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Characterization and in vitro antioxidation of papain hydrolysate from black-bone silky fowl (Gallus gallus domesticus Brisson) muscle and its fractions
Food Research International 44 (2011) 133–138
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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Characterization and in vitro antioxidation of papain hydrolysate from black-bone silky fowl (Gallus gallus domesticus Brisson) muscle and its fractions Jian-Hua Liu, Ying-Gang Tian, Yong Wang, Shao-Ping Nie, Ming-Yong Xie ⁎, Sheng Zhu, Chun-Yan Wang, Pan Zhang State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
a r t i c l e
i n f o
Article history: Received 24 July 2010 Accepted 31 October 2010 Keywords: Black-bone silky fowl (Gallus gallus domesticus Brisson) High-performance liquid chromatography Antioxidant activity Amino acid composition Molecular weight distribution
a b s t r a c t Black-bone silky fowl (Gallus gallus domesticus Brisson) (BSF) muscle was hydrolyzed by papain, and the hydrolysate was separated by preparative high performance liquid chromatography (HPLC). The amino acid composition of the BSF hydrolysate (BSFH) and its fractions was determined by HPLC precolumn derivation with 2,4-dinitrofluorobenzene. The molecular weight (MW) distribution of the BSFH and its fractions was measured by a peptide column on an HPLC system. Antioxidant activities of the BSFH and its fractions were studied by testing the reducing power and four radical scavenging systems: superoxide anion (O2•−), hydroxyl (·OH), 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radicals. The results demonstrated that the BSFH had strong antioxidant capacity to scavenge O2•−, DPPH• and ABTS•+, and displayed strong reducing power, but revealed less powerful ability to scavenge ·OH. Fraction II of the BSFH exhibited the highest activity in scavenging O2•− and DPPH•, and reducing power, whereas fraction I displayed the strongest ·OH scavenging ability. Besides Glu, Asp and Gly, the rich amino acids of Ala and Leu played an important role in antioxidant activity. The small-size peptides with MW ranging from approximately 200–6000 Da probably contributed to higher antioxidant activity. Results from this study indicated that BSFH and its fractions could be used as food additives and diet nutrients. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Free radical-mediated oxidation and antioxidants are receiving more and more attention in many current research areas. In the ground state, the oxygen molecule is stable until exposed to environmental pollutants, radiation, UV, etc. At this time, reactive oxygen species (ROS) and free radicals are easily formed, which can induce oxidative damage to biomacromolecules, including DNA, proteins, membrane lipids and carbohydrates (Wiseman & Halliwell, 1996; Lai & Piette, 1977; Kellogg & Fridovich, 1975). ROS and free radicals are reported to be involved in the occurrence of numerous diseases, such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging (Halliwell, 1991; Pryor, 1982). In food industry, lipid or fatty acid oxidation has been receiving great concerns because it can result in quality deterioration (development of undesirable off-flavor, discoloration, nutrition loss, formation of toxins), reducing the shelf-life of food products. To retard lipid or fatty acid oxidation, various antioxidants with a strong antioxidant capacity have been developed. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), can be used to preserve food containing fats and oils. However, because of their
⁎ Corresponding author. Tel./fax: +86 791 3969009. E-mail address: [email protected] (M.-Y. Xie). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.10.050
potential health risks and negative consumer perception (Yu et al., 2002), natural antioxidants have become appealing alternatives and are in great demand in the food industry. For example, tocopherols, ascorbate, flavonoids, carotenoids and phenolic compounds from plants are the most commonly used natural antioxidants in the processed food (Pokorný, 1991). In recent years, it has been recognized that dietary proteins provide a rich source of naturally occurring antioxidants. These proteins are of wide distribution in plants, animals and fungi, such as soybean protein (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998), maize zein (Zhu, Chen, Tang, & Xiong, 2008), potato protein (Wang & Xiong, 2005), whey protein (Hernández-Ledesma, Dávalos, Bartolomé, & Amigo, 2005), grass carp muscle protein (Ren et al., 2008) and fermented mushroom protein (Sun, He, & Xie, 2004). Protein hydrolysates and peptides exhibit strong antioxidant efficiency in both model and in situ systems, including radical scavenging, reducing, and metal ion chelating activity (Elias, Kellerby, & Decker, 2008). Black-bone silky fowl (Gallus gallus domesticus Brisson) (BSF), black in skin, meat, bones, and white in feathers, is a unique breed of chicken in China. It has been well known for its health functions, such as treating diabetes and anemia, curing women's diseases like menoxenia and postpartum complications. With the expansion of BSF breeding, effective development and utilization of BSF are emergent. Carnosine is a natural antioxidative di-peptide, present in
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a remarkably high content within BSF (Tian et al., 2007). However, little has been known about different antioxidant properties of hydrolysates and peptides derived from BSF muscle. Because of antioxidant peptides production ability (Wang, Zhao, Zhao, & Jiang, 2007), easy availability and low commercial price, papain was used to hydrolyze BSF muscle. The antioxidant activities assessment can be achieved by means of determining their radical scavenging activity and reducing power. In the present study, the antioxidant properties of papain hydrolysates from BSF and its fractions separated by preparative HPLC were investigated in four radical scavenging systems (O2•−, ·OH, DPPH• and ABTS•+) and reducing power using carnosine and ascorbate as comparison. In addition, the MW distribution and amino acid composition of the BSFH and its fractions were studied. 2. Materials and methods 2.1. Materials and reagents Black-bone silky fowl (75–90 days old, half male and half female) were provided by the Taihe Original Black-Bone Silky Fowl Hennery (Jiangxi, China). Papain (320,000 U/g) was purchased from Beijing Fangshan Enzyme Factory (Beijing, China). Cytochrome c, 1,1diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reduced and oxidized glutathione and 17 amino acid standards (Asp, Glu, Ser, Arg, Gly, Thr, Pro, Ala, Val, Met, Cys, Ile, Leu, Phe, His, Lys, Tyr) were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Acetonitrile was of chromatographic grade. Other reagents used in this study were of analytical grade.
(0.1, 1, 10 and 50 mg/mL for the BSFH; 1 mg/mL for each fraction) was mixed with 1.8 mL of 50 mmol/L Tris–HCl buffer (pH 8.2). The mixture was incubated at 25 °C for 10 min, and then 0.1 mL of 10 mmol/L pyrogallol (dissolved in 10 mmol/L HCl) was added. The absorbance of the solution at 320 nm was measured up to 4 min. The oxidation rate of pyrogallol for samples was calculated as the change of the absorbance (ΔA1). The autoxidation rate of pyrogallol for control was measured with 1.0 mL of ultrapure water (ΔA0). For comparison, the O2•− scavenging activity of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) was also tested. The O2•− scavenging activity was calculated as [(ΔA0 − ΔA1) / ΔA0] × 100%. 2.5. Hydroxyl radical (·OH) scavenging activity The ·OH scavenging assay of the BSFH and its fractions were carried out using the method described by Li, Jiang, Zhang, Mu, and Liu (2008) with some modifications. The sample tubes were filled out with the following solutions accordingly: 1 mL of 0.4 mol/L phosphate buffer (pH 7.4), 1 mL of 2.5 mmol/L 1,10-phenanthroline, 1 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction), 1 mL of 2.5 mmol/L FeSO4 and 0.5 mL of H2O2 (1%). The mixture was incubated at 37 °C for 1 h, and the absorbance was measured at 536 nm. The ·OH scavenging assay of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The ·OH scavenging activity was calculated as [(A2 – A0) / (A1 – A0)] × 100%, where A2 is the absorbance of the sample; A0 is the absorbance of the blank solution using ultrapure water (1 mL) instead of sample solution (1 mL); and A1 is the absorbance of the control solution using ultrapure water (1.5 mL) instead of sample solution (1 mL) and H2O2 (0.5 mL).
2.2. Preparation of BSFH
2.6. DPPH radical (DPPH•) scavenging activity
The enzymatic hydrolysis method optimized by uniform design was used to prepare BSFH. Briefly, BSF muscle (1/6, w/v) was hydrolyzed with papain (3‰, w/v) at 55 °C and pH 7.0 for 1 h to achieve a strong antioxidant activity. The enzyme was inactivated by heating at 90 °C for 20 min. The resulting BSFH solution was centrifuged at 3000×g for 10 min. The lipids and melanin were removed by chloroform-methanol (2:1, v/v) extraction and centrifugation. The supernatant was subjected to vacuum drying at 60 °C, sealed in plastic bags, and stored in a desiccator until use.
