Antioxidant Peptides from the Protein Hydrolysate of Spanish Mackerel (Scomberomorous niphonius) Muscle by in Vitro Gastrointestinal Digestion and Their In Vitro Activities

For the full use of Spanish mackerel (Scomberomorous niphonius) muscle to produce antioxidant peptides, the proteins of Spanish mackerel muscle were separately hydrolyzed under five kinds of enzymes and in vitro gastrointestinal digestion, and antioxidant peptides were isolated from the protein hydrolysate using ultrafiltration and multiple chromatography methods. The results showed that the hydrolysate (SMPH) prepared using in vitro GI digestion showed the highest degree of hydrolysis (27.45 ± 1.76%) and DPPH radical scavenging activity (52.58 ± 2.68%) at the concentration of 10 mg protein/mL among the six protein hydrolysates, and 12 peptides (SMP-1 to SMP-12) were prepared from SMPH. Among them, SMP-3, SMP-7, SMP-10, and SMP-11 showed the higher DPPH radical scavenging activities and were identified as Pro-Glu-Leu-Asp-Trp (PELDW), Trp-Pro-Asp-His-Trp (WPDHW), and Phe-Gly-Tyr-Asp-Trp-Trp (FGYDWW), and Tyr-Leu-His-Phe-Trp (YLHFW), respectively. PELDW, WPDHW, FGYDWW, and YLHFW showed high scavenging activities on DPPH radical (EC50 1.53, 0.70, 0.53, and 0.97 mg/mL, respectively), hydroxyl radical (EC50 1.12, 0.38, 0.26, and 0.67 mg/mL, respectively), and superoxide anion radical (EC50 0.85, 0.49, 0.34, and 1.37 mg/mL, respectively). Moreover, PELDW, WPDHW, FGYDWW, and YLHFW could dose-dependently inhibit lipid peroxidation in the linoleic acid model system and protect plasmid DNA (pBR322DNA) against oxidative damage induced by H2O2 in the tested model systems. In addition, PELDW, WPDHW, FGYDWW, and YLHFW could retain their high activities when they were treated under a low temperature (<60 °C) and a moderate pH environment (pH 5–9). These present results indicate that the protein hydrolysate, fractions, and isolated peptides from Spanish mackerel muscle have strong antioxidant activity and might have the potential to be used in health food products.


Introduction
Food nutrition is intricately linked with human health because they can provide the necessary bioactive substances and cause specific physiological responses in the human body [1]. Among all the
The specificity of the protease applied for the hydrolysis process is the key factor for the production of APs because the protein hydrolysates displayed very different spectra of substrate specificity, such as DH, biological activity, and nutritive values [3]. Wang et al. reported that the neutrase hydrolysate of blue mussel (Mytilus edulis) protein showed the highest DPPH· scavenging activity compared to the hydrolysates prepared using alcalase, neutrase, pepsin, and papain [9]. Agrawal et al. reported that the DH (17.47 ± 0.63%) of trypsin hydrolysate of finger millet protein was higher than that of the pepsin hydrolysate (13.73 ± 0.18%) [19]. The EC 50 value (0.945 mg/mL) of papain hydrolysate of purple sea urchin (Strongylocentrotus nudus) gonad on DPPH· was significantly higher than those of trypsin (1.699 mg/mL) and dual-enzymatic (papain + trypsin) hydrolysates (2.481 mg/mL) [20]. Fish gelatin hydrolysate (FSGH) of Nile tilapia skin using ginger protease exhibited higher DH (13.08%), lipid peroxidation (48.46%), and DPPH· scavenging activity (97.21%) than hydrolysate using pepsin-pancreatin did [21]. Therefore, the protein hydrolysate (SMPH) of Spanish mackerel muscles prepared using in vitro GI digestion showed the highest DH and DPPH· scavenging activity and was chosen for further experiment.

