www.elsevier.com/locate/foodres

Food Research International 38 (2005) 45–50

Antioxidant activity of a peptide isolated from Alaskapollack (Theragra chalcogramma) frame protein hydrolysate

Jae-Young Je, Pyo-Jam Park, Se-Kwon Kim *

Department of Chemistry, Pukyong National University, Busan 608-737, Republic of Korea

Received 30 April 2004; accepted 28 July 2004

Abstract

Alaska pollack frame protein, which is normally discarded as an industrial by-product in the process of fish plant, was hydro-

lyzed with mackerel intestine crude enzyme (MICE). Alaska pollack frame protein hydrolysate (APH) was fractionated according to

the molecular basis into five major types of APH-I (30–10 kDa), APH-II (10–5 kDa), APH-III (5–3 kDa), APH-IV (3–1 kDa), and

APH-V (below 1 kDa) using an ultrafiltration (UF) membrane bioreactor system. The antioxidative activity of the APHs was inves-

tigated and compared with that of a natural antioxidant, a-tocopherol, used as a reference. The fraction, APH-V, exhibited the high-

est antioxidative activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex

G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. The sequence of the purified

peptide was Leu-Pro-His-Ser-Gly-Tyr and molecular weight was 672 Da. In addition, the purified peptide scavenged 35% on hyd-

roxyl radical at 53.6 lM using electron spin resonance (ESR) spectroscopy.

� 2004 Elsevier Ltd. All rights reserved.

Keywords: Antioxidative activity; Alaska pollack frame protein; Hydrolysate; Electron spin resonance

1. Introduction

Reactive oxygen species (ROS) and free radicals play

an important role in many diseases such as cancer (Lean-

derson, Faresjo, & Tagesson, 1997; Muramatsu et al.,

1995), gastric ulcers (Debashis, Bhattacharjee, & Bane-

rjee, 1997; Sussman&Bulkley, 1990), Alzheimer�s, arthri-tis and ischemic reperfusion (Vajragupta, Boonchoong,&

Wongkrajang, 2000). Formation of free radicals such as

superoxide anion radical ðO��2 Þ and hydroxyl radical

(ÆOH) is an unavoidable consequence in aerobic organ-

isms during respiration. These radicals are very unstable

and react rapidly with the other groups or substances in

the body, leading to cell or tissue injury. Free radical scav-

enger is a preventive antioxidant. Antioxidants can act at

0963-9969/$ - see front matter � 2004 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodres.2004.07.005

* Corresponding author. Tel.: +82 51 620 6375; fax: +82 51 628

8147.

E-mail address: sknkim@mail.pknu.ac.kr (S.-K. Kim).

different levels in an oxidative sequence. Thismay be illus-

trated considering one of the many mechanism by which

oxidative stress can cause damage by stimulating the free

radical chain reaction of lipid peroxidation.

Lipid peroxidation is of great concern to the food

industry and consumers because it leads to the develop-

ment of undesirable off-flavors and potentially toxicreaction products (Maillard, Soum, Meydani, & Berset,

1996). Many synthetic antioxidants such as butylated

hydroxyanisole, butylated hydroxytoluene, t-butyl-

hydroquinone and propyl gallate may be used to retard

lipid peroxidation in a lot of fields (Wanita & Lorenz,

1996). However, the use of synthetic antioxidants is un-

der strict regulation due to the potential health hazards

caused by such compounds (Hettiarachchy, Glenn,Gnanasambandam, & Johnson, 1996; Park, Jung,

Nam, Shahidi, & Kim, 2001). Therefore, the search for

natural antioxidants as alternatives to synthetic ones is

of great interest among researchers. Recently, several

46 J.-Y. Je et al. / Food Research International 38 (2005) 45–50

studies have described the antioxidative activity of

hydrolysates such as milk casein (Yamaguchi, Naito,

Yokoo, & Fujimaki, 1980), soy protein (Pratt, 1972),

bovine serum albumin (Yukami, 1972), oil seed protein

(Rhee, Ziprin, & Rhee, 1979), egg-yolk protein (Park

et al., 2001), yellowfin sole frame protein (Jun, Park,Jung, & Kim, 2004), and pork protein (Carlsen, Ras-

mussen, Kjeldsen, Westergaard, & Skibsted, 2003).