The DPPH• scavenging activity of the BSFH and its fractions was measured by using the procedure described by Chen, Xie, Nie, Li, and Wang (2008). The 0.2-mmol/L solution of DPPH in 95% ethanol was prepared daily before UV measurements. 1 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was thoroughly mixed with 2 mL of freshly prepared DPPH and 2 mL of 95% ethanol. The mixture was shaken vigorously and allowed to stand for 30 min in the dark, and then the absorbance was measured at 517 nm against a blank. The DPPH• scavenging activity of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The DPPH• scavenging activity was calculated as [1 − (A2 − A1) / A0] × 100%, where A0 is the absorbance of DPPH solution without sample (2 mL DPPH + 3 mL 95% ethanol), A2 is the absorbance of the sample mixed with DPPH solution (1 mL sample + 2 mL DPPH + 2 mL 95% ethanol) and A1 is the absorbance of the sample without DPPH solution (1 mL of sample + 4 mL 95% ethanol).
2.3. Preparative HPLC separation The separation of BSFH was performed on a Waters DeltaPrep 400 preparative chromatography system equipped with Waters Prep LC controller and Waters 2487 dual λ absorbance detector (Waters, Milford, MA, USA). The preparative HPLC was performed on a Bondapak C18 preparative column (300 × 30 mm I.D., 10 μm). For the simple and fast separation, ultrapure water was selected as the mobile phase. The BSFH concentration was 50 mg/mL. The hydrolysate solution was filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 5 mL. The flow rate was 25 mL/ min, and the detected wavelength was 254 nm. The preparative HPLC equipment was controlled by Waters Empower 2 chromatography data software. The BSFH fractions were manually collected, reduced to 10 mL under reduced pressure rotary evaporation at 50 °C and then, freeze-dried, sealed, and stored in a desiccator before use. 2.4. Superoxide radical (O2•−) scavenging activity The assay of O2•− scavenging activity of the BSFH and its fractions were determined according to the autoxidation of a pyrogallol method described by Marklund and Marklund (1974). Briefly, 1.0 mL of samples
2.7. ABTS radical cation (ABTS•+) scavenging activity The ABTS•+ scavenging activity of the BSFH and its fractions was analyzed by the method described by Re et al. (1999). This method is based on ABTS•+ production by reacting ABTS stock solution (7 mmol/L) with potassium persulfate (2.45 mmol/L, final concentration). The mixture was left in the dark at room temperature for 12 h before use. One milliliter of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was mixed with 4 mL of ABTS•+ solution, and the absorbance was recorded at 734 nm after 6 min. Carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The ABTS•+ scavenging activity was calculated as [(A0 − A1) / A0] × 100%, where A0 is the absorbance of the blank solution without sample and A1 is the absorbance of the sample solution.
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2.8. Reducing power
2.11. Statistical analysis
The reducing power of the BSFH and its fractions was measured by the procedure described by Wu, Chen, and Shiau (2003). Briefly, 2 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was mixed with 2 mL of 0.2 mol/L phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min, and then, 2 mL of 10% TCA was added. The mixtures were centrifuged for 10 min at 3000×g, and 2 mL of the supernatant was mixed with 2 mL of ultrapure water and 0.4 mL of 0.1% FeCl3. After reaction for 10 min, the absorbance of the solution was read at 700 nm. For comparison, carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested.
All the experiments were performed with three independent trials and all the determinations were triplicated. The results were represented as mean ± standard deviation. Analysis of variance (ANOVA) was performed to identify significant differences (P b 0.05).
2.9. Amino acid composition analysis Amino acid composition of the BSFH and its fractions was determined by HPLC precolumn derivation method through amino acid reacting with 2,4-dinitrofluorobenzene (Sanger, 1945). To prepare amino acids solution, 20 mg of samples was hydrolyzed at 110 °C for 24 h with 10 mL of 6 mol/L HCl, and the resulting solution was made up to 50 mL with 0.05 mol/L boric acid buffer (pH 9.0). The brown volumetric flask (50 mL) was filled with the following solutions accordingly: 5 mL of amino acid standard solution (5 mg of each amino acid standard was dissolved into 100 mL of ultrapure water) or amino acids solution, 5 mL of 0.05 mol/L boric acid buffer (pH 9.0) and 5 mL of 2,4-dinitrofluorobenzene. The resulting solution was shaken and sealed up, and then placed into water bath (60 °C) for 1 h to react in the dark. After cooled down to room temperature, the solution was diluted with 0.05 mol/L phosphate buffer (pH 7.0) up to 50 mL. The derivative solutions were separated by a Waters HPLC system (UK6 injector and 515 HPLC pump) equipped with a Kromasil C18 (250 × 4.6 mm I.D., 5 μm) and a Waters 2996 Photodiode Array Detector. The mobile phase A was 50 mmol/L sodium acetate buffer solution (pH 6.8) containing 1% (v/v) N,N-dimethylformamide. The mobile phase B was 50% (v/v) acetonitrile in water. Gradient elution was carried out according to the following procedure: 0–0.3 min, 16% B; 0.3–4 min, 16–31% B; 4–9.5 min, 31–36% B; 9.5–17 min, 36–55% B; 17–28 min, 55–65% B, 28–34 min, 65–100% B, 34–36 min, 100–16% B. The derivative solutions were filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 20 μL. The flow rate was 1 mL/min, and the detected wavelength was 360 nm. The amino acid contents were calculated according to the HPLC peak area ratio between samples and amino acid standards. The results were expressed in terms of milligram of amino acid per gram sample.
3. Results and discussion 3.1. BSFH and its fractions The BSFH was prepared by enzymatic hydrolysis with papain and its fractions were obtained by preparative HPLC separation on C18 column. As shown in Fig. 1, fractions I, II, III and IV were obtained. Of four fractions, fraction II is in the majority (yield), followed by fraction III. The hydrophobicity of the peptides can be predicted by the retention time (Chen et al., 1998; Meek, 1980). Fraction with a long retention time has the weakest hydrophilicity. Therefore, the hydrophilicity of fractions was as follows: fraction I N fraction II N fraction III N fraction IV. RP-HPLC was widely used in separation of proteins and peptides because of its many advantages, such as higher resolution, better repeatability and greater recovery rate. In the present experiment, preparative HPLC with C18 column provided a simple, fast and large-scale preparative approach to fractionate the BSFH.
3.2. O2•− scavenging activity O2•−, a highly toxic free radical, can be easily generated by numerous biological reactions. Because it is potential precursors of highly reactive species, such as ·OH, the development of antioxidants to scavenge this radical is emergent (Kanatt, Chander, & Sharma, 2007). As shown in Table 1, the BSFH exhibited a strong ability to scavenge O2•−. With the increased concentration of BSFH, the scavenging activity against O2•− was elevated. At 10 mg/mL, the O2•− scavenging activity (72.3%) of BSFH was stronger than that of carnosine at 1 mg/mL (55.9%), and was nearly the same to ascorbate at 0.1 mg/mL (77.0%). Using the same method, BSFH showed far more powerful O2•− scavenging activity than zein hydrolysate reported by Tang et al. (2009) at 10 mg/mL (72.3% versus 11.5%). Fraction II revealed the highest activity (25.9%), followed by fraction I (23.8%), which exhibited stronger O2•− scavenging activity than BSFH (22.6%) at the same concentration (P b 0.05).
2.10. Determination of MW distribution The MW distribution of the BSFH and its fractions was evaluated using a Waters HPLC system (UK6 injector and 515 HPLC pump) equipped with a Superdex peptide 10/300 GL column (300 × 10 mm I. D., 13–15 μm) and a Waters 2996 Photodiode Array Detector. According to the column instructions, the choice of eluent was 0.05 mol/L phosphate buffer (pH 7.0) containing 0.15 mol/L NaCl. The concentration of the BSFH and its fractions was 10 mg/mL. The sample solutions were filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 20 μL. The flow rate was 1 mL/ min, and the detected wavelength was 210 nm. Cytochrome c (12384 Da), reduced glutathione (615 Da), oxidized glutathione (310 Da) and glycine (75 Da) were used as the MW markers. In Fig. 2(A), according to the retention time of four MW markers, the regression equation is logMW = −0.265 t + 7.066, in which R2 = 0.9793, MW represented molecular weight, t represented retention time.
Fig. 1. Fractions of the BSFH eluted by C18 column on preparative HPLC system. Four fractions were identified as fraction I, II, III and IV.
