Eighteen Novel Bioactive Peptides from Monkfish (Lophius litulon) Swim Bladders: Production, Identification, Antioxidant Activity, and Stability

In the study, papain was chosen from five proteases to hydrolyze proteins of monkfish swim bladders for effectively utilizing monkfish (Lophius litulon) processing byproducts, and the hydrolysis conditions of papain were optimized as hydrolysis temperature of 65 °C, pH 7.5, enzyme dose 2.5% and time 5 h using single-factor and orthogonal experiments. Eighteen peptides were purified from the swim bladder hydrolysate of monkfish by ultrafiltration and gel permeation chromatography methods and identified as YDYD, QDYD, AGPAS, GPGPHGPSGP, GPK, HRE, GRW, ARW, GPTE, DDGGK, IGPAS, AKPAT, YPAGP, DPT, FPGPT, GPGPT, GPT and DPAGP, respectively. Among eighteen peptides, GRW and ARW showed significant DPPH· scavenging activities with EC50 values of 1.053 ± 0.003 and 0.773 ± 0.003 mg/mL, respectively; YDYD, QDYD, GRW, ARW and YPAGP revealed significantly HO· scavenging activities with EC50 values of 0.150 ± 0.060, 0.177 ± 0.035, 0.201 ± 0.013, 0.183 ± 0.0016 and 0.190 ± 0.010 mg/mL, respectively; YDYD, QDYD, ARW, DDGGK and YPAGP have significantly O2−· scavenging capability with EC50 values of 0.126 ± 0.0005, 0.112 ± 0.0028, 0.127 ± 0.0002, 0.128 ± 0.0018 and 0.107 ± 0.0002 mg/mL, respectively; and YDYD, QDYD and YPAGP showed strong ABTS+· scavenging ability with EC50 values of 3.197 ± 0.036, 2.337 ± 0.016 and 3.839 ± 0.102 mg/mL, respectively. YDYD, ARW and DDGGK displayed the remarkable ability of lipid peroxidation inhibition and Ferric-reducing antioxidant properties. Moreover, YDYD and ARW can protect Plasmid DNA and HepG2 cells against H2O2-induced oxidative stress. Furthermore, eighteen isolated peptides had high stability under temperatures ranging from 25–100 °C; YDYD, QDYD, GRW and ARW were more sensitive to alkali treatment, but DDGGK and YPAGP were more sensitive to acid treatment; and YDYD showed strong stability treated with simulated GI digestion. Therefore, the prepared antioxidant peptides, especially YDYD, QDYD, GRW, ARW, DDGGK and YPAGP from monkfish swim bladders could serve as functional components applied in health-promoting products because of their high-antioxidant functions.


Introduction
Bioactive peptides (BPs) comprise 3-30 amino acid (AA) residues with molecular weights (MWs) ranging from 500 to 1850 Da and are generated from diversified protein resources by enzymatic hydrolysis, chemical degradation and microbial fermentation methods [1][2][3]. In addition to their widely accepted nutritive value, BPs have also been proven to have important applications in promoting human health due to their significant kDa) from monkfish muscle could improve the antioxidant capacity of the liver to alleviate non-alcoholic fatty liver disease (NAFLD) progression mainly through modulating the intestinal flora and AMPK and Nrf2 pathways [42][43][44]. APs prepared from monkfish muscle hydrolysate, including EWPAQ, FLHRP, LMGQW, EDIVCW, MEPVW and YWDAW, could concentration-dependently scavenge free radicals and control lipid peroxidation [18,41]. Low MW peptides from monkfish roes could enhance the immune regulatory effect in immunosuppressive mice by activating the signaling pathways of NF-κB/MAPK in spleen tissues [45]. Collagen peptides from monkfish skin could protect mice against the kidney damage induced by a high-fat diet by regulating the signaling pathways of Nrf2/NLRP3 [46]. However, there is no study on BPs from monkfish swim bladders. Therefore, the objectives of the study were to produce and characterize APs from the swim bladder hydrolysate of monkfish for efficient utilization of monkfish processing byproducts. Moreover, we comprehensively determined and evaluated the antioxidant capability and stability of eighteen prepared APs (MSP1 to MSP18).

