Purication of chicken breast protein hydrolysate and analysis

of its antioxidant activity

Yangying Sun
a
, Daodong Pan
a,b,
, Yuxing Guo
a
, Junjiang Li
a
a
Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, PR China
b
Food Science Department of Marine Science School, Ningbo University, Ningbo 315211, PR China
a r t i c l e i n f o
Article history:
Received 27 April 2012
Accepted 23 July 2012
Available online 1 August 2012
Keywords:
Chicken breast protein hydrolysate
Antioxidant activity
In vitro and in vivo systems
Transmission electron microscope
a b s t r a c t
Chicken breast protein was hydrolyzed by papain under optimal conditions. The antioxidant activity of
the chicken breast protein hydrolysate was then evaluated in vitro and in vivo using different measure-
ments, including reducing power and DPPH radical scavenging assays. The reducing power of the hydro-
lysate was 0.5 at 2.37 mg/mL. The DPPH radical scavenging assay showed that the EC
50
value of the
hydrolysate was 1.28 mg/mL. In antioxidant assays in vivo, D-galactose-induced aging mice administrated
the fraction peptides of chicken breast protein hydrolysate showed signicantly increased antioxidant
enzyme activities, while malondialdehyde levels decreased both in serums and livers. Under a transmis-
sion electron microscope (TEM), the ultramicrostructure of hepatic tissue was observed and we found
that the hydrolysate may play a part in inhibiting oxidative stress in hepatocytes in vivo. Therefore, we
concluded that chicken breast protein hydrolysate exhibits signicant antioxidant activity.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Research has indicated that free radicals may cause a range of
serious health problems in humans, including diabetes and various
cardiovascular diseases. Lipid oxidation can generate free radicals,
which are unstable and rapidly react with other substances (Teng
et al., 2011). Therefore, to prevent serious disease, it is crucial to
control lipid peroxidation. One method is to use antioxidants,
which can reduce the oxidative damage associated with many dis-
eases (Bougatef et al., 2010).
In recent years, protein hydrolysates have been found to exhibit
strong antioxidant activity, such as wheat germ (Zhu et al., 2006),
loach (Misgurnus anguillicaudatus) (You et al., 2010a) and pea
(Pownal et al., 2011).
It has been reported that the antioxidant activity of protein
hydrolysates is related to their amino acid composition (Thians-
ilakul et al., 2007). The residues of hydrophobic amino acid, such
as Val and Leu, can increase the presence of hydrolysates at the
waterlipid interface, and thereby facilitate access to allow the
scavenging of free radicals generated in the lipid phase (Kumar
et al., 2011; Ren et al., 2008). In addition, aromatic amino acid
residues can exhibit radical scavenging activity (Rajapakse et al.,
2005). The hydrolysates, which contain largely acidic amino acid
residues (Glu, Asp) also display high antioxidant properties (Saiga
et al., 2003). The antioxidant capacities of protein hydrolysates
may be attributed to the amino acid proles of the hydrolysates
and cooperation between various amino acids.
Chicken meat is well accepted by consumers all over the world.
Chicken protein, which is rich in essential amino acids, is an effec-
tive source of protein. It also may be a potential source of antiox-
idants for human consumption, since chicken protein offers a
well-balanced amino acid composition combined with the abun-
dant supply and high nutritional value of chicken (Cui et al.,
2009; Sallam, 2007)
As far as we know, few studies have analyzed the antioxidant
properties of chicken breast protein hydrolysate in vitro or
in vivo. This study reports on the antioxidant properties of chicken
breast protein hydrolysate hydrolyzed by papain under optimal
conditions which were obtained in our previous work. Reducing
power and DPPH radical scavenging ability were evaluated
in vitro on the crude hydrolysate, while antioxidant enzyme activ-
ities and malondialdehyde (MDA) levels were assayed invivo, using
puried peptides, on serums and livers of mice in an aging model
using D-galactose. Under a transmission electron microscope
(TEM), the ultramicrostructure of hepatic tissue was observed.
Chicken breast protein hydrolysate was also isolated using gel
chromatography on a Sephadex G-25. Changes in the composition
of amino acids during purication were also analyzed to determine
the relationships between their antioxidant capacities.
0278-6915/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2012.07.047

Corresponding author at: Food Science and Nutrition Department, Nanjing

Normal University, Nanjing 210097, PR China. Tel.: +86 25 83598771; fax: +86 25
83707623.
