In vitro antioxidative activities of squid (Ommastrephes bartrami) viscera autolysates and identification of active peptides

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In vitro antioxidative activities of squid (Ommastrephes bartrami) viscera autolysates and identification of active peptides Ru Song a,∗ , Kai-qiang Zhang a , Rong-bian Wei b,∗ a Key Laboratory of Health Risk Factors for Seafood of Zhejiang Province, College of Food Science and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China b College of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China

a r t i c l e

i n f o

Article history: Received 25 December 2015 Received in revised form 7 June 2016 Accepted 14 June 2016 Available online xxx Keywords: Squid viscera Autolysis Antioxidative activity Peptide identification Amino acid sequence

a b s t r a c t Squid viscera, one of the major by-products in squid processing, were hydrolyzed to generate bioactive autolysates using their endogenous proteases. In vitro antioxidative activities of squid viscera autolysates (SVAs) were evaluated. The SVAs demonstrated strong activity on scavenging 1,1-diphenyl-2-picrylhydrazil (DPPH) and hydroxyl radicals, as well as reducing power ability. The SVAs were purified using size exclusion chromatography and RP-HPLC. Nineteen peptides were identified in the active fraction SVAs3 by liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-QTOF-MS/MS). Among these identified peptides, six peptides with relatively small molecular weights (700–800 Da), LLAPPER, FPGLADR, WVAPLK, FFNPVH, FNVVLK and LELPLK, were synthesized for assaying antioxidative activity in vitro. Peptide WVAPLK demonstrated strong scavenging effects on free radicals, with the IC50 values of 0.82 ± 0.08 mg/mL (or 1.14 ± 0.11 mM) for DPPH radical and 1.85 ± 0.04 mg/mL (or 2.60 ± 0.06 mM) for hydroxyl radical, respectively. The low molecular weight and hydrophobic residue W at the N-terminus and basic residue K at the C-terminus as well as specific residues P and L within sequence should play a key role for the high antioxidative activity of WVAPLK. The results suggested that the SVAs could be used as a source of antioxidants and peptides. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Squid viscera, which are one of the predominant by-products and account for more than 20% of the whole body weight, are generated accompanying squid processing [1]. Squid viscera contain abundant natural proteins, lipids, and minerals [2]. The disposal of squid viscera will cause environmental pollution because of their high organic content [2,3]. Furthermore, the improper treatment of squid viscera will retard the instant processing for squid industry in market places [4]. Therefore, it is essential to develop efficient methods to convert squid viscera into more profitable and marketable products. Enzymatic hydrolysis is considered as a safety approach to transform protein into functional hydrolysates [5]. Recently, protein hydrolysates produced from aquatic products and by-products have become popular in food industry due to utilization of these

∗ Corresponding authors at: 1 Ocean University S. Rd, Lincheng New District, Zhoushan, Zhejiang, 316022, PR China. E-mail addresses: [email protected] (R. Song), [email protected] (R.-b. Wei).

under-utilized marine sources into acceptable food supplements with enhanced functional properties [6–13]. In addition, aquatic products and by-products hydrolysates, such as abalone viscera [14], threadfin bream surimi processing byproduct [15], patin myofibrillar [16], grass carp skin [17], tuna dark muscle protein byproduct [18] and bluefin leatherjacket heads [19], have also been proven to be good sources of antioxidant peptides. Aquatic invertebrates and vertebrates viscera are rich sources of various enzymes, including proteases, lipase, and amylase [4,20]. Proteases represent one of the three largest groups of industrial enzymes, and play vital roles in food, pharmaceutical and leather industries, as well as in bioremediation [4]. The proteases of viscera have been found to possess pepsin, trypsin, chymotrypsin, collagenase and elastase [20]. In fact, aquatic invertebrates or vertebrates viscera have been reported to digest proteins to produce bioactive protein hydrolysates, such as autolysis-assisted Pacific hake protein hydrolysate [21], sardine viscera hydrolysate [22], and bovine muscle protein and gelatin hydrolysates digested by catfish viscera [23]. Hence, it is reasonable to believe that autolysis would be a much more economical and efficient way to resolve the problem of reutilization and valorization of aquatic byproducts in large scale

http://dx.doi.org/10.1016/j.procbio.2016.06.015 1359-5113/© 2016 Elsevier Ltd. All rights reserved.