Optimization of Hydrolysis Conditions of Papain
The hydrolysis conditions of papain including hydrolysis temperature (A), time (B), pH (C) and enzyme dose (D) on the influence of the radical scavenging activity of monkfish swim bladder hydrolysates were optimized using the single-factor experiment (Figure 2). Figure 2A depicted that hydrolysis temperature significantly affected the radical scavenging capability of swim bladder hydrolysates, and the DPPH· scavenging rate (43.47 ± 1.41%) of prepared hydrolysate at 65 °C was significantly higher than those of

Optimization of Hydrolysis Conditions of Papain
The hydrolysis conditions of papain including hydrolysis temperature (A), time (B), pH (C) and enzyme dose (D) on the influence of the radical scavenging activity of monkfish swim bladder hydrolysates were optimized using the single-factor experiment ( Figure 2). Figure 2A depicted that hydrolysis temperature significantly affected the radical scavenging capability of swim bladder hydrolysates, and the DPPH· scavenging rate (43.47 ± 1.41%) of prepared hydrolysate at 65 • C was significantly higher than those of prepared hydrolysates at other temperature (p < 0.05). The DPPH· clearance rate of prepared hydrolysate showed a decreasing trend when the temperature was lower or higher than 65 • C. Figure 2B displayed that DPPH· clearance rates of swim bladder hydrolysates increased gradually with the prolongation of hydrolysis time and reached the maximum value (43.53 ± 0.96%) Mar. Drugs 2023, 21,169 4 of 24 at 4 h. Prolongation of hydrolysis time caused a persistent decrease in the activity of hydrolysates. Figure 2C indicated that DPPH· scavenging rate reached the maximum value (45.58 ± 0.15%) at pH 7.5. The inappropriate pH value of enzymolysis solution can affect the binding of enzyme and substrate by destroying the active center or spatial structure of papain, which further reduced its catalytic activity. Figure 2D displayed that the activity curve showed a trend of rapid rise at the enzyme dose was 1-2.0% and a slow decline when the dose was higher than 2.0%. The highest DPPH· clearance rate was 45.68 ± 0.38% when the enzyme followed by was 2.0%. According to the above experimental results, the ranges of hydrolytic conditions for papain were narrowed down to 60-70 • C, 3-5 h, 7.0-8.0, 1.5-2.5% and 3-5 h for hydrolysis temperature, time, pH and enzyme dose, respectively. prepared hydrolysates at other temperature (p < 0.05). The DPPH· clearance rate of prepared hydrolysate showed a decreasing trend when the temperature was lower or higher than 65 °C. Figure 2B displayed that DPPH· clearance rates of swim bladder hydrolysates increased gradually with the prolongation of hydrolysis time and reached the maximum value (43.53 ± 0.96%) at 4 h. Prolongation of hydrolysis time caused a persistent decrease in the activity of hydrolysates. Figure 2C indicated that DPPH· scavenging rate reached the maximum value (45.58 ± 0.15%) at pH 7.5. The inappropriate pH value of enzymolysis solution can affect the binding of enzyme and substrate by destroying the active center or spatial structure of papain, which further reduced its catalytic activity. Figure 2D displayed that the activity curve showed a trend of rapid rise at the enzyme dose was 1-2.0% and a slow decline when the dose was higher than 2.0%. The highest DPPH· clearance rate was 45.68 ± 0.38% when the enzyme followed by was 2.0%. According to the above experimental results, the ranges of hydrolytic conditions for papain were narrowed down to 60-70°C, 3-5 h, 7.0-8.0, 1.5-2.5% and 3-5 h for hydrolysis temperature, time, pH and enzyme dose, respectively. The orthogonal test L9(3) 4 was designed for optimizing the hydrolysis conditions of papain (Table 1). Following the R values, the conditions interfering with the antioxidant activity of monkfish swim bladder hydrolysates were listed in decreasing order: C (enzyme dose) > B (pH) > D (hydrolysis time) > A (hydrolysis temperature). The enzyme dose was recognized as the most important condition influencing the antioxidant activity of swim bladder hydrolysates. By verification experiments, the maximum DPPH· scavenging ratio of monkfish swim bladder hydrolysate was 47.13 ± 1.15% at 5.0 mg/mL on the optimal enzymolysis level of A2B2C3D3, that is, the optimum conditions of papain for producing monkfish swim bladder hydrolysate were hydrolysis temperature 65 °C, pH 7.5, enzyme dose 2.5% and time 5 h. In addition, the monkfish swim bladder hydrolysate produced under the optimal conditions of papain was named MSBH. The orthogonal test L 9 (3) 4 was designed for optimizing the hydrolysis conditions of papain (Table 1). Following the R values, the conditions interfering with the antioxidant activity of monkfish swim bladder hydrolysates were listed in decreasing order: C (enzyme dose) > B (pH) > D (hydrolysis time) > A (hydrolysis temperature). The enzyme dose was recognized as the most important condition influencing the antioxidant activity of swim bladder hydrolysates. By verification experiments, the maximum DPPH· scavenging ratio of monkfish swim bladder hydrolysate was 47.13 ± 1.15% at 5.0 mg/mL on the optimal enzymolysis level of A2B2C3D3, that is, the optimum conditions of papain for producing monkfish swim bladder hydrolysate were hydrolysis temperature 65 • C, pH 7.5, enzyme dose 2.5% and time 5 h. In addition, the monkfish swim bladder hydrolysate produced under the optimal conditions of papain was named MSBH. The radical scavenging rates of MSBH and its four ultrafiltration fractions (MSBH-I, MW < 1 kDa; MSBH-II, 1 kDa < MW < 3.5 kDa; MSBH-III, 3.5 kDa < MW < 10 kDa; MSBH-IV, MW > 10 kDa) at 5.0 mg/mL were measured ( Figure 3). The data manifested that DPPH· and HO· scavenging rates of MSBH-I were 51 The radical scavenging rates of MSBH and its four ultrafiltration fractions (M MW < 1 kDa; MSBH-II, 1 kDa < MW < 3.5 kDa; MSBH-III, 3.5 kDa < MW < 10 kDa; IV, MW > 10 kDa) at 5.0 mg/mL were measured ( Figure 3). The data manifest DPPH· and HO· scavenging rates of MSBH-І were 51.57 ± 1.45% and 76.96 ± 2.40 rates of MSBH-І were significantly greater than those of MSBH (47.13 ± 1.18% and 3.17%), MSBH-II (49.23 ± 0.42% and 68.29 ± 1.141%), MSBH-III (48.86 ± 1.00% and 1.92%) and MSBH-IV (46.11 ± 0.42% and 57.54 ± 0.81%) (p < 0.05). Then, MSBH-І w sen for subsequent separation.
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Determination of the AA Sequences and MWs of Eighteen Isolated APs (MSP1-MSP18)
The MWs and sequences of APs in HPLC chromatographic peaks (P1-P16) were determined by protein sequencer and ESI-MS ( Table 2). Peaks of P1 and P10 contained two peptides. Therefore, eighteen APs (MSP1-MSP18) from chromatographic peaks of P1-P16 were identified as Tyr-Asp   Figure 6A and Table 3 showed that the EC 50 values of MSP7 and MSP8 on DPPH· were 1.053 ± 0.003 and 0.773 ± 0.003 mg/mL, which were significantly lower than those of the other sixteen isolated APs (p < 0.05).