E-mail address: daodongpan@163.com (D. Pan).
Food and Chemical Toxicology 50 (2012) 33973404
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology
j our nal homepage: www. el sevi er . com/ l ocat e/ f oodchemt ox
2. Materials and methods
2.1. Materials and chemicals
Chicken breast meat was purchased from Suguo supermarket in Nanjing, China.
After removing fat and connective tissue with the scalpel, the meat was minced and
sealed in polyethylene bags and stored at 20 C for further use. Papain with an
activity of 800 U/mg was obtained from Novozyme Nordisk (Denmark). 1,1-diphe-
nyl-2-picrylhydrazyl (DPPH) and D-galactose were obtained from SigmaAldrich
(St. Louis, MO, USA). Pills of six ingredients with rehmannia were obtained from
Nanjing Tongrentang Pharmaceutical Co. (Nanjing, China). Assay kits for superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), MDA and
protein were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing,
China). All other chemicals used were analytical grade and purchased from Nanjing
Rongshide Trading Co. (Nanjing, China).
2.2. Preparation of chicken breast protein hydrolysate
Chicken breast meat (50 g) was homogenated with distilled water (100 mL) and
the mixture hydrolyzed by papain for 6.15 h at 51.2 C. The enzyme-to-substrate-
protein ratio was 1.5:1,000 (w/w). The material-to-water ratio was 1:2 (g/mL).
The homogenate was placed in a water bath shaker at a pH of 6.5. After hydrolysis,
the solutions were immediately heated at 100 C for 15 min to stop the hydrolysis.
The hydrolysate was then centrifuged (4000 rpm, 20 min), and after ltering the
soluble supernatants, they were lyophilized and stored at 20 C for further use.
2.3. Measurement of in vitro antioxidant capacities of chicken protein hydrolysate
2.3.1. Reducing power assay
The reducing power of chicken protein hydrolysate was determined according
to the method of Wu et al. (2003). Different concentrations (0.5, 1.0, 1.5, 2.0 and
2.5 mg/mL) of chicken protein hydrolysate were dissolved in distilled water. A vol-
ume of 0.2 mL of the sample solution in different concentrations was mixed with
2.0 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 2.0 mL of 1% (w/v) potassium
ferricyanide. Then the mixture was allowed to react at 50 C for 20 min and then
2.0 mL of 10% (w/v) trichloroacetic acid was added. The mixture was then centri-
fuged (3,000 rpm, 20 min) and 2.0 mL of distilled water and 0.4 mL of 0.1% (w/v)
ferric chloride were mixed with 2.0 mL of the soluble supernatant. After allowing
a reaction for 10 min, the solutions were determined at 700 nm. The blank was pre-
pared by using distilled water instead of the sample. Increased reducing power was
indicated by the increased absorbance of the reaction solution. In addition, RP
0.5AU
was dened as the concentration of compound producing an absorbance of 0.5 at
700 nm.
2.3.2. DPPH scavenging activity assay
DPPH scavenging activity was measured by the method described by You et al.
(2009). Distilled water was used to prepare different concentrations (1.0, 2.0, 3.0,
4.0 and 5.0 mg/mL) of hydrolysate solutions. The solutions were obtained by mixing
2.0 mL of sample with 2.0 mL of 0.1 mM DPPH dissolved in 95% ethanol and kept in
the dark to react for 30 min. Absorbance was measured at 517 nm. A higher DPPH
scavenging activity was represented by a lower absorbance. The following equation
was used to calculate scavenging activity:
Scavenging activity% 100 1 A
sample
A
blank
=A
control

While sample is prepared by mixing 2 mL of sample solution with DPPH solu-

tion, blank is prepared by mixing 2 mL of sample solution with 2 mL of 95% ethanol
and control is prepared by mixing 2 mL of 95% ethanol with DPPH solution.
2.4. In vivo antioxidant activities of chicken protein hydrolysate (fraction peptides)
2.4.1. Animal preparation
ICR mice [male, 8 weeks old, weighing 20 2 g, SCXK(Su)2008-0004] were ob-
tained from Nanjing Medical University, Laboratory Animal Center (Nanjing, China).
