Professional Documents
Culture Documents
DOI 10.1007/s00217-004-0882-9
ORIGINAL PAPER
Introduction
An antioxidant is defined as any substance that significantly 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
21
were those which passed through the 1 kDa membrane. All the
YFPHs recovered were lyophilized on a freeze-drier for 5 days.
Measurement of antioxidative activity
The antioxidative activity of the YFPHs was measured in a linoleic
acid model system according to the methods of Osawa et al. [20]. A
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
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 thiocyanate values. The TBA value was measured using a modified
version 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 of
8.1% 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 C
for 1 h in the dark. The TBA value was measured by reading the
absorbance at 532 nm. The ferric thiocyanate value was measured
according to the method of Mitsuda et al. [22]. The reaction solution (100 ml) incubated in the linoleic acid model system described
above [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 chloride
solution in 3.5% HCl. After 3 min, the PV was measured by reading
the absorbance at 500 nm following color development with FeCl2
and thiocyanate at different intervals during the incubation period
at 401 C . All analyses were run in triplicate and averaged.
Molecular weight distribution profile
Molecular weight distributions of the hydrolysates were determined
by gel permeation chromatography (GPC) using a high-performance 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), were
connected in series, and the hydrolysates were chromatographed
and monitored at 230 nm at room temperature.
Purification of the antioxidative peptide and determination
of amino acid sequence
The lyophilized YFPH-I was dissolved in 20 mM sodium acetate
buffer (pH 4.0) and fractionated by ion-exchange chromatography
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
collected at a flow rate of 60 ml/h. The fractions showing antioxidative activity were pooled and lyophilized. The lyophilized fraction was dissolved in 50 mM sodium phosphate buffer (pH 7.0) and
loaded onto a Sephadex G-75 gel filtration column (2.590 cm)
which had previously been equilibrated with the same buffer. The
column was then eluted with the same buffer, and 5-ml fractions
were collected at a flow rate of 60 ml/h. The fractions exhibiting
antioxidative activity were pooled and lyophilized. The antioxidative fraction was dissolved in distilled water and separated using
reversed-phase HPLC on a Primesphere 10 C18-HC 120 (10 mm,
1.025 cm; Phenomenex, Macclesfield, UK) column using a linear
gradient of acetonitrile (050% in 40 min) in 0.1% trifluoroacetic
acid (TFA) at a flow rate of 2.0 ml/min. The elution peaks were
monitored at 215 nm, and their antioxidative activities were measured using the same method. The active peaks were concentrated
using a centrifugal evaporator. The peaks representing the antioxidative activity were rechromatographed on the same column
using 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 antioxidative
peptide was determined by automated Edman degradation with a
PerkinElmer 491 protein sequencer (Branchburg, NJ., USA).