Lipid Peroxidation Inhibition Ability
In organic tissues, lipid peroxidation is generally described as a process in which oxidants attack lipids containing polyunsaturated fatty acids (PUFAs), which has an important role in human health because lipid peroxidation products, including MDA and 4-HNE, play a vital cytotoxic role in promoting cell death and controlling gene expression [47]. Therefore, the assay was applied to comprehensively evaluate the activities of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 ( Figure 7A). In the linoleic acid emulsion system, the values at 500 nm of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 were significantly smaller than that of blank control (without peptide and GSH) during 7 days. The finding demonstrated that MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 could effectively inhibit the reaction rate and efficiency of lipid peroxidation in the experimental system by reacting with H 2 O 2 . Moreover, the MSP8 showed a similar inhibiting capability to that of GSH, followed by MSP1, MSP10 and MSP13.

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay reflects the ability of compounds that serve as electron donors to decrease the oxidized intermediates in the lipid peroxidation process, and it has been used as a preferred method to evaluate the "total antioxidant content" of functional molecules [48,49]. As shown in Figure 6B, MSP8 showed a higher ability to convert Fe 3+ /ferricyanide complex into Fe 2+ form than the other five determined peptides, followed by MSP1, MSP10 and MSP7. However, the reducing power of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 was lower than that of glutathione (GSH). Mar. Drugs 2023, 21, x FOR PEER REVIEW 10 of 24

