Eur Food Res Technol (2004) 219:2026
DOI 10.1007/s00217-004-0882-9
ORIGINAL PAPER
Shin-Young Jun Pyo-Jam Park Won-Kyo Jung
Se-Kwon Kim
Purification and characterization of an antioxidative peptide
from enzymatic hydrolysate of yellowfin sole (Limanda aspera)
frame protein
Received: 10 November 2003 / Revised: 29 December 2003 / Published online: 31 March 2004

Springer-Verlag 2004
Abstract In order to utilize yellowfin sole (Limanda
aspera) frame protein (YFP), which is normally discarded
as industrial waste in the process of fish manufacture,
yellowfin sole frame protein hydrolysates (YFPHs) were
fractionated 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 with
molecular weight cut-offs of 30, 10, 5, 3, and 1 kDa,
respectively. The antioxidative activity of the YFPHs was
investigated and compared with that of a natural antiox-
idant, a-tocopherol, used as a reference. Furthermore, the
fraction showing strong antioxidative activity was iso-
lated from the YFPHs using consecutive chromatographic
methods on an SP-Sephadex C-25 column, on a Sephadex
G-75 column, and high-performance liquid chromatog-
raphy (HPLC) on an octadecylsilane column. The mo-
lecular mass of the antioxidant was identified as 13 kDa
using HPLC on a gel permeation chromatography (GPC)
column, and the antioxidative peptide was composed of
10 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 when
present at low concentrations compared to that of an
oxidizable substrate. Antioxidants can act at different
S.-Y. Jun P.-J. Park W.-K. Jung S.-K. Kim ())
Department of Chemistry,
Pukyong National University,
Busan 608737, Korea
e-mail: sknkim@mail.pknu.ac.kr
Tel.: +82-51-620 6375
Fax: +82-51-628 8147
levels in an oxidative sequence. This can be illustrated by
considering one of the many mechanisms by which oxi-
dative stress can cause damage by stimulating the free
radical chain reaction of lipid peroxidation. Free radical
chain reactions within a material can be inhibited either
by adding chemicals that retard the formation of free
radicals or by introducing substances that compete with
the existing radicals and remove them from the reaction
medium.
Lipid oxidation is of great concern to the food industry
and 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 products
by retarding discoloration and deterioration as a result of
oxidation. Therefore, antioxidants are increasingly used
as a means of enhancing shelf-life and to improve the
stability of lipid and lipid-containing foods. Synthetic
antioxidants such as butylated hydroxyanisole (BHA),
butylated hydroxytoluene (BHT), tertiary butylhydro-
quinone, and propyl gallate are added to food products to
retard 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 a
number 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, the
search for natural antioxidants as alternatives to synthetic
ones is of great interest among researchers. Several
studies 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]. Amino
acids have also been reported to exhibit antioxidant
properties against linoleic acid oxidation in the freeze-
dried emulsion condition [18]. However, little is known
 21