Mice were housed under controlled conditions [SYXK(Su)2008-0007] with 55 5%
relative humidity at a temperature of 22 1 C (12 h light/dark cycle). The experi-
ments were carried out according to the Chinese legislation on the use and care
of laboratory animals. Mice were given free access to pellets and drinking water
during the experiment. After 5 days acclimation, the mice were randomly assigned
to six groups with 10 mice in each group. Group I (normal mice) were given distilled
water by gavage; Group II (model mice) were given distilled water by gavage;
Group III (positive control group mice) were given distilled water containing pills
of six ingredients with rehmannia by gavage (750 mg/kg body weight); Group IV
(normal mice) were given distilled water containing peptides (125 mg/kg body
weight) by gavage; Group V (normal mice) were given distilled water containing
peptides (250 mg/kg body weight) by gavage; and Group VI (normal mice) were gi-
ven distilled water containing peptides (500 mg/kg body weight) by gavage. All
mice were given the substances with the same volume of 0.2 mL. Mice in Group I
were given saline (10 mL/kg body weight) via abdominal injection, while the other
groups were treated with D-galactose (500 mg/kg body weight) via abdominal
injection. All groups were treated once per day every day at approximately the
same time over 42 days.
2.4.2. Biochemical determinations
Mice were weighed and sacriced 24 h after the last administration. Their
whole blood was centrifuged at 3000 rpm for 10 min (4 C) to separate the serum
and kept at 80 C for further use. The liver was washed with ice-cold physiological
saline and stored at 80 C immediately before analysis. Furthermore, 10% (w/v)
homogenate was prepared by homogenizing the liver with icecold physiological
saline. Then the supernatant of the homogenate, which was centrifuged at
3000 rpm for 10 min (4 C), was obtained for further analysis.
The activities of CAT, SOD and GSH-Px, as well as the MDA level and the protein
content were determined using assay kits according to their instructions (Product
Nos. A007, A001-1, A005, A003-2 and A045-2, respectively, production batch num-
ber: 20110709, Nanjing Jiancheng Bioengineering Institute).
Inbrief, CATactivitywas measuredat 405 nmduetotheyellowH
2
O
2
-ammonium
molybdate complex. SOD activity was measured by determining its absorbance at
550 nm, because the superoxide radicals generatedinthe xanthinexanthine oxidase
system can inhibit hydroxylamine oxidation. GSH-Px activity was determined by
monitoring the reduction of 5,5-dithiobis (2-nitrobenzoic acid) at 412 nm. The activ-
ities of CAT, SODand GSH-Px were determined using the method described by Zhang
et al. (2011). The MDA level was determined at an absorbance of 532 nm using the
TBARS method (Roghani and Baluchnejadmojarad, 2010), while the protein content
of the liver was measured according to the Bradford method. Enzyme activities were
expressed as U/mL in serum or U/mg protein in liver, while the MDA level was ex-
pressed as nmol/mL in serum or nmol/mg protein in liver (Liu et al., 2010).
2.4.3. Ultramicrostructure of hepatic tissue
Livers of mice were cut into 1 mm
3
specimens and xed in 4% (v/v) glutaralde-
hyde at 4 C for 2 h. After rinsing in 0.2 M phosphate buffer (pH 7.4) four times
(15 min/step), specimens were xed in 1% (w/v) OsO4 at 4 C for 2 h, rinsed twice
with phosphate buffer (5 min/step) and dehydrated in a graded acetone series
[15 min/step; (507090100)% (v/v)]. Each specimen was treated with an ace-
tone/epoxy resin mixture with a ratio of 1:1 for 1.5 h, and then an acetone/epoxy
resin mixture with a ratio of 1:2 for 1.5 h. Each specimen was infused in pure epoxy
resin (Epon618) for 3 h at 37 C. Then each specimen was infused in epoxy resin and
polymerized for 36 h at 60 C. Each specimen was cut using an ultramicrotome (Lei-
ca Ultracut R, Wetzlar, Germany) into ultra-thin specimens and stained with uranyl
acetate for 15 min. The specimens were thrice rinsed with distilled water (15 min/
step) and stained using lead citrate for 5 min. The specimens were then thrice
rinsed again with distilled water (15 min/step) before their examination using a
TEM (Wang et al., 2008; Xu and Wei, 2006). A JEOL JEM-1010 electron microscope
(JEOL, Tokyo, Japan) was used to capture the micrographs at an acceleration voltage
of 80 kV.
2.5. Purication of the chicken breast protein hydrolysate
2.5.1. Ultraltration
The hydrolysates were fractionated at an operating temperature of 25 C and at
a pressure of 0.1 MPa using ultraltration membranes with a molecular weight cut-
off of 5 kDa MWCO (Shanghai Mosutech., China). The obtained fractions were
lyophilized and kept at 20 C for further use.