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compared with the conventional method of adding commercial proteases. In a latest study by Arias-Moscoso et al. (2015), squid by-products were produced by autolysis using a mixture of endogenous proteases [24]. However, to the best of our knowledge, there are very few studies on the antioxidative activities of squid viscera autolysates. In our previous study, the autolysis conditions were optimized to prepare antioxidative squid viscera autolysates (SVAs) (data not published). In this study, the SVAs were evaluated for antioxidative activity using various assays in vitro, with the aim to assess the potential application used as natural antioxidants. Furthermore, the features of the most active peptide fraction were analyzed to investigate the possible influence of peptide composition on antioxidant properties.

Where AC was the absorbance of control with sample replaced by an equivalent volume of distilled water, AB was the absorbance of blank with the same volume of 99.5% ethanol replacing DPPH solution.

2. Materials and methods

2.3.2. Hydroxyl radical-scavenging activity assay Hydroxyl radical scavenging activity was measured by the method as reported by De Avellar et al. [27] with slight modifications. First, the reaction mixture, including 70 ␮L of 0.75 mM 1, 10-phenanthroline, 140 ␮L of sodium phosphate (pH 7.4), 70 ␮L of FeSO4 , and 70 ␮L of sample, were mixed vigorously. Before incubating at 37 ◦ C for 1 h, the mixture was added with 70 ␮L of 0.12% (v/v) H2 O2 , and then cooled quickly to room temperature with running tap water. The absorbance was determined at 536 nm (721G-100 Vis-spectrophotometer, Shanghai, China). The hydroxyl radical-scavenging activity was expressed as:

2.1. Materials and chemicals

Hydroxyl radical- scavenging activity(%) =

Fresh squid viscera of North Pacific squid (Ommastrephes bartrami) were provided by Zhejiang Fudan Tourism Food Co., Ltd. Squid viscera were transported to our laboratory at 4 ◦ C within 30 min. After removal of ink sac, the visceral masses were portioned, sealed separately in plastic bags (200 g), and stored at −20 ◦ C until use. 1,1-diphenyl-2-picryl-hydrazil (DPPH) and reduced Lglutathione (GSH) were purchased from Shanghai Sigma Chemical Co., Ltd (Shanghai, China). L-carnosine was purchased from Aladdin Industrial Corporation (Shanghai, China). Acetonitrile and formic acid were of chromatographic grade, purchased from Thermo Fisher Scientific Co., Ltd (Shanghai, China). All other chemicals and reagents used in this study were of analytical grade.

Where AS , the absorbance of sample; AP , replacing sample with equivalent volume of distilled water; AB , replacing sample and 0.12% (v/v) H2 O2 with equivalent volume of distilled water.

2.2. Preparation of squid viscera autolysates (SVAs) Frozen squid viscera were thawed with distilled water. The proximate composition of squid viscera used in this study was determined (water 73.06 ± 0.10%, crude protein 17.45 ± 0.69%, crude fat 8.08 ± 1.98% and ash 1.28 ± 0.01%) following the official methods of the AOAC [25]. Then, 100 g of squid viscera was mixed with distilled water at a ratio of 1:2 (w/v), and homogenized at a speed of 10,000 g for 1 min using a homogenizer (TM-767, Zhongshan, China). The pH of the mixture was adjusted to 7.0 using 6 M NaOH, followed by incubation at 50 ◦ C for 100 min to autolysis. After autolysis, the mixtures were heated at 100 ◦ C for 10 min to inactivate the endogenous enzymes, and then centrifuged at 5000g for 10 min (TD5A-WS, Changsha, China) to remove the insoluble substrate and upper layer of fat. The soluble squid viscera autolysates, designated as SVAs, were lyophilized, and stored at −20 ◦ C for further study. 2.3. Determination of antioxidative activity of SVAs 2.3.1. DPPH radical scavenging activity assay The abilities of SVAs and separated fractions or purified peptides to scavenge DPPH radical were evaluated according to our previous protocol [26] with slight modifications. Briefly, 600 ␮L of sample was mixed with 300 ␮L of 99.5% ethanol, followed by addition of 30 ␮L 0.02% DPPH (dissolved in 99.5% ethanol). After blending vigorously, the reaction mixtures were kept at room temperature for 60 min in dark. The absorbance of the resulting solution (AS ) was measured at 517 nm (721G-100 Vis-spectrophotometer, Shanghai, China). The DPPH radical scavenging activity was calculated with the following equation. DPPH radical- scavenging activity(%) =

