Purification and Identification of Antioxidant Peptides from Enzymatic Hydrolysates of Tilapia (Oreochromis niloticus) Frame Protein

Tilapia frame protein was hydrolyzed by different proteases, including properase E, pepsin, trypsin, flavourzyme, neutrase, gc106 and papain, to obtain antioxidant peptides. The tilapia frame protein hydrolysate (TFPH) obtained by trypsin exhibited the highest degree of hydrolysis and antioxidant activity. Three series of peptides (TFPH1, TFPH 2 and TFPH 3) were obtained by ultrafiltration of TFPH through molecular weight cut-off membranes of 5, 3 and 1 kDa, respectively, and their IC50 values on scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide anion radical (•O2), hydrogen peroxides (H2O2) and hydroxyl radical (•OH) activities were determined and compared with glutathione (GSH). The results showed that TFPH1 had the highest antioxidant activity. TFPH1 was further purified using ion exchange chromatography, gel filtration chromatography, and reversed phase high performance liquid chromatography (RP-HPLC). Finally, two antioxidant peptides were identified and the amino acid sequences were identified as Asp-Cys-Gly-Tyr (456.12 Da) and Asn-Tyr-Asp-Glu-Tyr (702.26 Da), respectively. The IC50 values of two peptides on hydroxyl radical scavenging activity were 27.6 and 38.4 μg/mL, respectively.


Introduction
Reactive oxygen species (ROS) are unavoidable metabolic byproducts of normal aerobic metabolism [1]. ROS can attack macromolecules such as membrane lipids, proteins, and DNA, leading to many health disorders such as hypertension, cardiovascular, cancer, diabetes mellitus, and neurodegenerative and inflammatory diseases with severe tissue injuries [2]. Antioxidants may have a positive effect on human health as they can protect the human body against damage by ROS.
Tilapia (Oreochromis niloticus) is important species in freshwater aquaculture. It is the third most widely cultured fish, after carp and salmonids [16]. Over the past years, the production of tilapia has been increased steadily and has become one of China's leading aquatic product exports. The tilapia fish processing industry generates a wide variety of by-products such as heads, skins, viscera and frames. In view of utilizing these fish industry wastes, tilapia by-product proteins and protein hydrolysates are being studied by several researchers all over the World. Jamilah et al. [17] isolated gelatin from tilapia skin and studied its properties. Tilapia skin gelatin hydrolysates were proved to be a good antioxidant resource [18], and Dekkers et al. [19] showed that tilapia protein hydrolysates in mahi mahi red muscle had good oxidative stability.
However, there are few reports on antioxidant peptides from tilapia frame protein. Fish frame is one of the major fractions of fish wastes (accounting for more than 15% of the fish weight), and it contains around 30% protein [20]. This protein could be a good candidate for nutraceuticals. Therefore, the objective of this study was to prepare tilapia protein hydrolysates using different proteases, evaluate their antioxidant properties using different in vitro systems, and further purify antioxidant peptides.

Preparation of Tilapia Frame Protein Hydrolysates (TFPH)
In the present study, tilapia frame protein was separately hydrolyzed by properase E, pepsin, trypsin, flavourzyme, neutrase, gc106 and papain, for the production of antioxidant peptides. As shown in Table 1, the degree of hydrolysis (DH) of the hydrolysates were determined and was observed to be 13.8, 15.1 and 12.7% for properase E, trypsin and flavourzyme, respectively, the other proteolytic enzymes showed DH values lower than 10%. Values not sharing a common letter are significantly different at p < 0.05.
The antioxidant activity of a substance can be identified by assessing scavenging activities on free radicals generated in oxidative systems. As shown in Table 1, peptides from seven hydrolysates were evaluated for their antioxidant activities using scavenging DPPH, • O 2 , H 2 O 2 and •OH activity assays. Seven hydrolysates could act as significant scavengers of the four radicals, and the hydrolysates from trypsin showed stronger antioxidant activities. Based on the results, trypsin was chosen to prepare tilapia frame protein hydrolysates (TFPH).