However, little information about antioxidative peptide

from fish by-product was available until now.

In this study, we examined the antioxidative effect of

Alaska pollack frame protein hydrolysate (APH), which

is normally discarded industrial waste in the process of

fish manufacture.

2. Materials and methods

2.1. Materials

Alaska pollack frame was donated by Daerim Co.

(Busan, Korea). Mackerel intestine was obtained froma local fish market and stored at �20 �C until use.

Ammonium thiocyanate, linoleic acid, a-tocopherol,SP-Sephadex C-25 and Sephadex G-25 were purchased

from Sigma Chemical Co. (St. Louis, MO). The ultrafil-

tration (UF) membrane bioreactor system (MinitanTM)

and membranes for the fractionation of APH, based

on molecular weights, were purchased from Millipore

Co. (Bedford, MA). All other reagents were of the high-est grade available commercially.

2.2. Extraction of mackerel intestine crude enzyme

(MICE)

The crude proteinase from mackerel intestine was ex-

tracted according to the method of Kim et al. (2003).

Briefly, the mackerel intestine was added to two volumesof 20 mM Tris–HCl buffer (pH 7.0) containing 5 mM

CaCl2, and homogenized twice at 12,000 rpm for 2

min using an homogenizer (Ace homogenizer, Nissei

AM-7, Nihonseiki Kaisha, Tokyo, Japan). The homo-

genate was incubated at 37 �C for 2 h, and centrifuged

at 9500g for 20 min. After adjusting the supernatant

to a 50% saturated solution with cold acetone (v/v), it

was centrifuged again as described above. To removeinsoluble protein from the precipitated protein, the same

volume of distilled water was added, and the mixture

was centrifuged at 9500g for 10 min. The supernatant

was lyophilized and stored at �20 �C until use.

2.3. Preparation of APH by MICE

Alaska pollack frame protein was hydrolyzed withMICE as adjusting substrate/enzyme ratio to 50:1 (w/

w) under optimal conditions (50 �C, 12 h, and pH

10.0). APH was subsequently boiled for 10 min to inac-

tivate the enzyme. The resultant APH was fractionated

through five different UF membrane bioreactor system

having a range of molecular weight cut-offs (MWCO)

of 30, 10, 5, 3, and 1 kDa, repectively. The fractionates

were designed as follows: APH-I, APH permeated the30 kDa membrane but not permeated the 10 kDa mem-

brane; APH-II, APH permeated the 10 kDa membrane

but not permeated the 5 kDa membrane; APH-III, APH

permeated the 5 kDa membrane but not permeated the

3 kDa membrane; APH-IV, APH permeated the 3 kDa

membrane but not permeated the 1 kDa membrane; and

APH-V was permeated the 1 kDa membrane. All APHs

recovered were lyophilized in a freeze-drier for 5 days.

2.4. Measurement of antioxidative acitivty

The antioxidative activity of the APHs was measured

in a linoleic acid model system according to the methods

of Osawa and Namiki (1985). Briefly, a sample (1.3 mg)

was dissolved in 10 ml of 50 mM phosphate buffer (pH

7.0), and added to a solution of 0.13 ml of linoleic acidand10ml of 99.5%ethanol. Then the total volumewas ad-

justed to 25ml with distilled water. Themixture was incu-

bated in a conical flask with a screw cap at 40 ± 1 �C in a

dark room and the degree of oxidation was evaluated by

measuring the ferric thiocyanate values. The ferric thiocy-

anate value was measured according to the method of

Mitsuta,Yasumoto, and Iwami (1996). The reaction solu-

tion (100 ll) incubated in the linoleic acid model systemdescribed above (Osawa&Namiki, 1985) was mixed with

4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocy-

anate, and 0.1 ml of 2 · 10�2 M ferrous chloride solution

in 3.5%HCl. After 3min, the thiocyanate valuewasmeas-

ured by reading the absorbance at 500 nm following color

development with FeCl2 and thiocyanate at different

intervals during the incubation period at 40 ± 1 �C.