about the structure of antioxidative peptides from various were those which passed through the 1 kDa membrane. All the
food proteins. YFPHs recovered were lyophilized on a freeze-drier for 5 days.
In this study, we purified an antioxidative peptide
derived from enzymatic hydrolysate of yellowfin sole Measurement of antioxidative activity
frame protein (YFP), which is normally discarded as in-
dustrial waste in the process of fish manufacture, and The antioxidative activity of the YFPHs was measured in a linoleic
acid model system according to the methods of Osawa et al. [20]. A
determined the amino acid sequence. sample (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 and
10 ml of 99.5% ethanol. Then the total volume was adjusted to
Materials and methods 25 ml with distilled water. The solution was incubated in a conical
flask with a screw cap at 401 C in a dark room, and the degree of
oxidation was evaluated by measuring the TBA and ferric thiocy-
Materials
anate values. The TBA value was measured using a modified
version of the method of Ohkawa et al. [21]. The reaction mixture
Fresh samples of yellowfin sole frame (moisture, 79%) were do-
(50 ml) was added to a mixture of 0.8 ml distilled water, 0.2 ml of
nated by Daerim Co. (Busan, Korea), and stored at 20 C until
8.1% sodium dodecyl sulfate, and 1.5 ml of 0.8% TBA solution.
use. Mackerel intestine was obtained from a local fish market and
The mixture was incubated at 5 C for 1 h, and then heated at 95 C
stored at 20 C until use. Alcalase 0.6 L (0.6 AU/g) and Neutrase
for 1 h in the dark. The TBA value was measured by reading the
0.5 L (0.5 AU/g) were acquired from Novo Co. (Novo Nordisk,
absorbance at 532 nm. The ferric thiocyanate value was measured
Bagsvaerd, Denmark), and papain from papaya latex (type IV),
according to the method of Mitsuda et al. [22]. The reaction solu-
pepsin from porcine stomach mucosa, trypsin from bovine pancreas
tion (100 ml) incubated in the linoleic acid model system described
(type II), a-chymotrypsin from bovine pancreas (type II), pronase E
above [20] was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 30%
from Streptomyces griseus (type XIV), 2-thiobarbituric acid (TBA),
ammonium thiocyanate, and 0.1 ml of 210-2 M ferrous chloride
ammonium thiocyanate, linoleic acid, a-tocopherol. SP-Sephadex
solution in 3.5% HCl. After 3 min, the PV was measured by reading
C-25, and Sephadex G-75 were purchased from Sigma Chemical
the absorbance at 500 nm following color development with FeCl2
Co. (St. Louis, MO., USA). The ultrafiltration membrane (UF)
and thiocyanate at different intervals during the incubation period
reactor (Minitan) system and membranes for the fractionations of
at 401 C . All analyses were run in triplicate and averaged.
yellowfin sole hydrolysate, based on molecular weights, were from
Millipore Co. (Bedford, USA). All other reagents were of the
highest grade available commercially.
Molecular weight distribution profile

Molecular weight distributions of the hydrolysates were determined

Extraction of mackerel intestine crude enzyme (MICE)
by gel permeation chromatography (GPC) using a high-perfor-
mance liquid chromatography (HPLC) system (Hewlett-Packard,
The crude proteinase from mackerel intestine was extracted ac-
Palo Alto, CA). Two GPC columns, Zorbax PSM 300 and 60
cording to the method of Kim et al. [19]. Briefly, the minced in-
(Hewlett-Packard), with exclusion limits of 3103 to 3105 Da
testine was added to two volumes of 20 mM Tris-HCl buffer (pH
(6.2 mm, 254.6 cm) and 110103 Da (6.2 mm, 254.6 cm), were
7.0) containing 5 mM CaCl2, and homogenized twice at 12,000 rpm
connected in series, and the hydrolysates were chromatographed
for 2 min using an homogenizer (Ace homogenizer, Nissei AM-7,
and monitored at 230 nm at room temperature.
Nihonseiki Kaisha, Tokyo, Japan). The homogenate was incubated
at 37 C for 2 h, and centrifuged at 9,500g for 20 min. After
adjusting the supernatant to a 50% saturated solution with cold
Purification of the antioxidative peptide and determination
acetone (v/v), it was centrifuged again as described above. To re-
of amino acid sequence
move insoluble protein from the precipitated protein, the same
volume of distilled water was added, and the mixture was cen-
The lyophilized YFPH-I was dissolved in 20 mM sodium acetate
trifuged at 9,500g for 10 min. The supernatant was lyophilized
buffer (pH 4.0) and fractionated by ion-exchange chromatography
and stored at 20 C until use.
on a SP-Sephadex C-25 column (440 cm). The column was
equilibrated with the same buffer and fractions eluted with a linear
gradient of NaCl concentration (01.0 M). Fractions of 5 ml were
Preparation of yellowfin sole frame protein hydrolysates (YFPH)
collected at a flow rate of 60 ml/h. The fractions showing antiox-
idative activity were pooled and lyophilized. The lyophilized frac-
Eight proteases (Alcalase, a-chymotrypsin, MICE, Neutrase, pa-
tion was dissolved in 50 mM sodium phosphate buffer (pH 7.0) and
pain, pepsin, pronase E, trypsin) were used for the digestion of
loaded onto a Sephadex G-75 gel filtration column (2.590 cm)
YFP. YFP was hydrolyzed with each protease for 6 h in a batch
which had previously been equilibrated with the same buffer. The
reactor under optimal conditions and then heated at 98 C for
column was then eluted with the same buffer, and 5-ml fractions
10 min to inactivate the proteases. The resulting hydrolysates were
were collected at a flow rate of 60 ml/h. The fractions exhibiting
lyophilized, and assayed for antioxidative activity. To purify the
antioxidative activity were pooled and lyophilized. The antioxida-
antioxidative peptides, YFP was hydrolyzed with MICE (enzyme to
tive fraction was dissolved in distilled water and separated using
substrate ratio, 1:50) for 3 h at pH 10.0 and 50 C, and subsequently
reversed-phase HPLC on a Primesphere 10 C18-HC 120 (10 mm,
prepared with pepsin (enzyme to substrate, 1:50) for 3 h at pH 2.0
1.025 cm; Phenomenex, Macclesfield, UK) column using a linear
and 37 C, after the pH of the solution had been adjusted to 2.0
gradient of acetonitrile (050% in 40 min) in 0.1% trifluoroacetic
with conc. HCl. Finally, the resultant hydrolysate was fractionated
acid (TFA) at a flow rate of 2.0 ml/min. The elution peaks were
through five different UF membranes having a range of molecular
monitored at 215 nm, and their antioxidative activities were mea-
weight cut-offs (MWCO), i.e., 30, 10, 5, 3, and 1 kDa. Those
sured using the same method. The active peaks were concentrated
YFPHs which passed through the 30 kDa membrane but not
using a centrifugal evaporator. The peaks representing the anti-
through the 10 kDa membrane were categorized as YFPH-I. Those
oxidative activity were rechromatographed on the same column
which passed through the 10 kDa membrane but not passed through
using a linear gradient of acetonitrile (030% in 40 min) in 0.1%
the 5 kDa membrane were YFPH-II. Those that passed through
TFA at a flow rate of 2.0 ml/min. The sequence of antioxidative
the 5 kDa membrane but not through the 3 kDa membrane were
peptide was determined by automated Edman degradation with a
YFPH-III. Those which passed through the 3 kDa membrane but
PerkinElmer 491 protein sequencer (Branchburg, NJ., USA).
not through the 1 kDa membrane were YFPH-IV, and YFPH-V
22

