III-1 exhibited strongest an-tioxidative activity (Fig. 5B). This fraction was furtherseparated by reversed-phase HPLC using a 0.1% TFA-acetonitrile system and fractionated to III-11, III-12,III-13, and III-14. The subfraction III-11 possessed thehighest antioxidative activity (Fig. 5C). Subfraction III-11 was further separated by RP-HPLC using the samesolvent system. Two portions were finally obtained fromthe hydrolysate of YFP with the MICE and pepsin com-bination, and the antioxidative activity was investigated;III-11a fraction was higher than that of III-11b fraction(Fig. 5D). Therefore, we determined the N-terminalamino acid sequence of III-11a, and the antioxidativepeptide was composed of 10 amino acid residues, Arg-Pro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr. In addition, themolecular weight of the antioxidative peptide was deter-mined to be 13 kDa by HPLC on GPC columns as abovedescribed (data not shown). The amino acid residues atthe N- termini of dipeptides have been demonstrated to beantioxidative in an oil system [33]. It is probable that theamino acid residues play a role in increasing the inter-action between peptides and fatty acids. The antioxidativepeptide contained tyrosine residue, which is a potent hy-drogen donor. Previously, many proteins have been re-ported to have strong antioxidative activity against theperoxidation of lipid or fatty acid systems [26, 34]. As a

Fig. 4 Synergistic effects of a-tocopherol and hydrolysates of YFPby MICE and pepsin combination in linoleic acid autoxidationsystem measured by the TBA method (A), and by the ferric thio-cyanate method (B). l,control; , a-tocopherol; t, a-tocopheroland YFPH-I combination; n, a-tocopherol and YFPH-II combi-nation; o, a-tocopherol and YFPH-III combination; u, a-toco-pherol and YFPH-IV combination; }, a-tocopherol and YFPH-Vcombination

Fig. 5 Purification of antioxidative peptides from the YFPH-I. (A)SP-Sephadex C-25 chromatography (lower panel) and antioxidativeactivities of the fractions (upper panel) measured by TBA methodafter 6 days. Elution was performed at a flow of 60 mL/h with alinear NaCl gradient (01 M) in 20 mM sodium acetate buffer, pH4.0. (B) Re-chromatography of fraction III from Fig. 4A onSephadex G-75 gel chromatography (lower panel) and antioxida-tive activity of the fraction (upper panel) measured by the TBAmethod after 6 days. Elution was done at a flow rate of 60 mL/h in50 mM sodium phosphate buffer (pH 7.0). (C) Reversed-phase

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HPLC pattern on a Primesphere 10 C18 column of fraction III-1from Fig. 4B was eluted on the gel chromatography (lower panel)and antioxidative activities of the fractions (upper panel) measuredby the TBA method after 6 days. HPLC operation was carried outwith 50% acetonitrile as mobile phase at a flow rate of 2 ml/minusing an UV detector at 215 nm. (D) Further separation of sub-

fraction III-11 reversed phase HPLC. Elution profiles (lowerpanel) and antioxidative activities of the fractions (upper panel)measured by the TBA method after 6 days. The HPLC operationwas carried out with 30% acetonitrile as mobile phase at a flow rateof 2 mL/min using a UV detector at 215 nm

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result, antioxidative activity of peptide is thought to berelated to their molecular weight and amino acid se-quence.

Acknowledgments This work was supported by the MOST, BusanMetropolitan City, and Daerim Co. in Korea.

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