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Characterization of antioxidative peptide purified from black eelpout (Lycodes diapterus) hydrolysate

Characterization of antioxidative peptide purified from black eelpout (Lycodes diapterus)... The functional peptides from protein hydrolysates of various fishery sources have been identified such as antioxidant activity. The main intention of this study was purification and characterization of antioxidative peptide from black eelpout muscle. The antioxidative peptides were purified from black eelpout (Lycodes diapterus) muscle using different proteases. Antioxidant activity of black eelpout hydrolysates was evaluated using DPPH radical scavenging activity. Among six hydrolysates, the pepsin hydrolysate had the highest antioxidant activity compared to the other hydrolysates. Therefore, it was further purified and a peptide with seven amino acid residues of DLVKVEA (784 Da) was identified by amino acid sequence analysis. The EC value for scavenging DPPH radicals by purified peptide was 688.77 μM. Additionally, the purified peptide exhibited protective effect against DNA damage induces by oxidation in mouse macrophages (RAW 264.7 cells). The results of this study suggest that black eelpout muscle protein hydrolysate could potentially contribute to development of bioactive peptides in basic research. Keywords: Antioxidant, DPPH radical scavenging, Peptide, Hydrolysates, Pepsin, Black eelpout muscle Background oxidative stress (McCord 1993). Steady-state maintenance Free radicals are highly reactive species with their single of ROS/antioxidant ratio is vital for avoiding oxidative and unbalanced electrons. The oxidation by free radicals stress (Somani and Rybak 1996). Synthetic antioxidants in the body may cause many chronic diseases such as (butylated hydroxyanisole (BHA), tbutylhydroquinone cardiovascular diseases, diabetes, cancer, and neurode- (TBHQ), butylated hydroxytoluene (BHT), and propyl generative disorders (Dong et al. 2008). Fatty acids and gallate) have been widely used as food preservatives as lipids oxidation induced by free radicals deteriorate the they delay the discoloration and deterioration caused by food quality (Liceaga-Gesualdo and Li-Chan 1999). oxidation (Wanita and Lorenz 1996). So, the use of these Reactive oxygen species (ROS) (O (superoxide anion), synthetic antioxidants has been limited in some countries � OH (hydroxyl radical), and H O (hydrogen peroxide)) due to their potential health hazard (Becker 1993). 2 2 are metabolic by-products of normal aerobic metabolism Recently, enzymatic hydrolysis with proteases has gar- (Castro and Freeman 2001). Nevertheless, the body is sup- nered much attention. Protein hydrolysates or peptides ported with several antioxidant defense systems where affect health-related functions such as antioxidant func- they can scavenge and transform ROS or free radicals into tion (Clemente 2000). Therefore, various antioxidant harmless species (Yeung et al. 2002). The antioxidant peptides have been isolated from marine organisms defense system includes catalase (CAT), glutathione per- through enzymatic hydrolysis, including abalone muscle oxidase (GSH-Px), superoxide dismutase (SOD), and (Haliotis discus hannai Ino) and scallop (Patinopecten glutathione reductase (GR). Enzymatic and non-enzymatic yessoensis) (Zhou et al. 2012), threadfin bream surimi antioxidants team up to scavenge and eradicate the (Wiriyaphan et al. 2012), croaker (Otolithes ruber) muscle (Nazeer et al. 2012), sand eel (Lee et al. 2011a, 2011b), sardinelle (Sardinella aurita) (Bougatef et al. * Correspondence: hgbyun@gwnu.ac.kr 2010), tuna liver (Je et al. 2009), marine rotifer (Byun Department of Marine Biotechnology, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea et al. 2009), and algae protein waste (Sheih et al. 2009). Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 2 of 7 Enzymatic hydrolysates exhibited several advantages wavelength at 254 nm and a flow rate of 1.0 mL/min. All when incorporated into foods, by improving water- analyses were carried out in triplicate. binding ability, solubility of protein, emulsifying stability, heat stability of myofibrillar protein, and the nutritional Preparation of black eelpout muscle hydrolysates quality of foods. Thus, enzymatic hydrolysis has become To prepare black eelpout muscle hydrolysates, enzymatic an appreciated tool for modifying the applicability of hydrolysis was performed using various enzymes (Alca- proteins (Korhonen et al. 1998). Normally, bioactive lase, α-chymotrypsin, Neutrase, papain, pepsin, and tryp- peptides remain inactive within the parent protein mol- sin) at their optimal conditions. Black eelpout muscle ecule until they are released by hydrolysis. Most of bio- was hydrolyzed separately using various enzymes with a active peptides are composed with 2–20 amino acids. substrate to enzyme ratio of 1:100 for 6 h, under Amino acids arrangement of the peptides plays a critical optimum pH and temperature conditions (Table 1). At role in its bioactivity (Himaya et al. 2012). the end of 6 h, hydrolysates were filtered by glass filter The black eelpout, Lycodes diapterus, is distributed in and lyophilized and stored at − 80 °C until use. The yield the Northwest Pacific/North of central East Sea of Korea of hydrolysate from black eelpout muscle was calculated and the Sea of Okhotsk and inhabits sand and mud bot- as follows: toms in deep water of 150–200 m depth. Black eelpout is a traditional food that is rich in protein, essential weight of the black eelpout hydrolysates YieldðÞ % ¼  100 amino acids, omega-3 polyunsaturated fatty acids, and weight of the black eelpout vitamins. In the present study, we investigated the 2,2- diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scav- enging activity of enzymatically prepared black eelpout muscle protein hydrolysate to isolate a potent antioxi- Determination of DPPH radical scavenging activity dant peptide. And the protective effect of the purified DPPH radical scavenging activity (RSA) was assessed by peptide against deoxyribonucleic acid (DNA) oxidation using the method of Yen and Hsieh (1995) with minor induced by the hydroxyl radical was verified further. modifications. The sample was mixed with 120 μLof methanol and 40 μL of 0.15 mM DPPH in methanol was Materials and methods added. The mixture was incubated at room temperature Materials in the dark for 30 min. The absorbance of the mixture Fresh samples of black eelpout (Lycodes diapterus) were was measured at 517 