Fractionation of SMPH Using Membrane Ultrafiltration
SMPH was fractionated gradually by molecular weight (MW) Cut Off (MWCO) membranes of 3, 5, and 10 kDa, and four fractions including SMPH-I (MW < 3 kDa), SMPH-II (3 kDa < MW < 5 kDa), SMPH-III (5 kDa < MW < 10 kDa), and SMPH-II (>10 kDa) were prepared. As shown in Figure 1, DPPH· scavenging activity of SMPH-I was 68.24 ± 3.29% at the concentration of 10.0 mg protein/mL, and this was significantly higher than those of SMP (14.26 ± 1.03%), SMPH (52.58 ± 2.68%), SMPH-II (44.68 ± 3.65%), SMPH-III (28.74 ± 1.41%), and SMPH-IV (21.62 ± 1.52%), respectively (p < 0.05). The differences in the activities among SMPH and its fractions are mainly because of the chain length, and the amino acid composition and sequence, which led to the diversity in the mechanisms of action [3]. The data agreed with the reports that the MW distribution of protein hydrolysates was negative relative to their antioxidant activity [18,22]. In addition, the lowest MW fractions from protein hydrolysates of blue-spotted stingray [23], buffalo and bovine casein [24], and Tergillarca granosa [25] showed the highest antioxidant activity. Therefore, SMPH-I with small MW was selected for the subsequent separation. Figure 1. DPPH· scavenging activity of SMPH and its fractions by ultrafiltration at the concentration of 10.0 mg protein/mL. All data are presented as the mean ± SD (n = 3). a-e Values with the same superscripts indicate no significant difference (p > 0.05).

No.
Retention

No.
Half Elimination Ratio (EC 50

Lipid Peroxidation Inhibition Activity
The inhibiting abilities of SMP-3, SMP-7, SMP-10, and SMP-11 on the lipid peroxidation system were expressed as the absorbance of 500 nm, and the higher absorbance of the sample group illustrated lower antioxidant capacity [31]. Figure 8 shows that the absorbance of the SMP-10 group was significantly lower than those of SMP-3, SMP-7, SMP-11, and the negative control (without antioxidant), but slightly higher than that of the positive control of GSH. The data indicate that SMP-10 had the highest ability of lipid peroxidation inhibition among four APs.
The peroxidation of the membrane lipids caused by ROS can lead to cell injury and eventually unprogrammed apoptosis, and it is a crucial step in the pathogenesis of several disease states in adult and infant patients [38][39][40]. In addition, lipid peroxidation is also an important factor in high-fat food spoilage [28,36]. Therefore, the lipid peroxidation inhibition assay in the linoleic acid model system has been widely applied to evaluate the comprehensive ability of APs from seafoods, such as bluefin leatherjacket [8,36], miiuy croaker [4,7], red stingray [28], and monkfish [41]. SMP-10 can dramatically inhibit the peroxidation of linoleic acid over 7 days of incubation and has significant potential applications in food and medicine.