2.5.2. Gel ltration chromatography
The fraction (100 mg) which exhibited the highest antioxidant activity was dis-
solved with 2 mL of distilled water. The obtained solution was separated using
Sephadex G-25 and a gel ltration column (1.6 cm 50 cm) that was eluted with
distilled water. The ow rate was 36 mL/h. Each fraction was measured at
280 nm. Fractions with active peaks were collected and lyophilized for antioxidant
activity assay.
2.5.3. High performance liquid chromatography (HPLC)
The desired peak after GFC purication was prepared by dissolving it in distilled
water. The solution was applied onto a semi-preparative RP-HPLC column
(9.4 mm 250 mm, 5-lm particles, ZorBax 300SB-C18, Agilent Technologies,
USA) with a linear gradient of acetonitrile (040% in 40 min) containing 0.1% (v/
v) triuoroacetic acid (TFA) with a ow rate of 2 mL/min. Peaks were monitored
at 215 nm. The active peak (data not shown) was collected and lyophilized
immediately.
The active peak from the semi-preparative column was further subjected to an
analysis using an RP-HPLC column (4.6 mm 250 mm, 5-lm particles, ZorBax
Eclipse XDB-C18, Agilent Technologies, USA).
The mobile phases for the analysis of chromatography were: (A) 0.1% triuoro-
acetic acid (TFA) in acetonitrile; and (B) 0.1% TFA in water. Gradient elution was as
follows: 010 min, linear gradient 9895% B; 1015 min, isocratic gradient 95% B;
the ow rate: 0.5 mL/min; the detection wavelength 215 nm.
3398 Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 33973404
2.6. Analysis of amino acid compositions
The composition of the amino acids were analyzed by RP-HPLC (Agilent 1100)
using a Hypersil ODS column (4.6 mm 250 mm). The total amino acids (except
for tryptophan) were determined after hydrolysis at 110 C for 24 h with 6 M HCl,
prior to derivatization with o-phthaldialdehyde (OPA) and 9-uorenylmethyl chlo-
roformate (FMOC). The measurement was performed at 40 C and at a ow rate of
1.0 mL/min. The detection wavelength was 338 nm, and the fractions were loaded
for RP-HPLC analysis (Agilent1100, USA).
2.7. Statistical analysis
Experiments were carried out at least in triplicate. Data was averaged and the
standard deviation calculated. The statistical analysis was performed using SPSS
17.0 software. The signicant difference was determined by ANOVA with a 95% con-
dence interval (P < 0.05).
3. Results and discussion
3.1. Antioxidant activities of chicken protein hydrolysate in vitro
3.1.1. Reducing power assay
The reduction of the Fe
3+
/ferricyanide complex to ferrous form
was measured in the presence of the chicken protein hydrolysates.
The Fe
2+
complex can be detected by determining the formation of
Perls Prussian blue at an absorbance of 700 nm. The potential anti-
oxidant activity of the compound is shown by its reducing capacity
(Hsu et al., 2006). It has been reported that with higher reducing
power, hydrolysates display a greater ability to donate electrons
and free radicals in order to form stable substances, thereby inter-
rupting the free-radical chain reactions. That is to say, the hydrol-
ysates have an excellent ability to donate electrons, which are
involved in antioxidant activity (Pan et al., 2011).
Fig. 1 shows that the chicken protein hydrolysate possessed
remarkable reducing power with concentration-dependent effects
and a high correlation index (r
2
= 0.9948). Moure et al. (2006) re-
ported that the reducing power exhibited an obvious effect on both
protein size and concentration, with the latter playing an impor-
tant role in the fractions that had a smaller molecular weight. Sev-
eral works have indicated that increasing the concentration of
samples can increase reducing power (Xie et al., 2008; Zhu et al.,
2006).
The reducing power (RP
0.5AU
) of the chicken protein hydrolysate
was 2.37 mg/mL. Compared with wheat germ protein hydrolysate,
the reducing power of the chicken protein hydrolysate was much
higher (Zhu et al., 2006). These results indicate that chicken pro-
tein hydrolysate displays signicant antioxidant activity. Chicken
protein hydrolysate may exhibit donating capacity by neutralizing
and converting free radicals to more stable products and, thereby,
may terminate the chain reactions initiated by free radicals (Arde-
stani and Yazdanparast, 2007).