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Table 1 Antioxidant activities of the BSFH, fraction I, II, III and IV, carnosine and ascorbate. Antioxidant
BSFH
Fraction I Fraction II Fraction III Fraction IV Carnosine
Ascorbate
Concentration (mg/mL) 50 10 1 0.1 1 1 1 1 10 1 0.1 1 0.1
Scavenging activity (%) O2•−
·OH
DPPH•
ABTS•+
≥100 72.3 ± 2.3 22.6 ± 1.5 0.8 ± 0.3 23.8 ± 0.3 25.9 ± 0.5 17.0 ± 1.3 21.6 ± 0.7 ≥100 55.9 ± 0.3 20.3 ± 1.7 ≥100 77.0 ± 0.7
57.1 ± 0.6 24.7 ± 1.5 12.7 ± 0.7 6.8 ± 0.2 28.5 ± 0.7 10.5 ± 0.5 11.7 ± 0.3 10.7 ± 0.8 89.5 ± 0.9 20.3 ± 0.5 9.4 ± 1.1 96.7 ± 1.5 10.1 ± 1.3
≥100 73.0 ± 2.2 10.0 ± 2.8 2.9 ± 1.2 25.7 ± 1.3 36.7 ± 1.6 10.6 ± 2.4 11.0 ± 1.9 7.4 ± 0.2 6.4 ± 0.3 5.9 ± 0.5 96.6 ± 0.6 94.4 ± 1.1
85.9 ± 0.2 79.5 ± 0.9 18.5 ± 1.3 6.3 ± 0.5 11.0 ± 0.3 15.5 ± 0.3 16.4 ± 0.9 17.3 ± 0.6 59.8 ± 0.7 12.1 ± 0.6 2.4 ± 0.2 97.5 ± 0.2 97.2 ± 0.1
A700 reducing power 2.504 ± 0.100 1.196 ± 0.050 0.237 ± 0.010 0.093 ± 0.003 0.206 ± 0.010 0.731 ± 0.030 0.205 ± 0.010 0.196 ± 0.008 0.097 ± 0.002 0.069 ± 0.002 0.062 ± 0.003 3.221 ± 0.100 1.909 ± 0.070
The results were represent as mean ± standard deviations from three replications (n = 3).
3.3. ·OH scavenging activity
3.5. ABTS•+ scavenging activity
Among ROS, the chemical toxicity of ·OH is the strongest because it is able to easily reacts with biomolecules, such as amino acids, proteins and DNA (Cacciuttoloa, Trinha, Lumpkina, & Rao, 1993). Therefore, the removal of ·OH is probably one of the most effective defenses of a living body against various diseases (Je, Park, & Kim, 2005). Table 1 demonstrated that the BSFH displayed a certain activity to quench ·OH and the effect was concentration dependent. Although ·OH scavenging activity of BSFH was weaker than carnosine and ascorbate, an elevated scavenging activity fraction was found after fractionation. At 1 mg/mL, fraction I showed the strongest ·OH scavenging activity (28.5%) among four fractions, and its activity was significantly stronger than BSFH (12.7%). In the present study, the ·OH scavenging activity of BSFH was weaker than that of cottonseed protein hydrolysate (47.33% at 10 mg/mL) (Gao, Cao, & Li, 2010). Kim, Je, and Kim (2006) reported four fractions from hoki frame protein hydrolysate exhibited extremely strong ·OH scavenging activity (69.18% or more) at 0.5 mg/mL. These different results could be due to the different ·OH scavenging activity assay methods used, different protein sources, and different fractionation methods used.
ABTS•+ is generated by oxidation of ABTS with potassium persulfate (absorption maxima at 734 nm) and is reduced in the presence of hydrogen-donating antioxidants (decolorization) (Re et al., 1999). As shown in Table 1, BSFH showed strong scavenging ability against ABTS•+, and the effect was concentration dependent. The 79.5% of ABTS•+ can be neutralized by BSFH at 10 mg/mL. It was noteworthy that the ABTS•+ scavenging ability of the BSFH was stronger than that of carnosine at 0.1, 1 and 10 mg/mL, respectively. At 1 mg/mL, however, BSFH and its fractions exhibited less than 19% of ABTS•+ scavenging ability. Fraction I displayed the weakest ABTS•+ scavenging ability (11.0%) among four fractions, while the other three fractions showed nearly the same ability (15.5%, 16.4% and 17.3%). Tang et al. (2009) reported that zein hydrolysate revealed 64.3% of ABTS•+ scavenging activity at 1 mg/mL, while its fractions displayed no more than 35% of activity at the same concentration. This was in good agreement with the present study: at 1 mg/mL, the ABTS•+ scavenging activity of the BSFH was stronger than its four fractions (Table 1). Nonetheless, a fraction or peptide with higher ABTS•+ scavenging ability could be predicted after further purification.
3.4. DPPH• scavenging activity DPPH• assay was the simplest and most accurate method to measure the ability of antioxidant to intercept free radicals (SánchezMoreno, 2002). It is an organic nitrogen radical with a UV-vis absorption in the range of 515–520 nm, and its solution color fades upon reduction. DPPH• scavenging activities of BSFH and its four fractions are shown in Table 1. At 10 mg/mL, BSFH displayed 73.0% of DPPH• scavenging activity, while it completely scavenged DPPH• at 50 mg/mL. Fraction II showed the most powerful DPPH• scavenging activity (36.7%), followed by Fraction I (25.7%), and these two fractions exhibited significantly stronger activity than BSFH at 1 mg/ mL (P b 0.05). In recent years, Gao et al. (2010) and Li, Han, and Chen (2008) also reported the DPPH•-scavenging peptides derived from food protein hydrolysates. In our research, as comparison, ascorbate displayed strong activity in scavenging DPPH• (94.4% at 0.1 mg/mL), while carnosine showed weak (7.4% at 10 mg/mL). Although carnosine is a histidine-containing natural antioxidant, its low dispersibility in ethanol solution probably hinder the electron transfer between antioxidants and DPPH•. Our results suggested that the BSFH and its fractions were better natural antioxidants than carnosine in scavenging DPPH•.
3.6. Reducing power The antioxidant activity has been reported to be accompanied by the reducing power (Tanaka, Kuei, Nagashima, & Taguchi, 1988). The results of the reducing power of BSFH and its fractions are also shown in Table 1. The reducing power was expressed as the absorbance value at 700 nm, and the increased absorbance value indicated an increased reducing power. With the increasing concentration from 0.1 mg/mL to 50 mg/mL, the absorbance value also greatly increased from 0.093 to 2.504, indicating enhancement of BSFH reducing power. Thus, the reducing power of BSFH was dose dependent. Fraction II displayed the strongest reducing power among four fractions, and the effect was significantly stronger than BSFH (the absorbance value of 0.731 versus 0.237) at 1 mg/mL. The reducing power of BSFH and fraction II were even stronger than alcalase-treated zein hydrolysates (the absorbance value was no more than 0.8 at 8 mg/mL) (Zhu et al., 2008). For comparison, the reducing power of ascorbate soared from 1.909 to 3.221, while carnosine displayed the reducing power no more than 0.1. It can be concluded that the reducing power of the BSFH was tremendously stronger than carnosine, and the reducing power of 50 mg/mL of the BSFH can surpass the ascorbate reducing power at 0.1 mg/mL. The reducing power determination in the present study was carried out in an aqueous environment. The antioxidative and hydrophilic peptides with good solubility were capable of donating electrons easily, and thus antioxidant activity increased.
J.-H. Liu et al. / Food Research International 44 (2011) 133–138 Table 2 Amino acid composition of the BSFH and its fractions determined by HPLC precolumn derivation with 2,4-dinitrofluorobenzene. “–” denotes this amino acid was not found.
