Advance Journal of Food Science and Technology 4(1): 34-38, 2012
ISSN: 2042-4876
© Maxwell Scientific Organization, 2012
Submitted: November 25, 2011
Accepted: December 27, 2011
Published: February 15, 2012
Partial Purification of an Antioxidizing Component in Silky Fowl Egg
Toshiyuki Toyosaki
Department of Foods and Nutrition, Koran Women’s Junior College, Fukuoka, 811-1311, Japan
Abstract: Gel filtration was used to partially purify the antioxidizing component of a crude extracted solution
of silky fowl egg, and its properties were studied. The antioxidizing effect did not decrease after dialysis,
however, the antioxidizing effect significant decrease by heating of albumen. The finding of no change after
dialysis suggested that the compound was of high molecular weight, estimated at 49.6 kDa by gel filtration
chromatography. The antioxidizing component of this 49.6 kDa fraction may be a protein-bound tocopherol.
We found an antioxidizing components in silky fowl egg. This antioxidizing components is estimated at 49.6
kDa fraction may be a protein-bound tocopherol.
Key words: Antioxidizing component, gel filtration, SDS-polyacrylamide gel electrophoresis (PAGE), silky
fowl egg, tocopherol-bound protein
CO., Ltd. (Tokyo, Japan). Diethylaminoethyl (DEAE)
cellulose (DE 52) were purchased from Whatman, Ltd.,
great Britain. Macro-Prep 25Q Strong Anion Exchange
support (Macro-Prep 25 Q, 200 mL), Macro-Prep 25 S
Strong Cation Exchange support (Macro-Prep 25 S, 10
mL), and Bio-Scale Empty Column (MT20, 15 x 113 mm,
19.4-21.9 mL) were purchased from Bio-Rad Laboratories
(Tokyo, Japan). All other chemicals used were analytical
grade and obtained from Nacalai Tesque Inc. (Kyoto,
Japan). Eggs of silky fowl were a kind gift from Canaly
21 Co., Ltd., Gifu, Japan. Each fresh egg fraction was
obtained from the eggs collected within a day after laying
and immediately used for these experiments. Eggs were
collected from flocks of 40 silky fowls.
INTRODUCTION
Many natural antioxidaizing substances besides
polyphenols or carotenoids are known (Tagliazucchi
et al., 2010; Alikkunjhi et al., 2010; Faller and Flaiho,
2009; Castro et al., 2005), but their potential harm to the
human body their use as food additives. Components of
common foods are less likely to cause unexpected health
problems than newly synthesized compounds. The eggs
of the silky fowl are well known in the Orient and for
thousands of years have been credited with medicinal and
health-promoting values. However, a modern scientific
approach has only recently been applied to determine
their medicinal, chemical, and biochemical components
(Sakakibara et al., 2000; Ferrand and L'hermite, 1985).
Recently, we reported that silky fowl eggs are a chemical
storehouse and an excellent source of sialic acid (Koketsu
et al., 2003), which is an important component for the
protection of life (Koketsu et al., 1995; Koketsu et al.,
1997), and that silky fowl eggs are an excellent food
material (Koketsu and Toyosaki, 2004; Toyosaki and
Koketsu, 2004; Toyosaki and Koketsu, 2007). In a
preliminary experiment (unpublished), we found an
antioxidizing component in a silky fowl egg. The purpose
of this study, was to isolate and identify the component,
and investigate its suitability as a food additive.
Dialysis and heat of crude extracted samples: Crude
solubility of albumen (100 mL) of silky fowl egg was
dialyzed with a tube-shaped dialysis membrane (diameter,
27 mm; wall thickness, 0.0203 mm; pores, 24 A; Visking
Co., USA) at 4ºC against 1500 mL of either 40mM TrisHCl buffer, pH 8.0, or 40 mM sodium phosphate buffer
containing 0.154M NaCl, pH 9.5, for 24 h. Different
crude solubility fraction was heated in a water stirred for
30 min. The antioxidizing effects of these were measured.