AC − (AS − AB ) × 100 AC

AS − AP × 100 AB

2.3.3. Reducing power assay The ability of sample to reduce Fe3+ /ferric cyanide complex to the ferrous form was determined according to our previous method [26] with minor modifications. In short, the reaction mixtures, including 100 ␮L of sample, 50 ␮L of 0.2 M sodium phosphate buffer (pH 6.6), and 50 ␮L of 1% (w/v) potassium ferricyanide, were incubated at 50 ◦ C for 20 min. Subsequently, 50 ␮L of 10% trichloroacetic acid (v/v) was added to the mixture, and centrifuged at 3000g for 10 min (TGL-16C, Shanghai, China). The supernatant was added with 50 ␮L of 0.1% (w/v) ferric chloride, followed by vigorous mixing. After keeping at room temperature for 10 min, the absorbance of the reaction mixture was measured at 700 nm using a spectrophotometer (721G-100, Shanghai, China). An aliquot of 100 ␮L of distilled water instead of the sample was used as the blank. The higher the absorbance, the stronger the reducing power. 2.4. Amino acid composition analysis Amino acid composition was determined for the SVAs. Ten milligrams of freeze-dried SVAs was previously digested with 15 mL of 6 mol/L HCl under a nitrogen atmosphere at 110 ◦ C for 22 h. The digested samples were subsequently diluted to 25 mL by adding distilled water. Then, one milliliter of diluted digestions was completely dried under a nitrogen atmosphere. The dried sample was re-dissolved by adding 1.0 mL distilled water and subsequently dried under a nitrogen atmosphere. Finally, the dried sample was then re-dissolved in 1.0 mL of 0.02 mol/L HCl. After filtrating with a 0.45 ␮m filter membrane, 20 ␮L of sample filtration was used for assaying amino acid composition in an automatic amino acid analyzer (Hitachi L-8900, Tokyo, Japan). Results were determined as mg per g of sample. 2.5. Isolation and purification of antioxidative peptides from SVAs 2.5.1. Gel filtration chromatography The lyophilized SVAs (0.5 g) was dissolved in 2 mL of distilled water, then fractioned on a Sephadex G 25 gel chromatography (1.6 × 50 cm, 5 ␮m) column pre-equilibrated and eluted with distilled water. The elution rate was 1.3 mL/min. Eluents were collected with an interval of 3 min. The absorbance was measured at 280 nm. All fractions were pooled, concentrated and lyophilized. The active fraction SVAs3 was collected for further purification. Molecular weight (MW) standards, bovine serum albumin (BSA)