Aspartic acid Glutamic acid Serine Arginine Glycine Threonine Proline Alanine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Histidine Lysine Tyrosine Total
mg amino acid/g BSFH or its fractions BSFH
Fraction I
Fraction II
Fraction III
Fraction IV
71.4 132.7 19.4 61.3 66.7 74.0 33.8 45.4 33.5 31.2 58.9 28.8 50.4 14.6 24.1 71.1 51.6 868.9
58.8 155.0 33.4 36.8 51.2 33.7 16.0 54.2 24.3 13.8 4.1 12.9 34.1 7.0 19.5 46.2 7.5 608.5
59.2 124.3 26.9 45.8 66.4 28.8 33.3 53.6 22.7 12.7 1.6 26.5 49.8 26.4 24.1 38.9 19.1 660.1
66.4 105.0 22.7 50.8 81.8 35.5 54.5 35.5 27.4 10.8 2.0 25.3 41.6 27.2 26.9 40.1 15.3 668.8
77.1 120.1 27.5 109.5 73.2 33.2 59.4 50.1 30.9 – – 30.8 51.9 31.4 30.4 56.6 17.2 799.3
Absorbance at 210 nm (AU)
A
3.7. Amino acid composition The amino acid composition of the BSFH and its fractions was analyzed in order to evaluate the possible effect of the amino acid profile on its antioxidant activity. As shown in Table 2, BSFH contained mainly Glu, Thr, Asp, Lys and Gly. All of these amino acids were hydrophilic and the hydrophobic amino acids were 30.24% in total, indicating that the hydrophilic amino acids in the sequence of the BSFH peptides were mainly responsible for the antioxidant activity. The amino acids of fraction I mainly consist of Glu, Asp, Ala, Gly and Lys; for fraction II, they were mainly Glu, Gly, Asp, Ala, and Leu; for fraction III, they were mainly Glu, Gly, Asp, Pro, and Arg; and for fraction IV, they were mainly Glu, Arg, Asp, Gly, and Pro. Therefore, the strongest ·OH scavenging capacity of fraction I was attributed to abundant Ala and Lys. Fraction II, which exhibited the strongest scavenging capacity against O2•− and DPPH•, and reducing power, contained more Ala and Leu. Although Ala and Leu were highly hydrophobic, they played an important role in antioxidant ability. Kim et al. (2001) reported that Ala and Leu contributed to free radicals scavenging activity. The antioxidant activity of carnosine (Ala-His) is much greater than the individual or combined activity of Ala and His
B 0.90
oxidized glutathione
MW markers
0.75 0.60 reduced glutathione
0.45 0.30
cytochrome C
0.15
glycine
0.00 0
5
10
15
20
25
Absorbance at 210 nm (AU)
Amino acids
0.6
0.4 0.3 0.2 0.1 0.0
30
0
5
1
2
3 4
10
15
20
25
30
Time (min)
D 1.0
Fraction I
No. MW Range Content (Da) (%) 1 5443-255 53.08 2 255-74 35.19 74 11.73 3
0.8 0.6 0.4 0.2
1
2 3
15
20
0.0 0
5
10
25
Absorbance at 210 nm (AU)
C Absorbance at 210 nm (AU)
BSFH
No. MW Range Content (Da) (%) 1 95733-6070 18.60 2 6070-291 39.76 3 291-82 23.44 82 18.20 4
0.5
Time (min)
1.0
Fraction II
No. MW Range Content (Da) (%) 1 5069-210 44.20 2 210-84 14.60 3 84 41.20
0.8 0.6 0.4 0.2 0.0
30
1 0
5
Time (min)
10
15
2 3 20
25
30
Time (min)
F 0.4
Fraction III
No. MW Range Content (Da) (%) 1 10121-223 52.95 2 223-33 29.22 33 17.83 3
0.3
0.2
0.1
1
0.0 0
5
10
15
2 20
Time (min)
3 25
30
Absorbance at 210 nm (AU)
E Absorbance at 210 nm (AU)
137
0.5
Fraction IV
No. MW Range Content (Da) (%) 1 8868-230 58.70 2 230-30 27.92 30 13.38 3
0.4 0.3 0.2 0.1
1
0.0 0
5
10
15
2 3 20
25
30
Time (min)
Fig. 2. MW distribution of the MW markers (A), BSFH (B), fraction I (C), fraction II (D), fraction III (E) and fraction IV (F). (Inset) Relative components (peptides or amino acids) content in BSFH, fraction I, II, III and IV. Components are indicated by numbers.
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(Decker, 1995), suggesting a synergistic effect of Ala. Tang et al. (2009) isolated two radical scavenging peptide, Tyr-Ala and LeuMet-Cys-His, from zein protein. The presence of Ala and Leu played an important role in the antioxidant activity. Fraction III and IV, which were less in Ala and Leu contents, showed weak free radicals scavenging activity and reducing power. The results demonstrated that the abundance of Glu, Asp, Gly, Ala and Leu contributed to the strong antioxidant activity. 3.8. MW distribution The MW distribution was recognized as an important factor to determine the overall antioxidant activity of the hydrolyzed proteins. Li, Jiang et al. (2008) revealed that the peptide fraction from chickpea protein hydrolysate with the MW distribution from 200 to 3000 Da was associated with high antioxidant activity. Wu et al. (2003) claimed that a peptide from mackerel protein hydrolysate with MW of approximately 1400 Da possessed stronger antioxidant activity than higher MW peptides. Fig. 2 showed the MW distribution of MW markers, BSFH and its four fractions. Fig. 2(A) showed the retention time of four MW markers. In Fig. 2(B), BSFH contained mainly smallsize peptides (291–6070 Da), which was determinant components for its antioxidant activity. In Fig. 2(C) and (D), the major components of fraction I and II were also small-size peptides (255–5443 Da and 210–5069 Da, respectively), displaying narrow MW distribution. By contrast, in Fig. 2(E) and (F), the major components of fraction III and IV shared wide MW range (223–10121 Da and 230–8868 Da). Therefore, the considerable small-size peptides were responsible for the strong antioxidant activity of fraction I and II. These results showed that the peptides with MW ranging from approximately 200–6000 Da were probably associated with higher antioxidant activity. 4. Conclusions BSFH was prepared by papain hydrolysis and its four fractions (I, II, III and IV) were successfully obtained by preparative HPLC separation. The BSFH (10 mg/mL) and fraction II (1 mg/mL) exhibited strong antioxidant activities than carnosine in scavenging DPPH• and ABTS•+, and reducing power. Fraction I (1 mg/mL) displayed strong antioxidant activities than carnosine in scavenging ·OH. Glu, Asp, Gly, Ala and Leu played an important role in antioxidant activity. The MW of peptides responsible for higher antioxidant activity probably ranged from 200 to 6000 Da. Results from this study suggested that the antioxidant BSFH and its fractions (especially fraction II) could be used as food additives and diet nutrients. Nonetheless, further purification is needed to identify the individual peptides responsible for the antioxidant activity of the BSFH. Acknowledgements This study was supported financially by the National Natural Science Foundation of China (No. 20862012), the Objective-Oriented Project of State Key Laboratory of Food Science and Technology (SKLFMB-200806), and the Cheung Kong Scholars Program and Innovative Research Team in University (No. IRT0540). References Cacciuttoloa, M. A., Trinha, L., Lumpkina, J. A., & Rao, G. (1993). Hyperoxia induces DNA damage in mammalian cells. Free Radical Biological and Medicine, 14, 267−276. Chen, H. M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998). Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, 46, 49−53. Chen, Y., Xie, M. Y., Nie, S. P., Li, C., & Wang, Y. X. (2008). Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chemistry, 107, 231−241.
