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Purification of chicken breast protein hydrolysate and analysis of its antioxidant activity
Food and Chemical Toxicology 50 (2012) 3397–3404
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Purification 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 b
Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, PR China 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 measurements, including reducing power and DPPH radical scavenging assays. The reducing power of the hydrolysate was 0.5 at 2.37 mg/mL. The DPPH radical scavenging assay showed that the EC50 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 significantly increased antioxidant enzyme activities, while malondialdehyde levels decreased both in serums and livers. Under a transmission 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 significant antioxidant activity. Ó 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 diseases (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 (Thiansilakul et al., 2007). The residues of hydrophobic amino acid, such as Val and Leu, can increase the presence of hydrolysates at the water–lipid 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
⇑ 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: [email protected] (D. Pan). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.07.047
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 profiles 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 effective source of protein. It also may be a potential source of antioxidants for human consumption, since chicken protein offers a well-balanced amino acid composition combined with the abundant 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 activities and malondialdehyde (MDA) levels were assayed in vivo, using purified 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 purification were also analyzed to determine the relationships between their antioxidant capacities.
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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-diphenyl-2-picrylhydrazyl (DPPH) and D-galactose were obtained from Sigma–Aldrich (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-substrateprotein 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 filtering 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 volume 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 centrifuged (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 prepared by using distilled water instead of the sample. Increased reducing power was indicated by the increased absorbance of the reaction solution. In addition, RP0.5AU was defined 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 activityð%Þ ¼ 100 ½1 ðAsample Ablank Þ=Acontrol While sample is prepared by mixing 2 mL of sample solution with DPPH solution, 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 obtained 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 experiments 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 given 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 sacrificed 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 ice–cold 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 number: 20110709, Nanjing Jiancheng Bioengineering Institute). In brief, CAT activity was measured at 405 nm due to the yellow H2O2-ammonium molybdate complex. SOD activity was measured by determining its absorbance at 550 nm, because the superoxide radicals generated in the xanthine–xanthine 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 activities of CAT, SOD and 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 expressed 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 mm3 specimens and fixed in 4% (v/v) glutaraldehyde at 4 °C for 2 h. After rinsing in 0.2 M phosphate buffer (pH 7.4) four times (15 min/step), specimens were fixed 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; (50–70–90–100)% (v/v)]. Each specimen was treated with an acetone/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 (Leica 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. Purification of the chicken breast protein hydrolysate 2.5.1. Ultrafiltration The hydrolysates were fractionated at an operating temperature of 25 °C and at a pressure of 0.1 MPa using ultrafiltration membranes with a molecular weight cutoff of 5 kDa MWCO (Shanghai Mosutech., China). The obtained fractions were lyophilized and kept at 20 °C for further use.
2.5.2. Gel filtration chromatography The fraction (100 mg) which exhibited the highest antioxidant activity was dissolved with 2 mL of distilled water. The obtained solution was separated using Sephadex G-25 and a gel filtration column (1.6 cm 50 cm) that was eluted with distilled water. The flow 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 purification 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 (0–40% in 40 min) containing 0.1% (v/ v) trifluoroacetic acid (TFA) with a flow 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% trifluoroacetic acid (TFA) in acetonitrile; and (B) 0.1% TFA in water. Gradient elution was as follows: 0–10 min, linear gradient 98–95% B; 10–15 min, isocratic gradient 95% B; the flow rate: 0.5 mL/min; the detection wavelength 215 nm.
Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 3397–3404 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-fluorenylmethyl chloroformate (FMOC). The measurement was performed at 40 °C and at a flow 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 significant difference was determined by ANOVA with a 95% confidence 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 Fe3+/ferricyanide complex to ferrous form was measured in the presence of the chicken protein hydrolysates. The Fe2+ complex can be detected by determining the formation of Perl’s Prussian blue at an absorbance of 700 nm. The potential antioxidant 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 interrupting the free-radical chain reactions. That is to say, the hydrolysates 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 (r2 = 0.9948). Moure et al. (2006) reported that the reducing power exhibited an obvious effect on both protein size and concentration, with the latter playing an important role in the fractions that had a smaller molecular weight. Several works have indicated that increasing the concentration of samples can increase reducing power (Xie et al., 2008; Zhu et al., 2006). The reducing power (RP0.5AU) 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 protein hydrolysate displays significant 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 (Ardestani and Yazdanparast, 2007).
Fig. 1. Reducing power of different concentrations of chicken breast protein hydrolysate. Each value is expressed as mean ± S.D. (n = 9).
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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 activity, is a relatively stable free radical. The color of its ethanolic solution changes from modena to yellow when free radicals are scavenged and its absorbance gradually decreases (Xie et al., 2008). Therefore, a substance’s 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 concentrations. The chicken protein hydrolysate showed a concentrationdependent increase in antioxidative activity for concentrations up to 3 mg/mL. DPPH radical scavenging activity increased with increasing hydrolysate concentrations until about 90%, and thereafter 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 EC50 (the concentration that scavenged 50% of the initial DPPH radicals) can be expressed to determine the efficiency of an antiradical; the lower the EC50, the higher the free radical scavenging ability. The regression equation of the scavenging activity was calculated and the EC50 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 revealed 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 antioxidant. 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 enough to promote lipid oxidation (Elias et al., 2008). Protein hydrolysates have good antioxidant activity, and it is pivotal to elucidate how the composition of protein hydrolysate influences 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 abilities. 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 significantly (P < 0.05)
Fig. 2. DPPH radical scavenging activity of different concentrations of chicken breast protein hydrolysate. Each value is expressed as mean ± S.D. (n = 9).
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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 II III IV V VI
322.13 ± 27.16 255.61 ± 65.04 293.30 ± 46.64 264.47 ± 47.11 331.81 ± 49.38** 338.64 ± 29.13**
58.49 ± 6.02 47.98 ± 6.32 54.36 ± 9.30* 57.00 ± 7.43** 60.75 ± 6.09** 57.56 ± 6.41**
323.43 ± 19.36 288.14 ± 24.24 335.14 ± 26.04** 360.72 ± 25.04** 377.14 ± 24.72** 376.43 ± 17.56**
3.74 ± 0.58 4.17 ± 0.63 3.76 ± 0.33* 3.75 ± 0.31* 3.59 ± 0.20** 3.73 ± 0.36*
Data were expressed as means ± SD (n = 10) and evaluated by one-way ANOVA. Differences were considered to be statistically significant 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 II III IV V VI
16.74 ± 0.93 15.84 ± 0.93 16.37 ± 0.25 16.97 ± 0.43** 17.43 ± 0.92** 17.88 ± 0.36**
47.04 ± 4.03 41.19 ± 3.10 45.60 ± 2.58* 43.64 ± 5.51 48.45 ± 1.65** 50.11 ± 2.32**
103.59 ± 10.06 93.96 ± 9.00 102.57 ± 8.11* 106.93 ± 6.84** 113.24 ± 7.92** 111.45 ± 6.38**
1.24 ± 0.18 1.55 ± 0.18 1.26 ± 0.26** 1.27 ± 0.26** 1.06 ± 0.09** 1.24 ± 0.13**
Data were expressed as means ± SD (n = 10) and evaluated by one-way ANOVA. Differences were considered to be statistically significant if P < 0.05. * P < 0.05, compared with the model group. ** P < 0.01, compared with the model group.