3.1.2. DPPH radical scavenging activity assay
DPPH, which shows maximum absorbance at 517 nm in ethanol
and has commonly been used in the analysis of antioxidant activ-
ity, is a relatively stable free radical. The color of its ethanolic solu-
tion changes from modena to yellow when free radicals are
scavenged and its absorbance gradually decreases (Xie et al.,
2008). Therefore, a substances DPPH radical scavenging ability
can be used to measure its antioxidant activity.
Fig. 2 shows that the hydrolysate prepared by papain hydrolysis
exhibited the ability to scavenge DPPH radicals at various concen-
trations. The chicken protein hydrolysate showed a concentration-
dependent increase in antioxidative activity for concentrations up
to 3 mg/mL. DPPH radical scavenging activity increased with
increasing hydrolysate concentrations until about 90%, and there-
after reached a plateau. Moreover, at concentrations between 3
and 5 mg/mL, the chicken protein hydrolysate was able to quench
about 90% of the radicals.
The EC
50
(the concentration that scavenged 50% of the initial
DPPH radicals) can be expressed to determine the efciency of
an antiradical; the lower the EC
50
, the higher the free radical scav-
enging ability. The regression equation of the scavenging activity
was calculated and the EC
50
value of chicken protein hydrolysate
was expressed as 1.28 mg/mL, which is comparable to that of
wheat germ protein hydrolysate (Zhu et al., 2006). The results re-
vealed that chicken protein hydrolysate can terminate the radical
chain reaction by converting free radicals into more stable
products.
Although a protein may have free radical scavenging activity,
this ability is not conclusive evidence that the protein is an antiox-
idant. For a free-radical scavenger to be considered an antioxidant
in foods, it must be more oxidatively labile than unsaturated fatty
acids, and the resulting protein radical must not be powerful en-
ough to promote lipid oxidation (Elias et al., 2008). Protein hydrol-
ysates have good antioxidant activity, and it is pivotal to elucidate
how the composition of protein hydrolysate inuences their ability
to scavenge free radicals.
3.2. Chicken protein hydrolysate (fraction peptides) antioxidant
activities in vivo
In our present study, ICR mice used as an aging animal model
were treated with D-galactose to evaluate in vivo antioxidant abil-
ities. At the end of the study, there was no difference in body
weight gain between the animals. However, mice in the model
group showed signs of dullness, depression and exhibited sparse,
dull hair, while mice in the normal group had shining hair and
smooth fur. Mice in the other groups were similar to the normal
group, but displayed less activity than the normal group. As shown
in Tables 1 and 2, the model group exhibited signicantly (P < 0.05)
Fig. 1. Reducing power of different concentrations of chicken breast protein
hydrolysate. Each value is expressed as mean S.D. (n = 9).
Fig. 2. DPPH radical scavenging activity of different concentrations of chicken
breast protein hydrolysate. Each value is expressed as mean S.D. (n = 9).
Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 33973404 3399
decreased SOD, CAT and GSH-Px activities, and increased MDA lev-
els compared with the normal group both in serums and livers.
These ndings indicate that the D-galactose induced aging mice
model was successful. Peptides (125, 250 and 500 mg/kg) signi-
cantly (P < 0.05) increased enzymatic activities compared with
the D-galactose induced aging mice group, while MDA levels de-
creased signicantly (P < 0.05) with a few exceptions between
the mice who received peptides (125 mg/kg) and the model group.
SOD activities in serums and CAT activities in livers did not signif-
icantly differ (P > 0.05). The enzymatic activities and the MDA lev-
els of the positive control group were signicantly (P < 0.05)
different from those of the model group, except for SOD activities
in serums and livers. To some extent, the positive control group
in experimental animals was useful. For the three dose levels, the
enzymatic activities and the MDA levels were comparable to one
another. In the present study, we found no statistical signicant
difference between the three doses; however, the results indicate
that the peptides displayed potential antioxidant properties.
D-galactose, which has the ability to induce oxidative stress
in vivo, can be used to mimic natural aging in order to screen
antioxidants (Luo et al., 2010; Qiao et al., 2009). It has been dem-
onstrated that aging, as a result of diminished antioxidant protec-
tion, is associated with a reduction in antioxidants and incremental
increases in lipid peroxidation (Katrin et al., 2006). The major anti-
oxidant enzymes, acting as the rst line of defense against antiox-
idants, can prevent the formation of toxic compounds so as to
protect against oxidative stress and tissue damage (Chen et al.,
2011).