Protective Effect on Plasmid DNA Damaged by H 2 O 2
In the assay, HO· was produced from the decomposition of H 2 O 2 mediated by iron when FeSO 4 and H 2 O 2 were added to the sample solutions, and the resulted HO· subsequently broke the supercoiled DNA and converted the supercoiled form into the open circular and/or linear form. Therefore, the protective effects of four APs (SMP-3, SMP-7, SMP-10, and SMP-11) on the oxidative damage of pBR322DNA induced by H 2 O 2 were measured, and the results are shown in Figure 9.
The results indicate that the plasmid DNA (pBR322DNA) was mainly of the supercoiled form under normal conditions ( Figure 9A). An open circular form was generated when one phosphodiester chain of a supercoiled form of plasmid DNA was broken by HO· ( Figure 9B). However, almost no linear form of DNA was found in Figure 9B, which indicates that the HO· produced from iron-mediated decomposition of H 2 O 2 might be too little to break some double-strand of DNA in the assay. As shown in Figure 9C-E, the contents of the open circular form of DNA was obvious lower than that of Figure 9B, which indicates that four APs (SMP-3, SMP-7, SMP-10, and SMP-11) and the positive control of GSH have different protective effects on DNA damaged by oxidation, and the protective effect of SMP-10 was slightly higher than that of SMP-7 and significantly higher than those of SMP-3 and SMP-7. In addition, the image of SMP-10 and SMP-7 was similar to those of the positive control of GSH ( Figure 9C) and the normal control ( Figure 9A). Therefore, four APs (SMP-3, SMP-7, SMP-10, and SMP-11), especially SMP-10 and SMP-11, have high abilities to guard the supercoiled pBR322DNA against HO·-dependent strand breaks. In the organism, DNA damage is a key step in ROS-induced degenerative processes, such as premature aging, hepatopathy, and diabetes, cancer, atherosclerosis, and neurodegenerative diseases [42,43]. The present results indicate that SMP-10 had a potential ability to protect pBR322DNA from oxidative damage, and our future experiment will be performed on the cell and in vivo.  Figure 10A shows that the effects of temperature on HO· scavenging activity of SMP-3, SMP-7, SMP-10, and SMP-11 (expressed as an EC 50 value). No significant difference in EC 50 values of SMP-3 and SMP-11 was found when the treated temperature was 20, 40, and 60 • C (p > 0.05), but their EC 50 values significantly increased when the treated temperatures increased to 80 and 100 • C (p < 0.05). Compared with SMP-3 and SMP-11, thermal treatment had stronger effects on SMP-7 and SMP-10 because their EC 50 values treated at 60 • C were significantly (p > 0.05) higher than those of SMP-7 and SMP-10 treated at 20 and 40 • C (p < 0.05). The results indicate that SMP-3 and SMP-11 could retain their antioxidant activity when the treated temperature was lower than 60 • C, but SMP-7 and SMP-10 would lose their function at the same processing temperature. Figure 10B shows the EC 50 values of SMP-3, SMP-7, SMP-10, and SMP-11 on HO· when they were treated at a pH value ranging from 3 to 11. No significant difference on EC 50 values of SMP-3, SMP-7, SMP-10, or SMP-11 was found when pH value ranged from 5 to 9, but pH values of 3 and 11 significantly affected the EC 50 values of SMP-3, SMP-7, SMP-10, and SMP-11 (p < 0.05). Thermal and pH treatments are popular processing methods of food products for altering their taste, physicochemical properties, nutritional ingredients, and safety. Therefore, the stability of APs on thermal and pH treatments is closely related to their application scopes [32,44,45]. Thermal treatment can eliminate the majority of spoilage and pathogenic microorganisms, and APs can effectively inhibit lipid peroxidation if they have strong heat-resistant properties. A combination of APs and heat treatment will significantly prolong the shelf life of products. In addition, APs with broad acid-alkali tolerance properties can be used in more food products [23]. Two antioxidant hexapeptides (WAFAPA and MYPGLA) from the hydrolysate of blue-spotted stingray showed high stability because their EC 50 values on HO· were not significantly different when they were treated at 25-100 • C or at pH values of 3-11 (p > 0.05) [23]. Yang et al. reported that MDLFTE and WPPD from protein hydrolysate of Tergillarca granosa could not stand the high-temperature (>80 • C) and strong basic (pH > 9.0) processing [25]. Similarly, Jang et al. reported that ATSHH from hydrolysate of sandfish incubated at 50-90 • C reduced its partial DPPH· scavenging activity. In addition, ATSHH lost some biological activity when it was treated at strong basicity (pH [10][11][12] or acidity (pH 2) [46]. Our results indicate that SMP-3 (PELDW), SMP-7 (WPDHW), SMP-10 (FGYDWW), and SMP-11 (YLHFW) had similar thermal and pH stability with MDLFTE, WPPD, and ATSHH because they could only keep their high activity when they were treated under a low temperature (<60 • C) and a moderate pH environment (pH 5-9).