Ultrafiltration
Ultrafiltration is a technique used commonly both on the laboratory and commercial scale to fractionate, purify, and concentrate proteins and peptides [21]. It has been reported to be a useful method for improving the antioxidant activities of protein hydrolysates [13,22,23]. In this study, TFPH was separated into three fractions (TFPH 1 with MW < 1 kDa, TFPH 2 with 1 kDa < MW < 3 kDa and TFPH 3 with 3 kDa < MW < 5 kDa) by ultrafiltration. Based on nitrogen contents, TFPH1, TFPH 2, and TFPH 3 represented 41.9%, 32.7% and 25.4% of hydrolysates, respectively. TFPH1 had the highest yield among all the fractions.
In order to evaluate the antioxidant activities, the IC 50 values of three fractions on scavenging activities against the four radicals were calculated and compared with GSH. As shown in Figure 1, the IC 50 values of TFPH1 and GSH on the DPPH-scavenging activity were 1.92 and 0.69 mg/mL, respectively. TFPH1 had significantly higher DPPH-scavenging activity than other fractions (p < 0.05). However, the activity of TFPH1 was lower than that of GSH (p < 0.05). You et al. [5] reported the IC 50 value of DPPH-scavenging activity of loach protein hydrolysate was 2.64 mg/mL, which was similar to our result. The • O 2 -scavenging activity of TFPH1 and GSH were 2.01 and 0.37 mg/mL, respectively. The activity of TFPH1 was significant higher than TFPH2 and TFPH3 (p < 0.05). Furthermore, TFPH1 also showed high scavenging H 2 O 2 and •OH activities, and its IC 50 values were 2.44 and 0.98 mg/mL, respectively, while, the IC 50 values of GSH were 0.42 and 0.24 mg/mL, respectively. This indicated that TFPH1 might contain peptides which are more easily accessible to the hydroxyl radicals and allows these peptides to trap the radicals more easily [16]. Several studies have looked at the contribution of molecular size of peptide mixtures in protein hydrolysates to their bioactivity. These studies showed that low molecular weight fractions in general contained more potent antioxidative peptides [24][25][26].

Purification of Antioxidant Peptides
Hydroxyl radical is the most reactive radical and can be formed from superoxide anion and hydrogen peroxide in the presence of metal ions, such as copper or iron. The hydroxyl radical has been demonstrated to be highly damaging species in free radical pathology, attacking almost every molecule in living cells. Therefore, the removal of hydroxyl radical is probably one of the most effective defence of a living body against various diseases. Base on this, the scavenging •OH activity was selected as the indicator of purification of antioxidant peptides in the study [16].

Purification of Antioxidant Peptide Using Ion-Exchange Chromatography
SP Sephadex C-25 (main functional group: sulphopropyl) was one of the strong cation exchangers and it was widely utilized for separating antioxidant peptides [16,24]. In order to prepare the antioxidant peptides, TFPH1 was separated by SP-Sephadex C-25 ion exchange chromatography, showing six different peaks, named A, B, C, D, E and F ( Figure 2a). As shown in Figure 2b, fraction C was found to possess the strongest hydroxyl radical scavenging activity, among six fractions, with the IC 50 value of 0.47 mg/mL. Different letters indicate significant differences (p < 0.05).

Purification of Antioxidant Peptide Using Gel Filtration
Peptide length was considered to be closely related to biological activities. Earlier studies have shown that peptide length had a significant effect on antioxidant activity [25,26], so fraction C was further fractionated by gel filtration chromatography on a Sephadex G-25 column, showing four different peaks, named C1, C2, C3, and C4 (Figure 3a). The C3 fraction exhibited stronger antioxidant activity than the other fractions (p < 0.05), with the IC 50 value being 280.97 μg/mL (Figure 3b). Different letters indicate significant differences (p < 0.05).

Purification of Antioxidant Peptide Using RP-HPLC
The C3 fraction was further isolated by RP-HPLC on the semi-preparative C 18 column using a linear gradient of acetonitrile containing 0.1% TFA. As shown in Figure 4a, fifteen peaks, designated as C3-1 to C3-15, were collected separately. Each fraction was pooled and concentrated and the C3-9 and C3-11 showed the higher scavenging •OH activity at a concentration of 50 μg/mL. Fractions C3-9 and C3-11 were further identified by RP-HPLC on the Zorbax SB-C 18 analysis using a linear gradient of acetonitrile containing 0.1% TFA. One main peak was obtained from C3-9 ( Figure 4b

Identification of Amino Acid Sequence of Antioxidant Peptide
As shown in Figure 5, the two peptides (C3-9 and C3-11) were obtained from TPFH and the molecular weights were 456.12 and 702.26 Da, respectively. The amino acid sequences were Asp-Cys-Gly-Tyr and Asn-Tyr-Asp-Glu-Tyr, respectively.  In the sequences of our purified peptides, direct proton-donators such as Tyr, Cys, Gly, Glu and Asp are present, which is very important to the radical-scavenging activity of peptides due to their ability to quench unpaired electrons or radicals by donating protons [27].
Some studies have reported that peptide sequences containing Tyr showed strong antioxidant activity. Especially, when the presence of Tyr was at terminals of the peptide sequence, such as C3-9