nm using a spectrophotometer obtained from East Sea Fisheries Research Institute, (JASCO, Japan). The control sample was prepared in the Gangneung, South Korea. The bones and viscera were same manner where methanol was used instead of the removed from the black eelpout. Then the separated 40 μL sample volume. DPPH radical scavenging activity muscle was stored at − 80 °C until use. Several commer- was calculated as follows: cial enzymes, such as α-chymotrypsin, papain, pepsin, and trypsin, were obtained from Sigma Chemical Co. A −A (St. Louis, MO). Alcalase and Neutrase enzymes were control sample RSAðÞ % ¼  100 obtained from Novo Co. (Novo Nordisk, Bagsvaerd, control Denmark). DPPH was obtained from Wako Chemical Co. All other reagents used in this study were reagent where A is the absorbance of sample and A is sample control grade chemicals. the absorbance of the control. The EC value is defined as an effective concentration of peptide that is required Analysis of proximate compositions to scavenge 50% of radical activity. Crude protein content of black eelpout was determined Table 1 Optimal conditions for enzymatic hydrolysis of various by the Kjeldahl method (Auto Kjeldahl system, Buchi B- enzymes 324/435/412, Switzerland). Ether extraction method was Enzyme Buffer pH Temperature (°C) used to determine the crude lipid content. Moisture Alcalase 50 mM Na HPO -NaH PO 7.0 50 2 4 2 4 content was determined by oven drying at 105 °C for 24 α-Chymotrypsin 50 mM Na HPO -NaH PO 7.0 37 h. Ash content was determined by a muffler furnace at 2 4 2 4 550 °C for 4 h (Association of Official Analytical Chemist Neutrase 50 mM Na HPO -NaH PO 7.0 50 2 4 2 4 (AOAC) 2000). Amino acids were analyzed using an Papain 50 mM Na HPO -NaH PO 7.0 37 2 4 2 4 automatic analyzer (Hitachi Model 835-50, Japan) with a Pepsin 20 mM HCl 2.0 37 C18 column (5 μm, 4.6 × 250 mm, Watchers, MA). The Trypsin 50 mM Na HPO -NaH PO 7.0 37 2 4 2 4 reaction was carried out at 38 °C, with the detection Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 3 of 7 Purification and identification of antioxidant peptides Statistical analysis The black eelpout muscle hydrolysate was dissolved in Data were analyzed for statistical significance using analysis distilled water and loaded onto a Sephadex G-25 gel fil- of variance (ANOVA) followed by Dunnett’smultiplecom- tration column (2.5 × 70 cm) which had been previously parison test with statistical package for the social sciences equilibrated with distilled water. The column was then (SPSS) software (version 14). All values obtained from three eluted with distilled water at a flow rate of 1.5 mL/min different experiments were expressed as the mean value ± (fraction volume 7.5 mL) and separated fractions were standard deviation (SD). monitored at 215 nm, collected at a volume of 7.5 mL, and measured for DPPH radical scavenging activity. Results and discussion Highest active fraction was injected into a preparative Proximate composition of black eelpout muscle reverse-phase high performance liquid chromatography Proximate composition of black eelpout muscle showed (RP-HPLC) column (Grom-Sil 120 ODS-5ST, ø 10 × the 20.81% moisture content, 8.63% lipid content, 4.09% 250 mm, 5 μm, Grom™, Germany) and was separated ash, 2.46% carbohydrate, and 64.02% protein content using linear gradient of acetonitrile (0–20% v/v) contain- (Table 2). The protein content was the highest among all ing 0.1% trifluoroacetic acid (TFA) on an RP-HPLC sys- the composition contents. However, the low lipid and ash tem (Agilent Technologies, USA). Elution peaks were content suggests that the extraction processes by enzym- monitored at 280 nm on diode array detector (DAD). atic hydrolysis of biofunctional peptide is effective. The The purified fractions from preparative column were most abundant amino acids in black eelpout muscle were monitored at 280 nm and purified by RP-HPLC on a glycine, alanine, lysine, and leucine which accounted for C18 analytical column (ø 4.6 × 250 mm, 5 μm, Waters, 20.82%, 17.13%, 8.1%, and 6.24%, respectively (Table 3). Milford, MA, USA) using an acetonitrile gradient of 5– Generally, fish and other mammalian skin have higher 30% (v/v) at a flow rate of 0.5 mL/min for 40 min. Fi- percentage of Gly, Leu, and Pro compared to muscle pro- nally, the fraction with the highest DPPH radical scaven- teins (Gomez-Guillen et al. 2002). ging activity was collected and lyophilized followed by the amino acid sequence identification. Antioxidant activity of black eelpout muscle hydrolysates Black eelpout muscle protein hydrolysates were prepared by using commercial proteases including Alcalase, α- Determination of molecular weight and amino acid chymotrypsin, Neutrase, papain, pepsin, and trypsin. sequence The hydrolysis yields were 68.28%, 66.85%, 66.14%, and Molecular weight and amino acid sequence of purified 58.76% for papain, Alcalase, pepsin, and trypsin, respect- peptide from black eelpout muscle protein were deter- ively (Table 4). Among six hydrolysates, pepsin hydrolys- mined by quadrupole time-of-flight (Q-TOF) mass spec- ate exhibited the greatest DPPH radical scavenging trometry (Micromass, Altrincham, UK) coupled with activity relative to the other hydrolysates. In terms of the electrospray ionization (ESI) source. The purified peptide DPPH radical scavenging activation (Fig. 1), the lowest dissolved in methanol/water (1:1, v/v) was infused into the EC value was exhibited by the pepsin hydrolysate at ESI source and the molecular mass was determined by 0.83 mg/mL. Thus the pepsin hydrolysate may contain 2+ doubly charged (M+ 2H) state in the mass spectrum. bioactive compounds that could react with free radicals Following molecular mass determination, the peptide was to transform them into more stable products and ter- automatically selected for fragmentation and sequence in- minate the radical chain reaction. Peptides with antioxi- formation was obtained by tandem MS analysis. dative activity have been obtained by enzymatic hydrolysis of various marine organisms (Je et al. 2007). Protective potential by the hydroxyl radical-induced DNA Several studies have suggested that the variation of anti- damage oxidant activity of a peptide is due to its amino acid se- To assess the protective effects of the hydrolysate against quence and length (Kim et al. 2001). However, DPPH DNA damage caused by hydroxyl radicals, the reaction radical scavenging activity of pepsin hydrolysate was was induced by placing the following reagents in an Table 2 Proximate compositions of black eelpout muscle Eppendorf tube: 5 μL of genomic DNA (RAW 264.7 cell Components Content (%) line), 2 mM FeSO , and various concentrations of the Moisture 20.81 purified peptide from black eelpout hydrolysate. The mixture was then incubated at 37 °C for 30 min, followed Protein 64.02 by the addition of 4 μLof10mM H O (Dávalos et al. 2 2 Lipid 8.63 2004). Finally, the mixture was subjected to 1.0% agarose Ash 4.09 gel electrophoresis and DNA bands were stained with Carbohydrate 2.46 ethidium bromide. Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 4 of 7 Table 3 Amino acid contents of black eelpout muscle Amino acids Contents (%) Tau 0.78 Asp 6.08 Thr 4.07 Ser 3.93 Glu 2.21 Gly 20.82 Ala 17.13 Val 3.61 Cys 0.34 Met 1.96 Ile 2.43 Fig. 1 EC values for DPPH radical scavenging activity of black eelpout muscle hydrolysates. Statistical significance was determined Leu 6.24 by ANOVA Try 1.71 Phe 2.33 secondary fractions contain small-molecular-size peptides. Lys 8.10 According to Pihlanto (2000), numerous bioactive peptides His 1.80 are found between 2 and 20 amino acids in length with a Arg 5.13 small-molecular-size. Therefore, the secondary fractions Total 100.00 were assumed to have the greatest potential bioactivity. Fraction B was further separated by RP-HPLC using an ODS column and subsequently fractionated into three frac- lower than that of synthetic antioxidants BHA and BHT. tions (F1–F3) (Fig. 2II). Among separated fractions, the The next stage in analysis required the use of HPLC for fraction F1 showed the highest DPPH radical scavenging purifying the antioxidant peptide from pepsin hydrolys- activity with the EC value of 87.45 μg/mL (Fig. 2II). Frac- ate of black eelpout muscle. tion F1–1, with the strongest DPPH radical scavenging ac- tivity was purified further by using RP-HPLC on the C18 Purification of antioxidant peptide analytical column a linear gradient of acetonitrile (5–30%) To identify the antioxidant peptide from pepsin hydrolysate for 40 min at a flow rate of 0.5 mL/min (Fig. 2III). The EC of black eelpout muscle, the use of different chromato- value of the purified peptide was 51.12 μg/mL, 16.24–fold graphic techniques is required. As shown in Fig. 2,chroma- compared to the pepsin hydrolysate (0.83 mg/mL) using tographic profiles were obtained during different the three-step purification procedure (Table 5). A single purification steps of black eelpout muscle hydrolysate. In peptide fraction that demonstrated DPPH radical scaven- the first step, pepsin hydrolysate was separated into four ging activity was purified on an analytical HPLC column fractions (A–D) on a Sephadex G-25 chromatography col- and their amino acid sequences were determined by N- umn (Fig. 2I). Among separated fractions, the B fraction terminal sequencing analysis. had the highest DPPH radical scavenging activity at 0.65 mg/mL (Fig. 2I). Sephadex G-25 column chromatography Characterization of purified antioxidant peptide separates according to molecular-size, where the primary The purified fraction F1–1 was analyzed by electrospray fractions contain large-molecular-size peptides, and ionization mass spectrometry (ESI-MS) for molecular mass determination and ESI-MS/MS for the peptide Table 4 Yields of various hydrolysates from black eelpout characterization. Amino acid sequence of purified antioxi- muscle dant peptide was identified as Asp-Leu-Val-Lys-Val-Glu-Ala Hydrolysates Yields (%) with EC value and molecular weight of 688.77 μMand Alcalase 66.85 784 Da, respectively (Fig. 3). These results support for the α-Chymotrypsin 51.30 general finding that short peptides with 2–10 amino acids demonstrate greater bioactive properties such as antioxidant Neutrase 39.47 activity compared to their parent native proteins or large Papain 68.28 polypeptides (Li et al. 2007). In this study, the purified anti- Pepsin 66.14 oxidant peptide was found to have a similar sequence with Trypsin 58.76 the other reports, including the sardinelle (Sardinellaaurita) Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 5 of 7 Fig. 2 Steps for the purification of DPPH radical scavenging activity peptide from black eelpout muscle hydrolysate. I Sephadex G-25 Gel filtration chromatogram of hydrolysates. Gel filtration chromatogram of hydrolysates prepared with black eelpout muscle. Separation was performed with 1.5 mL/min and collected at a fraction volume of 7.5 mL. The fractions isolated by Sephadex G-25 Gel column were separated (A–D) and DPPH radical scavenging activity was determined as upper panel. II, III Reverse phase-HPLC chromatograms of the potent DPPH radical scavenging activity fractions from the previous steps. The lower panels of each pair show the chromatography results of separated fractions while the top panels of each pair represent the DPPH radical scavenging activity of separated fractions in terms of their EC values expressed in mg/mL (I)or μg/mL (II, III). Statistical significance was determined by ANOVA (Gly-Ala-Trp-Ala, RSA = 52 ± 1.44% at 150 μg/mL) (Bouga- of histidine-containing peptides was accredited to the tef et al. 2010), Nile tilapia (Oreochromis niloticus)(Asp-Pro- proton-donation ability of the histidine imidazole group. Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe, IC = Also, histidine and proline take part in the antioxidant activ- 8.82 μM) (Ngo et al. 2010), black pomfret (Parastromateus ity of designed peptides tests,among Pro-His-Hisexhibited niger) (Ala-Met-Thr-Gly-Leu-Glu-Ala, RSA = 78.6%) (Jai the greatest antioxidant activity(Tsugeet al. 1991). As re- Ganesh et al. 2011), and croaker (Gly-Asn-Arg-Gly-Phe- ported by Dávalos et al. (2004), among amino acids, tyrosine, Ala-Cys-Arg-His-Ala) (Samaranayaka and Li-chan 2011) tryptophan, and methionine exhibited the highest antioxi- (Lee et al. 2011a, 2011b). According to previous reports, the dant activity, followed by histidine, cysteine, and phenylalan- antioxidant peptides possess some metal chelation or hydro- ine. The antioxidant activity of peptides containing histidine gen/electron donating activity, thereby allowing them to has been accredited to the chelating and lipid radical- interact with free radicals and to terminate the radical chain trapping ability of the imidazole ring (Murase et al. 1993; reaction or prevent their formation (Ren et al. 2008;You Park et al. 2001). However, the active peptide in our study et al. 2010). Amino acid constituents and sequence of pep- did not have hydrophobic amino acids. Since, our peptide tides are vital for their antioxidant activity. Hydrophobic yielded larger EC values. amino acids and one or more residues of cysteine, methio- nine, histidine, tyrosine, tryptophan, proline, and phenylalan- Prevention of oxidation-induced DNA damage by a black ine have been identified to enhance the activities of the eelpout peptide antioxidant peptides (Ren et al. 2008;Jeetal. 