decreased SOD, CAT and GSH-Px activities, and increased MDA levels compared with the normal group both in serums and livers. These findings indicate that the D-galactose induced aging mice model was successful. Peptides (125, 250 and 500 mg/kg) significantly (P < 0.05) increased enzymatic activities compared with the D-galactose induced aging mice group, while MDA levels decreased significantly (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 significantly differ (P > 0.05). The enzymatic activities and the MDA levels of the positive control group were significantly (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 significant 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 demonstrated that aging, as a result of diminished antioxidant protection, is associated with a reduction in antioxidants and incremental increases in lipid peroxidation (Katrin et al., 2006). The major antioxidant enzymes, acting as the first line of defense against antioxidants, 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 defense of active oxygen compounds (Li et al., 2007; Pan and Mei, 2010). Meanwhile, MDA is a cytotoxic product of lipid peroxidation. 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 Dgalactose induced aging mice, while the MDA levels both in serums and livers significantly increased. In vitro, the peptide concentration can affect antioxidant activities as measured by an assay, while antioxidant activities in vivo may be influenced by metabolism, digestibility and the bioavailability 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 oxidative-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 membranes and cells were clear, and the nuclear pores were clearly observed. 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 because the organelles merged with each other. The rough endoplasmic 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 membranes were obscured and the nucleoli were loose. The cytoplasm was homogeneous since the organelles merged with each other. The rough endoplasmic reticula disappeared. Numerous mitochondria were found in the cytoplasm. Little difference was found between 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 reticulum. 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 inhibiting the oxidative stress of hepatocytes in vivo. 3.4. Purification of chicken protein hydrolysate The protein content of chicken protein hydrolysate (lyophilizate) 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
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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. Magnification: 10000.
Fig. 4. Elution profile of chicken breast protein hydrolysate separated by gel filtration on Sephadex G-25.
until use. It was isolated using Sephadex G-25 and three peaks were obtained: F1, F2 and F3 (Fig. 4). Gel filtration was applied to isolate the protein hydrolysates according to their molecular weight (You et al., 2010b). All peaks were collected, lyophilized and the antioxidant activities 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 scavenging 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 RPHPLC on a ZorBax 300SB-C18 column with a linear gradient of acetonitrile (0–40%). The desired peak was purified on an analytical HPLC column (Fig. 5).
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×10 7
Active peak
6
Abs. 215nm
5 4 3 2 1 0 1
2
3
4
5
6
7
8
9
10
11
Time (min) Fig. 5. RP-HPLC on a ZorBax EclipseXDB-C18 analytical column. Flow rate: 0.5 mL/min. Detection wavelength: 215 nm.
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 filtration. 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 filtration seemed to greatly influence 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 alanine. However, the amounts of free amino acids, including arginine and leucine, seemed to increase after filtration, 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 hydrophobic amino acids facilitate interaction with free radicals by increasing 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 containing 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 significant amount of antioxidant amino acids found in it. In summary, the amino acid composition of a hydrolysate seems to have a direct influence on its antioxidative properties. However, further detailed studies focused on how the amino acid composition influences the antioxidant activity of a hydrolysate are needed.