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay reflects the ability of compounds that serve as electron donors to decrease the oxidized intermediates in the lipid peroxidation process, and it has been used as a preferred method to evaluate the "total antioxidant content" of functional molecules [48,49]. As shown in Figure 6B, MSP8 showed a higher ability to convert Fe 3+ /ferricyanide complex into Fe 2+ form than the other five determined peptides, followed by MSP1, MSP10 and MSP7. However, the reducing power of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 was lower than that of glutathione (GSH).  In addition, the viability of H2O2injured HepG2 cells in MSP1 and MSP8 groups was 82.14 ± 3.28% and 81.32 ± 2.45%, which were significantly higher than that of the model group (51.08 ± 1.97%). Therefore, MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 presented cytoprotective function to H2O2-injuried HepG2 cells by increasing the cell viability.    In addition, the viability of H2O2injured HepG2 cells in MSP1 and MSP8 groups was 82.14 ± 3.28% and 81.32 ± 2.45%, which were significantly higher than that of the model group (51.08 ± 1.97%). Therefore, MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 presented cytoprotective function to H2O2-injuried HepG2 cells by increasing the cell viability.   Table 4 indicated that MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 showed significant differences in tolerance to acid and alkali treatment (pH 3 to 11). MSP1, MSP2, MSP7 and MSP8 kept the highest O − 2 · scavenging activity at pH 7.0, but MSP10 and MSP13 kept the highest activity at pH 11.0 and 9.0, respectively. In addition, MSP1, MSP2, MSP7 and MSP8 were more sensitive to alkali treatment because their O − 2 · scavenging rates   Table 4 indicated that MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 showed significant differences in tolerance to acid and alkali treatment (pH 3 to 11). MSP1, MSP2, MSP7 and MSP8 kept the highest O − 2 · scavenging activity at pH 7.0, but MSP10 and MSP13 kept the highest activity at pH 11.0 and 9.0, respectively. In addition, MSP1, MSP2, MSP7 and MSP8 were more sensitive to alkali treatment because their O − 2 · scavenging rates dropped by 35.26%, 73.07%, 69.19% and 83.05%, respectively, at pH 11.0. Conversely, MSP10 and MSP13 were more sensitive to acid treatment because their O − 2 · scavenging rates dropped by 26.26% and 33.05% at pH 3.0. Those data suggested that MSP1, MSP2, MSP7 and MSP8 are appropriate for the application to products in a neutral environment, but MSP10 and MSP13 are appropriate for the application to products in an alkali environment. dropped by 35.26%, 73.07%, 69.19% and 83.05%, respectively, at pH 11.0. Conversely, MSP10 and MSP13 were more sensitive to acid treatment because their O − 2 · scavenging rates dropped by 26.26% and 33.05% at pH 3.0. Those data suggested that MSP1, MSP2, MSP7 and MSP8 are appropriate for the application to products in a neutral environment, but MSP10 and MSP13 are appropriate for the application to products in an alkali environment.   Figure 11, and the results manifested that six APs have high-temperature tolerance. Compared with MSP1, MSP7, MSP8 and MSP13, MSP2 and MSP10 were relatively affected by high temperature (100 °C), and their O − 2 · scavenging activity, respectively, decreased by 3.84% and 2.60%. Those data suggested that MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 are appropriate for the application to products treated by high temperatures because of their high-temperature tolerance.  The O − 2 · scavenging activity of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 treated using different temperatures (25, 37, 60, 80 and 100 • C) was present in Figure 11, and the results manifested that six APs have high-temperature tolerance. Compared with MSP1, MSP7, MSP8 and MSP13, MSP2 and MSP10 were relatively affected by high temperature (100 • C), and their O − 2 · scavenging activity, respectively, decreased by 3.84% and 2.60%. Those data suggested that MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 are appropriate for the application to products treated by high temperatures because of their high-temperature tolerance. 2 · scavenging activities of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 decreased gradually when they were treated with pepsin and trypsin in turn. Among six peptides, the O − 2 · scavenging rates of MSP2, MSP7 and MSP10 showed the most seriously affected after simulated GI digestion and decreased by 47.59%, 49.19% and 53.33%, respectively; the O − 2 · scavenging rates of MSP8 and MSP13 were also affected and decreased by 28.62% and 35.83%, respectively (Table 5). However, MSP1 showed good tolerance and its O − 2 · scavenging rate only decreased by 4.1% (Table 6). Those results suggested that MSP1 showed high stability when it was treated with simulated GI digestion.  (Table 5). However, MSP1 showed good tolerance and its O − 2 · scavenging rate only decreased by 4.1% (Table 6). Those results suggested that MSP1 showed high stability when it was treated with simulated GI digestion.    (Table 5). However, MSP1 showed good tolerance and its O − 2 · scavenging rate only decreased by 4.1% (Table 6). Those results suggested that MSP1 showed high stability when it was treated with simulated GI digestion.