Decker, E. A. (1995). The role of phenolics, conjugated linoleic acid, carnosine, and pyrroloquinoline quinone as nonessential dietary antioxidants. Nutrition Reviews, 53, 49−58. Elias, R. J., Kellerby, S. S., & Decker, E. A. (2008). Antioxidant activity of proteins and peptides. Critical Reviews in Food Science and Nutrition, 48, 430−441. Gao, D. D., Cao, Y. S., & Li, H. X. (2010). Antioxidant activity of peptide fractions derived from cottonseed protein hydrolysate. Journal of the Science of Food and Agriculture, 90, 1855−1860. Halliwell, B. (1991). The biological toxicity of free radicals and other reactive oxygen species. In O. I. Aruoma, & B. Halliwell (Eds.), Free radicals and food additives (pp. 23−27). PA: Taylor & Francis Inc. Hernández-Ledesma, B., Dávalos, A., Bartolomé, B., & Amigo, L. (2005). Preparation of antioxidant enzymatic hydrolysates from α-lactalbumin and β-lactoglobulin. Identification of active peptides by HPLC-MS/MS. 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Review: methods used to evaluate the free radical scavenging activity in foods and biological systems. Food Science and Technology International, 8, 121−137. Sanger, F. (1945). The free amino groups of insulin. Biochemical Journal, 39, 507−515. Sun, J., He, H., & Xie, B. J. (2004). Novel antioxidant peptides from fermented mushroom Ganoderma lucidum. Journal of Agricultural and Food Chemistry, 52, 6646−6652. Tanaka, M., Kuei, C. W., Nagashima, Y., & Taguchi, T. (1988). Application of antioxidative maillard reaction products from histidine and glucose to sardine products. Nippon Suisan Gakkashi, 54, 1409−1414. Tang, X. Y., He, Z. Y., Dai, Y. F., Xiong, Y. L., Xie, M. Y., & Chen, J. (2009). Peptide fractionation and free radical scavenging activity of zein hydrolysate. Journal of Agricultural and Food Chemistry, 58, 587−593. Tian, Y. G., Xie, M. Y., Wang, W. Y., Wu, H. J., Fu, Z. H., & Lin, L. (2007). Determination of carnosine in Black-Bone Silky Fowl (Gallus gallus domesticus Brisson) and common chicken by HPLC. European Food Research and Technology, 226, 311−314. Wang, L. L., & Xiong, Y. L. (2005). Inhibition of lipid oxidation in cooked beef patties by hydrolyzed potato protein is related to its reducing and radical scavenging ability. Journal of Agricultural and Food Chemistry, 53, 9186−9192. Wang, J. S., Zhao, M. M., Zhao, Q. Z., & Jiang, Y. M. (2007). Antioxidant properties of papain hydrolysates of wheat gluten in different oxidation systems. Food Chemistry, 101, 1658−1663. Wiseman, H., & Halliwell, B. (1996). Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochemical Journal, 313, 17−29. Wu, H. C., Chen, H. M., & Shiau, C. Y. (2003). Free amino acids and peptides as related to antioxidant properties in protein hydrolysates of mackerel (Scomber austriasicus). Food Research International, 36, 949−957. Yu, L. 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Food Research International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f o o d r e s
Characterization and in vitro antioxidation of papain hydrolysate from black-bone silky fowl (Gallus gallus domesticus Brisson) muscle and its fractions Jian-Hua Liu, Ying-Gang Tian, Yong Wang, Shao-Ping Nie, Ming-Yong Xie ⁎, Sheng Zhu, Chun-Yan Wang, Pan Zhang State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
a r t i c l e
i n f o
Article history: Received 24 July 2010 Accepted 31 October 2010 Keywords: Black-bone silky fowl (Gallus gallus domesticus Brisson) High-performance liquid chromatography Antioxidant activity Amino acid composition Molecular weight distribution
a b s t r a c t Black-bone silky fowl (Gallus gallus domesticus Brisson) (BSF) muscle was hydrolyzed by papain, and the hydrolysate was separated by preparative high performance liquid chromatography (HPLC). The amino acid composition of the BSF hydrolysate (BSFH) and its fractions was determined by HPLC precolumn derivation with 2,4-dinitrofluorobenzene. The molecular weight (MW) distribution of the BSFH and its fractions was measured by a peptide column on an HPLC system. Antioxidant activities of the BSFH and its fractions were studied by testing the reducing power and four radical scavenging systems: superoxide anion (O2•−), hydroxyl (·OH), 1,1-diphenyl-2-picrylhydrazyl (DPPH•) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+) radicals. The results demonstrated that the BSFH had strong antioxidant capacity to scavenge O2•−, DPPH• and ABTS•+, and displayed strong reducing power, but revealed less powerful ability to scavenge ·OH. Fraction II of the BSFH exhibited the highest activity in scavenging O2•− and DPPH•, and reducing power, whereas fraction I displayed the strongest ·OH scavenging ability. Besides Glu, Asp and Gly, the rich amino acids of Ala and Leu played an important role in antioxidant activity. The small-size peptides with MW ranging from approximately 200–6000 Da probably contributed to higher antioxidant activity. Results from this study indicated that BSFH and its fractions could be used as food additives and diet nutrients. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Free radical-mediated oxidation and antioxidants are receiving more and more attention in many current research areas. In the ground state, the oxygen molecule is stable until exposed to environmental pollutants, radiation, UV, etc. At this time, reactive oxygen species (ROS) and free radicals are easily formed, which can induce oxidative damage to biomacromolecules, including DNA, proteins, membrane lipids and carbohydrates (Wiseman & Halliwell, 1996; Lai & Piette, 1977; Kellogg & Fridovich, 1975). ROS and free radicals are reported to be involved in the occurrence of numerous diseases, such as cancer, atherosclerosis, diabetes, neurodegenerative disorders and aging (Halliwell, 1991; Pryor, 1982). In food industry, lipid or fatty acid oxidation has been receiving great concerns because it can result in quality deterioration (development of undesirable off-flavor, discoloration, nutrition loss, formation of toxins), reducing the shelf-life of food products. To retard lipid or fatty acid oxidation, various antioxidants with a strong antioxidant capacity have been developed. Synthetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), can be used to preserve food containing fats and oils. However, because of their
⁎ Corresponding author. Tel./fax: +86 791 3969009. E-mail address: [email protected] (M.-Y. Xie). 0963-9969/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.10.050
potential health risks and negative consumer perception (Yu et al., 2002), natural antioxidants have become appealing alternatives and are in great demand in the food industry. For example, tocopherols, ascorbate, flavonoids, carotenoids and phenolic compounds from plants are the most commonly used natural antioxidants in the processed food (Pokorný, 1991). In recent years, it has been recognized that dietary proteins provide a rich source of naturally occurring antioxidants. These proteins are of wide distribution in plants, animals and fungi, such as soybean protein (Chen, Muramoto, Yamauchi, Fujimoto, & Nokihara, 1998), maize zein (Zhu, Chen, Tang, & Xiong, 2008), potato protein (Wang & Xiong, 2005), whey protein (Hernández-Ledesma, Dávalos, Bartolomé, & Amigo, 2005), grass carp muscle protein (Ren et al., 2008) and fermented mushroom protein (Sun, He, & Xie, 2004). Protein hydrolysates and peptides exhibit strong antioxidant efficiency in both model and in situ systems, including radical scavenging, reducing, and metal ion chelating activity (Elias, Kellerby, & Decker, 2008). Black-bone silky fowl (Gallus gallus domesticus Brisson) (BSF), black in skin, meat, bones, and white in feathers, is a unique breed of chicken in China. It has been well known for its health functions, such as treating diabetes and anemia, curing women's diseases like menoxenia and postpartum complications. With the expansion of BSF breeding, effective development and utilization of BSF are emergent. Carnosine is a natural antioxidative di-peptide, present in
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a remarkably high content within BSF (Tian et al., 2007). However, little has been known about different antioxidant properties of hydrolysates and peptides derived from BSF muscle. Because of antioxidant peptides production ability (Wang, Zhao, Zhao, & Jiang, 2007), easy availability and low commercial price, papain was used to hydrolyze BSF muscle. The antioxidant activities assessment can be achieved by means of determining their radical scavenging activity and reducing power. In the present study, the antioxidant properties of papain hydrolysates from BSF and its fractions separated by preparative HPLC were investigated in four radical scavenging systems (O2•−, ·OH, DPPH• and ABTS•+) and reducing power using carnosine and ascorbate as comparison. In addition, the MW distribution and amino acid composition of the BSFH and its fractions were studied. 2. Materials and methods 2.1. Materials and reagents Black-bone silky fowl (75–90 days old, half male and half female) were provided by the Taihe Original Black-Bone Silky Fowl Hennery (Jiangxi, China). Papain (320,000 U/g) was purchased from Beijing Fangshan Enzyme Factory (Beijing, China). Cytochrome c, 1,1diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reduced and oxidized glutathione and 17 amino acid standards (Asp, Glu, Ser, Arg, Gly, Thr, Pro, Ala, Val, Met, Cys, Ile, Leu, Phe, His, Lys, Tyr) were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Acetonitrile was of chromatographic grade. Other reagents used in this study were of analytical grade.
(0.1, 1, 10 and 50 mg/mL for the BSFH; 1 mg/mL for each fraction) was mixed with 1.8 mL of 50 mmol/L Tris–HCl buffer (pH 8.2). The mixture was incubated at 25 °C for 10 min, and then 0.1 mL of 10 mmol/L pyrogallol (dissolved in 10 mmol/L HCl) was added. The absorbance of the solution at 320 nm was measured up to 4 min. The oxidation rate of pyrogallol for samples was calculated as the change of the absorbance (ΔA1). The autoxidation rate of pyrogallol for control was measured with 1.0 mL of ultrapure water (ΔA0). For comparison, the O2•− scavenging activity of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) was also tested. The O2•− scavenging activity was calculated as [(ΔA0 − ΔA1) / ΔA0] × 100%. 2.5. Hydroxyl radical (·OH) scavenging activity The ·OH scavenging assay of the BSFH and its fractions were carried out using the method described by Li, Jiang, Zhang, Mu, and Liu (2008) with some modifications. The sample tubes were filled out with the following solutions accordingly: 1 mL of 0.4 mol/L phosphate buffer (pH 7.4), 1 mL of 2.5 mmol/L 1,10-phenanthroline, 1 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction), 1 mL of 2.5 mmol/L FeSO4 and 0.5 mL of H2O2 (1%). The mixture was incubated at 37 °C for 1 h, and the absorbance was measured at 536 nm. The ·OH scavenging assay of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The ·OH scavenging activity was calculated as [(A2 – A0) / (A1 – A0)] × 100%, where A2 is the absorbance of the sample; A0 is the absorbance of the blank solution using ultrapure water (1 mL) instead of sample solution (1 mL); and A1 is the absorbance of the control solution using ultrapure water (1.5 mL) instead of sample solution (1 mL) and H2O2 (0.5 mL).