2007;You We evaluated the protective activity of purified antioxidant et al. 2010). As it has been confirmed, functional peptides peptide against hydroxyl radical-induced DNA damage in rely on amino acid sequence and structure (Elias et al. in vitro studies by using RAW 264.7 cell line. As shown in 2008). Li et al. (2007) reported that the antioxidant activity Fig. 4, the purified peptide had a protective effect against DNA oxidation induced by hydroxyl radical with increasing peptide concentrations ranging from 50 to 200 μM. These Table 5 Purification of antioxidant peptide from black eelpout results indicate that black eelpout peptide purified, exerted muscle hydrolysate by pepsin treatment a adequate protective effects on radical-mediated DNA dam- Purification step EC value (μg/mL) Purification fold age. Furthermore, our results clearly explain the fact that Pepsin hydrolysate 830.01 ± 0.05 1.00 purified peptide can inhibit oxidative damage to DNA when Sephadex gel filtration (B) 650.32 ± 0.14 1.28 2+ exposed to OH radical generated by Fe(II)/H O .Fe 2 2 RP−HPLC (F1) 87.45 ± 0.05 9.49 catalyzes the conversion of H O to OH radical in physical 2 2 Purified peptide (F1–1) 56.12 ± 0.01 16.24 systems. The OH radical highly reacted leading to damage Relative value of reciprocal of DPPH radical scavenging activity by EC of both the purine and pyrimidine base and also 50 Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 6 of 7 Fig. 3 Identification of molecular mass and amino acid sequence of the purified peptides from black eelpout muscle hydrolysate by HPLC. MS/ MS experiments were performed on a Q-TOF tandem mass spectrometer equipped with a nano-ESI source deoxyribose backbone lesion for DNA (Ngo et al. 2009). Conclusion DNA is another sensitive bio-target for ROS-mediated In this study, black eelpout muscle protein was hydro- oxidative damage (Martinez et al. 2003)asit isknown to lyzed using enzymatic hydrolysis with various enzymes. initiate carcinogenesis or pathogenesis in neurodegenerative The antioxidant activity of the different enzyme hydroly- diseases such as Parkinson’s disease and Alzheimer’s sates was determined and compared. Pepsin hydrolysate disease. Therefore, ROS, a hydroxyl radical, has been recog- showed the highest antioxidant activity and thus it was nized as a DNA-damaging agent of physiological signifi- further purified using chromatography. A seven-amino cance (You et al. 2002). Bioactive peptides with various acid residue peptide with antioxidant activity was identi- biological activities such as antioxidative activity can be uti- fied from the pepsin hydrolysate of black eelpout muscle. lized in order to develop pharmaceutical and nutraceutical Collectively, the results of this study suggest that black products in industrial scale (Abuine et al. 2019). eelpout muscle protein hydrolysate could potentially contribute to development of bioactive peptides in basic research. Abbreviations ANOVA: Analysis of variance; BHA: Butylated hydroxyanisole; BHT: Butylated hydroxytoluene; CAT: Catalase; DAD: Diode array detector; DNA: Deoxyribonucleic acid; DPPH: 2,2-Diphenyl-1-picryl-hydrazyl-hydrate; ESI-MS: Electrospray ionization mass spectrometry; GR: Glutathione reductase; GSH-Px: Glutathione peroxidase; H O : Hydrogen peroxide; O −: Superoxide 2 2 2 anion; � OH: Hydroxyl radical; Q-TOF: Quadrupole time-of-flight; ROS: Reactive oxygen species; RP-HPLC: Reverse-phase high performance liquid chromatography; SEM: Scanning electron microscope; SOD: Superoxide dismutase; SPSS: Statistical package for the social sciences; TBHQ: Tbutylhydroquinone; TFA: Trifluoroacetic acid Acknowledgements This research was supported by a grant from Marine Bioprocess Research Fig. 4 Protective effect on oxidation-induced DNA damage of Center of the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime, Republic of Korea. purified peptide from black eelpout at various concentrations. Blank: untreated sample and H O , FeSO . Control: distilled water instead 2 2 4 Authors’ contributions of sample. Sample: Treated sample, H O and FeSO (+, treatment; 2 2, 4. HGB and LJK conceived and designed the study and helped to draft the −, not treatment) manuscript and revised the manuscript. LJK performed the experiments, Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 7 of 7 analyzed the data, and drafted the manuscript. All authors read and Kim SK, Kim YT, Byun HG, Nam KS, Joo DS, Shahidi F. Isolation and approved the final manuscript. characterization of antioxidative peptides from gelatin hydrolysate of Alaska Pollack skin. J Agric Food Chem. 2001;49:1984–9. Korhonen M, Pihlanto-Leppala A, Tupasela T. Impact of processing on bioactive Funding proteins and peptides. Trends Food Sci Technol. 1998;9:307–19. The design of the study; collection, analysis, and interpretation of the data; Lee WS, Jeon JK, Byun HG. Characterization of a novel antioxidative peptide from and writing of the manuscript were funded by a grant from Marine the sand eel Hypoptychus dybowskii. Process Biochem. 2011b;46:1207–11. Bioprocess Research Center of the Marine Biotechnology Program funded by Lee WS, Kim YT, Byun HG. Antioxidant activities of steamed extract from squid the Ministry of Land. (Todarodes pacificus) muscle. J Food Sci Nutr. 2011a. Li B, Chen F, Wang X, Ji B, Wu Y. Isolation and identification of antioxidative peptides Availability of data and materials from porcine collagen hydrolysate by consecutive chromatography and All datasets generated during and/or analyzed during the current study are electrospray ionization–mass spectrometry. Food Chem. 2007;102(4):1135–43. available from the corresponding author on reasonable request. Liceaga-Gesualdo AM, Li-Chan ECY. Functional properties of fish protein hydrolysate from herring (Clupea harengus). J Food Sci. 1999;64:1000–4. 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Food Res Int. 2009;42:1266–72. Je JY, Qian Z, Byun HG, Kim SK. Purification and characterization of an antioxidant Publisher’sNote peptide obtained from tuna backbone protein by enzymatic hydrolysis. Springer Nature remains neutral with regard to jurisdictional claims in Process Biochem. 2007;42:840–6. published maps and institutional affiliations. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Fisheries and Aquatic Sciences Springer Journals

