Article pubs.acs.org/JAFC
Identification and Characterization of Antioxidant Peptides from Enzymatic Hydrolysates of Duck Meat Lu-Sha Wang, Ji-Chao Huang, Yu-Lian Chen, Ming Huang,* and Guang-Hong Zhou Synergetic Innovation Center of Food Safety and Nutrition, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China S Supporting Information *
ABSTRACT: The objective of this study was to prepare antioxidant peptides from duck meat hydrolysate (DMH) using Protamex. The DPPH• scavenging activity, hydroxyl radical (•OH) scavenging activity, and Fe2+-chelating ability of DMH were investigated. DMH was separated into three groups, MWCO-1 (69.57%), MWCO-2 (9.53%), and MWCO-3 (8.21%), by ultrafiltration. MWCO-3 exhibited the highest DPPH• scavenging activity (83.17 ± 0.73%) and was subsequently fractionated by using gel filtration chromatography to obtain fraction B (40.90%). Fraction B5 (6.71%) obtained from ion exchange chromatography exhibited the highest DPPH• scavenging activity (93.63 ± 0.13%) and contained seven peptides which were characterized by LC−MS/MS. Among these peptides, LQAEVEELRAALE showed the highest DPPH• scavenging activity (93.36 ± 0.53%) and Fe2+-chelating ability (87.13 ± 0.47%) and IEDPFDQDDWGAWKK exhibited the highest •OH scavenging activity (46.51 ± 0.16%). The results presented here indicated that DMH could serve as a suitable source of antioxidant peptides. KEYWORDS: duck meat hydrolysate, duck meat derived antioxidant peptides, antioxidant capacity of duck peptides, duck meat protein enzymatic hydrolysis
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pharmaceuticals.14 Various antioxidant peptides derived from vegetables are allowed to be incorporated into specific foods in the United States as food additives and are allowed as food ingredients in most countries.14,15 Antioxidant peptides from meat might have such an application, although a limited number of studies have been conducted to date for assessing the biological antioxidant potential of peptides for human clinical use.14 Nevertheless, several studies showed that some antioxidant peptides could have an impact on reducing oxidative stress as well as the risk of various diseases. The peptide GGFDMG derived from Japanese flounder skin gelatin hydrolysate could protect membrane lipids, proteins, and DNA from reactive oxygen species (ROS)-mediated damage.16 The loach peptides prepared by papain have antioxidant and antiproliferative activities for cancer cell lines.17 Croaker muscle peptide (KTFCGRH) fed to Wistar rat helps to neutralize ethanol-induced oxidative stress.18 Peptides from duck skin byproducts have been reported to exhibit high antioxidant activities in vitro and in vivo.19−21 However, there is little information about antioxidant peptides from duck meat. Therefore, the main objective of this study was to (1) investigate the antioxidant activities of duck meat hydrolysate (DMH) prepared using Protamex and (2) purify and characterize the antioxidant peptides from DMH.