Results and discussion

Every year, about 100 million tons of fish are harvested;
however, 30% of the total catch is transformed into
fishmeal [23, 24]. Over 50% of the harvest is processing
waste which includes bone, skin, fins, internal organs,
heads, and so on [25]. In Korea, the annual yellowfin sole
harvest exceeds 13,828 tons, and 1,327 tons were pro-
cessed in Korean fish plants in 2002. In particular, fish
frames obtained after filleting include bones, heads and
tails. 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 investigated
the antioxidative activity of enzymatic hydrolysate of
YFP, a by-product, which is normally transformed into
fishmeal or discarded by fish processing plants.

Digestion of YFP with various enzymes

and their antioxidative activity

In order to select suitable proteases for the hydrolysis of

YFP, the YFP was independently hydrolyzed with MICE,
alcalase, a-chymotrypsin, papain, pepsin, pronase E,
neutrase, and trypsin using a batch reactor. The degree of
hydrolysis of YFP by MICE was the highest (67%), and
the degree of hydrolysis of YFP by pepsin was the lowest
(22%) compared to hydrolysis using commercial pro-
teases (data not shown). In addition, the antioxidative
activities of the resultant YFPHs were measured and
compared with that of a-tocopherol. As shown in Fig. 1,
the oxidation of linoleic acid was markedly inhibited by
YFPHs derived from YFP with various proteases. Among
the hydrolysates resulting from various enzymes, the Fig. 1 Antioxidative activities of hydrolysates from yellowfin sole
highest antioxidative activity was observed in the pepsin frame protein (YFP) from various proteases in a linoleic acid au-
hydrolysate, which exhibited about 70% inhibition of li- toxidation system measured by the thiobarbituric acid (TBA)
noleic acid peroxidation. Therefore, these results indicate method (A), and by the ferric thiocyanate method (B). l,control;
that the hydrolysates of YFP seemed to contain some
, a-tocopherol; t, mackerel intestine crude enzyme (MICE)hy-
drolysate; 5, alcalse hydrolysate; n, a-chymotrypsin hydrolysate;
antioxidative peptides. o, papain hydrolysate; u, pepsin hydrolysate; }, pronase E hy-
drolysate; s, neutrase hydrolysate; 4, trypsin hydrolysate. All
experiments were carried out in triplicate, and the results were
Preparation and molecular weight profiles of YFPHs expressed as means