4. Conclusion In the present study, chicken breast protein was hydrolysed by papain and the hydrolysate’s antioxidant activities were investigated 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
Table 3 Comparative free amino acid profiles of hydrolysate and peptide fractions separated by G-25 (g/100 g protein). Amino acid
Hydrolysate
Fraction I
Fraction II
Fraction III
Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Alanine Arginine Tryosine Cystine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Total essential free amino acids Total hydrophobic free amino acids
0.901 2.176 0.824 0.531 0.467 0.836 0.876 3.863 1.137 0.239 1.152 1.143 1.363 1.306 2.958 1.617 10.374 9.059
1.257 2.704 1.677 0.659 0.985 1.739 1.265 7.552 0.188 0.482 2.351 2.436 1.583 2.481 6.286 2.589 19.464 15.324
0.031 0.317 0.549 1.238 0.501 0.523 0.325 1.316 4.290 0.216 0.589 1.077 16.031 0.587 1.354 0.481 20.641 23.928
0.000 0.080 0.074 0.882 0.107 0.047 0.200 0.368 0.344 0.000 0.072 0.086 0.249 0.097 0.239 0.224 1.014 1.087
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Chicken breast meat
Hydrolysate
Fraction I
Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Alanine Arginine Tryosine Cystine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Proline Total essential amino acids Total hydrophobic amino acids Total amino acids
1.533 2.881 0.770 0.735 0.735 0.714 0.959 0.791 0.662 0.060 0.973 0.483 0.655 0.840 1.638 1.915 0.868 7.218 6.119 17.212
8.693 14.368 2.653 5.769 3.816 4.696 5.422 6.086 2.677 0.233 5.048 2.505 3.299 4.284 7.625 8.311 2.589 35.768 28.027 88.074
7.079 11.880 2.255 6.788 3.177 5.643 5.839 5.608 1.711 0.466 5.250 2.659 3.496 4.036 7.884 6.804 2.387 35.772 27.423 82.962
hydrolysate significantly increased the activity of antioxidant enzymes, while decreasing MDA levels both in serums and livers. Under 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 antioxidative peptides, further research should be conducted to identify the amino acid sequences of the individual antioxidative peptides. In addition, further research is required to evaluate the cytotoxicity of the specific peptides.
Conflict of Interest The authors declare that there are no conflict of interest. Acknowledgements This work was supported by the Modern Agricultural Technical System Foundation of China (CARS-43-17), Natural Science Program of China (No. 30972130 and 31101309), and the High School Natural Science Program of Jiangsu Province (No. 10KJB550003). References Anthony, F., Edmond, R., Christian, R., 2008. Is the in vitro antioxidant potential of whole-grain cereals and cereal products well reflected in vivo? J. Cereal Sci. 48, 258–276. Ardestani, A., Yazdanparast, R., 2007. Antioxidant and free radical scavenging potential of Achillea santolina extracts. Food Chem. 104, 21–29. Bougatef, A., Arroume, N.N., Manni, L., Ravallec, R., Barkia, A., Guillochon, D., Nasri, M., 2010. Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins. Food Chem. 118, 559–565. Chen, Y., Huang, B., He, J.S., Han, L., Zhan, Y.C., Wang, Y.W., 2011. In vitro and in vivo antioxidant effects of the ethanolic extract of Swertia chirayita. J. Ethnopharmacol. 136, 309–315. Cui, C., Zhou, X.S., Zhao, M.M., Yang, B., 2009. Effect of thermal treatment on the enzymatic hydrolysis of chicken proteins. Innov. Food Sci. Emerg. Technol. 10, 37–41.
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Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Purification 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 b
Food Science and Nutrition Department, Nanjing Normal University, Nanjing 210097, PR China 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 measurements, including reducing power and DPPH radical scavenging assays. The reducing power of the hydrolysate was 0.5 at 2.37 mg/mL. The DPPH radical scavenging assay showed that the EC50 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 significantly increased antioxidant enzyme activities, while malondialdehyde levels decreased both in serums and livers. Under a transmission 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 significant antioxidant activity. Ó 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 diseases (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 (Thiansilakul et al., 2007). The residues of hydrophobic amino acid, such as Val and Leu, can increase the presence of hydrolysates at the water–lipid 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
⇑ 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: [email protected] (D. Pan). 0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2012.07.047
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 profiles 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 effective source of protein. It also may be a potential source of antioxidants for human consumption, since chicken protein offers a well-balanced amino acid composition combined with the abundant 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 activities and malondialdehyde (MDA) levels were assayed in vivo, using purified 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 purification were also analyzed to determine the relationships between their antioxidant capacities.