SOD dismutates superoxide radicals to form hydrogen peroxide.
CAT and GSH-Px prevent the formation of hydroxyl radicals by
decomposing hydrogen peroxide into water and oxygen. Therefore,
these enzymes act cooperatively against free radicals in the de-
fense of active oxygen compounds (Li et al., 2007; Pan and Mei,
2010). Meanwhile, MDA is a cytotoxic product of lipid peroxida-
tion. The formation of lipid peroxides can damage hepatic tissue
by causing incomplete cell membranes (Chen et al., 2011; Huang
et al., 2011). Our results showed that the antioxidant enzyme
activities in serums and livers were markedly decreased in the D-
galactose induced aging mice, while the MDA levels both in serums
and livers signicantly increased.
In vitro, the peptide concentration can affect antioxidant activ-
ities as measured by an assay, while antioxidant activities in vivo
may be inuenced by metabolism, digestibility and the bioavail-
ability of the substances (Anthony et al., 2008; Liu et al., 2010).
In vitro antioxidant assays usually ignore biological actions
in vivo, including the activity of antioxidant enzymes and oxida-
tive-related metabolism pathways, as well as the gene expression
of antioxidant substances and enzymes (Liu et al., 2010). Therefore,
antioxidant assays in vivo are required to determine the potential
antioxidant activity of peptides.
3.3. Ultramicrostructure of hepatic tissue
Fig. 3 shows the ultramicrostructure of the hepatic tissue of
experimental animals. In the normal group (Fig. 3a), the cells
stayed in good condition. The boundaries between the cell mem-
branes and cells were clear, and the nuclear pores were clearly ob-
served. The cells have abundant cytoplasm and vast rough
endoplasmic reticula, which were arranged in order with plenty
of ribosomes distributed all over them. Numerous mitochondrion
were evenly dispersed and the architecture of mitochondria stayed
intact. The mitochondrial bilayers and cristae were clearly
observed.
In the model group (Fig. 3b), obvious changes were found in the
nucleus. The nuclear membranes were irregular and the nucleoli
were dispersed and sparse. The cytoplasm was homogeneous be-
cause the organelles merged with each other. The rough endoplas-
mic reticula were fractured, degranulated and even disappeared,
with large vacuoles observed. The mitochondria swelled and the
mitochondrial bilayers were fuzzy and partially fused. The cristae
were thinner and fewer in number, with most of the crests unclear.
In the positive group (Fig. 3c), the cells stayed in good condition.
The boundaries between the cell membranes and the cells were
clear. The nucleoli and nuclear pores were clearly observed, and
the nucleoli were relatively concentrated. The ultrastructure of
the cells were similar to that of the normal control group.
In the low-dose and middle-dose groups (Fig. 3d and e), the
cells nuclear membranes were incomplete. The bilayer mem-
branes were obscured and the nucleoli were loose. The cytoplasm
was homogeneous since the organelles merged with each other.
The rough endoplasmic reticula disappeared. Numerous mitochon-
dria were found in the cytoplasm. Little difference was found be-
tween the low-dose and middle-dose groups.
In the high-dose group (Fig. 3f), the boundaries between cell
membranes and the cells were clear, and the nuclear pores were
clearly observed. The cells had abundant cytoplasm and vast rough
endoplasmic reticula were arranged in order with less breakage.
Many ribosomes were distributed on the rough endoplasmic retic-
ulum. The architecture of mitochondria remained intact, while
mitochondrial bilayers and cristae were clearly observed. The
integrity of the mitochondria was comparable to that of the normal
group, and was much better that that found in the model group.
TEM images showed that the hydrolysate may play a part in inhib-
iting the oxidative stress of hepatocytes in vivo.
3.4. Purication of chicken protein hydrolysate
The protein content of chicken protein hydrolysate (lyophiliz-
ate) was 91.36%. The molecular weight of the desired fraction
was less than 5 kDa, which made up 78.53% of the total amount
of hydrolysate. The fraction was lyophilized and kept at 20 C
Table 1
Effects of chicken breast protein hydrolysate on the activities of SOD (U/mL), CAT (U/
mL), GSH-Px (U/mL) and MDA levels (nmol/mL) in mice serums.