The pepsin hydrolysate showed the highest antioxidative
activity compared to those of other enzymatic hy-
drolysates. However, the degree of hydrolysis of YFP by
pepsin was the lowest. Therefore, we combined MICE,
which showed the highest degree of hydrolysis of YFP,
with pepsin, which exhibited the highest antioxidative
activity. The YFPH was prepared with MICE for 3 h at
pH 10.0 and 50C, and subsequently hydrolyzed with
pepsin for 3 h at pH 2.0 and 37C after the pH of the
solution had been adjusted to 2.0 with HCl. The antiox-
idative activity of the YFPH derived from the MICE and
pepsin combination was similar to or slightly higher than
that of pepsin hydrolysate (data not shown). To isolate an
antioxidative peptide from YFP with the MICE and
pepsin combination, five different kinds of YFPHs were
prepared by a batch system, and fractionated by passing
the protein through five UF membranes with molecular
weight cut-offs (MWCO) of 30, 10, 5, 3, and 1 kDa. The
YFPHs were named as YFPH-I, which passed through the
30 kDa membrane but not through the 10 kDa membrane;
YFPH-II, which passed through the 10 kDa membrane
but not through 5 kDa; YFPH-III, which passed through
the 5 kDa membrane but not through 3 kDa; YFPH-IV,
which passed through the 3 kDa membrane but not
passed through1 kDa, and YFPH-V, which passed
through 1 kDa. The molecular weight distributions varied
according 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. The
molecular weight distribution of YFPH-II was between 13
and 6 kDa, and the major peaks were at 9 and 7 kDa.
YFPH-III showed a major peak at 5 kDa. The molecular