Column chromatography with diethylaminoethyl
(DEAE) cellulose and gel filtration: The crude solubility
albumen fraction was separated by DEAE-cellulose
(DE 52) column chromatography as follows. A DEAEcellulose column (4.5×54 cm) was equilibrated with 50
mM Tris-HCl buffer (pH 8.0), washed with the same
buffer and 300 mL of the same buffer containing 0.4M
NaCl. The flow rate was 40 mL/h and fractions of 10mL
were collected. Each of the resulting fractions was
MATERIALS AND METHODS
Materials: Linoleic acid (more than 99% pure) were
purchased from Sigma Chemical Co. (St. Louis, MO,
USA). N’,N’-dimethylformamide, Chondroitin sulfate
sodium salt, hematin, 2’,7’-dichlorofluorescin diacetate
(DCFDA) were purchased from Tokyo Kasei Kogyo
34
Adv. J. Sci. Technol., 4(1): 34-38, 2012
where a is the test crude extracted sample or fraction from
gel filtration plus linoleic acid, b is the control sample
(linoleic acid only), and c and c’ = a and b, respectively,
before incubation.
desalted with Macro-Prep 25 S (Macro-Prep 25 S strong
cation exchange support) and concentrated by
ultrafiltration (Tokyo Ultrafilter UK-10, Japan). The
resulting concentrate was further purified by gel filtration
chromatography with a Bio-Scale Empty Column (MT20,
15×13 mm, 19.4-21.9 mL) and equilibrated with a 25 mM
sodium phosphate buffer, pH 7.2 (temperature, 4ºC; flow
rate, 10 mL/h; fraction volume, 5 mL). The protein
concentration of each fraction was measured by
absorbance at 280 nm.
Determination of tocopherols by High Performance
Liquid Chromatography (HPLC): Tocopherols were
extracted from crude albumen was as described by
Sheehy et al. (1994). In brief, samples (10 g) were
agitated with 500 :L of 25 mM pyrocatechol solution and
10 mL of a saturated methanoic solution of potassium
hydroperoxide, and saponified by heating in a water bath
at 80ºC for 15 min. Following saponification, the mixture
was mixed with 5.0 mL n-hexane and 5.0 mL water and
centrifuged, and an aliquot of the upper phase was
evaporated to dryness to be further reconstituted in
methanol and injected into the HPLC (Shimazu, Model
6AV, Tokyo, Japan). HPLC was carried out using a
normal phase silica column (Nucleosil C18, 5 mm, 250×
4.6 mm column), and a mobile phase of methanol/water
(97:3, v/v) that was delivered in the system at a flow rate
of 2 mL/min (Sheehy et al., 1994). Monitoring of the
column effluents was performed using a fluorimetric
detector set at an excitation wavelength of 290 nm and an
emission wavelength of 330 nm. Detector signals were
quantified using peak heights and a standard calibration
curve.
SDS-polyacrylamide gel electrophoresis (PAGE):
Polyacrylamide gel electrophoresis of fraction A an B was
done by the method of Laemmi (1979) under the
following condition. The gel were run at 2 mA/column for
90 min. After electrophoresis, each band from the
polyacrrylamide gel was isolated in a Canalco (USA)
Prep-Disc apparatus as described in the manual. A
constant voltage (350V) was applied to this system for
about 3 h at between 20 and 30 mA. The sample were
dialyzed and lyophilized. The protein concentration
samples of recovered bands from polyacrylamide gels
were measured with a total protein kit (Bio-Rad
Laboratories, Inc., Tokyo, Japan).
Molecular weight estimation: The molecular weight of
the antioxidizing component was estimated using the
method of Andrews (1965). A standard protein Kit for
molecular weight measurement (Bio-Rad Laboratories,
Inc., Tokyo, Japan) was used. The elution volume for the
alkaline phosphatase was found by the method of Garen
and Levinthal (1960), for ovalbumin by absorbance at 280
nm, and for cytochrome c by absorbance at 410 nm.
Statistical analysis: All data are presented as mean ±
standard deviation. Statistical comparison between
different treatments was performed by t-test
(kaleidaGraph, Ver. 4.0, Synergy software, PA, USA),
which was authorized using Duncan’s new multiple range
test (Steel and Torrie, 1980) and application software Stat
View 512 only for statistics + (Brain Power, Berkeley,
CA).