Characterization of antioxidative peptide purified from black eelpout (Lycodes diapterus) hydrolysate

Fisheries and Aquatic Sciences , Volume 22 (1) – Oct 29, 2019

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Life Sciences; Fish & Wildlife Biology & Management; Marine & Freshwater Sciences; Zoology; Animal Ecology
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Abstract

The functional peptides from protein hydrolysates of various fishery sources have been identified such as antioxidant activity. The main intention of this study was purification and characterization of antioxidative peptide from black eelpout muscle. The antioxidative peptides were purified from black eelpout (Lycodes diapterus) muscle using different proteases. Antioxidant activity of black eelpout hydrolysates was evaluated using DPPH radical scavenging activity. Among six hydrolysates, the pepsin hydrolysate had the highest antioxidant activity compared to the other hydrolysates. Therefore, it was further purified and a peptide with seven amino acid residues of DLVKVEA (784 Da) was identified by amino acid sequence analysis. The EC value for scavenging DPPH radicals by purified peptide was 688.77 μM. Additionally, the purified peptide exhibited protective effect against DNA damage induces by oxidation in mouse macrophages (RAW 264.7 cells). The results of this study suggest that black eelpout muscle protein hydrolysate could potentially contribute to development of bioactive peptides in basic research. Keywords: Antioxidant, DPPH radical scavenging, Peptide, Hydrolysates, Pepsin, Black eelpout muscle Background oxidative stress (McCord 1993). Steady-state maintenance Free radicals are highly reactive species with their single of ROS/antioxidant ratio is vital for avoiding oxidative and unbalanced electrons. The oxidation by free radicals stress (Somani and Rybak 1996). Synthetic antioxidants in the body may cause many chronic diseases such as (butylated hydroxyanisole (BHA), tbutylhydroquinone cardiovascular diseases, diabetes, cancer, and neurode- (TBHQ), butylated hydroxytoluene (BHT), and propyl generative disorders (Dong et al. 2008). Fatty acids and gallate) have been widely used as food preservatives as lipids oxidation induced by free radicals deteriorate the they delay the discoloration and deterioration caused by food quality (Liceaga-Gesualdo and Li-Chan 1999). oxidation (Wanita and Lorenz 1996). So, the use of these Reactive oxygen species (ROS) (O (superoxide anion), synthetic antioxidants has been limited in some countries � OH (hydroxyl radical), and H O (hydrogen peroxide)) due to their potential health hazard (Becker 1993). 2 2 are metabolic by-products of normal aerobic metabolism Recently, enzymatic hydrolysis with proteases has gar- (Castro and Freeman 2001). Nevertheless, the body is sup- nered much attention. Protein hydrolysates or peptides ported with several antioxidant defense systems where affect health-related functions such as antioxidant func- they can scavenge and transform ROS or free radicals into tion (Clemente 2000). Therefore, various antioxidant harmless species (Yeung et al. 2002). The antioxidant peptides have been isolated from marine organisms defense system includes catalase (CAT), glutathione per- through enzymatic hydrolysis, including abalone muscle oxidase (GSH-Px), superoxide dismutase (SOD), and (Haliotis discus hannai Ino) and scallop (Patinopecten glutathione reductase (GR). Enzymatic and non-enzymatic yessoensis) (Zhou et al. 2012), threadfin bream surimi antioxidants team up to scavenge and eradicate the (Wiriyaphan et al. 2012), croaker (Otolithes ruber) muscle (Nazeer et al. 2012), sand eel (Lee et al. 2011a, 2011b), sardinelle (Sardinella aurita) (Bougatef et al. * Correspondence: hgbyun@gwnu.ac.kr 2010), tuna liver (Je et al. 2009), marine rotifer (Byun Department of Marine Biotechnology, Gangneung-Wonju National University, Gangneung 25457, Republic of Korea et al. 2009), and algae protein waste (Sheih et al. 2009). Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 2 of 7 Enzymatic hydrolysates exhibited several advantages wavelength at 254 nm and a flow rate of 1.0 mL/min. All when incorporated into foods, by improving water- analyses were carried out in triplicate. binding ability, solubility of protein, emulsifying stability, heat stability of myofibrillar protein, and the nutritional Preparation of black eelpout muscle hydrolysates quality of foods. Thus, enzymatic hydrolysis has become To prepare black eelpout muscle hydrolysates, enzymatic an appreciated tool for modifying the applicability of hydrolysis was performed using various enzymes (Alca- proteins (Korhonen et al. 1998). Normally, bioactive lase, α-chymotrypsin, Neutrase, papain, pepsin, and tryp- peptides remain inactive within the parent protein mol- sin) at their optimal conditions. Black eelpout muscle ecule until they are released by hydrolysis. Most of bio- was hydrolyzed separately using various enzymes with a active peptides are composed with 2–20 amino acids. substrate to enzyme ratio of 1:100 for 6 h, under Amino acids arrangement of the peptides plays a critical optimum pH and temperature conditions (Table 1). At role in its bioactivity (Himaya et al. 2012). the end of 6 h, hydrolysates were filtered by glass filter The black eelpout, Lycodes diapterus, is distributed in and lyophilized and stored at − 80 °C until use. The yield the Northwest Pacific/North of central East Sea of Korea of hydrolysate from black eelpout muscle was calculated and the Sea of Okhotsk and inhabits sand and mud bot- as follows: toms in deep water of 150–200 m depth. Black eelpout is a traditional food that is rich in protein, essential weight of the black eelpout hydrolysates YieldðÞ % ¼  100 amino acids, omega-3 polyunsaturated fatty acids, and weight of the black eelpout vitamins. In the present study, we investigated the 2,2- diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scav- enging activity of enzymatically prepared black eelpout muscle protein hydrolysate to isolate a potent antioxi- Determination of DPPH radical scavenging activity dant peptide. And the protective effect of the purified DPPH radical scavenging activity (RSA) was assessed by peptide against deoxyribonucleic acid (DNA) oxidation using the method of Yen and Hsieh (1995) with minor induced by the hydroxyl radical was verified further. modifications. The sample was mixed with 120 μLof methanol and 40 μL of 