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 at
a flow rate of 1.0 mL/min. A, albumin (MW 66,000 Da); B, car-
bonic anhydrase (MW 29,000 Da); C, cytochrome C (MW
12,327 Da); D, aprotinin (MW 6,000 Da); E, pentaphenylalane
(MW 753.9 Da)
weight distribution of YFPH-IV was between 3 and
0.9 kDa, except for the appearance of peak at 0.8 kDa,
and a major peak at 2.6 kDa. YFPH-V showed two major
peaks at 0.8 and 0.5 kDa. The result of the molecular
weight profiles of each hydrolysate showed a distinct
23
Fig. 3 Antioxidative activities of hydrolysates by MICEpepsin
combination from YFP in the linoleic acid autoxidation system
measured by the TBA method (A), and by the ferric thiocyanate
method. l,control;
, a-tocopherol; t, YFPH-I; n, YFPH-II; o,
YFPH-III; u, YFPH-IV; }, YFPH-V
decrease of molecular weight according to the pore size of
the membrane.
Antioxidative activity of YFPHs
There are only a few reports on the antioxidative efficacy
of amino acids. Tryptophan and histidine showed high
antioxidant activity whereas glycine and alanine showed
only weak activity, and methionine and cysteine had an
antioxidative effect in Soybean oil [30]. However, all
amino acids have exhibited antioxidant activity in some
systems, which probably reflects the antioxidant nature of
the NH3R group [31]. The use of a protein or a hydrol-
ysate for the improvement of the antioxidative activity in
functional foods might be more practical than the use
of amino acids, because proteins and hydrolysates have
other desired functional properties. The antioxidative ac-
tivity of soybean protein hydrolysates has been docu-
mented [32]. In a recent paper, the antioxidative effect of
peptides derived from the enzymatic hydrolysates of fish
24
Fig. 4 Synergistic effects of a-tocopherol and hydrolysates of YFP
by MICE and pepsin combination in linoleic acid autoxidation
system measured by the TBA method (A), and by the ferric thio-
cyanate method (B). l,control;
, a-tocopherol; t, a-tocopherol
and 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-V
combination
skin gelatin was described [27]. In this study, the an-
tioxidative activity of five hydrolysates fractionated from
YFP 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 synergistic
effects of nonpeptidic antioxidants on antioxidative ac-
tivity have previously been demonstrated with the hy-
drolysates of a vegetable protein, yeast protein, Alaska
Pollack skin gelatin hydrolysates, and bovine serum al-
bumin [26, 27, 28]. Chen et al. [29] reported that the
hydrolysates of soybean protein showed a strong syner-
gistic effect with nonpeptidic antioxidants although some
hydrolysates had very low antioxidative activity. In this
study, the hydrolysates of YFP had both antioxidative
activity and a synergistic effect with a-tocopherol using
the linoleic acid in water/alcohol system. Therefore, we
focused on the isolation and structural characterization of
potent antioxidative peptides from the YFPH.
Purification of an antioxidative peptide and determination
of amino acid sequence
During the process of identifying the antioxidative pep-
tide derived from YFP, the protein was hydrolyzed with
the MICE and pepsin combination, and five hydrolysates
(YFPH-I, II, III, IV, and V) were obtained using UF
membranes with 30, 10, 5, 3, and 1 kDa MWCO. YFPH-I
was then separated using ion-exchange chromatography
on an SP-Sephadex C-25 column and fractionated into
three portions. When these fractions were tested for an-
tioxidative activity, fraction III was found to possess a
strong activity and was then lyophilized (Fig. 5A). The
lyophilized fraction III was subjected to size exclusion
chromatography on Sephadex G-75 and fractionated into
a major portion. Fraction III-1 exhibited strongest an-
tioxidative activity (Fig. 5B). This fraction was further
separated 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 the
highest antioxidative activity (Fig. 5C). Subfraction III-
11 was further separated by RP-HPLC using the same
solvent system. Two portions were finally obtained from
the 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-terminal
amino acid sequence of III-11a, and the antioxidative
peptide was composed of 10 amino acid residues, Arg-
Pro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr. In addition, the
molecular weight of the antioxidative peptide was deter-
mined to be 13 kDa by HPLC on GPC columns as above
described (data not shown). The amino acid residues at
the N- termini of dipeptides have been demonstrated to be
antioxidative in an oil system [33]. It is probable that the
amino acid residues play a role in increasing the inter-
action between peptides and fatty acids. The antioxidative
peptide contained tyrosine residue, which is a potent hy-
drogen donor. Previously, many proteins have been re-
ported to have strong antioxidative activity against the
peroxidation of lipid or fatty acid systems [26, 34]. As a
Fig. 5 Purification of antioxidative peptides from the YFPH-I. (A)
SP-Sephadex C-25 chromatography (lower panel) and antioxidative
activities of the fractions (upper panel) measured by TBA method
after 6 days. Elution was performed at a flow of 60 mL/h with a
linear NaCl gradient (01 M) in 20 mM sodium acetate buffer, pH
4.0. (B) Re-chromatography of fraction III from Fig. 4A on
Sephadex G-75 gel chromatography (lower panel) and antioxida-
tive activity of the fraction (upper panel) measured by the TBA
method after 6 days. Elution was done at a flow rate of 60 mL/h in
50 mM sodium phosphate buffer (pH 7.0). (C) Reversed-phase

HPLC pattern on a Primesphere 10 C18 column of fraction III-1
from Fig. 4B was eluted on the gel chromatography (lower panel)
and antioxidative activities of the fractions (upper panel) measured
by the TBA method after 6 days. HPLC operation was carried out
with 50% acetonitrile as mobile phase at a flow rate of 2 ml/min
using an UV detector at 215 nm. (D) Further separation of sub-
25
fraction III-11 reversed phase HPLC. Elution profiles (lower
panel) and antioxidative activities of the fractions (upper panel)
measured by the TBA method after 6 days. The HPLC operation
was carried out with 30% acetonitrile as mobile phase at a flow rate
of 2 mL/min using a UV detector at 215 nm
26
result, antioxidative activity of peptide is thought to be
related to their molecular weight and amino acid se-
quence.
Acknowledgments This work was supported by the MOST, Busan
Metropolitan City, and Daerim Co. in Korea.
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