Preparation of APs from MSBH
BPs are encapsulated in the sequence of proteins and keep inactive form and can be released by a variety of hydrolysis pathways [28]. In comparison with chemical and microbiological degradation, protease degradation is known as an effective, safe and rational method for the production of protein hydrolysates because of the high controllability and reproducibility of the enzymatic process, the mild and safe conditions of enzymatic protein digestion and the absence of side reactions in the enzymatic reaction. Then, protease degradation is more widely used in the food and pharmaceutical industries [3,50,51]. Therefore, proteases including papain, alcalase, pepsin, flavourzyme, Protamex ® , trypsin and their combinations are frequently used to manufacture BPs from marine organisms and their by-products [9,52,53]. In addition, many enzymes have specific cleavage sites (papain: Arg-, Lys-and Phe-; alcalase: Ala-, Leu-, Val-, Tyr-, Phe-and Try-; trypsin: Argand Lys-; pepsin: Phe-and Leu-), and different cleavage sites will have a certain effect on the activity of hydrolysates [54,55]. Ktari et al. found that hydrolysates from cuttlefish (Sepia officinalis) by-products obtained by alcalase and sardinelle crude enzyme exhibited the strongest activity among eight hydrolysates [52]. Alcalase hydrolysate of Antarctic Krill had the highest radical scavenging ability among the five hydrolysates [56]. Nangnoi strain hydrolysate generated by alcalase showed the highest activities among the three hydrolysates [57]. In the study, MSBH produced by papain shows the highest activity further proving this conclusion that specificity and conditions of proteases are the key factors for the generation of APs.
The hydrolysate profile is also influenced by enzyme concentration, digestion time, digestion temperature, ambient pH and other factors. At the optimum pH and temperature, the cleavage rate of protease is accelerated and non-specific digestion sites are less likely to occur. In addition, insufficient enzymatic digestion may occur with too short a digestion time and too low an enzyme concentration [3,12]. Jang et al. found that the optimal hydrolysis conditions for alcalase 2.4 L were pH 6.0, temperature 70 • C, enzyme concentration 5% (w/w), and hydrolysis time 3 h. The optimal hydrolysis conditions for Collupulin MG were pH 9.0, temperature 60 • C, enzyme dose 5% (w/w), and the DPPH· radical scavenging activity of the two hydrolysates under optimal conditions was 60.04 and 79.65%, respectively [58]. In the study, the optimum conditions of enzymatic hydrolysis of papain were temperature 65 • C, pH 7.5, enzyme dose 2.5%, enzymatic hydrolysis time 2 h and the DPPH· scavenging rate of enzymatic hydrolysis product was 47.13 ± 1.15%. It was further proved that the enzymatic hydrolysis condition of papain significantly influenced the generation of APs.
The roles of AA composition, especially hydrophobic/aromatic AAs, are often discussed in previous literature [3,12,28]. Hydrophobic/aromatic AAs can facilitate the binding between the peptides and ROS by improving the peptides' solubility in the reactive solution [21,70]. Ala residue should play an important role in the antioxidant activity of MSP8 (ARW) and MSP13 (YPAGP). Aromatic AAs contain a benzene ring structure, which can provide hydrogen ions to convert ROS into more stable phenoxy radicals and control the peroxide domino effects mediated by ROS [10,20,74]. Sheng et al. reported that Tyr and Phe residues in GEYGFE and Phe residue in IELFPGLP exerted key roles in their antioxidant activities [20]. Therefore, Tyr residue in MSP1 (YDYD), MSP2 (QDYD) and MSP13 (YPAGP) and Trp residue in MSP7 (GRW) and MSP8 (ARW) could positively affect their activity. In addition, Pro residue could improve the flexibility of peptides and act as proton/hydrogen donors to remove ROS directly [24,31,75]. Therefore, Pro residue should be important for the activity of MSP13 (YPAGP).
Hydrophilic AA residues also are necessary for the activity of APs. Acidic (Asp, Glu, Asn and Gln) and basic (Lys and Arg) AA residues have been proven as excellent chelating agents of metal-ions because the excessive electrons in their carboxylic group could improve electrostatic and ionic with metal-ion to play their excellent metal-chelating function [10,76]. Therefore, basic (Arg and Lys) and acidic (Glu and Asp) AA residues were frequently found in APs, such as LKPGN [29], VPR, IEPH, LEEEE and IEEEQ [11], LDEPDPLI and NTDGSTDY-GILQINSR [77], PHPR, VRDQY [54] and AEDKKLIQ [78]. Therefore, Asp residue in MSP1 (YDYD), Gln and Asp residues in MSP2 (QDYD), Arg residue in MSP7 (GRW) and MSP8 (ARW) and Asp and Lys residues in MSP10 (DDGGK) must be a great help to their antioxidant ability. Gly residue is often found in collagen peptides with antioxidant activity, such as GFRGTIGLVG, GPAGPAG, GFPSG [13], FTGMD, GFEPY, GFYAA, GIEWA [79], PFGPD, PYGAKG and YGPM [50] because it can maintain the high flexibility of peptide chain and act as a single hydrogen donor to neutralize ROS. Then, Gly residue presented in MSP7 (GRW), MSP10 (DDGGK) and MSP13 (YPAGP) are helpful for their activity.