22
23
24
skin gelatin was described [27]. In this study, the antioxidative activity of five hydrolysates fractionated from
YFP was investigated and compared with that of a-tocopherol, a widely used natural antioxidant. YFPH-I exhibited the highest activity (Fig. 3). In addition, the synergistic antioxidative effects of the YFPH with the nonpeptidic antioxidant a-tocopherol were studied. All hydrolysates of YFP from various enzymes exhibited synergistic effects with a-tocopherol (Fig. 4). The synergistic
effects of nonpeptidic antioxidants on antioxidative activity have previously been demonstrated with the hydrolysates of a vegetable protein, yeast protein, Alaska
Pollack skin gelatin hydrolysates, and bovine serum albumin [26, 27, 28]. Chen et al. [29] reported that the
hydrolysates of soybean protein showed a strong synergistic effect with nonpeptidic antioxidants although some
During the process of identifying the antioxidative peptide 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 antioxidative 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 antioxidative activity (Fig. 5B). This fraction was further
separated by reversed-phase HPLC using a 0.1% TFAacetonitrile 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 III11 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 combination, 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, ArgPro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr. In addition, the
molecular weight of the antioxidative peptide was determined 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 interaction between peptides and fatty acids. The antioxidative
peptide contained tyrosine residue, which is a potent hydrogen donor. Previously, many proteins have been reported 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 antioxidative 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
25
26
References
1. Maillard MG, Soum MH, Meydani SN, Berset C (1996) Food
Sci Technol 29:238244
2. Wanita A, Lorenz K (1996) J Food Process Preserv 20:417429
3. Hettiarachchy NS, Glenn KC, Gnanasambandam R, Johnson
MG (1996) J Food Sci 61:516519
4. Frlich I, Riederer P (1995) Drug Res 45:443449
5. Yamaguchi NS, Naito Y, Yokoo Y, Fujimaki M (1980) J Jpn
Soc Food Sci Technol 27:5659
6. Pratt DE (1972) J Food Sci 37:322323
7. Yukami S (1972) Agric Biol Chem 36:871874
8. Rhee KS, Ziprin YA, Rhee KC (1979) J Food Sci 44:1132
1135
9. Iwami K, Hattori M, Ibuki F (1987) J Agric Food Chem
35:628631
10. Chavan UD, Amarowicz R, Shahidi F (1999) J Food Lipids 9:
1-11
11. Shahidi F, Amarowicz R, He Y, Wettasinghe M (1997) J Food
Lipids 7:7586
12. Wang JY, Fujimoto K, Miyazawa T, Endo Y (1991) J Agric
Food Chem 39:351355
13. Park PJ, Jung WK, Nam KS, Shahidi F, Kim SK (2001) J Am
Oil Chem Soc 78:651656
14. Kim SK, Kim YT, Byun HG, Nam KS, Joo DS, Shahidi F
(2001) J Agric Food Chem 49:19841989
15. Park PJ, Je JY, Kim SK (2003) J Agric Food Chem 51:4624
4627
16. Podsedek, A, sosnowska D, Anders B (2003) Eur Food Res
Technol 217:296300
17. Carlsen CU, Rasmussen KT, Kjeldsen KK, Westergaard P,
Skibsted LH (2003) Eur Food Res Technol 217:195200
18. Gopala KAG, Prabhakar JV (1994) J Am Oil Chem Soc
71:645647
19. Kim SK, Park PJ, Byun HG, Je JY, Moon SH (2003) J Food
Biochem 27:255-266
20. Osawa T, Namiki M (1985) J Agric Food Chem 33:777780
21. Ohkawa H, Ohishi N, Yagi K (1979) Anal Biochem 95:351
358
22. Mitsuda H, Yasumoto K, Iwami K (1966) Eiyo to Shokuryo
19:210214
23. Kim SK, Jeon YJ, Byun HG, Kim YT, Lee CK (1997) Fish Sci
63:421528
24. Receca BD, Pena-Vera MT, Deaz-Castaneda M (1991) J Food
Sci 56:309314
25. Nair AL, Gopakumar K (1982) Fish Technol 19:101103
26. Bishov SJ, Henick AS (1975) J Food Sci 40:345348
27. Kim SK, Kim YT, Byun HG, Nam KS, Joo DS, Shahidi F
(2001) J Agric Food Chem 49:19841989
28. Hatate H, Nagata Y, Kochi M (1990) Yukagaku 39:4246
29. Chen HM, Muramoto K, Yamauchi F, Nokihara K (1996)
J Agric Food Chem 44:26192623
30. Riisom T, Sims RJ, Fiorti JA (1980) J Am Oil Chem Soc
57:351359
31. Taylor MJ, Richardson T (1980) J Food Sci 45:12231227
32. Yee JJ, Shipe WF, Kinsella JE (1980) J Food Sci 45:10821083
33. Kawashima K, Itoh H, Miyoshi M, Chibata I (1979) Chem
Pharm Bull 40:19121916
34. Bishov SJ, Henick AS (1972) Chem Pharm Bull 37:873875