Preparation of Protein Hydrolysate from Spanish Mackerel Muscle
The Spanish mackerel muscle was homogenized and blended with isopropanol at a ratio of 1:4 (w/v) and stand at 30 ± 2 • C for 6 h, and the isopropanol was changed each 2.0 h. Finally, the solution was filtered using a cheesecloth and the solid precipitate was air-dried at 35 ± 2 • C.
The hydrolytic process of the defatted muscle using five proteases was performed following the previous methods [9]. The dispersions of the defatted muscle (1%, w/v) were ultrasonic for 15 min and hydrolyzed separately on their optimal hydrolysis parameters ( Table 4). The hydrolytic process of the defatted muscle using in vitro gastrointestinal (GI) digestion was performed on the method described by Yang et al. [10]. Briefly, the defatted muscle powders dispersed in DW (pH 1.5, 1%) were ultrasonic for 15 min and firstly hydrolyzed by pepsin with a dosage of 1 g pepsin/100 g defatted powder under the conditions of 37.0 ± 2 • C and pH 1.5. Two hours later, the pH of the degraded solution was adjusted to 7.0 using a 1.0 M NaOH solution and further hydrolyzed using trypsin with a dosage of 1 g trypsin/100 g defatted powder for 2 h.
After 4 h of hydrolysis, the hydrolysis solutions were heated at 90 ± 2 • C for 20 min and centrifuged at 8000 g for 25 min at −4 • C. The resulting supernatant were freeze-dried and kept at −20 • C. The dispersions of the defatted muscle (1%, w/v) were ultrasonic for 15 min and freeze-dried, and the freeze-dried powder was referred to as SMP. The protein hydrolysate of Spanish mackerel muscle prepared using in vitro GI digestion method was referred to as SMPH. The concentrations of hydrolysates and their fractions were expressed as mg protein/mL and measured by the dye binding method of Bradford [47], and BSA was used as the standard protein. Figure 11 shows the flow diagram of isolating APs from SMPH. SMPH was fractionated using ultrafiltration with 3, 5, and 10 kDa MWCO membranes (Millipore, Hangzhou, China), and four fractions termed SMPH-I (MW < 3 kDa), SMPH-II (3 kDa < MW < 5 kDa), SMPH-III (5 kDa < MW < 10 kDa), and SMPH-IV (>10 kDa) were collected and lyophilized. Figure 11. The flow diagram of isolating APs from the hydrolysate (SMPH) of Spanish mackerel muscle prepared using in vitro GI digestion.

Chromatography Isolation of APs from SMPH-I
The chromatography isolation process was performed according to previous methods [48,49]. Five milliliters of an SMPH-I solution (40.0 mg/mL) were injected into a pre-equilibrated DEAE-52 cellulose column (1.6 × 80 cm) and separately eluted with 150 mL of DW, 0.1 M NaCl, 0.5 M NaCl, and 1.0 M NaCl solution at a flow rate of 1.0 mL/min. Each eluate (5.0 mL) was collected and detected at 214 nm. Finally, five fractions (SMPH-I-1 to SMPH-I-5) were prepared on the chromatogram. Five milliliters of SMPH-I-3 were separated on a column of Sephadex G-25 (2.6 × 160 cm) eluted with DW at a flow rate of 0.6 mL/min. Each eluate (3.0 mL) was collected and measured at 214 nm. Three fractions (SMPH-I-3a to SMPH-I-3c) were prepared on the chromatogram. Five milliliters of SMPH-I-3c were further purified using RP-HPLC with a Zorbax, SB C-18 column (4.6 × 250 mm, 5 µm) on an Agilent 1260 (Santa Rosa, CA, USA). The sample was eluated with a linear gradient of ACN (0-40% in 0-30 min) in 0.1% TFA at a flow rate of 1.0 mL/min. Twelve major peaks (SMP-1 to SMP-12) were isolated on the absorbance at 214 nm.

Analysis of Amino Acid Sequence and Molecular Mass
The amino acid sequences of four APs (SMP-3, SMP-7, SMP-10, and SMP-11) were measured on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc, Foster City, CA, USA). The molecular masses of four APs (SMP-3, SMP-7, SMP-10, and SMP-11) were measured using a Q-TOF mass spectrometer coupled with an ESI source (Waters, Los Angeles, CA, USA), respectively.