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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-diphenyl-2-picrylhydrazyl (DPPH) and D-galactose were obtained from Sigma–Aldrich (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-substrateprotein 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 filtering 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 volume 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 centrifuged (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 prepared by using distilled water instead of the sample. Increased reducing power was indicated by the increased absorbance of the reaction solution. In addition, RP0.5AU was defined 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 activityð%Þ ¼ 100 ½1 ðAsample Ablank Þ=Acontrol While sample is prepared by mixing 2 mL of sample solution with DPPH solution, 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 obtained 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 experiments 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 given 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 sacrificed 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 ice–cold 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 number: 20110709, Nanjing Jiancheng Bioengineering Institute). In brief, CAT activity was measured at 405 nm due to the yellow H2O2-ammonium molybdate complex. SOD activity was measured by determining its absorbance at 550 nm, because the superoxide radicals generated in the xanthine–xanthine 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 activities of CAT, SOD and 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 expressed 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 mm3 specimens and fixed in 4% (v/v) glutaraldehyde at 4 °C for 2 h. After rinsing in 0.2 M phosphate buffer (pH 7.4) four times (15 min/step), specimens were fixed 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; (50–70–90–100)% (v/v)]. Each specimen was treated with an acetone/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 (Leica 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. Purification of the chicken breast protein hydrolysate 2.5.1. Ultrafiltration The hydrolysates were fractionated at an operating temperature of 25 °C and at a pressure of 0.1 MPa using ultrafiltration membranes with a molecular weight cutoff of 5 kDa MWCO (Shanghai Mosutech., China). The obtained fractions were lyophilized and kept at 20 °C for further use.
2.5.2. Gel filtration chromatography The fraction (100 mg) which exhibited the highest antioxidant activity was dissolved with 2 mL of distilled water. The obtained solution was separated using Sephadex G-25 and a gel filtration column (1.6 cm 50 cm) that was eluted with distilled water. The flow 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 purification 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 (0–40% in 40 min) containing 0.1% (v/ v) trifluoroacetic acid (TFA) with a flow 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% trifluoroacetic acid (TFA) in acetonitrile; and (B) 0.1% TFA in water. Gradient elution was as follows: 0–10 min, linear gradient 98–95% B; 10–15 min, isocratic gradient 95% B; the flow rate: 0.5 mL/min; the detection wavelength 215 nm.
Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 3397–3404 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-fluorenylmethyl chloroformate (FMOC). The measurement was performed at 40 °C and at a flow 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 significant difference was determined by ANOVA with a 95% confidence 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 Fe3+/ferricyanide complex to ferrous form was measured in the presence of the chicken protein hydrolysates. The Fe2+ complex can be detected by determining the formation of Perl’s Prussian blue at an absorbance of 700 nm. The potential antioxidant 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 interrupting the free-radical chain reactions. That is to say, the hydrolysates 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 (r2 = 0.9948). Moure et al. (2006) reported that the reducing power exhibited an obvious effect on both protein size and concentration, with the latter playing an important role in the fractions that had a smaller molecular weight. Several works have indicated that increasing the concentration of samples can increase reducing power (Xie et al., 2008; Zhu et al., 2006). The reducing power (RP0.5AU) 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 protein hydrolysate displays significant 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 (Ardestani and Yazdanparast, 2007).
Fig. 1. Reducing power of different concentrations of chicken breast protein hydrolysate. Each value is expressed as mean ± S.D. (n = 9).
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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 activity, is a relatively stable free radical. The color of its ethanolic solution changes from modena to yellow when free radicals are scavenged and its absorbance gradually decreases (Xie et al., 2008). Therefore, a substance’s 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 concentrations. The chicken protein hydrolysate showed a concentrationdependent increase in antioxidative activity for concentrations up to 3 mg/mL. DPPH radical scavenging activity increased with increasing hydrolysate concentrations until about 90%, and thereafter 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 EC50 (the concentration that scavenged 50% of the initial DPPH radicals) can be expressed to determine the efficiency of an antiradical; the lower the EC50, the higher the free radical scavenging ability. The regression equation of the scavenging activity was calculated and the EC50 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 revealed 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 antioxidant. 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 enough to promote lipid oxidation (Elias et al., 2008). Protein hydrolysates have good antioxidant activity, and it is pivotal to elucidate how the composition of protein hydrolysate influences 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 abilities. 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 significantly (P < 0.05)
Fig. 2. DPPH radical scavenging activity of different concentrations of chicken breast protein hydrolysate. Each value is expressed as mean ± S.D. (n = 9).