Group SOD CAT GSH-Px MDA
I 322.13 27.16 58.49 6.02 323.43 19.36 3.74 0.58
II 255.61 65.04 47.98 6.32 288.14 24.24 4.17 0.63
III 293.30 46.64 54.36 9.30
*
335.14 26.04
**
3.76 0.33
*
IV 264.47 47.11 57.00 7.43
**
360.72 25.04
**
3.75 0.31
*
V 331.81 49.38
**
60.75 6.09
**
377.14 24.72
**
3.59 0.20
**
VI 338.64 29.13
**
57.56 6.41
**
376.43 17.56
**
3.73 0.36
*
Data were expressed as means SD (n = 10) and evaluated by one-way ANOVA.
Differences were considered to be statistically signicant if P < 0.05.
*
P < 0.05, compared with the model group.
**
P < 0.01, compared with the model group.
Table 2
Effects of chicken breast protein hydrolysate on the activities of SOD (U/mg protein),
CAT (U/mg protein), GSH-Px (U/mg protein) and MDA levels (nmol/mg protein) in
mice livers.
Group SOD CAT GSH-Px MDA
I 16.74 0.93 47.04 4.03 103.59 10.06 1.24 0.18
II 15.84 0.93 41.19 3.10 93.96 9.00 1.55 0.18
III 16.37 0.25 45.60 2.58
*
102.57 8.11
*
1.26 0.26
**
IV 16.97 0.43
**
43.64 5.51 106.93 6.84
**
1.27 0.26
**
V 17.43 0.92
**
48.45 1.65
**
113.24 7.92
**
1.06 0.09
**
VI 17.88 0.36
**
50.11 2.32
**
111.45 6.38
**
1.24 0.13
**
Data were expressed as means SD (n = 10) and evaluated by one-way ANOVA.
Differences were considered to be statistically signicant if P < 0.05.
*
P < 0.05, compared with the model group.
**
P < 0.01, compared with the model group.
3400 Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 33973404
until use. It was isolated using Sephadex G-25 and three peaks
were obtained: F1, F2 and F3 (Fig. 4). Gel ltration was applied
to isolate the protein hydrolysates according to their molecular
weight (You et al., 2010b).
All peaks were collected, lyophilized and the antioxidant activ-
ities were evaluated. Each peak was then kept at 20 C for further
use. We also determined the DPPH scavenging activity of all the
peaks and discovered that F1 exhibited the greatest DPPH scaveng-
ing activity. At a concentration of 1.5 mg/mL, DPPH scavenging
activities for the peaks F1, F2 and F3 were, respectively, 72.8%,
46.5% and 37.2% (P < 0.05). F1 was also analyzed for its reducing
power. Its absorbance at 700 nm reached 0.495 at a concentration
of 1.5 mg/mL.
F1, which showed strong antioxidant activity, was loaded to RP-
HPLC on a ZorBax 300SB-C18 column with a linear gradient of ace-
tonitrile (040%). The desired peak was puried on an analytical
HPLC column (Fig. 5).
Fig. 3. TEM photos of liver sections. (a): normal; (b): model; (c): positive; (d): low-dose; (e): middle dose; (f): high dose. 1: cell nuclear membrane; 2: mitochondria; 3: rough
endoplasmic reticulum. Magnication: 10000.
Fig. 4. Elution prole of chicken breast protein hydrolysate separated by gel
ltration on Sephadex G-25.
Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 33973404 3401
3.5. Analysis of amino acid compositions
The amino acid composition of the samples was evaluated to
determine whether the composition affected antioxidant activity.
