Purification and Characterization of Antioxidant Peptides Derived from Protein Hydrolysate of the Marine Bivalve Mollusk Tergillarca granosa

In this report, protein hydrolysate (TGH) of blood cockle (Tegillarca granosa) was prepared using a two-enzyme system (Alcalase treatment for 1.5 h following Neutrase treatment for 1.5 h). Subsequently, six antioxidant peptides were isolated from TGH using ultrafiltration and chromatography methods, and their amino acid sequences were identified as EPLSD, WLDPDG, MDLFTE, WPPD, EPVV, and CYIE with molecular weights of 559.55, 701.69, 754.81, 513.50, 442.48, and 526.57 Da, respectively. In which, MDLFTE and WPPD exhibited strong scavenging activities on DPPH radical (EC50 values of 0.53 ± 0.02 and 0.36 ± 0.02 mg/mL, respectively), hydroxy radical (EC50 values of 0.47 ± 0.03 and 0.38 ± 0.04 mg/mL, respectively), superoxide anion radical (EC50 values of 0.75 ± 0.04 and 0.46 ± 0.05 mg/mL, respectively), and ABTS cation radical (EC50 values of 0.96 ± 0.08 and 0.54 ± 0.03 mg/mL, respectively). Moreover, MDLFTE and WPPD showed high inhibiting ability on lipid peroxidation. However, MDLFTE and WPPD were unstable and could not retain strong antioxidant activity at high temperatures (>80 °C for 0.5 h), basic pH conditions (pH > 9 for 2.5 h), or during simulated GI digestion. In addition, the effect of simulated gastrointestinal digestion on TGP4 was significantly weaker than that on MDLFTE. Therefore, MDLFTE and WPPD may be more suitable for serving as nutraceutical candidates in isolated forms than as food ingredient candidates in functional foods and products.


Introduction
Toxic reactive oxygen species (ROS) induced by oxidative stress destroy structures of some functional biomacromolecules including DNA, proteins, and membrane lipids, which further lead to some chronic diseases, such as liver damage, type 2 diabetes, asthma, neurodegenerative diseases, and arthritis [1][2][3]. In addition, oxidative deterioration produces some off-flavors and harmful lipid metabolites, which negatively influence the food quality and somatic functions [2,4]. Therefore, eliminating superfluous ROS is important for keeping cellular homeostasis. At present, people often use synthetic antioxidants to prevent and intervene ROS damage, but the negative effects of synthetic antioxidants, including liver damage and carcinogenesis, limit their scope of usage and dosage [5,6].

Amino Acid Sequence Analysis and Mass Spectrometry of APs (TGP1-TGP6)
The amino acid sequences and molecular mass of six APs (TGP1-TGP6) were determined using a protein sequencer and electrospray ionization (ESI)-mass spectrometer (MS), and the data are shown in Table 1 (Table 1).

Lipid Peroxidation Inhibition Ability
As presented in Figure 6, the absorbance values of TGP3 and TGP4 solutions at 500 nm were significantly (p < 0.5) lower than those of other four APs (TGP1, TGP2, TGP5, and TGP6) and the blank control without antioxidant. However, the 500 nm absorbance values of TGP3 and TGP4 solutions were little greater than that of the positive control (GSH). The oxidative process is complicated in food and biological systems and embroiled in multifarious reactions for propagation of lipid radicals hydroperoxides [3,5]. Those data of TGP3 and TGP4 in the linoleic acid model system indicated that they have a strong ability to inhibit lipid peroxidation. In addition, these abilities of TGP3 and TGP4 were similar to that of GSH in the seven days incubation.
Molecular size plays a key role in the antioxidant capacities of APs [5,24]. Six APs (TGP1-TGP6) from protein hydrolysate of blood cockle (T. granosa) are tetrapeptide to hexapeptide with MWs ranging 442.48 Da-754.81 Da (Table 1). These data indicated that six APs could easily interact with free radicals to inhibit the lipid peroxidation. Furthermore, amino acid composition and sequence are believed to play major contributions to the activities of APs [5,29,36]. Hydrophobic amino acid residues, including aliphatic (Val, Leu, and Ile), aromatic (Phe, Trp, and Tyr), and sulfur-containing (Met and Cys) can play their functions on radical scavenging because of their high reactivity to hydrophobic PUFAs in lipid-rich foods [26,41]. Aromatic residues can donate protons to electron deficient radicals to keep ROS stable during the radical scavenging process [5]. Sulfur-containing amino acid residues (Met and Cys) might work as a reactive site, where the peptide could scavenge oxidants through the formation of a sulfoxide structure after oxidation to stop free-radical chain reactions [2,[42][43][44]. Therefore, hydrophobic/aromatic amino acid residues in TGP3 (Met, Leu, and Phe) and TGP4 (Trp and Pro) should contribute to their activity through helping them to contact target radicals. Giménez et al. [45] and Zhu et al. [46] found that polar amino acid residues (Glu, Asp, and Lys) played a critical role in antioxidant activity including metal ion chelating and hydroxyl radical scavenging activities. Gly residue can make the peptide skeleton more flexible and its single hydrogen atom can serve as proton-donating to neutralize free radicals [47][48][49]. Therefore, polar amino acids including Asp and Glu residues in TGP3, and Asp residues in TGP4 could play a critical role in the lipid peroxidation inhibition activities.