Measurement of antioxidant activity: Crude solubility
faction, and fractions of the gel filtration chromatography
were tested for antioxidizing effects against linoleic acid
autoxidation by the method of Mitsuda et al. (1996) with
2×0G3M linoleic acid, 0.1 M sodium phosphate buffer (pH
7.0), and 12 :M FeCl3. The total reaction volume was 5.0
mL, and the mixture was shaken at 37ºCfor 1 h. After
incubation, the generation of linoleic acid hydroperoxide
was measured by the method of Cathcart et al. (1984).
The amount of dichlorofluorescein (DCF) was measured
by monitoring of the fluorescence intensities at 470 nm
(excitation) and 550 nm (emission) with a 605-40
fluorescence spectrophotometer (Hitachi, Ltd., Japan).
The antioxidant activity remaining, with the control
activity (with linoleic acid only) as 100%, was calculated
from the equation:
RESULTS AND DISCUSSION
Antioxidant activity of silky fowl egg albumen and
yolk crude solubility fraction after dialysis, heat: The
results of various treatments of the silky fowl egg
albumen and yolk before fractionation are shown in Table
1. Dialysis did not decrease antioxidant activity, so the
antioxidizing component was of high molecular weight.
Heated crude extracted fraction of albumen retained more
than 2.4% of its activity, so the component was not an
enzyme. Protein itself has weak antioxidizing activity
(Kajimoto and Yoshida, 1972, 1975; Tappel, 1955;
Yukami, 1972). However, heat did not destroy most
of the antioxidizing activity, of the crude extracted
fraction as it would if the component were entirety
protein.
Antioxidant activity remaining (%) = 100
-[Amount of DCF (pmol) in a – Amount of DCF
(pmol) in c]/[Amount of DCF (pmol) in b – Amount
of DCF (pmol) in c’]× 100
35
Adv. J. Sci. Technol., 4(1): 34-38, 2012
II
Absorbance at 280 nm ( )
3.0
2.5
III
100
80
I
2.0
60
1.5
40
1.0
20
0.5
0.0
Antioxidant activity (%) (-O-)
Table1: Antioxidant activity of silky fowl egg crude solubility fraction
after dialysis, heat
Antioxidant activity (%)
------------------------------------Treatment
Albumen
Yolk
Dialyzed against
Tris-HCl buffer, pH 8.0
88.3±3.57
2.57±0.97
NaCl-phosphate buffer, pH 9.5
80.1±8.41
1.88±0.55
Heated for 15 min at 40ºC
60.6±9.43
0.39±0.06
Heated for 15 min at 60ºC
15.8±1.69
nd
Heated for 15 min at 80ºC
2.4±0.57
nd
nd: not detected
Fig. 4: Polyacrylamide gel electrophoresis of fraction A and B
using Bio-Scale Empty column chromatography
Isolation and purification of antioxidizing component
using DEAE-cellulose column chromatography: The
elution pattern, antioxidant activity, and tocopherol
content of fraction are shown in Fig. 1 and 2, after the
extraction of crude proteins from silky fowl egg, proteins
with antioxidizing activity were isolated and purified
using DEAE-cellulose column chromatography, done to
isolate the antioxidizing component gave fraction III with
antioxidant activity and tocopherol content.
0
0
20
40
60 80 100 120 140 160
Elution volume (mL)
II
III
8
Absorbance at 280 nm ()
3.0
7
I
2.5
6
2.0
5
4
1.5
3
1.0
2
0.5
0.0
1
0
0
20
40
60
80 100 120 140 160
Elution volume (mL)
Total tocopherol control (g/mL; -O-)
Fig. 1: Changes of antioxidant activity in a crude solubility
fraction of silky fowl egg using DEAE-Cellulose
column chromatography
Isolation and purification of antioxidizing component
using a Bio-Scale Empty column chromatography:
After the isolated of antioxidizing activity fraction using
DEAE-cellulose column chromatography were purified
using Bio-Scale Empty column chromatography. Fraction
III with antioxidizing activity were obtained, and their
elution patterns, antioxidant activity, are given in Fig. 3.