Table 3 presents the changes in the free amino acid composition
in the chicken breast protein hydrolysate and the three fractions
of the hydrolysate separated by gel ltration. Table 4 presents
the changes in the amino acid composition in the chicken breast
meat, its protein hydrolysate and Fraction I. As mentioned above,
F1 exhibited the greatest antioxidant activity both in terms of
DPPH scavenging activity and reducing power. Therefore, it was
important to analyze the amino acid composition of F1. Gel ltra-
tion seemed to greatly inuence the difference in the amino acid
composition between the hydrolysate and its fractions. The amino
acid compositions of the hydrolysate and the F1 fraction revealed
that they are both rich in arginine, leucine, glutamic acid, lysine,
isoleucine, methionine, valine, aspartic acid, threonine, and ala-
nine. However, the amounts of free amino acids, including arginine
and leucine, seemed to increase after ltration, while the amount
of tryosine decreased. In addition, the total hydrophobic amino
acids (leucine, isoleucine, valine, phenylalanine, methionine,
proline, and tryosine) accounted for 33.05% of the total amino acids
in F1, which is important since it has been reported that hydropho-
bic amino acids facilitate interaction with free radicals by increas-
ing their solubility in lipids (Je et al., 2007; Pan et al., 2011;
Rajapakse et al., 2005). Phe has been shown to act as a proton
donor, which scavenges radicals and maintains their stability via
resonance structures (Rajapakse et al., 2005). Although Phe was
found in relatively high amounts in F2, the amount of other amino
acids was relatively lower than in F1. For this reason, F2 showed
lower DPPH radical scavenging activity and reducing power than
F1. Furthermore, it has been demonstrated that peptides contain-
ing arginine, leucine, lysine, methionine, valine, aspartic acid, and
alanine possess strong antioxidant activity (Dong et al., 2008; Je
et al., 2007; Pan et al., 2011; Wang et al., 2007; Zhang et al.,
2010). Therefore, the antioxidant activity of the F1 fraction seems
to be related to the signicant amount of antioxidant amino acids
found in it. In summary, the amino acid composition of a hydroly-
sate seems to have a direct inuence on its antioxidative proper-
ties. However, further detailed studies focused on how the amino
acid composition inuences the antioxidant activity of a hydroly-
sate are needed.
4. Conclusion
In the present study, chicken breast protein was hydrolysed by
papain and the hydrolysates antioxidant activities were investi-
gated in vitro and in vivo. The hydrolysate showed strong reducing
power, as well as an ability to scavenge DPPH radicals. Antioxidant
testing in vivo showed that D-galactose-induced aging mice
administrated with the fraction peptides of chicken breast protein
10
2
0
1
2
3
4
5
6
7
Time (min)
1 2 3 4 5 6 7 8 9 10 11
A
b
s
.

2
1
5
n
m


Active peak
Fig. 5. RP-HPLC on a ZorBax EclipseXDB-C18 analytical column. Flow rate: 0.5 mL/min. Detection wavelength: 215 nm.
Table 3
Comparative free amino acid proles of hydrolysate and peptide fractions separated by G-25 (g/100 g protein).
Amino acid Hydrolysate Fraction I Fraction II Fraction III
Aspartic acid 0.901 1.257 0.031 0.000
Glutamic acid 2.176 2.704 0.317 0.080
Serine 0.824 1.677 0.549 0.074
Histidine 0.531 0.659 1.238 0.882
Glycine 0.467 0.985 0.501 0.107
Threonine 0.836 1.739 0.523 0.047
Alanine 0.876 1.265 0.325 0.200
Arginine 3.863 7.552 1.316 0.368
Tryosine 1.137 0.188 4.290 0.344
Cystine 0.239 0.482 0.216 0.000
Valine 1.152 2.351 0.589 0.072
Methionine 1.143 2.436 1.077 0.086
Phenylalanine 1.363 1.583 16.031 0.249
Isoleucine 1.306 2.481 0.587 0.097
Leucine 2.958 6.286 1.354 0.239
Lysine 1.617 2.589 0.481 0.224
Total essential free amino acids 10.374 19.464 20.641 1.014
Total hydrophobic free amino acids 9.059 15.324 23.928 1.087
3402 Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 33973404
hydrolysate signicantly increased the activity of antioxidant en-
zymes, while decreasing MDA levels both in serums and livers. Un-
der a TEM, the ultramicrostructure of hepatic tissue was observed
and we found that the hydrolysate may play a part in inhibiting the
oxidative stress of hepatocytes in vivo. The results indicate that the
hydrolysate has potential antioxidant capabilities. The hydrolysate
was further separated using Sephadex G-25 into three peaks.
According to the analysis of their amino acid compositions, it
was evident that higher amounts of basic amino acids combined
with hydrophobic amino acids may contribute to the antioxidant
activity of chicken breast protein hydrolysate. In order to elucidate
the relationships between the properties and structures of the anti-
oxidative peptides, further research should be conducted to iden-
tify the amino acid sequences of the individual antioxidative
peptides. In addition, further research is required to evaluate the
cytotoxicity of the specic peptides.
Conict of Interest
The authors declare that there are no conict of interest.
Acknowledgements
This work was supported by the Modern Agricultural Technical
System Foundation of China (CARS-43-17), Natural Science Pro-
gram of China (No. 30972130 and 31101309), and the High School
Natural Science Program of Jiangsu Province (No. 10KJB550003).
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