Effects of Thermal, pH, and Simulated Gastrointestinal (GI) Digestion Treatments on TGP3 and TGP4 Stability
As shown in Figure 7A, heat treatments could influence the hydroxyl radical scavenging activity (expressed as EC 50 ) of TGP3 and TGP4. When TGP3 and TGP4 were treated at 25, 37, and 60 • C for Heat treatment is a common method of food processing and APs are helpful to lengthen the food shelf-life if they can keep their activity after heating. Peptides with large-scale pH stability can be incorporated into diverse liquid products and keep their bioactivity. Therefore, thermal and pH stability of peptides are important indexes for their application in functional products, and characterization of those properties can help to design their potential processing parameters [15,50]. ATSHH from protein hydrolysate of sandfish (Arctoscopus japonicus) partially lost its DPPH radical scavenging activity when it was incubated at 50-90°C. In addition, ATSHH bored moderate losses of activity under basic (pH 10-12) and acidic (pH 2) conditions [50]. However, there are no significant (p > 0.05) differences when WAFAPA and MYPGLA from blue-spotted stingray are incorporated during heat (25-100°C) and pH (3)(4)(5)(6)(7)(8)(9)(10)(11) treatments [15]. In the experiment, EC50 values of TGP3 and TGP4 on hydroxyl radical significantly (p < 0.005) increased at temperatures above 80°C and pH values higher than 9, which indicated that TGP3 and TGP4 were not suitable for high temperature treatment and basic (pH > 9.0 for 2.5 h) food products. The capacity of TGP3 and TGP4 to resist GI digestion is one of the key requirements for their applications in vivo, which may tell whether they will be used as food ingredients or nutraceuticals in isolated forms. Then, simulated GI digestion is usually used to evaluate the fate of peptides before exploring their bioactivity and bioavailability in vivo [50]. In this assay, EC50 values of TGP3 and TGP4 with simulated GI digestion on hydroxyl radical were significantly (p < 0.05) increased ( Figure 7C), which reflected that TGP3 and TGP4 are partially susceptible to be degradated by GI digestive enzymes. In addition, Figure 7C shows that the effect of GI digestion on TGP4 was significantly weaker than that on TGP3. The results were in accordance with the report by Segura-Campos et al. that bioactive peptides containing Pro residues generally stand up to degradation by GI digestive enzymes [51]. Taken together, TGP3 and TGP4 are unstable under high thermal food processing and cannot retain bioactivity under basic pH conditions. The EC 50 values of TGP3 and TGP4 on hydroxyl radical treated at pH 3 to 11 were presented in Figure 7B. There were no significant (p > 0.05) differences when the pH changed from 3 to 7. However, EC 50 values of TGP3 and TGP4 on hydroxyl radical in acidic and neutral conditions were significantly (p < 0.05) lower than those of alkaline conditions (pH 9 to 11).
Heat treatment is a common method of food processing and APs are helpful to lengthen the food shelf-life if they can keep their activity after heating. Peptides with large-scale pH stability can be incorporated into diverse liquid products and keep their bioactivity. Therefore, thermal and pH stability of peptides are important indexes for their application in functional products, and characterization of those properties can help to design their potential processing parameters [15,50]. ATSHH from protein hydrolysate of sandfish (Arctoscopus japonicus) partially lost its DPPH radical scavenging activity when it was incubated at 50-90 • C. In addition, ATSHH bored moderate losses of activity under basic (pH 10-12) and acidic (pH 2) conditions [50]. However, there are no significant (p > 0.05) differences when WAFAPA and MYPGLA from blue-spotted stingray are incorporated during heat (25-100 • C) and pH (3)(4)(5)(6)(7)(8)(9)(10)(11) treatments [15]. In the experiment, EC 50 values of TGP3 and TGP4 on hydroxyl radical significantly (p < 0.005) increased at temperatures above 80 • C and pH values higher than 9, which indicated that TGP3 and TGP4 were not suitable for high temperature treatment and basic (pH > 9.0 for 2.5 h) food products. The capacity of TGP3 and TGP4 to resist GI digestion is one of the key requirements for their applications in vivo, which may tell whether they will be used as food ingredients or nutraceuticals in isolated forms. Then, simulated GI digestion is usually used to evaluate the fate of peptides before exploring their bioactivity and bioavailability in vivo [50]. In this assay, EC 50 values of TGP3 and TGP4 with simulated GI digestion on hydroxyl radical were significantly (p < 0.05) increased ( Figure 7C), which reflected that TGP3 and TGP4 are partially susceptible to be degradated by GI digestive enzymes. In addition, Figure 7C shows that the effect of GI digestion on TGP4 was significantly weaker than that on TGP3. The results were in accordance with the report by Segura-Campos et al. that bioactive peptides containing Pro residues generally stand up to degradation by GI digestive enzymes [51]. Taken together, TGP3 and TGP4 are unstable under high thermal food processing and cannot retain bioactivity under basic pH conditions.