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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 II III IV V VI
322.13 ± 27.16 255.61 ± 65.04 293.30 ± 46.64 264.47 ± 47.11 331.81 ± 49.38** 338.64 ± 29.13**
58.49 ± 6.02 47.98 ± 6.32 54.36 ± 9.30* 57.00 ± 7.43** 60.75 ± 6.09** 57.56 ± 6.41**
323.43 ± 19.36 288.14 ± 24.24 335.14 ± 26.04** 360.72 ± 25.04** 377.14 ± 24.72** 376.43 ± 17.56**
3.74 ± 0.58 4.17 ± 0.63 3.76 ± 0.33* 3.75 ± 0.31* 3.59 ± 0.20** 3.73 ± 0.36*
Data were expressed as means ± SD (n = 10) and evaluated by one-way ANOVA. Differences were considered to be statistically significant 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 II III IV V VI
16.74 ± 0.93 15.84 ± 0.93 16.37 ± 0.25 16.97 ± 0.43** 17.43 ± 0.92** 17.88 ± 0.36**
47.04 ± 4.03 41.19 ± 3.10 45.60 ± 2.58* 43.64 ± 5.51 48.45 ± 1.65** 50.11 ± 2.32**
103.59 ± 10.06 93.96 ± 9.00 102.57 ± 8.11* 106.93 ± 6.84** 113.24 ± 7.92** 111.45 ± 6.38**
1.24 ± 0.18 1.55 ± 0.18 1.26 ± 0.26** 1.27 ± 0.26** 1.06 ± 0.09** 1.24 ± 0.13**
Data were expressed as means ± SD (n = 10) and evaluated by one-way ANOVA. Differences were considered to be statistically significant if P < 0.05. * P < 0.05, compared with the model group. ** P < 0.01, compared with the model group.
decreased SOD, CAT and GSH-Px activities, and increased MDA levels compared with the normal group both in serums and livers. These findings indicate that the D-galactose induced aging mice model was successful. Peptides (125, 250 and 500 mg/kg) significantly (P < 0.05) increased enzymatic activities compared with the D-galactose induced aging mice group, while MDA levels decreased significantly (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 significantly differ (P > 0.05). The enzymatic activities and the MDA levels of the positive control group were significantly (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 significant 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 demonstrated that aging, as a result of diminished antioxidant protection, is associated with a reduction in antioxidants and incremental increases in lipid peroxidation (Katrin et al., 2006). The major antioxidant enzymes, acting as the first line of defense against antioxidants, 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 defense of active oxygen compounds (Li et al., 2007; Pan and Mei, 2010). Meanwhile, MDA is a cytotoxic product of lipid peroxidation. 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 Dgalactose induced aging mice, while the MDA levels both in serums and livers significantly increased. In vitro, the peptide concentration can affect antioxidant activities as measured by an assay, while antioxidant activities in vivo may be influenced by metabolism, digestibility and the bioavailability 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 oxidative-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 membranes and cells were clear, and the nuclear pores were clearly observed. 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 because the organelles merged with each other. The rough endoplasmic 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 membranes were obscured and the nucleoli were loose. The cytoplasm was homogeneous since the organelles merged with each other. The rough endoplasmic reticula disappeared. Numerous mitochondria were found in the cytoplasm. Little difference was found between 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 reticulum. 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 inhibiting the oxidative stress of hepatocytes in vivo. 3.4. Purification of chicken protein hydrolysate The protein content of chicken protein hydrolysate (lyophilizate) 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
Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 3397–3404
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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. Magnification: 10000.