2.2. Preparation of BSFH
2.6. DPPH radical (DPPH•) scavenging activity
The enzymatic hydrolysis method optimized by uniform design was used to prepare BSFH. Briefly, BSF muscle (1/6, w/v) was hydrolyzed with papain (3‰, w/v) at 55 °C and pH 7.0 for 1 h to achieve a strong antioxidant activity. The enzyme was inactivated by heating at 90 °C for 20 min. The resulting BSFH solution was centrifuged at 3000×g for 10 min. The lipids and melanin were removed by chloroform-methanol (2:1, v/v) extraction and centrifugation. The supernatant was subjected to vacuum drying at 60 °C, sealed in plastic bags, and stored in a desiccator until use.
The DPPH• scavenging activity of the BSFH and its fractions was measured by using the procedure described by Chen, Xie, Nie, Li, and Wang (2008). The 0.2-mmol/L solution of DPPH in 95% ethanol was prepared daily before UV measurements. 1 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was thoroughly mixed with 2 mL of freshly prepared DPPH and 2 mL of 95% ethanol. The mixture was shaken vigorously and allowed to stand for 30 min in the dark, and then the absorbance was measured at 517 nm against a blank. The DPPH• scavenging activity of carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The DPPH• scavenging activity was calculated as [1 − (A2 − A1) / A0] × 100%, where A0 is the absorbance of DPPH solution without sample (2 mL DPPH + 3 mL 95% ethanol), A2 is the absorbance of the sample mixed with DPPH solution (1 mL sample + 2 mL DPPH + 2 mL 95% ethanol) and A1 is the absorbance of the sample without DPPH solution (1 mL of sample + 4 mL 95% ethanol).
2.3. Preparative HPLC separation The separation of BSFH was performed on a Waters DeltaPrep 400 preparative chromatography system equipped with Waters Prep LC controller and Waters 2487 dual λ absorbance detector (Waters, Milford, MA, USA). The preparative HPLC was performed on a Bondapak C18 preparative column (300 × 30 mm I.D., 10 μm). For the simple and fast separation, ultrapure water was selected as the mobile phase. The BSFH concentration was 50 mg/mL. The hydrolysate solution was filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 5 mL. The flow rate was 25 mL/ min, and the detected wavelength was 254 nm. The preparative HPLC equipment was controlled by Waters Empower 2 chromatography data software. The BSFH fractions were manually collected, reduced to 10 mL under reduced pressure rotary evaporation at 50 °C and then, freeze-dried, sealed, and stored in a desiccator before use. 2.4. Superoxide radical (O2•−) scavenging activity The assay of O2•− scavenging activity of the BSFH and its fractions were determined according to the autoxidation of a pyrogallol method described by Marklund and Marklund (1974). Briefly, 1.0 mL of samples
2.7. ABTS radical cation (ABTS•+) scavenging activity The ABTS•+ scavenging activity of the BSFH and its fractions was analyzed by the method described by Re et al. (1999). This method is based on ABTS•+ production by reacting ABTS stock solution (7 mmol/L) with potassium persulfate (2.45 mmol/L, final concentration). The mixture was left in the dark at room temperature for 12 h before use. One milliliter of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was mixed with 4 mL of ABTS•+ solution, and the absorbance was recorded at 734 nm after 6 min. Carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested for comparison. The ABTS•+ scavenging activity was calculated as [(A0 − A1) / A0] × 100%, where A0 is the absorbance of the blank solution without sample and A1 is the absorbance of the sample solution.
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2.8. Reducing power
2.11. Statistical analysis
The reducing power of the BSFH and its fractions was measured by the procedure described by Wu, Chen, and Shiau (2003). Briefly, 2 mL of samples (0.1, 1, 10 and 50 mg/mL of the BSFH; 1 mg/mL for each fraction) was mixed with 2 mL of 0.2 mol/L phosphate buffer (pH 6.6) and 2 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min, and then, 2 mL of 10% TCA was added. The mixtures were centrifuged for 10 min at 3000×g, and 2 mL of the supernatant was mixed with 2 mL of ultrapure water and 0.4 mL of 0.1% FeCl3. After reaction for 10 min, the absorbance of the solution was read at 700 nm. For comparison, carnosine (0.1, 1 and 10 mg/mL) and ascorbate (0.1 and 1 mg/mL) were also tested.
All the experiments were performed with three independent trials and all the determinations were triplicated. The results were represented as mean ± standard deviation. Analysis of variance (ANOVA) was performed to identify significant differences (P b 0.05).
2.9. Amino acid composition analysis Amino acid composition of the BSFH and its fractions was determined by HPLC precolumn derivation method through amino acid reacting with 2,4-dinitrofluorobenzene (Sanger, 1945). To prepare amino acids solution, 20 mg of samples was hydrolyzed at 110 °C for 24 h with 10 mL of 6 mol/L HCl, and the resulting solution was made up to 50 mL with 0.05 mol/L boric acid buffer (pH 9.0). The brown volumetric flask (50 mL) was filled with the following solutions accordingly: 5 mL of amino acid standard solution (5 mg of each amino acid standard was dissolved into 100 mL of ultrapure water) or amino acids solution, 5 mL of 0.05 mol/L boric acid buffer (pH 9.0) and 5 mL of 2,4-dinitrofluorobenzene. The resulting solution was shaken and sealed up, and then placed into water bath (60 °C) for 1 h to react in the dark. After cooled down to room temperature, the solution was diluted with 0.05 mol/L phosphate buffer (pH 7.0) up to 50 mL. The derivative solutions were separated by a Waters HPLC system (UK6 injector and 515 HPLC pump) equipped with a Kromasil C18 (250 × 4.6 mm I.D., 5 μm) and a Waters 2996 Photodiode Array Detector. The mobile phase A was 50 mmol/L sodium acetate buffer solution (pH 6.8) containing 1% (v/v) N,N-dimethylformamide. The mobile phase B was 50% (v/v) acetonitrile in water. Gradient elution was carried out according to the following procedure: 0–0.3 min, 16% B; 0.3–4 min, 16–31% B; 4–9.5 min, 31–36% B; 9.5–17 min, 36–55% B; 17–28 min, 55–65% B, 28–34 min, 65–100% B, 34–36 min, 100–16% B. The derivative solutions were filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 20 μL. The flow rate was 1 mL/min, and the detected wavelength was 360 nm. The amino acid contents were calculated according to the HPLC peak area ratio between samples and amino acid standards. The results were expressed in terms of milligram of amino acid per gram sample.
3. Results and discussion 3.1. BSFH and its fractions The BSFH was prepared by enzymatic hydrolysis with papain and its fractions were obtained by preparative HPLC separation on C18 column. As shown in Fig. 1, fractions I, II, III and IV were obtained. Of four fractions, fraction II is in the majority (yield), followed by fraction III. The hydrophobicity of the peptides can be predicted by the retention time (Chen et al., 1998; Meek, 1980). Fraction with a long retention time has the weakest hydrophilicity. Therefore, the hydrophilicity of fractions was as follows: fraction I N fraction II N fraction III N fraction IV. RP-HPLC was widely used in separation of proteins and peptides because of its many advantages, such as higher resolution, better repeatability and greater recovery rate. In the present experiment, preparative HPLC with C18 column provided a simple, fast and large-scale preparative approach to fractionate the BSFH.
3.2. O2•− scavenging activity O2•−, a highly toxic free radical, can be easily generated by numerous biological reactions. Because it is potential precursors of highly reactive species, such as ·OH, the development of antioxidants to scavenge this radical is emergent (Kanatt, Chander, & Sharma, 2007). As shown in Table 1, the BSFH exhibited a strong ability to scavenge O2•−. With the increased concentration of BSFH, the scavenging activity against O2•− was elevated. At 10 mg/mL, the O2•− scavenging activity (72.3%) of BSFH was stronger than that of carnosine at 1 mg/mL (55.9%), and was nearly the same to ascorbate at 0.1 mg/mL (77.0%). Using the same method, BSFH showed far more powerful O2•− scavenging activity than zein hydrolysate reported by Tang et al. (2009) at 10 mg/mL (72.3% versus 11.5%). Fraction II revealed the highest activity (25.9%), followed by fraction I (23.8%), which exhibited stronger O2•− scavenging activity than BSFH (22.6%) at the same concentration (P b 0.05).