Preparation of Protein Hydrolysate (TGH) of Blood Cockle (T. granosa)
The defatting process of blood cockle was carried out according to the previous methods [2,17]. Blood cockle internal organs were removed, and the resulting meat was rinsed and homogenized using a JJ-2 Kinematica (Jiangsu Jiangling Co., Ltd., Yancheng, China). The homogenate and isopropanol were mixed in a ratio of 1:4 (w/v) and stirred uninterrupted at 35 • C for 2.5 h, and the defatting process was performed three times. After that, the degreasing mixture was centrifuged at 9000 g for 20 min at 4 • C. The supernatant was removed, and the sediment was freeze-dried and stored at −20 • C.
The hydrolysis process was carried out using a two-enzyme system (Alcalase treatment for 1.5 h following Neutrase treatment for 1.5 h). The defatted precipitate (100 g) was dissolved (5%, w/v) in 0.05 M Tris-HCl buffer solution (pH 8.5) and hydrolyzed using Alcalase at 50.0 • C with enzyme dose 1.5% (w/w) for 1.5 h. After that, the pH of dispersions was changed with HCl solution (1.0 M) and hydrolyzed using Neutrase at pH 7.0, 55.0 • C with enzyme dose 1.5% (w/w) for 1.5 h. Afterwards, the hydrolysate was kept in a 95 • C water bath for 10 min to inactivate proteases and centrifuged at 12,000 g for 15 min. The resulted supernatant, referred to as TGH, was freeze-dried and stored at −20 • C.
The concentrations of TGH and hydrolysate fractions were expressed as mg protein/mL and determined by the dye binding method of Bradford (1976) with BSA as the standard protein [52]. Finally, TGH-I-5B3 was purified on an Agilent 1260 HPLC system (Agilent Ltd., Santa Rosa, CA, USA) with a Zorbax C-18 column (4.6 × 250 mm). The sample was eluated at a flow rate of 0.8 mL/min with a linear gradient of acetonitrile from 0% to 50% in 0-25 min in 0.1% TFA. Six APs (TGP1 to TGP6) were isolated on 214 nm absorbance and lyophilized.

Degree of Hydrolysis (DH)
DH analysis was performed according to the previously described method [6]. The hydrolysate (50µL) was mixed with 0.5 mL of 0.2 M phosphate buffered saline (PBS), pH 8.2 and 0.5 mL of 0.05% 2,4,6-trinitrobenzene sulfonic acid (TNBS) reagent. TNBS was freshly prepared before use by diluting with DW water. The mixture was incubated at 50 • C for 1 h in a water bath. The reaction was stopped by adding 1 mL of 0.1 M HCl and incubated at room temperature for 30 min. The absorbance was monitored at 420 nm. l-Leucine was used as a standard. To determine the total amino acid content, TGH was completely hydrolyzed with 6 M HCl with a sample to acid ratio of 1:100 at 120 • C for 24 h. DH (%) was calculated using the following equation: where A t is the amount of a-amino acids released at time t, A 0 is the amount of amino acids in the supernatant at 0 h, and Amax is the total amount of a-amino acids obtained after acid hydrolysis at 120 • C for 24 h.

Radical Scavenging Activity
The DPPH radical, hydroxyl radical, superoxide anion radical, and ABTS cation radical scavenging activities were measured according to the previous methods [6,53,54]. The results were expressed as a half elimination ratio (EC 50 ) defined as the concentration by which a sample caused a 50% decrease of the initial concentration of DPPH radical, hydroxyl radical, superoxide anion radical, and ABTS cation radical, respectively, and the calculation method of EC 50 was according to linear relationship of radical scavenging rates and concentrations of samples [6,26].