0.15 mM DPPH in methanol was Materials and methods added. The mixture was incubated at room temperature Materials in the dark for 30 min. The absorbance of the mixture Fresh samples of black eelpout (Lycodes diapterus) were was measured at 517 nm using a spectrophotometer obtained from East Sea Fisheries Research Institute, (JASCO, Japan). The control sample was prepared in the Gangneung, South Korea. The bones and viscera were same manner where methanol was used instead of the removed from the black eelpout. Then the separated 40 μL sample volume. DPPH radical scavenging activity muscle was stored at − 80 °C until use. Several commer- was calculated as follows: cial enzymes, such as α-chymotrypsin, papain, pepsin, and trypsin, were obtained from Sigma Chemical Co. A −A (St. Louis, MO). Alcalase and Neutrase enzymes were control sample RSAðÞ % ¼  100 obtained from Novo Co. (Novo Nordisk, Bagsvaerd, control Denmark). DPPH was obtained from Wako Chemical Co. All other reagents used in this study were reagent where A is the absorbance of sample and A is sample control grade chemicals. the absorbance of the control. The EC value is defined as an effective concentration of peptide that is required Analysis of proximate compositions to scavenge 50% of radical activity. Crude protein content of black eelpout was determined Table 1 Optimal conditions for enzymatic hydrolysis of various by the Kjeldahl method (Auto Kjeldahl system, Buchi B- enzymes 324/435/412, Switzerland). Ether extraction method was Enzyme Buffer pH Temperature (°C) used to determine the crude lipid content. Moisture Alcalase 50 mM Na HPO -NaH PO 7.0 50 2 4 2 4 content was determined by oven drying at 105 °C for 24 α-Chymotrypsin 50 mM Na HPO -NaH PO 7.0 37 h. Ash content was determined by a muffler furnace at 2 4 2 4 550 °C for 4 h (Association of Official Analytical Chemist Neutrase 50 mM Na HPO -NaH PO 7.0 50 2 4 2 4 (AOAC) 2000). Amino acids were analyzed using an Papain 50 mM Na HPO -NaH PO 7.0 37 2 4 2 4 automatic analyzer (Hitachi Model 835-50, Japan) with a Pepsin 20 mM HCl 2.0 37 C18 column (5 μm, 4.6 × 250 mm, Watchers, MA). The Trypsin 50 mM Na HPO -NaH PO 7.0 37 2 4 2 4 reaction was carried out at 38 °C, with the detection Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 3 of 7 Purification and identification of antioxidant peptides Statistical analysis The black eelpout muscle hydrolysate was dissolved in Data were analyzed for statistical significance using analysis distilled water and loaded onto a Sephadex G-25 gel fil- of variance (ANOVA) followed by Dunnett’smultiplecom- tration column (2.5 × 70 cm) which had been previously parison test with statistical package for the social sciences equilibrated with distilled water. The column was then (SPSS) software (version 14). All values obtained from three eluted with distilled water at a flow rate of 1.5 mL/min different experiments were expressed as the mean value ± (fraction volume 7.5 mL) and separated fractions were standard deviation (SD). monitored at 215 nm, collected at a volume of 7.5 mL, and measured for DPPH radical scavenging activity. Results and discussion Highest active fraction was injected into a preparative Proximate composition of black eelpout muscle reverse-phase high performance liquid chromatography Proximate composition of black eelpout muscle showed (RP-HPLC) column (Grom-Sil 120 ODS-5ST, ø 10 × the 20.81% moisture content, 8.63% lipid content, 4.09% 250 mm, 5 μm, Grom™, Germany) and was separated ash, 2.46% carbohydrate, and 64.02% protein content using linear gradient of acetonitrile (0–20% v/v) contain- (Table 2). The protein content was the highest among all ing 0.1% trifluoroacetic acid (TFA) on an RP-HPLC sys- the composition contents. However, the low lipid and ash tem (Agilent Technologies, USA). Elution peaks were content suggests that the extraction processes by enzym- monitored at 280 nm on diode array detector (DAD). atic hydrolysis of biofunctional peptide is effective. The The purified fractions from preparative column were most abundant amino acids in black eelpout muscle were monitored at 280 nm and purified by RP-HPLC on a glycine, alanine, lysine, and leucine which accounted for C18 analytical column (ø 4.6 × 250 mm, 5 μm, Waters, 20.82%, 17.13%, 8.1%, and 6.24%, respectively (Table 3). Milford, MA, USA) using an acetonitrile gradient of 5– Generally, fish and other mammalian skin have higher 30% (v/v) at a flow rate of 0.5 mL/min for 40 min. Fi- percentage of Gly, Leu, and Pro compared to muscle pro- nally, the fraction with the highest DPPH radical scaven- teins (Gomez-Guillen et al. 2002). ging activity was collected and lyophilized followed by the amino acid sequence identification. Antioxidant activity of black eelpout muscle hydrolysates Black eelpout muscle protein hydrolysates were prepared by using commercial proteases including Alcalase, α- Determination of molecular weight and amino acid chymotrypsin, Neutrase, papain, pepsin, and trypsin. sequence The hydrolysis yields were 68.28%, 66.85%, 66.14%, and Molecular weight and amino acid sequence of purified 58.76% for papain, Alcalase, pepsin, and trypsin, respect- peptide from black eelpout muscle protein were deter- ively (Table 4). Among six hydrolysates, pepsin hydrolys- mined by quadrupole time-of-flight (Q-TOF) mass spec- ate exhibited the greatest DPPH radical scavenging trometry (Micromass, Altrincham, UK) coupled with activity relative to the other hydrolysates. In terms of the electrospray ionization (ESI) source. The purified peptide DPPH radical scavenging activation (Fig. 1), the lowest dissolved in methanol/water (1:1, v/v) was infused into the EC value was exhibited by the pepsin hydrolysate at ESI source and the molecular mass was determined by 0.83 mg/mL. Thus the pepsin hydrolysate may contain 2+ doubly charged (M+ 2H) state in the mass spectrum. bioactive compounds that could react with free radicals Following molecular mass determination, the peptide was to transform them into more stable products and ter- automatically selected for fragmentation and sequence in- minate the radical chain reaction. Peptides with antioxi- formation was obtained by tandem MS analysis. dative activity have been obtained by enzymatic hydrolysis of various marine organisms (Je et al. 2007). Protective potential by the hydroxyl radical-induced DNA Several studies have suggested that the variation of anti- damage oxidant activity of a peptide is due to its amino acid se- To assess the protective effects of the hydrolysate against quence and length (Kim et al. 2001). However, DPPH DNA damage caused by hydroxyl radicals, the reaction radical scavenging activity of pepsin hydrolysate was was induced by placing the following reagents in an Table 2 Proximate compositions of black eelpout muscle Eppendorf tube: 5 μL of genomic DNA (RAW 264.7 cell Components Content (%) line), 2 mM FeSO , and various concentrations of the Moisture 20.81 purified peptide from black eelpout hydrolysate. The mixture was then incubated at 37 °C for 30 min, followed Protein 64.02 by the addition of 4 μLof10mM H O (Dávalos et al. 2 2 Lipid 8.63 2004). Finally, the mixture was subjected to 1.0% agarose Ash 4.09 gel electrophoresis and DNA bands were stained with Carbohydrate 2.46 ethidium bromide. Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 4 of 7 Table 3 Amino acid contents of black eelpout muscle Amino acids Contents (%) Tau 0.78 Asp 6.08 Thr 4.07 Ser 3.93 Glu 2.21 Gly 20.82 Ala 17.13 Val 3.61 Cys 0.34 Met 1.96 Ile 2.43 Fig. 1 EC values for DPPH radical scavenging activity of black eelpout muscle hydrolysates. Statistical significance was determined Leu 6.24 by ANOVA Try 1.71 Phe 2.33 secondary fractions contain small-molecular-size peptides. Lys 8.10 According to Pihlanto (2000), numerous bioactive peptides His 1.80 are found between 2 and 20 amino acids in length with a Arg 5.13 small-molecular-size. Therefore, the secondary fractions Total 100.00 were assumed to have the greatest potential bioactivity. Fraction B was further separated by RP-HPLC using an ODS column and subsequently fractionated into three frac- lower than that of synthetic antioxidants BHA and BHT. tions (F1–F3) (Fig. 2II). Among separated fractions, the The next stage in analysis required the use of HPLC for fraction F1 showed the highest DPPH radical scavenging purifying the antioxidant peptide from pepsin hydrolys- activity with the EC value of 87.45 μg/mL (Fig. 2II). Frac- ate of black eelpout muscle. tion F1–1, with the strongest DPPH radical scavenging ac- tivity was purified further by using RP-HPLC on the C18 Purification of antioxidant peptide analytical column a linear gradient of acetonitrile (5–30%) To identify the antioxidant peptide from pepsin hydrolysate for 40 min at a flow rate of 0.5 mL/min (Fig. 2III). The EC of black eelpout muscle, the use of different chromato- value of the purified peptide was 51.12 μg/mL, 16.24–fold graphic techniques is required. As shown in Fig. 2,chroma- compared to the pepsin hydrolysate (0.83 mg/mL) using tographic profiles were obtained during different the three-step purification procedure (Table 5). A single purification steps of black eelpout muscle hydrolysate. In peptide fraction that demonstrated DPPH radical scaven- the first step, pepsin hydrolysate was separated into four ging activity was purified on an analytical HPLC column fractions (A–D) on a Sephadex G-25 chromatography col- and their amino acid sequences were determined by N- umn (Fig. 2I). Among separated fractions, the B fraction terminal sequencing analysis. had the highest DPPH radical scavenging activity at 0.65 mg/mL (Fig. 2I). Sephadex G-25 column chromatography Characterization of purified antioxidant peptide separates according to molecular-size, where the primary The purified fraction F1–1 was analyzed by electrospray fractions contain large-molecular-size peptides, and ionization mass spectrometry (ESI-MS) for molecular mass determination and ESI-MS/MS for the peptide Table 4 Yields of various hydrolysates from black eelpout characterization. Amino acid sequence of purified antioxi- muscle dant peptide was identified as Asp-Leu-Val-Lys-Val-Glu-Ala Hydrolysates Yields (%) with EC value and molecular weight of 688.77 μMand Alcalase 66.85 784 Da, respectively (Fig. 3). These results support for the α-Chymotrypsin 51.30 general finding that short peptides with 2–10 amino acids demonstrate greater bioactive properties such as antioxidant Neutrase 39.47 activity compared to their parent native proteins or large Papain 68.28 polypeptides (Li et al. 2007). In this study, the purified anti- Pepsin 66.14 oxidant peptide was found to have a similar sequence with Trypsin 58.76 the other reports, including the sardinelle (Sardinellaaurita) Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 5 of 7 Fig. 2 Steps for the purification of DPPH radical scavenging activity peptide from black eelpout muscle hydrolysate. I Sephadex G-25 Gel filtration chromatogram of hydrolysates. Gel filtration chromatogram of hydrolysates prepared with black eelpout muscle. Separation was performed with 1.5 mL/min and collected at a fraction volume of 7.5 mL. The fractions isolated by Sephadex G-25 Gel column were separated (A–D) and DPPH radical scavenging activity was determined as upper panel. II, III Reverse phase-HPLC chromatograms of the potent DPPH radical scavenging activity fractions from the previous steps. The lower panels of each pair show the chromatography results of separated fractions while the top panels of each pair represent the DPPH radical scavenging activity of separated fractions in terms of their EC values expressed in mg/mL (I)or μg/mL (II, III). Statistical significance was determined by ANOVA (Gly-Ala-Trp-Ala, RSA = 52 ± 1.44% at 150 μg/mL) (Bouga- of histidine-containing peptides was accredited to the tef et al. 2010), Nile tilapia (Oreochromis niloticus)(Asp-Pro- proton-donation ability of the histidine imidazole group. Ala-Leu-Ala-Thr-Glu-Pro-Asp-Pro-Met-Pro-Phe, IC = Also, histidine and proline take part in the antioxidant activ- 8.82 μM) (Ngo et al. 2010), black pomfret (Parastromateus ity of designed peptides tests,among Pro-His-Hisexhibited niger) (Ala-Met-Thr-Gly-Leu-Glu-Ala, RSA = 78.6%) (Jai the greatest antioxidant activity(Tsugeet al. 1991). As re- Ganesh et al. 2011), and croaker (Gly-Asn-Arg-Gly-Phe- ported by Dávalos et al. (2004), among amino acids, tyrosine, Ala-Cys-Arg-His-Ala) (Samaranayaka and Li-chan 2011) tryptophan, and methionine exhibited the highest antioxi- (Lee et al. 2011a, 2011b). According to previous reports, the dant activity, followed by histidine, cysteine, and phenylalan- antioxidant peptides possess some metal chelation or hydro- ine. The antioxidant activity of peptides containing histidine gen/electron donating activity, thereby allowing them to has been accredited to the chelating and lipid radical- interact with free radicals and to terminate the radical chain trapping ability of the imidazole ring (Murase et al. 1993; reaction or prevent their formation (Ren et al. 2008;You Park et al. 2001). However, the active peptide in our study et al. 2010). Amino acid constituents and sequence of pep- did not have hydrophobic amino acids. Since, our peptide tides are vital for their antioxidant activity. Hydrophobic yielded larger EC values. amino acids and one or more residues of cysteine, methio- nine, histidine, tyrosine, tryptophan, proline, and phenylalan- Prevention of oxidation-induced DNA damage by a black ine have been identified to enhance the activities of the eelpout peptide antioxidant peptides (Ren et al. 2008;Jeetal. 