C3-11
Leu-Pro-His-Ser-Gly-Tyr, Leu-His-Tyr, Val-Thr-Pro-Tyr and Asp-Val-Cys-Gly-Arg-Asp-Val-Asn-Gly-Tyr. The antioxidant activity of Tyr may be explained by the special capability of phenolic groups to serve as hydrogen donors, which is one mechanism of inhibiting the radical-mediated peroxidizing chain reaction [28,29]. Our results were similar to these previous studies, and the two peptides obtained from TPFH contained Tyr at the terminus.
Previous studies show Cys is hydrophobic in nature and can interact directly with free radicals by donating the sulfur hydrogen [9], so the presence of Cys is one of the reasons for the good antioxidant activity of the isolated peptide. Moreover, hydroxyl radicals could be formed from superoxide anion and hydrogen peroxide in the presence of transition metal ions, such as Fe 2+ and Cu 2+ , so chelating metal ions may inhibit the formation of the hydroxyl radical [16]. Acidic amino acids such as Asn and Glu play important roles in the chelation of metal ions by their side chains [30,31]. In addition, Rajapakse et al. [32] reported that presence of Asp seemed to play a vital role irrespective of its position as observed in several antioxidative peptide sequences. It was assumed that the presence of both Cys and Asp residues within the sequence of C3-9, and Asn, Asp and Glu within the sequences of C3-11 seemed to play a vital role for their antioxidant activities.

Enzymatic Hydrolysis
Tilapia frame was treated with high-pressure cooking at 120 °C for 30 min to soften the backbone and then homogenized in a high speed tissue homogenizer (Cany Precision Instrument Co., Ltd., Shanghai, China).
The tilapia frame protein was hydrolyzed by seven enzymes, respectively. Tilapia frame homogenate (protein concentration 25 mg/mL) was adjusted to optimum (based on the manufacturer recommendations) of the respective enzyme used ( Table 2). The enzymatic hydrolysis process was without pH control and the enzyme inactivation was accomplished by heating for 3 min in boiling water. The obtained hydrolysates were centrifuged at 8,000 × g for 10 min and the supernatant was collected.

Determination of the Degree of Hydrolysis
Degree of hydrolysis (DH) was evaluated according to ninhydrin colorimetric method based on the equation: DH (%) = h (mmol/g)/h tot (mmol/g) × 100%, where h is the number of broken peptide bonds per unit weight; and h tot is the total number of peptide bonds per unit weight. The h tot for tilapia frame protein was 9.3 mmol per gram protein [33,34].

1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity Assay
The DPPH radical-scavenging activity was measured according to the method of Huang and Mau [35]. An aliquot of 1 mL of hydrolysates was mixed with 1 mL of methanol solution containing 1 mM DPPH radicals. The mixture was allowed to stand for 40 min in the dark, and the absorbance was monitored at 517 nm. Distilled water was used instead of hydrolysates as a blank. GSH was used as a positive control. Scavenging DPPH activity was calculated according to the following equation [Equation (1)]: where A 1 is sample absorbance and A 0 is blank absorbance.

Superoxide Anion ( • O 2 ) Scavenging Assay
• O 2 scavenging activity was assessed by chemiluminescence analysis of a pyrogallol luminol system with a slight modification [36]. A volume of 50 μL of freshly prepared pyrogallol solution (0.625 mM), 100 μL of hydrolysates and 850 μL of luminal solutions (1 mM, in 0.1 M NaCO 3 ) were mixed in a reaction tube. Luminol chemiluminescence of the system was measured using an ultraweak luminescence analyzer (BPCL, Beijing, China). Distilled water was used instead of hydrolysates as a blank. GSH was used as a positive control. Scavenging • O 2 activity was calculated according to Equation (2): where C 1 is sample chemiluminescence and C 0 is blank chemiluminescence.

Hydrogen Peroxide (H 2 O 2 ) Scavenging Assay
H 2 O 2 scavenging activity of hydrolysates was determined according to the method described by Zhuang et al. [37] with a few modifications. A solution of hydrogen peroxide (4 mM) was prepared in phosphate buffered saline (0.1 M, pH 7.4). Volumes of 200 μL of samples in distilled water were mixed with 2.8 mL of hydrogen peroxide solution. Absorbance of the mixture was measured at 230 nm after 10 min against the blank solution in phosphate buffer without hydrogen peroxide. Distilled water was used instead of hydrolysates as control. GSH was used as a positive control. The percentage of H 2 O 2 scavenging of hydrolysates was calculated according to the Equation 1.