2.10. Determination of MW distribution The MW distribution of the BSFH and its fractions was evaluated using a Waters HPLC system (UK6 injector and 515 HPLC pump) equipped with a Superdex peptide 10/300 GL column (300 × 10 mm I. D., 13–15 μm) and a Waters 2996 Photodiode Array Detector. According to the column instructions, the choice of eluent was 0.05 mol/L phosphate buffer (pH 7.0) containing 0.15 mol/L NaCl. The concentration of the BSFH and its fractions was 10 mg/mL. The sample solutions were filtered through a 0.45-μm Millipore membrane before injection. The injection volume was 20 μL. The flow rate was 1 mL/ min, and the detected wavelength was 210 nm. Cytochrome c (12384 Da), reduced glutathione (615 Da), oxidized glutathione (310 Da) and glycine (75 Da) were used as the MW markers. In Fig. 2(A), according to the retention time of four MW markers, the regression equation is logMW = −0.265 t + 7.066, in which R2 = 0.9793, MW represented molecular weight, t represented retention time.
Fig. 1. Fractions of the BSFH eluted by C18 column on preparative HPLC system. Four fractions were identified as fraction I, II, III and IV.
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Table 1 Antioxidant activities of the BSFH, fraction I, II, III and IV, carnosine and ascorbate. Antioxidant
BSFH
Fraction I Fraction II Fraction III Fraction IV Carnosine
Ascorbate
Concentration (mg/mL) 50 10 1 0.1 1 1 1 1 10 1 0.1 1 0.1
Scavenging activity (%) O2•−
·OH
DPPH•
ABTS•+
≥100 72.3 ± 2.3 22.6 ± 1.5 0.8 ± 0.3 23.8 ± 0.3 25.9 ± 0.5 17.0 ± 1.3 21.6 ± 0.7 ≥100 55.9 ± 0.3 20.3 ± 1.7 ≥100 77.0 ± 0.7
57.1 ± 0.6 24.7 ± 1.5 12.7 ± 0.7 6.8 ± 0.2 28.5 ± 0.7 10.5 ± 0.5 11.7 ± 0.3 10.7 ± 0.8 89.5 ± 0.9 20.3 ± 0.5 9.4 ± 1.1 96.7 ± 1.5 10.1 ± 1.3
≥100 73.0 ± 2.2 10.0 ± 2.8 2.9 ± 1.2 25.7 ± 1.3 36.7 ± 1.6 10.6 ± 2.4 11.0 ± 1.9 7.4 ± 0.2 6.4 ± 0.3 5.9 ± 0.5 96.6 ± 0.6 94.4 ± 1.1
85.9 ± 0.2 79.5 ± 0.9 18.5 ± 1.3 6.3 ± 0.5 11.0 ± 0.3 15.5 ± 0.3 16.4 ± 0.9 17.3 ± 0.6 59.8 ± 0.7 12.1 ± 0.6 2.4 ± 0.2 97.5 ± 0.2 97.2 ± 0.1
A700 reducing power 2.504 ± 0.100 1.196 ± 0.050 0.237 ± 0.010 0.093 ± 0.003 0.206 ± 0.010 0.731 ± 0.030 0.205 ± 0.010 0.196 ± 0.008 0.097 ± 0.002 0.069 ± 0.002 0.062 ± 0.003 3.221 ± 0.100 1.909 ± 0.070
The results were represent as mean ± standard deviations from three replications (n = 3).
3.3. ·OH scavenging activity
3.5. ABTS•+ scavenging activity
Among ROS, the chemical toxicity of ·OH is the strongest because it is able to easily reacts with biomolecules, such as amino acids, proteins and DNA (Cacciuttoloa, Trinha, Lumpkina, & Rao, 1993). Therefore, the removal of ·OH is probably one of the most effective defenses of a living body against various diseases (Je, Park, & Kim, 2005). Table 1 demonstrated that the BSFH displayed a certain activity to quench ·OH and the effect was concentration dependent. Although ·OH scavenging activity of BSFH was weaker than carnosine and ascorbate, an elevated scavenging activity fraction was found after fractionation. At 1 mg/mL, fraction I showed the strongest ·OH scavenging activity (28.5%) among four fractions, and its activity was significantly stronger than BSFH (12.7%). In the present study, the ·OH scavenging activity of BSFH was weaker than that of cottonseed protein hydrolysate (47.33% at 10 mg/mL) (Gao, Cao, & Li, 2010). Kim, Je, and Kim (2006) reported four fractions from hoki frame protein hydrolysate exhibited extremely strong ·OH scavenging activity (69.18% or more) at 0.5 mg/mL. These different results could be due to the different ·OH scavenging activity assay methods used, different protein sources, and different fractionation methods used.
ABTS•+ is generated by oxidation of ABTS with potassium persulfate (absorption maxima at 734 nm) and is reduced in the presence of hydrogen-donating antioxidants (decolorization) (Re et al., 1999). As shown in Table 1, BSFH showed strong scavenging ability against ABTS•+, and the effect was concentration dependent. The 79.5% of ABTS•+ can be neutralized by BSFH at 10 mg/mL. It was noteworthy that the ABTS•+ scavenging ability of the BSFH was stronger than that of carnosine at 0.1, 1 and 10 mg/mL, respectively. At 1 mg/mL, however, BSFH and its fractions exhibited less than 19% of ABTS•+ scavenging ability. Fraction I displayed the weakest ABTS•+ scavenging ability (11.0%) among four fractions, while the other three fractions showed nearly the same ability (15.5%, 16.4% and 17.3%). Tang et al. (2009) reported that zein hydrolysate revealed 64.3% of ABTS•+ scavenging activity at 1 mg/mL, while its fractions displayed no more than 35% of activity at the same concentration. This was in good agreement with the present study: at 1 mg/mL, the ABTS•+ scavenging activity of the BSFH was stronger than its four fractions (Table 1). Nonetheless, a fraction or peptide with higher ABTS•+ scavenging ability could be predicted after further purification.
3.4. DPPH• scavenging activity DPPH• assay was the simplest and most accurate method to measure the ability of antioxidant to intercept free radicals (SánchezMoreno, 2002). It is an organic nitrogen radical with a UV-vis absorption in the range of 515–520 nm, and its solution color fades upon reduction. DPPH• scavenging activities of BSFH and its four fractions are shown in Table 1. At 10 mg/mL, BSFH displayed 73.0% of DPPH• scavenging activity, while it completely scavenged DPPH• at 50 mg/mL. Fraction II showed the most powerful DPPH• scavenging activity (36.7%), followed by Fraction I (25.7%), and these two fractions exhibited significantly stronger activity than BSFH at 1 mg/ mL (P b 0.05). In recent years, Gao et al. (2010) and Li, Han, and Chen (2008) also reported the DPPH•-scavenging peptides derived from food protein hydrolysates. In our research, as comparison, ascorbate displayed strong activity in scavenging DPPH• (94.4% at 0.1 mg/mL), while carnosine showed weak (7.4% at 10 mg/mL). Although carnosine is a histidine-containing natural antioxidant, its low dispersibility in ethanol solution probably hinder the electron transfer between antioxidants and DPPH•. Our results suggested that the BSFH and its fractions were better natural antioxidants than carnosine in scavenging DPPH•.
3.6. Reducing power The antioxidant activity has been reported to be accompanied by the reducing power (Tanaka, Kuei, Nagashima, & Taguchi, 1988). The results of the reducing power of BSFH and its fractions are also shown in Table 1. The reducing power was expressed as the absorbance value at 700 nm, and the increased absorbance value indicated an increased reducing power. With the increasing concentration from 0.1 mg/mL to 50 mg/mL, the absorbance value also greatly increased from 0.093 to 2.504, indicating enhancement of BSFH reducing power. Thus, the reducing power of BSFH was dose dependent. Fraction II displayed the strongest reducing power among four fractions, and the effect was significantly stronger than BSFH (the absorbance value of 0.731 versus 0.237) at 1 mg/mL. The reducing power of BSFH and fraction II were even stronger than alcalase-treated zein hydrolysates (the absorbance value was no more than 0.8 at 8 mg/mL) (Zhu et al., 2008). For comparison, the reducing power of ascorbate soared from 1.909 to 3.221, while carnosine displayed the reducing power no more than 0.1. It can be concluded that the reducing power of the BSFH was tremendously stronger than carnosine, and the reducing power of 50 mg/mL of the BSFH can surpass the ascorbate reducing power at 0.1 mg/mL. The reducing power determination in the present study was carried out in an aqueous environment. The antioxidative and hydrophilic peptides with good solubility were capable of donating electrons easily, and thus antioxidant activity increased.