2007;You We evaluated the protective activity of purified antioxidant et al. 2010). As it has been confirmed, functional peptides peptide against hydroxyl radical-induced DNA damage in rely on amino acid sequence and structure (Elias et al. in vitro studies by using RAW 264.7 cell line. As shown in 2008). Li et al. (2007) reported that the antioxidant activity Fig. 4, the purified peptide had a protective effect against DNA oxidation induced by hydroxyl radical with increasing peptide concentrations ranging from 50 to 200 μM. These Table 5 Purification of antioxidant peptide from black eelpout results indicate that black eelpout peptide purified, exerted muscle hydrolysate by pepsin treatment a adequate protective effects on radical-mediated DNA dam- Purification step EC value (μg/mL) Purification fold age. Furthermore, our results clearly explain the fact that Pepsin hydrolysate 830.01 ± 0.05 1.00 purified peptide can inhibit oxidative damage to DNA when Sephadex gel filtration (B) 650.32 ± 0.14 1.28 2+ exposed to OH radical generated by Fe(II)/H O .Fe 2 2 RP−HPLC (F1) 87.45 ± 0.05 9.49 catalyzes the conversion of H O to OH radical in physical 2 2 Purified peptide (F1–1) 56.12 ± 0.01 16.24 systems. The OH radical highly reacted leading to damage Relative value of reciprocal of DPPH radical scavenging activity by EC of both the purine and pyrimidine base and also 50 Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 6 of 7 Fig. 3 Identification of molecular mass and amino acid sequence of the purified peptides from black eelpout muscle hydrolysate by HPLC. MS/ MS experiments were performed on a Q-TOF tandem mass spectrometer equipped with a nano-ESI source deoxyribose backbone lesion for DNA (Ngo et al. 2009). Conclusion DNA is another sensitive bio-target for ROS-mediated In this study, black eelpout muscle protein was hydro- oxidative damage (Martinez et al. 2003)asit isknown to lyzed using enzymatic hydrolysis with various enzymes. initiate carcinogenesis or pathogenesis in neurodegenerative The antioxidant activity of the different enzyme hydroly- diseases such as Parkinson’s disease and Alzheimer’s sates was determined and compared. Pepsin hydrolysate disease. Therefore, ROS, a hydroxyl radical, has been recog- showed the highest antioxidant activity and thus it was nized as a DNA-damaging agent of physiological signifi- further purified using chromatography. A seven-amino cance (You et al. 2002). Bioactive peptides with various acid residue peptide with antioxidant activity was identi- biological activities such as antioxidative activity can be uti- fied from the pepsin hydrolysate of black eelpout muscle. lized in order to develop pharmaceutical and nutraceutical Collectively, the results of this study suggest that black products in industrial scale (Abuine et al. 2019). eelpout muscle protein hydrolysate could potentially contribute to development of bioactive peptides in basic research. Abbreviations ANOVA: Analysis of variance; BHA: Butylated hydroxyanisole; BHT: Butylated hydroxytoluene; CAT: Catalase; DAD: Diode array detector; DNA: Deoxyribonucleic acid; DPPH: 2,2-Diphenyl-1-picryl-hydrazyl-hydrate; ESI-MS: Electrospray ionization mass spectrometry; GR: Glutathione reductase; GSH-Px: Glutathione peroxidase; H O : Hydrogen peroxide; O −: Superoxide 2 2 2 anion; � OH: Hydroxyl radical; Q-TOF: Quadrupole time-of-flight; ROS: Reactive oxygen species; RP-HPLC: Reverse-phase high performance liquid chromatography; SEM: Scanning electron microscope; SOD: Superoxide dismutase; SPSS: Statistical package for the social sciences; TBHQ: Tbutylhydroquinone; TFA: Trifluoroacetic acid Acknowledgements This research was supported by a grant from Marine Bioprocess Research Fig. 4 Protective effect on oxidation-induced DNA damage of Center of the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime, Republic of Korea. purified peptide from black eelpout at various concentrations. Blank: untreated sample and H O , FeSO . Control: distilled water instead 2 2 4 Authors’ contributions of sample. Sample: Treated sample, H O and FeSO (+, treatment; 2 2, 4. HGB and LJK conceived and designed the study and helped to draft the −, not treatment) manuscript and revised the manuscript. LJK performed the experiments, Lee and Byun Fisheries and Aquatic Sciences (2019) 22:22 Page 7 of 7 analyzed the data, and drafted the manuscript. All authors read and Kim SK, Kim YT, Byun HG, Nam KS, Joo DS, Shahidi F. Isolation and approved the final manuscript. characterization of antioxidative peptides from gelatin hydrolysate of Alaska Pollack skin. J Agric Food Chem. 2001;49:1984–9. Korhonen M, Pihlanto-Leppala A, Tupasela T. Impact of processing on bioactive Funding proteins and peptides. Trends Food Sci Technol. 1998;9:307–19. The design of the study; collection, analysis, and interpretation of the data; Lee WS, Jeon JK, Byun HG. Characterization of a novel antioxidative peptide from and writing of the manuscript were funded by a grant from Marine the sand eel Hypoptychus dybowskii. Process Biochem. 2011b;46:1207–11. Bioprocess Research Center of the Marine Biotechnology Program funded by Lee WS, Kim YT, Byun HG. Antioxidant activities of steamed extract from squid the Ministry of Land. (Todarodes pacificus) muscle. J Food Sci Nutr. 2011a. Li B, Chen F, Wang X, Ji B, Wu Y. Isolation and identification of antioxidative peptides Availability of data and materials from porcine collagen hydrolysate by consecutive chromatography and All datasets generated during and/or analyzed during the current study are electrospray ionization–mass spectrometry. Food Chem. 2007;102(4):1135–43. available from the corresponding author on reasonable request. Liceaga-Gesualdo AM, Li-Chan ECY. Functional properties of fish protein hydrolysate from herring (Clupea harengus). J Food Sci. 1999;64:1000–4. 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Fisheries and Aquatic SciencesSpringer Journals

Published: Oct 29, 2019

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