Optimization of Hydrolysis Conditions of Papain
A single-factor experiment was used to optimize the hydrolysis conditions of papain. Hydrolysis temperature (55,60,65,70 [80]. MSBH-I showed the highest radical scavenging activity.

Purification of APs from MSBH-I by Chromatography Methods
MSBH-I solutions (5 mL, 50.0 mg/mL) were loaded into the Sephadex G-15 column (2.0 × 120 cm) and eluted using ultrapure water at a flow rate of 0.8 mL/min. The eluent was collected each 2 min, and three fractions (MSBH-Ia, MSBH-Ib and MSBH-Ic) were collected according to the chromatographic diagram at 220 nm.
MSBH-Ib was further isolated using a Zorbax, SB C-18 column (4.6 × 250 mm, 5 µm) in the HPLC system. The Zorbax column was eluted by a linear gradient of acetonitrile (0-50% in 0-30 min) in 0.1% TFA. The eluent at a flow rate of 1.0 mL/min was detected at 214 and 254 nm. Finally, sixteen peaks (P1 to P16) were prepared on the HPLC chromatograms at 214 and 254 nm and freeze-dried.

Identification of Eighteen Isolated APs (MSP1-MSP18)
The AA sequences of eighteen peptides (MSP1-MSP18) from monkfish swim bladders were determined by a 494-protein sequencer of Applied Biosystems (Perkin Elmer Co., Ltd. Foster City, CA, USA). The MWs of eighteen peptides (MSP1-MSP18) were determined by a Q-TOF mass spectrometer with an ESI source (Micromass, Waters, Milford, MA, USA) [81,82] After incubating at 37 • C for 60 min, the absorbance of the reaction mixture was measured at 536 nm against a reagent blank. The reaction mixture without any antioxidants 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.

DPPH· Scavenging Activity
A total of 2.0 mL of sample solution consisting of distilled water and different concentrations of the analytes was added 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· with the following equation: where A s is the absorbance rate of the sample, A c is the control group absorbance and A b is the blank absorbance.

O −
2 · Scavenging Activity Superoxide anions were generated in 1 mL of nitrotetrazolium blue chloride (NBT) (2.52 mM), 1 mL of NADH (624 mM) and 1 mL of different sample concentrations. The reaction was initiated by adding 1 mL of phenazine methosulphate (PMS) solution (120 µM) to the reaction mixture. The absorbance was measured at 560 nm against the corresponding blank after incubation for 5 min at 25 • C. The scavenging capacity of the O − 2 · was calculated using the following equation: where A c is the absorbance without the sample and A s is the absorbance with the sample.

ABTS + · Scavenging Activity
The ABTS + · 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 + · solution was diluted in 5 mM phosphate buffered saline (PBS) pH 7.4, to an absorbance of 0.70 ± 0.02 at 734 nm. One milliliter of diluted ABTS + · solution was mixed with one milliliter of different concentrations of samples. Ten minutes 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 c was the absorbance without the sample and A s was the absorbance with the sample.