DPPH Radical Scavenging Activity
Two millilitres 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 antioxidant activity of the sample was evaluated using the inhibition percentage of the DPPH radical with the following equation: where A is the absorbance rate of the sample, A 0 is the control group absorbance, and A b is the blank absorbance.
Hydroxyl Radical Scavenging Activity First, 1.0 mL of a 1.87 mM 1,10-phenanthroline solution and 2.0 mL of the sample were added to a screw-capped tube and mixed. Then, 1.0 mL of a FeSO 4 ·7H 2 O solution (1.87 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 being incubated 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 hydroxyl radical 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.

Superoxide Anion Radical Scavenging Activity
The superoxide anions were generated in 1 mL of nitrotetrazolium blue chloride (2.52 mM), 1 mL of nicotinamide adenine dinucleotide hydride (NADH) (624 mM) and 1 mL of different sample concentrations. The reaction was initiated by adding 1 ml of phenazine methosulphate 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 superoxide anion radical scavenging capacity was calculated using the following equation: Superoxide anion radical scavenging activity (%) = [(A control − A sample )/A control ] × 100% (4) where A control is the absorbance without sample and A sample is the absorbance with sample.

ABTS Cation Radical Scavenging Activity
The ABTS radical cation was generated by mixing ABTS stock solution (7 mM) with potassium persulphate (2.45 mM). The mixture was left in the dark at room temperature for 16 h. The ABTS radical solution was diluted in 5 mM PBS pH 7.4, to an absorbance of 0.70 ± 0.02 at 734 nm. One milliliter of diluted ABTS radical solution was mixed with one milliliter of different concentrations of samples. 10 min later, the absorbance was measured at 734 nm against the corresponding blank. The ABTS scavenging activity of samples was calculated using the following equation: where A control is the absorbance without sample and A sample is the absorbance with sample.

Lipid Peroxidation Inhibition Assay
The lipid peroxidation inhibition and radical scavenging assays of TGP1 to TGP6 were measured according to the previous method [6,55]. In brief, a sample (5.0 mg) was dissolved in 10 mL of 50 mM phosphate buffer (pH 7.0), and added to a solution of 0.13 mL of linoleic acid and 10 mL of 99.5% ethanol. Then, 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 the 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 by reading the absorbance at 500 nm following color development with FeCl 2 and thiocyanate at different intervals during the incubation period at 40 • C.

Amino Acid Sequence and Molecular Mass Analysis
Amino acid sequences and molecular masses of TGP1 to TGP6 were measured on the previous method [26,56]. TGP1 to TGP6 were subjected to N-terminal amino acid sequencing on an Applied Biosystems 494 protein sequencer (Perkin Elmer/Applied Biosystems Inc., Foster City, CA, USA). Edman degradation was performed according to the standard program supplied by Applied Biosystems. Accurate molecular masses of TGP1 to TGP6 were determined using a Q-TOF mass spectrometer (Micromass, Waters, Milford, MA, USA) coupled with an ESI source.

Stability Properties of TGP3 and TGP4 against Heat, pH, and Simulated GI Digestion Treatments
Stability of TGP3 and TGP4 were measured according to the previous method with minor modifications [49]. A temperature-controlled water bath at 25, 37, 60, 80, or 100 • C for 0.5 h was used to measure thermostability of TGP3 and TGP4. Effects of pH treatments (pH 3, 5, 7, 9, or 11) of sample solutions incubated at 25 • C for 2.5 h were assessed to analyze the pH stability of TGP3 and TGP4. A two-stage digestion model (pepsin for 1.0 h + pancreatin for 2.0 h) was applied to simulate GI digestion of TGP3 and TGP4. Hydroxyl radical scavenging activities (EC 50 value) of the treated TGP3 and TGP4 were measured according to the previous methods [6,26].

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

Conclusions
In the experiment, blood cockle (T. granosa) was hydrolyzed under a two-enzyme system (Alcalase treatment for 1.5 h following Neutrase treatment for 1.5 h) and six APs (TGP1-TGP6) were isolated from the resulting hydrolysate (TGH) and identified as EPLSD (TGP1), WLDPDG (TGP2), MDLFTE (TGP3), WPPD (TGP4), EPVV (TGP5), and CYIE (TGP6), respectively. Six APs (TGP1-TGP6), especially TGP3 and TGP4, exhibited high radical scavenging and lipid peroxidation inhibition capabilities. However, TGP3 and TGP4 are unstable and cannot retain antioxidant activity at high temperatures (>80 • C for 0.5 h), basic pH conditions (pH > 9 for 2.5 h), or during simulated GI digestion. Therefore, TGP3 and TGP4 may be more suitable to serve as nutraceutical candidates in isolated forms than as food ingredient candidates. In addition, in vivo experiments to elucidate the antioxidant mechanisms of the six APs (TGP1-TGP6) need to be performed in future.