2.5. Synergistic effects of fractionated APHs

Synergistic effects of fractionatedAPHsweremeasured

as follows: briefly, a sample (1.3 mg) and a-tocopherol(0.13mg)weredissolved in10mlof 50mMphosphatebuf-

fer (pH 7.0), and added to a solution of 0.13 ml of linoleic

acid and 10 ml of 99.5% ethanol. Then the total volume

was adjusted to 25 ml with distilled water. The solutionwas incubated in a conical flask with a screw cap at

40 ± 1 �C in a dark room, and the degree of oxidation

was evaluated bymeasuring the ferric thiocyanate values.

2.6. Isolation of an antioxidative peptide from APH and

determination of amino acid sequence

The lyophilized APH-V with the highest antioxidativeactivity among the APHs, was loaded onto a SP-

Sephadex C-25 ion-exchange column (4.0 · 40 cm)

Incubation time (day)0 2 4 6

Abs

orba

nce

at 5

00 n

m

0

1

2

3

4

APH-IAPH-IIAPH-IIIAPH-IVAPH-VControlα−Tocopherol

Fig. 1. Antioxidative activities of fractionated APHs in linoleic acid

autoxidation system measured by the ferric thiocyanate method.

Values represent means ± SE (n = 3).

rban

ce a

t 50

0 nm

2

3

4

Toco + APH-IToco + APH-IIToco + APH-IIIToco + APH-IVToco + APH-VControlα−Tocopherol

J.-Y. Je et al. / Food Research International 38 (2005) 45–50 47

equilibrated with 20 mM sodium acetate buffer (pH 4.0),

and eluted with a linear gradient of NaCl (0–1 M) in the

same buffer at a flow rate of 60 ml/h. The active fractions

were pooled and lyophilized immediately. The lyophiliz-

ate was further purified on Sephadex G-25 gel filtration

column (2.5 · 90 cm) equilibrated with distilled water.The column was eluted with distilled water and 5.0 ml

of fractions were collected at a flow rate of 60 ml/h. The

fraction exhibiting antioxidative activitywas further puri-

fied using reversed-phase high performance liquid chro-

matography (RP-HPLC) on a Primesphere 10 C18 (20

mm · 250 mm) column with a linear gradient of acetonit-

rile (0–35% in 30min) containing 0.1% trifluoroacetic acid

(TFA) at a flow rate of 2.0 ml/min. The active peak wasconcentrated using a centrifugal evaporator. The peak

representing the antioxidative activity was rechromato-

graphed on a Capcell Pak C18 UG-120 (10 mm · 250

mm) column using a linear gradient of acetonitrile (0–

15% in 30 min) containing 0.1% TFA at a flow rate of

2.0ml/min. Finally, the sequence of an isolated antioxida-

tive peptide was determined by automated Edman degra-

dation with a Perkin–Elmer 491 protein sequencer(Branchburg, NJ, USA).

2.7. Hydroxyl radical scavenging activity

Hydroxyl radicals were generated by iron-catalyzed

Haber–Weiss reaction (Fenton drivenHaber–Weiss reac-

tion) and the generated hydroxyl radicals rapidly reacted

with nitrone spin trap DMPO (Rosen & Rauckman,1984). The resultant DMPO-OH adduct was detectable

with an ESR spectrometer. The purified peptide (0.2 ml)

was mixed with DMPO (0.3 M, 0.2 ml), Fe2SO4 (10

mM, 0.2 ml) and H2O2 (10 mM, 0.2 ml) in a phosphate

buffer solution (pH 7.2), and then transferred into a

100 ll quartz capillary tube. After 2.5 min, the ESR spec-

trum was recorded using an ESR spectrometer. Experi-

mental conditions as follows: magnetic field, 336.5 ± 5mT; power, 1 mW; modulation frequency, 9.41 GHz;

amplitude, 1 · 200; sweep time, 4 min.