Determination of Reducing Power
Generally, 2.0 mL of each sample dissolved in distilled water was mixed with 2.5 mL of 1% aqueous potassium hexacyanoferrate [K 3 Fe(CN) 6 ] solution. After 30 min incubation at 50 • C, 1.5 mL of 10% trichloroacetic acid was added. Finally, 2.0 mL of the upper layer was mixed with 2.0 mL of distilled water and 0.5 mL of 0.1% aqueous FeCl 3 and the absorbance was recorded at 700 nm. The higher absorbance of the reaction mixture indicated the stronger reducing power.

Lipid Peroxidation Inhibition Assay
Briefly, a sample (5.0 mg) was dissolved in 10 mL of 50 mM PBS (pH 7.0) and added to 0.13 mL of a solution of linoleic acid and 10 mL of 99.5% ethanol. Then, the total volume was adjusted to 25 mL with deionized water. 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. The higher absorbance of the solution means lower lipid peroxidation inhibition capacity. The protective functions of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 on plasmid DNA (pBR322) were determined using the previous method [83]. In short, peptide (MSP1, MSP2, MSP7, MSP8, MSP10 or MSP13, respectively) were added to the test tubes containing FeSO 4 (2 µL, 1.0 mM), pBR322 (1 µL, 0.5 µg) and H 2 O 2 (2 µL, 1.0 mM). A total of 15 µL of the manufactured reaction solution was incubated at 37 • C. After 30 min, 2 µL of loading buffer was added to the solution. Then, the solution was subsequently electrophoresed on 1% agarose gel containing 0.5 µg/mL EtBr at 60 V for 50 min. Finally, the DNA in the agarose gel was photographed and recorded under ultraviolet light.  [84][85][86]. Briefly, the HepG2 cells were incubated in a 96-well plate for 24 h. The supernatant in a 96-well plate was aspirated, and peptide solution (100 µL, 100.0 µM) was added into the sample groups and incubated for 8 h. After removing peptides, H 2 O 2 was added to the sample, GSH and model groups. After 24 h, the 96-wells were rinsed twice with PBS and used MTT method to determine the cell viability: Cell viability = (OD sample /OD control ) × 100%. The stability of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 was determined according to the previous method with a light modification [87][88][89]. The O − 2 · scavenging activity (%) of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 at 5.0 mg/mL were measured to evaluate their stability.
The pH values (3, 5, 7, 9 and 11) were set to evaluate the acid and alkali stability properties of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13 at 25 • C, and the incubating time with different pH solutions was set to 2 h. Two-stage simulated GI digestion model (2 h of pepsin digestion followed by 2 h of trypsin digestion) was designed to evaluate the influence of simulated GI digestion on the stability of MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13.

Statistical Analysis
All the data are expressed as the mean ± SD (n = 3). The experimental data were analyzed by an ANOVA test using SPSS 19.0. Significant differences were determined by Duncan's multiple range test (p < 0.05, 0.01, and 0.001).

Conclusions
In conclusion, the conditions of papain for hydrolyzing the protein of monkfish (L. litulon) swim bladders were optimized as hydrolysis temperature 65 • C, pH 7.5, enzyme dose 2.5% and time 5 h through single factor and orthogonal experiments, and eighteen APs (MSP1 to MSP18) were purified from the monkfish swim bladder hydrolysate and identified as YDYD, QDYD, AGPAS, GPGPHGPSGP, GPK, HRE, GRW, ARW, GPTE, DDGGK, IGPAS, AKPAT, YPAGP, DPT, FPGPT, GPGPT, GPT and DPAGP, respectively. In general, YDYD, ARW and DDGGK exhibited high ability on radical scavenging, lipid peroxidation inhibition, Ferric reducing antioxidant power, and protective function on oxidation-damaged Plasmid DNA and HepG2 cells. The antioxidant activity of eighteen isolated peptides (MSP1 to MSP18) was highly stable under high temperatures, but remarkably influenced by different pH and simulated GI digestion. In brief, the present finding provides a good perspective for monkfish processing byproducts-swim bladders as the high-quality biological resources to produce BPs, and the generated peptides could serve as antioxidative ingredients applied in health-promoting products. Moreover, the antioxidant mechanism of peptides (MSP1, MSP2, MSP7, MSP8, MSP10 and MSP13) and the therapeutic effects of these peptides on HepG2 cells and mice after oxidative damage will be systematically researched in our follow-up study.