SDS-polyacrylamide gel electrophoresis: Figure 4
shows the results of SDS-polyacrylamide gel
electrophoresis for fractions A, and B collected using
elution buffer. Fractions A and B of No.3 had the
strongest antioxidizing action and contained tocopherol;
its molecular weight was estimated by gel filtration to be
about 49.6 kDa. This band was not identified but it is, we,
think, a protein that contains tocopherol.
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
100
90
80
70
60
50
40
30
20
B
A
10
0
0
10
20
30
40
50
60
Elution volume (mL)
70
Antioxidant avtivity (%, -()-)
Absorbance at 280 nm ()
Fig. 2: Changes of protein elution in a crude solubility fraction
of silky fowl egg using DEAE-Cellulose column
chromatography
Antioxidant activity and tocopherol content of No. 1-8
gel fraction bands: Antioxidant activity and tocopherol
content of No. 1-8 was shown in Table 2. Also and
tocopherol content was confirmed a strong antioxidant
activity to the band of No.3. The molecular weight of
No.3 was 49.6 kDa.
Relationship between the antioxidant activity and the
tocopherol content of No.3 band: In a separate
experiment, we irradiated No. 3 band in with heated for
15 min at 40-80ºC (Fig. 5 and 6). Antioxidizing activity
80
Fig. 3: Changes of antioxidant activity in fraction III using BioScale Empty column chromatography
36
Adv. J. Sci. Technol., 4(1): 34-38, 2012
Table 2:Total tocopherol contents and antioxidant activity in A and B
fractions (No. 1-8 bands) of using SDS-polyacrylamide gel
electrophoresis
No. Total tocopherol contents (mg/band)a Antioxidant activity (%)
1
nd
nd
2
nd
nd
3
1.58±0.02
89.3±9.27
4
0.34±0.04
4.1±0.86
5
nd
nd
6
nd
nd
7
nd
nd
8
nd
nd
nd: not detected
component in silky fowl egg was estimated at 49.6 kDa
fraction may be a protein-bound tocopherol. This result
can submit knowledge to the food science field. From
now on, you have to examine the structural analysis of
protein-bound tocopherol, which showed antioxidation.
REFERENCES
Alikkunjhi, P., F. Korengath, C. Preethi and R. Kuttan,
2010. Antioxidant potential of meso-zeaxanthin a
semi synthetic carotenoid. Food Chem., 120:
1096-1101.
Andrews, O., 1965. The gel-filtration behavior of proteins
related to their molecular weights over a wide range.
Biochem. J., 96: 595-599.
Castro, I.A., S.B. Barros, U.M. Lanfer Marquez,
M. Motizuki and T.C. Higashi Sawada, 2005.
Optimization of the antioxidant capacity of a mizuter
of carotenoids and " - tocopherol in the development
of a nutritional. Food Res. Int., 38: 861-866.
Cathcart, R.E., E. Scheirs and B.H. Ames, 1984.
Detection of picomole levels of lipid peroxidation
using a dichlorofluorescein fluorescent assay. In:
Parker, (Ed.), Methods in Enzymology, Academic
Press, New York, 105: 352.
Faller, A.L.K. and E. Flaiho, 2009. The antioxidant
capacity and polyphenol content of organic and
conventional retall vegetables after demestic. Food
Res. Int., 42: 210-215.
Ferrand, R. and A. L'hermite, 1985. Experimental
analysis of the extensive pigmentation in the silkie
fowl embryo: Evidence for an environmental
regulatory process. Experientia, 41: 512-514.
Garen, A. and C. Levinthal, 1960. A fine-structure
genetic and chemical study of the enzyme alkaline
phospatase of E coli I. Purification and
characterization of alkaline phosphatase. Biochim.
Biophys. Acta, 38: 470-476.
Kajimoto, G. and H. Yoshida, 1972. Studies on the metalprotein complex. III. Relationship between the
binding ability of various inorganic metal-protein
complex and activity of metal-catalyzed autoxidation.
J. Jpn. Oil Chem. Soc., 21: 842-846.
Kajimoto, G. and H. Yoshida, 1975. Studies on the metalprotein complex IV. Effects of the inorganic metalfood constituents complex on the rancidification of
oil. J. Jpn. Oil Chem. Soc., 23: 83-85.