2.8. Statistics

The data presented are means ± SE of three

determinations.

Incubation time (day)0 2 4 6

Abs

o

0

1

Fig. 2. Synergistic effects of a-tocopherol and fractionated APHs

combination in linoleic acid autoxidation system measured by the

ferric thiocyanate method. Values represent means ± SE (n = 3).

3. Results and discussion

Every year, about 100 million tons of fish are har-

vested. However, 30% of the total catch is transformed

into fishmeal (Kim, Jeon, Byun, Kim, & Lee, 1997; Rec-

eca, Pena-Vera, & Deaz-Castaneda, 1991). Over 50% of

the harvest is a processing by-product, which includesbone, skin, fins, internal organs, heads, and so on (Nair

& Gopakumar, 1982). In particular, fish frames ob-

tained after filleting include bones, heads, and tails.

Alaska pollack frame contains approximately 61% of

protein, which can be used as potential bioactive sub-

stances. In addition, some antioxidants were reported

from fish processing by-product such as yellowfin sole

skin gelatin hydrolysate (Kim, Lee, Byun, & Jeon,1996), yellowfin sole frame protein hydrolysate (Jun

et al., 2004), Alaska pollack skin gelatin hydrolysate

(Kim et al., 2001), and cod frame protein hydrolysate

(Jeon, Byun, & Kim, 1999) in our laboratory. Therefore,

we investigated antioxidative activity of the enzymatic

hydrolysate from Alaska pollack frame protein.

Fig. 3. Purification of antioxidative peptide from the APH-V. (A) SP-Sephadex C-25 chromatography (lower panel) and antioxidative activities of

the fractions (upper panel) measured by the ferric thiocyanate method after 6 days. Elution was performed at 60 ml/h of flow rate with a linear

gradient of NaCl (0–1 M) in 20 mM sodium acetate buffer, pH 4.0. (B) Rechromatography of activity fraction from Fig. 3A on Sephadex G-25 gel

chromatography (lower panel) and antioxidative activities of the fractions (upper panel) measured by ferric thiocyanate method after 6 days. Elution

was done at 60 ml/h with distilled water. (C) Reversed-phase HPLC pattern on a Primesphere 10 C18 column of active fraction from Fig. 3B (lower

panel) and antioxidative activities of the fractions (upper panel) measured by ferric thiocyanate method after 6 days. HPLC operation was carried out

with 35% acetonitrile as mobile phase at 2 ml/min of a flow rate using UV detector at 215 nm. (D) Further separation of active fraction on Capcell

Pak C18 UG-120 reversed-phase HPLC column. Elution profiles (lower panel) and antioxidative activities of the fractions (upper panel) measured by

the ferric thiocyanate method after 6 days. HPLC operation was carried out with 15% acetonitrile as mobile phase at 2 ml/min of a flow rate using

UV detector at 215 nm.

48 J.-Y. Je et al. / Food Research International 38 (2005) 45–50

3.1. Antioxidative activity of fractionated APHs

Alaska pollack frame protein was hydrolyzed with

MICE under optimal conditions and fractionated into

five portions using an UF membrane bioreactor system

with MWCO of 30, 10, 5, 3, and 1 kDa, respectively.