INTRODUCTION Free radicals are resultant products of metabolism which usually exist in balance with biological antioxidants.1 However, breaking of this critical balance could result in oxidative stress, leading to many diseases, such as cancer, atherosclerosis, diabetes, arthritis, coronary heart disease, and Alzheimer’s disease.2 Free radicals are also the predominant cause of lipid peroxidation, which generates rancid flavor, undesired taste, and shortening of shelf life, as well as the production of potentially toxic reaction products.2 To prevent foods from undergoing deterioration and to protect from serious diseases, it is significant to inhibit oxidation occurring in foodstuffs and the living body. Recently, interest has been emerging to investigate antioxidant peptides from animal sources.3 Antioxidant peptides are inactive within the sequence of the parent protein and can be released by enzymatic hydrolysis.4 Once they are liberated, these peptides may exert antioxidant activities. Various studies have identified and characterized antioxidant peptides from animal sources by enzymatic hydrolysis, such as yellowfin sole,5 Alaska pollack,6 venison,7 hoki,8 loach,9 and chicken.10 Saiga et al.11 reported that hydrolysates obtained from porcine myofibrillar proteins exhibited high levels of lipid peroxidation inhibition, in which the peptide sequenced as DAQEKL showed the highest antioxidant activity. Antioxidant peptides (PVMGA and QHGV) isolated from oysters had activities to scavenge hydroxyl and DPPH radicals.12 An antioxidant peptide of GKFBV purified from jinhua ham exhibited higher DPPH• scavenging activity than butylated hydroxytoluene (BHT).13 Antioxidant peptides have great expectations in commercial use. They can be added as functional ingredients in food systems to reduce oxidative changes or used as nutraceuticals or © XXXX American Chemical Society
Received: September 12, 2014 Revised: February 15, 2015 Accepted: February 20, 2015
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DOI: 10.1021/jf506120w J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
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MATERIALS AND METHODS
hydroxyl radical scavenging activity (%) =
Materials. Duck (Anas platyrhynchos) breasts were purchased from Suguo supermarket in Nanjing, China, and stored at −20 °C until use. Protamex (1.5 AU of N/g) was purchased from Novo Co. (Novozyme Nordisk, Bagsvaerd, Denmark). 1,1-Diphenyl-2-picrylhydrazyl free radical (DPPH•) and L-glutathione reduced (GSH) were obtained from Sigma Chemical Co. (St. Louis, MO). Acetonitrile (ACN) and formic acid were of chromatographic grade, and other chemicals used were of analytical grade. Preparation of Enzymatic Hydrolysate from Duck Meat. Frozen duck breasts were thawed at 4 °C overnight, and then the skin and connective tissue were removed. The meat was chopped into pieces and mixed with distilled water to give a final protein content of 4% (w/v). The mixture was then homogenized for 1 min at 8000 rpm using a homogenizer (IKA T25 digital ultraturrax, IKA, Germany) with cooling in ice. The pH of the mixture was adjusted to 6.0 with a pH meter (FiveEasy 20, Mettler Toledo, Swiss), and Protamex was added at an enzyme-to-substrate ratio of 0.75/100 (w/w) after a preincubation at 50 °C for 10 min. During the reaction, the pH of the mixture was adjusted to 6.0 every 20 min by addition of 4 mol/L HCl solution. Enzymatic hydrolysis of the meat proteins was performed for 4 h to achieve an optimum level of hydrolysis and was then immediately heated at 100 °C for 10 min to terminate the hydrolysis. The reaction mixture was cooled to room temperature and then centrifuged (Allegra 64R centrifuge, Bechman Coulter, United States) at 10000g for 10 min. The supernatant was collected, lyophilized, and stored at 4 °C for further use. Measurement of DPPH• Scavenging Activity. The DPPH• scavenging activity was evaluated according to the procedure described by Ajila et al.22 with a slight modification. The sample group was obtained by mixing 0.5 mL of sample (1.0 mg/mL) with 0.5 mL of 0.2 mmol/L DPPH• dissolved in ethanol, the blank group consisted of aliquots of samples mixed 1:1 (v/v) with ethanol, and the control group consisted of aliquots of ethanol mixed 1:1 (v/v) with 0.2 mmol/ L DPPH•. The mixtures were shaken and kept in the dark for 30 min at room temperature. Then the absorbance was measured at 517 nm using a multifunctional microplate reader (model Spectral Max M2e, Molecular Devices, United States). The DPPH• scavenging activity was calculated as follows:
where As is the absorbance of the samples, Ad is the absorbance of the damaged group without sample solution, and An is the absorbance of the nondamaged group without H2O2. Peptide Fractionation by Ultrafiltration. A 2.0 g mass of the lyophilized DMH was dissolved in 350 mL of distilled water and then fractionated by centrifuge tubes fixed with ultrafiltration membranes having molecular weight cutoffs (MWCOs) of 30000 and 10000 (Amicon Ultra-15, Millipore Co., Billerica, MA), consecutively. MWCO-1, MWCO-2, and MWCO-3 represent the fractions with molecular weight distributions of >30000, 30000−10000, and 30000), MWCO-2 (MW = 10000−30000), and MWCO-3 (MW < 10000). The DPPH• scavenging activities were 29.01 ± 0.60%, 34.42 ± 1.61%, and 83.17 ± 0.73% at 1.0 mg/mL for MWCO1, MWCO-2, and MWCO-3, respectively (Table 2). The lower
Fmoc-protected amino acid synthesis method. These peptides were provided by Gen Script (Nanjing) Co., Ltd. Their purity was >95%. Statistical Analysis. All the tests were repeated in triplicate. Data were evaluated using SAS 8.1 software. Values were expressed as the mean ± standard deviation (SD). The mean values were analyzed using the one-way analysis of variance (ANOVA) test. Duncan’s multiple range tests were employed to determine any significant difference between treatments. The significant difference was determined with a 95% confidence interval (p < 0.05).