Koketsu, M., T. Nitoda., L.R. Juneja., M. Kim.,
N. Kashimura and T. Yamamoto, 1995.
Sialyloligosaccharides from egg yolk as an inhibitor
of rotaviral infection. J. Agric. Food Chem., 43:
858-861.
Koketsu, M., T. Nitoda, H. Sugino, L.R. Juneja, M. Kim,
T. Yamamoto, N. Abe, T. Kajimoto and C.H. Wong,
1997. Synthesis of a novel sialic acid derivative
(sialylphospholipid) as an antirotaviral agent. J. Med.
Chem., 40: 3332-3335.
Antioxidant activity (%)
100
90
80
70
60
50
40
30
20
10
0
5
0
10
15
20
25
Incubation time (min)
30
35
Fig. 5: Temperature effects on antioxidant activity of fraction
III. Each point represents the mean of four trials, given
with the standard deviation. Symbols were as follow:
-"- 40ºC; -!-, 60ºC; -G-, 80ºC
Tocopherol content (%)
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
Incubation time (min)
30
35
Fig. 6: Temperature effects on tocopherol content of fraction
III. Each point represents the mean of four trials, given
with the standard deviation. Symbols were as follow:
-"-, 40ºC; -!-, 60ºC; -G-, 80ºC
tended to decrease with longer heat, with a parallel
decrease in tocopherol content. These finding correspond
with the results shown in Table1; that is, the antioxidizing
action of band B was degraded rapidly by heat. Based on
these results, we concluded that tocopherol is probably
related to the antioxidizing activity of the silky fowl egg
component.
CONCLUSION
The current research demonstrated that new
antioxidant activity in silky fowl egg. From the results
of this study it will be concluded that the antioxidant
37
Adv. J. Sci. Technol., 4(1): 34-38, 2012
Koketsu, M., E. Sakuragawa., R.J. Linhardt and
H. Ishihara, 2003. Distribution of N-acetylneuraminic
acid and sialylglycan in eggs of the Silky fowl.
British Poul. Sci., 44: 145-148.
Koketsu, M. and T. Toyosaki, 2004. Nutritive
constituents of silky fowl eggs: Comparison with hen
eggs of White Leghorn origin. Anim. Sci. J., 75:
67-69.
Laemmi, U.K., 1979. Cleavage of structural proteins
during the assembly of the head of bacteriophage T4.
Nature, 227: 680-685.
Mitsuda, H., K. Yasumoto and K. Iwami, 1996.
Antioxidation action of indole compounds during the
autoxidation of linoleic acid. Eiyo Shokuryo, 19:
210-216.
Sakakibara, K., S. Tabta., N. Shiba., T. Gotoh.,
S. Nishimura and H. Iwamoto, 2000. Myofibre
composition and total collagen content in
Miliotibialis lateralis and M. pectoralis of Silkie and
White Leghorn chickens. Br. Poul. Sci., 41:
570-574.
Sheehy, P.J.A., P.A. Morrist and A. Flynn, 1994.
Consumption of thermally-oxidized sunflower oil by
chicks reduces "-tocopherol status and increase
susceptibility of tissues to lipid oxidation. Br. J.
Nutri., 70: 53-65.
Steel, R.G.D. and J.H. Torrie, 1980. Principles and
Procedures of Statistics: A Biometrical Approach.
2nd Edn., McGraw-Hill, New York.
Tagliazucchi, D., E. Verzelloni, D. Bertolini and
A. Conte, 2010. In vitro bio-accessibllity and
antioxidant activity of grape polyphenols. Food
Chem., 120: 599-606.
Tappel, A.L., 1955. Studies of the mechanism of vitamin
E action III. In vitro copolymerization of oxidized
fats with protein. Arch. Biochem. Biophys., 54:
266-280.
Toyosaki, T. and K. Koketsu, 2004. Oxidative stability of
silky fowl eggs. Comparison with hen eggs. J. Agric.
Food Chem., 52: 1328-1330.
Toyosaki, T. and K. Koketsu, 2007. Antioxidant effects
of the water-soluble fraction of baked sponge cake
made with silky fowl egg: comparison with White
Leghorn egg. Br. Poul. Sci., 48: 449-453.
Yukami, S., 1972. Autoxidation of sodium linoleate
in the protein solution. Agric. Biol. Chem., 36:
464-469.
38