Hydroxyl Radical (•OH) Scavenging Assay
•OH scavenging activity was assessed using an ascorbic acid-Cu 2+ -hydrogen superoxide-yeast suspension system with a slight modification [38]. A volume of 200 μL of freshly prepared ascorbic acid solution (2 mM), 0.4 mL of CuSO 4 (2 mM), 50 μL of luminal (1 mM, in 0.1 M NaCO 3 ), 200 μL of yeast suspension (7.5 g per 100 mL) and 600 μL of hydrolysates were injected into the reaction tube and kept in a water bath at 37 °C for 30 min, Then 600 μL of H 2 O 2 solution (6.8 mM) were added to start the reaction. Luminol chemiluminescence of the system was measured using an ultraweak luminescence analyzer. Distilled water was used instead of hydrolysates as a blank. GSH was used as a positive control. •OH scavenging activity was calculated according to Equation (2).

Ultrafiltration
The supernatant of hydrolysates showing the strongest antioxidant activity were fractioned using three different molecular weight cut-off (MWCO, 5, 3, 1 kDa) membranes (Yadong Hitech Co. Ltd, Shanghai, China). The fractioned temperature was kept at 4 °C and the pressure was adjusted to 0.15 MPa. Three series of peptides were obtained, noting TFPH 1 with MW < 1 kDa, TFPH 2 with 1 kDa < MW < 3 kDa and TFPH 3 with 3 kDa < MW < 5 kDa. The peptides were lyophilized and stored at −20 °C until future use. Hydrolysate nitrogen contained all nitrogens derived from proteins, peptides, and free amino acids. Protein content was measured according to the Kjeldahl procedure and multiplying the nitrogen value by 6.25.

Ion-Exchange Chromatography
TFPH was loaded onto a cationic exchange column (2.6 × 50 cm) with a SP Sephadex C-25 equilibrated with sodium acetate buffer (20 mM, pH 4.0). The column was washed with the same buffer and eluted with a linear gradient of NaCl concentrations from 0 to 1.0 M at a flow rate of 0.5 mL/min and monitored at 220 nm. Every 5 mL of eluted solution was collected. Fractions with the desire peak were pooled and lyophilized.

Gel filtration Chromatography
The fraction with the highest hydroxyl radical scavenging activity obtained from SP Sephadex C-25 was fractionated through Sephadex G-25 gel filtration column (2.0 × 100 cm) using distilled water as the eluting solvent at 0.5 mL/min flow rate and monitored at 220 nm. Every 5 mL of eluted solution was collected. Fractions with the desire peaks were pooled and lyophilized.

High-Performance Liquid Chromatography (HPLC)
The fraction with the highest hydroxyl radical scavenging activity obtained from Sepahadex G-25 was further separated by reversed phase high performance liquid chromatography (RP-HPLC) on a Zorbax semi-preparative C18 (9.4 × 250 mm) column (Agilent Technologies, Palo Alto, CA, USA), using a linear gradient of acetonitrile containing 0.1% TFA (5-30%, in 30 min) at a flow rate of 2.0 mL/min. The fractions showing the high antioxidant activity were identified on a Zorbax analytical C18 (4.6 × 250 mm) column (Agilent Technologies) at a flow rate of 1.0 mL/min with a linear gradient of acetonitrile containing 0.1% TFA (5-25%, in 20 min). The purification procedures were repeated until enough samples were collected for the activity assay and sequence identification.

Amino Acid Sequence of the Purified Peptides
An accurate molecular mass and amino acid sequence of the purified peptides were determined using a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled with an electrospray ionization (ESI) source.

Statistical Analysis
All results were expressed as means ± standard deviation and analyzed by the SPSS 11.5 statistical software. Data were analyzed using one-way analysis of variance (ANOVA). p < 0.05 indicated statistical significance.

Conclusions
Seven proteases were selected to hydrolyze tilapia frame protein. The tilapia frame protein hydrolysate (TFPH) obtained by trypsin showed the highest degree of hydrolysis and antioxidant activity. TFPH was ultrafiltered through the different MWCO membranes and three series of peptides, TFPH 1 with MW < 1 kDa, TFPH 2 with 1 kDa < MW < 3 kDa and TFPH 3 with 3 kDa < MW < 5 kDa, were obtained, respectively. TFPH1 had higher radicals-scavenging activity than TFPH2 and TFPH3 with the highest yield, so TFPH 1 was further fractionated using ion exchange chromatography, gel chromatography and RP-HPLC. Two antioxidant peptides were purified, and their sequences were identified as Asp-Cys-Gly-Tyr (456.12 Da) and Asn-Tyr-Asp-Glu-Tyr (702.26 Da). They displayed high hydroxyl radical scavenging activity with the IC 50 values of 27.6 and 38.4 μg/mL. Based on these results, these two peptides have the potential to be developed into new health foods.