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Table 2. Purification of Antioxidant Peptides from DMH (1.0 mg/mL)
RESULTS AND DISCUSSION Antioxidant Activity of DMH. The antioxidant properties of peptides result from their free radical scavenging, metal ion chelating, and single oxygen quenching activities.25 In this study, the antioxidant activities of DMH were evaluated by DPPH• scavenging activity, •OH scavenging activity, and Fe2+chelating ability (Table 1). DPPH• is a stable free radical that shows maximum absorbance at 517 nm. When DPPH radicals encounter proton-donating substrates such as antioxidants, the radicals are scavenged and the absorbance is reduced.26 DMH showed high DPPH• scavenging activity at a concentration of 1.0 mg/ mL (82.09%), whereas 1.0 mg/mL GSH exhibited nearly 95% scavenging activity on the DPPH radical (Table 1). This value is somewhat higher than that of many other protein hydrolysates. The chicken breast protein hydrolysate prepared by papain exhibited about 50% DPPH• scavenging activity at a concentration of 1.28 mg/mL.10 The DPPH• scavenging activities in various hydrolysates (1.0 mg/mL) of round scad muscle were as follows: Alcalase (39.36%), Nnutral (32.33%), trypsin (39.37%), papain (40.21%), and pepsin (32.63%). These values are lower than that found for DMH.27 Bougatef et al.28 reported that smooth hound muscle hydrolysates prepared using low molecular weight (LMW) alkaline protease exhibited 76.7% DPPH• scavenging activity at a concentration of 3.0 mg/ mL. Thus, we can infer that DMH contained antioxidant peptides that were capable of scavenging radicals. • OH is the most reactive radical that can induce severe damage to adjacent biomolecules and thereby cause aging, cancer, and other diseases.29 Therefore, removal of •OH could be one of the most effective defenses against these various diseases. The •OH scavenging activity for GSH (1.0 mg/mL) was about 75%. DMH exhibited a comparable higher •OH scavenging capacity, reaching a scavenging activity of 36.54% at a concentration of 1.0 mg/mL, comparable to that of bullfrog skin protein hydrolysate (34.5%) prepared using papain at a concentration of 1.5 mg/mL,30 but lower than the pepsin hydrolysate from duck skin byproducts with about 50% •OH scavenging activity at a concentration of 0.55 mg/mL.19 Thus, we can infer that DMH contained some antioxidant peptides which could convert free radicals to more stable products and terminate the radical chain reactions. Transition-metal ions such as iron and copper ions are strong agents to catalyze the generation of free radicals via the Fenton reaction.25 Thus, the chelation of metal ions contributes to antioxidation. As shown in Table 1, the hydrolysate exhibited an effective chelating effect on Fe2+ (78.57%).
step crude ultrafiltration >30 kDa 10−30 kDa