J.-H. Liu et al. / Food Research International 44 (2011) 133–138 Table 2 Amino acid composition of the BSFH and its fractions determined by HPLC precolumn derivation with 2,4-dinitrofluorobenzene. “–” denotes this amino acid was not found.
Aspartic acid Glutamic acid Serine Arginine Glycine Threonine Proline Alanine Valine Methionine Cysteine Isoleucine Leucine Phenylalanine Histidine Lysine Tyrosine Total
mg amino acid/g BSFH or its fractions BSFH
Fraction I
Fraction II
Fraction III
Fraction IV
71.4 132.7 19.4 61.3 66.7 74.0 33.8 45.4 33.5 31.2 58.9 28.8 50.4 14.6 24.1 71.1 51.6 868.9
58.8 155.0 33.4 36.8 51.2 33.7 16.0 54.2 24.3 13.8 4.1 12.9 34.1 7.0 19.5 46.2 7.5 608.5
59.2 124.3 26.9 45.8 66.4 28.8 33.3 53.6 22.7 12.7 1.6 26.5 49.8 26.4 24.1 38.9 19.1 660.1
66.4 105.0 22.7 50.8 81.8 35.5 54.5 35.5 27.4 10.8 2.0 25.3 41.6 27.2 26.9 40.1 15.3 668.8
77.1 120.1 27.5 109.5 73.2 33.2 59.4 50.1 30.9 – – 30.8 51.9 31.4 30.4 56.6 17.2 799.3
Absorbance at 210 nm (AU)
A
3.7. Amino acid composition The amino acid composition of the BSFH and its fractions was analyzed in order to evaluate the possible effect of the amino acid profile on its antioxidant activity. As shown in Table 2, BSFH contained mainly Glu, Thr, Asp, Lys and Gly. All of these amino acids were hydrophilic and the hydrophobic amino acids were 30.24% in total, indicating that the hydrophilic amino acids in the sequence of the BSFH peptides were mainly responsible for the antioxidant activity. The amino acids of fraction I mainly consist of Glu, Asp, Ala, Gly and Lys; for fraction II, they were mainly Glu, Gly, Asp, Ala, and Leu; for fraction III, they were mainly Glu, Gly, Asp, Pro, and Arg; and for fraction IV, they were mainly Glu, Arg, Asp, Gly, and Pro. Therefore, the strongest ·OH scavenging capacity of fraction I was attributed to abundant Ala and Lys. Fraction II, which exhibited the strongest scavenging capacity against O2•− and DPPH•, and reducing power, contained more Ala and Leu. Although Ala and Leu were highly hydrophobic, they played an important role in antioxidant ability. Kim et al. (2001) reported that Ala and Leu contributed to free radicals scavenging activity. The antioxidant activity of carnosine (Ala-His) is much greater than the individual or combined activity of Ala and His
B 0.90
oxidized glutathione
MW markers
0.75 0.60 reduced glutathione
0.45 0.30
cytochrome C
0.15
glycine
0.00 0
5
10
15
20
25
Absorbance at 210 nm (AU)
Amino acids
0.6
0.4 0.3 0.2 0.1 0.0
30
0
5
1
2
3 4
10
15
20
25
30
Time (min)
D 1.0
Fraction I
No. MW Range Content (Da) (%) 1 5443-255 53.08 2 255-74 35.19 74 11.73 3
0.8 0.6 0.4 0.2
1
2 3
15
20
0.0 0
5
10
25
Absorbance at 210 nm (AU)
C Absorbance at 210 nm (AU)
BSFH
No. MW Range Content (Da) (%) 1 95733-6070 18.60 2 6070-291 39.76 3 291-82 23.44 82 18.20 4
0.5
Time (min)
1.0
Fraction II
No. MW Range Content (Da) (%) 1 5069-210 44.20 2 210-84 14.60 3 84 41.20
0.8 0.6 0.4 0.2 0.0
30
1 0
5
Time (min)
10
15
2 3 20
25
30
Time (min)
F 0.4
Fraction III
No. MW Range Content (Da) (%) 1 10121-223 52.95 2 223-33 29.22 33 17.83 3
0.3
0.2
0.1
1
0.0 0
5
10
15
2 20
Time (min)
3 25
30
Absorbance at 210 nm (AU)
E Absorbance at 210 nm (AU)
137
0.5
Fraction IV
No. MW Range Content (Da) (%) 1 8868-230 58.70 2 230-30 27.92 30 13.38 3
0.4 0.3 0.2 0.1
1
0.0 0
5
10
15
2 3 20
25
30
Time (min)
Fig. 2. MW distribution of the MW markers (A), BSFH (B), fraction I (C), fraction II (D), fraction III (E) and fraction IV (F). (Inset) Relative components (peptides or amino acids) content in BSFH, fraction I, II, III and IV. Components are indicated by numbers.
138
J.-H. Liu et al. / Food Research International 44 (2011) 133–138
(Decker, 1995), suggesting a synergistic effect of Ala. Tang et al. (2009) isolated two radical scavenging peptide, Tyr-Ala and LeuMet-Cys-His, from zein protein. The presence of Ala and Leu played an important role in the antioxidant activity. Fraction III and IV, which were less in Ala and Leu contents, showed weak free radicals scavenging activity and reducing power. The results demonstrated that the abundance of Glu, Asp, Gly, Ala and Leu contributed to the strong antioxidant activity. 3.8. MW distribution The MW distribution was recognized as an important factor to determine the overall antioxidant activity of the hydrolyzed proteins. Li, Jiang et al. (2008) revealed that the peptide fraction from chickpea protein hydrolysate with the MW distribution from 200 to 3000 Da was associated with high antioxidant activity. Wu et al. (2003) claimed that a peptide from mackerel protein hydrolysate with MW of approximately 1400 Da possessed stronger antioxidant activity than higher MW peptides. Fig. 2 showed the MW distribution of MW markers, BSFH and its four fractions. Fig. 2(A) showed the retention time of four MW markers. In Fig. 2(B), BSFH contained mainly smallsize peptides (291–6070 Da), which was determinant components for its antioxidant activity. In Fig. 2(C) and (D), the major components of fraction I and II were also small-size peptides (255–5443 Da and 210–5069 Da, respectively), displaying narrow MW distribution. By contrast, in Fig. 2(E) and (F), the major components of fraction III and IV shared wide MW range (223–10121 Da and 230–8868 Da). Therefore, the considerable small-size peptides were responsible for the strong antioxidant activity of fraction I and II. These results showed that the peptides with MW ranging from approximately 200–6000 Da were probably associated with higher antioxidant activity. 4. Conclusions BSFH was prepared by papain hydrolysis and its four fractions (I, II, III and IV) were successfully obtained by preparative HPLC separation. The BSFH (10 mg/mL) and fraction II (1 mg/mL) exhibited strong antioxidant activities than carnosine in scavenging DPPH• and ABTS•+, and reducing power. Fraction I (1 mg/mL) displayed strong antioxidant activities than carnosine in scavenging ·OH. Glu, Asp, Gly, Ala and Leu played an important role in antioxidant activity. The MW of peptides responsible for higher antioxidant activity probably ranged from 200 to 6000 Da. Results from this study suggested that the antioxidant BSFH and its fractions (especially fraction II) could be used as food additives and diet nutrients. Nonetheless, further purification is needed to identify the individual peptides responsible for the antioxidant activity of the BSFH. Acknowledgements This study was supported financially by the National Natural Science Foundation of China (No. 20862012), the Objective-Oriented Project of State Key Laboratory of Food Science and Technology (SKLFMB-200806), and the Cheung Kong Scholars Program and Innovative Research Team in University (No. IRT0540). References Cacciuttoloa, M. A., Trinha, L., Lumpkina, J. A., & Rao, G. (1993). Hyperoxia induces DNA damage in mammalian cells. Free Radical Biological and Medicine, 14, 267−276. Chen, H. M., Muramoto, K., Yamauchi, F., Fujimoto, K., & Nokihara, K. (1998). Antioxidative properties of histidine-containing peptides designed from peptide fragments found in the digests of a soybean protein. Journal of Agricultural and Food Chemistry, 46, 49−53. Chen, Y., Xie, M. Y., Nie, S. P., Li, C., & Wang, Y. X. (2008). Purification, composition analysis and antioxidant activity of a polysaccharide from the fruiting bodies of Ganoderma atrum. Food Chemistry, 107, 231−241.
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