Radical Scavenging Assays
The DPPH·, HO·, and O − 2 · scavenging assays of four APs (SMP-3, SMP-7, SMP-10, and SMP-11) were performed according to the previous methods [28,31], and the EC 50 was defined as the concentration where a sample caused a 50% decrease of the initial radical concentration.

DPPH· Scavenging Assay
Two milliliters of samples consisting of DW and different concentrations of the analytes were placed in cuvettes, and 500 µL of an ethanolic solution of DPPH (0.02%) and 1.0 mL of ethanol were added. A control sample containing the DPPH solution without the sample was also prepared. In the blank, the DPPH solution was substituted with ethanol. The DPPH· scavenging activity was calculated using the following formula: where A s is the absorbance rate of the sample, A c is the control group absorbance, and A b is the blank absorbance.

HO· Scavenging Assay
One milliliter of a 1.865 mM 1,10-phenanthroline solution and 2.0 mL of the sample were added to a screw-capped tube and mixed. Afterwards, 1.0 mL of a FeSO 4 ·7H 2 O solution (1.865 mM) was added to the mixture. The reaction was initiated by adding 1.0 mL of H 2 O 2 (0.03%, v/v). After incubating at 37 • C for 60 min in a water bath, the absorbance of the reaction mixture was measured at 536 nm against a reagent blank. The reaction mixture without any antioxidant was used as the negative control, and a mixture without H 2 O 2 was used as the blank. The HO· scavenging activity was calculated using the following formula: where A s , A n , and A b are the absorbance values determined at 536 nm of the sample, the negative control, and the blank after the reaction, respectively. O − 2 · Scavenging Assay Superoxide anions were generated in 1.0 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1.0 mL of NADH (624 mM), and 1.0 mL of different sample concentrations. The reaction was initiated by adding 1.0 mL of phenazine methosulphate (PMS) solution (120 µM) to the reaction mixture. The absorbance was measured at 560 nm against the corresponding blank after 5 min incubation at 25 • C. The O − 2 · scavenging activity was calculated using the following equation: where A c is the absorbance without sample, and A s is the absorbance with sample.

Lipid Peroxidation Inhibition Assay
The lipid peroxidation inhibition activity of the APs was measured in the linoleic acid model system according to the method of Wang et al. [28]. Briefly, a sample (5.0 mg) was dissolved in 10.0 mL of 50.0 mM phosphate buffer solution (PBS, pH 7.0) and added to 0.13 mL of a solution of linoleic acid and 10.0 mL of 99.5% ethanol. The total volume was adjusted to 25 mL with DW. The mixture was incubated in a conical flask with a screw cap at 40 • C in a dark room, and the degree of oxidation was evaluated by measuring ferric thiocyanate values. The reaction solution (100 µL) incubated in the linoleic acid model system was mixed with 4.7 mL of 75% ethanol, 0.1 mL of 30% ammonium thiocyanate, and 0.1 mL of 20 mM ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured at 500 nm following color development with FeCl 2 and thiocyanate at different intervals during the incubation period at 40 • C.

Statistical Analysis
The data are expressed as the mean ± SD (n = 3). ANOVA test for differences between means of each group was used to analyze data using SPSS 19.0 (SPSS Corporation, Chicago, IL, USA). A p-value of less than 0.05 was considered statistically significant.

Conclusions
In the experiment, the proteins of Spanish mackerel (S. niphonius) muscle were hydrolyzed under five kinds of enzymes and in vitro GI digestion, and four APs (SMP-3, SMP-7, SMP-10, and SMP-11) were isolated from the hydrolysate prepared using in vitro GI digestion and identified as PELDW, WPDHW, FGYDWW, and YLHFW, respectively. PELDW, WPDHW, FGYDWW, and YLHFW showed high radical scavenging activity, lipid peroxidation inhibition ability, and protective effects on plasmid DNA (pBR322DNA) against oxidative damage induced by H 2 O 2 . Moreover, four APs (PELDW, WPDHW, FGYDWW, and YLHFW) from protein hydrolysate of Spanish mackerel muscle might be applied as an ingredient in new functional foods and products under a normal temperature (<40 • C) and a moderate pH environment (pH 5-9).