The antioxdative activity of the fractionated APHs is

shown in Fig. 1. APH-V with molecular weight (MW)

of below 1 kDa showed the highest antioxidative activity,

which exhibited about 85% inhibition of linoleic acid per-

oxidation. In addition, the synergistic antioxidative effect

of the APHs with the nonpeptidic antioxidant, a-tocoph-erol, were studied. The most APHs exhibited synergistic

effect with a-tocopherol exceptAPH-I (Fig. 2). The syner-

J.-Y. Je et al. / Food Research International 38 (2005) 45–50 49

gistic effects of nonpeptidic antioxidants on antioxidative

activity have previously been demonstrated with the

hydrolysates of a vegetable protein, yeast protein, Alaska

pollack skin gelatin hydrolysate, and bovine serum albu-

min (Bishov & Henick, 1975; Kim et al., 2001; Hatate,

Nagata, & Kochi, 1990). Chen, Muramoto, Yamauchi,and Nokihara (1996) reported that the hydrolysates of

soybean protein showed a strong synergistic effect with

nonpeptidic antioxidants although some hydrolysates

had very low antioxidative activity. In this study, the

hydrolysates of APH had both antioxidative activity

and a synergistic effect with a-tocopherol using the lino-

leic acid in water/alcohol system. Therefore, we focused

on the isolation and structural characterization of potentantioxidative peptides from the APH.

3.2. Isolation of antioxidative peptide

APH-V was dissolved in sodium acetate buffer (pH

4.0), and loaded onto a SP-Sephadex C-25 column with

the linear gradient of NaCl (0–1.0 M), and fractionated

into six portions (Fig. 3A). Each fraction was pooled,lyophilized, and measured for antioxidative activity in

a linoleic acid model system. The lyophilized active frac-

tion was subjected to size-exclusion chromatography on

Sephadex G-25, and fractionated into four portions

(Fig. 3B). Each fraction was pooled, lyophilized, and

measured for antioxidative activity. This active fraction

was further separated by RP-HPLC on a Primesphere

10 C18 (20 mm · 250 mm) column using a linear gradi-ent of acetonitrile (0–35%) containing 0.1% TFA, and

the fractions were divided into three portions (Fig.

3C). Active fraction was pooled, and further purified

on a Capcell Pak C18 UG-120 (10 mm · 250 mm) using

a linear gradient of acetonitrile (0–15%) containing 0.1%

TFA (Fig. 3D). Finally, we obtained a purified peptide

Fig. 4. Hydroxyl radical scavenging activity of the purifie

with amino acid sequence as Leu-Pro-His-Ser-Gly-Tyr

(MW 672 Da).

Recently, the antioxidative activity of histidine-

containing peptides has been reported (Uchida & Kaw-

akishi, 1992; Murase, Nagao, & Terao, 1993; Park et al.,

2001). This activity may be attributed to the chelatingand lipid radical-trapping ability of the imidazole ring.

In this study, the antioxidative peptide contained a his-

tidine residue. In addition, this peptide was contained

tyrosine residue, which is a potent hydrogen donor in

its sequence. These results suggest that antioxidative

activity of the isolated peptide was dependent on their

amino acid residue and molecular weight.

3.3. Hydroxyl radical scavenging effect of purified peptide

Hydroxyl radicals generated in Fe2+/H2O2 system

were trapped by DMPO forming spin adduct, which

could be detected by an ESR spectrometer. The typical

1:2:2:1 ESR signal of the DMPO-OH adduct was ob-

served as shown in Fig. 4. The height of the third peak

of the spectrum represents the relative amounts ofDMPO-OH adduct. The purified peptide quenched

about 35% of hydroxyl radical at 53.6 lM.

Free radicals with the major species of reactive oxy-

gen species (ROS) are unstable, and react readily with

other groups or substances in the body, resulting in cell

damage and hence human disease (Halliwell & Gutter-

idge, 1989). Especially, the chemical activity of hydroxyl

radical is the strongest among the ROS. It is easily reactwith biomolecules such as amino acids, proteins, and

DNA (Cacciuttolo, Trinh, Lumpkin, & Rao, 1993).

Therefore, the removal of hydroxyl radical is probably

one of the most effective defence of a living body against

various diseases.

d peptide at 53.6 lM. (A) Control and (B) Peptide.

50 J.-Y. Je et al. / Food Research International 38 (2005) 45–50

Acknowledgements

This research was funded by the MOMAF-SGRP,

Kitto Life Co., and this work was also supported by

the Brain Korea 21 project in 2003.

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