Fig. 4. Elution profile of chicken breast protein hydrolysate separated by gel filtration on Sephadex G-25.
until use. It was isolated using Sephadex G-25 and three peaks were obtained: F1, F2 and F3 (Fig. 4). Gel filtration was applied to isolate the protein hydrolysates according to their molecular weight (You et al., 2010b). All peaks were collected, lyophilized and the antioxidant activities 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 scavenging 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 RPHPLC on a ZorBax 300SB-C18 column with a linear gradient of acetonitrile (0–40%). The desired peak was purified on an analytical HPLC column (Fig. 5).
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×10 7
Active peak
6
Abs. 215nm
5 4 3 2 1 0 1
2
3
4
5
6
7
8
9
10
11
Time (min) Fig. 5. RP-HPLC on a ZorBax EclipseXDB-C18 analytical column. Flow rate: 0.5 mL/min. Detection wavelength: 215 nm.
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 filtration. 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 filtration seemed to greatly influence 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 alanine. However, the amounts of free amino acids, including arginine and leucine, seemed to increase after filtration, 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 hydrophobic amino acids facilitate interaction with free radicals by increasing 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 containing 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 significant amount of antioxidant amino acids found in it. In summary, the amino acid composition of a hydrolysate seems to have a direct influence on its antioxidative properties. However, further detailed studies focused on how the amino acid composition influences the antioxidant activity of a hydrolysate are needed.
4. Conclusion In the present study, chicken breast protein was hydrolysed by papain and the hydrolysate’s antioxidant activities were investigated 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
Table 3 Comparative free amino acid profiles of hydrolysate and peptide fractions separated by G-25 (g/100 g protein). Amino acid
Hydrolysate
Fraction I
Fraction II
Fraction III
Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Alanine Arginine Tryosine Cystine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Total essential free amino acids Total hydrophobic free amino acids
0.901 2.176 0.824 0.531 0.467 0.836 0.876 3.863 1.137 0.239 1.152 1.143 1.363 1.306 2.958 1.617 10.374 9.059
1.257 2.704 1.677 0.659 0.985 1.739 1.265 7.552 0.188 0.482 2.351 2.436 1.583 2.481 6.286 2.589 19.464 15.324
0.031 0.317 0.549 1.238 0.501 0.523 0.325 1.316 4.290 0.216 0.589 1.077 16.031 0.587 1.354 0.481 20.641 23.928
0.000 0.080 0.074 0.882 0.107 0.047 0.200 0.368 0.344 0.000 0.072 0.086 0.249 0.097 0.239 0.224 1.014 1.087
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Y. Sun et al. / Food and Chemical Toxicology 50 (2012) 3397–3404 Table 4 Comparative total amino acid profiles of chicken breast meat, its protein hydrolysate and Fraction I separated by G-25 (g/100 g protein). Amino acid
Chicken breast meat
Hydrolysate
Fraction I
Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Alanine Arginine Tryosine Cystine Valine Methionine Phenylalanine Isoleucine Leucine Lysine Proline Total essential amino acids Total hydrophobic amino acids Total amino acids
1.533 2.881 0.770 0.735 0.735 0.714 0.959 0.791 0.662 0.060 0.973 0.483 0.655 0.840 1.638 1.915 0.868 7.218 6.119 17.212
8.693 14.368 2.653 5.769 3.816 4.696 5.422 6.086 2.677 0.233 5.048 2.505 3.299 4.284 7.625 8.311 2.589 35.768 28.027 88.074
7.079 11.880 2.255 6.788 3.177 5.643 5.839 5.608 1.711 0.466 5.250 2.659 3.496 4.036 7.884 6.804 2.387 35.772 27.423 82.962
hydrolysate significantly increased the activity of antioxidant enzymes, while decreasing MDA levels both in serums and livers. Under 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 antioxidative peptides, further research should be conducted to identify the amino acid sequences of the individual antioxidative peptides. In addition, further research is required to evaluate the cytotoxicity of the specific peptides.
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