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(MW: 68000), aprotinin (MW: 6512), vitamin B12 (MW: 1355.37), and oxidized glutathione (GSSG) (MW: 612.63) were used to calibrate the molecular weight distribution. 2.5.2. Reverse phase high pressure liquid chromatography (RP-HPLC) Fraction SVAs3 (3 mg) was dissolved in 1 mL of eluent A (5% acetonitrile containing 0.1% (v/v) trifluoroacetic acid, TFA). Then, 20 ␮L of SVAs3 was loaded onto the RP-HPLC system equipped with a C18 column (4.6 × 250 mm, 5 ␮m, SunfireTM , Waters, MA, USA) for qualitative purification. A gradient elution was carried out as follows: 0–5 min, 5–10% eluent B (95% acetonitrile with 0.1% (v/v) TFA); 5–45 min, 10-40% eluent B; 45–50 min, 40–95% eluent B, with a flow rate of 1.0 mL/min. The absorbance of the eluents was measured at 215 and 280 nm. 2.6. Identification of peptides by LC-ESI-Q-TOF-MS/MS SVAs3 was separated into many sub-fractions in RP-HPLC, and some peaks of them were closed to each other. In order to identify all possible antioxidative peptides, SVAs3 was directly loaded onto liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI-Q-TOF-MS/MS). First, SVAs3 was filtered through a syringe filter (0.22 ␮m) and isolated on a Nano Aquity UPLC system (Waters Corporation, Milford, MA) connected to a quadrupole-Orbitrap mass spectrometer (Q-Exactive) (Thermo Fisher Scientific, Bremen, Germany) equipped with an online nanoelectrospray ion source. An aliquot of 8 ␮L of sample was separated on an analytical column with Michrom C18 reverse phase column (0.1 × 150 mm, 200 Å, Waters, MA, USA) by gradient elution with 5%–30% mobile phase B (acetonitrile/formic acid, 90%/0.1%, v/v) in 40 min at a flow rate of 300 nL/min. Mass spectrometer was performed in data-dependent mode to switch between full MS and MS/MS acquisition automatically. The accurate molecular weights of separated peaks were determined by their proton charged [M+H]+ states in the mass spectrum (m/z, 350–1500) under positive ionization mode, with spray voltage of 2.5 Kv, capillary temperature of 200 ◦ C, fragmentor voltage of 170 V, gas temperature of 320 ◦ C, and drying gas flow rate of 5 L/min. Following molecular weight determination, the peptide peak was automatically selected for fragmentation. De novo peptide automated spectrum processing was carried out using the PEAKS Studio software (7.0) (Bioinformatics Solutions Inc., Waterloo, Canada) (http://www.bioinfor.com). PEAKS Studio awards confidence scores for the entire range of amino acid sequences studied [28]. The false discovery rate (FDR) indicates the percentage of incorrect identification out of all correct identifications [29], whereas the average local confidence scores (ALC) and local confidence score indicate the probability that the peptide sequence is correct [28]. In this study, the FDR ≤5.0%, de novo sequence ALC ≥95%, and local confidence of each amino acid residue ≥90% in peptide sequence were set for validation of predicted peptide in SVAs3. Furthermore, manual interpretations were made in the spectra of MS/MS to confirm the validation of matched peptides. BLAST program was also employed for homology searches in NCBI non-redundant peptide database (http://www.ncbi.nlm. nih.gov/blast). 2.7. Antioxidative activity of synthetic peptides Peptides were chemically synthesized by GL Biochem Co., Ltd (Shanghai, China) based on amino acid sequences. The purity of all synthetic peptides was over 95% by HPLC analysis. The molecular weights were also determined by MS analysis under ESI positive or negative mode. The antioxidative activities of synthetic peptides were evaluated according to the method described in Section 2.3.

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2.8. Statistical analysis Data were represented as mean ± standard deviation (n = 3). Statistical analysis was performed using the SPSS® computer program (SPSS Statistical Software 19.0, Inc., Chicago, IL, USA). The significance between samples was verified with analysis of one-way ANOVA in post hoc mutiple comparisons by the Tukey’s test at P < 0.05.

3. Results and discussion 3.1. Antioxidative activity of SVAs The capacity of SVAs for DPPH and hydroxyl radical scavenging activities as well as reducing power assay compared with ascorbic acid, GSH, and L-carnosine was summarized in Table 1. The calculated IC50 value on DPPH radical, which is the concentration required to scavenge radicals by 50%, was determined to be 231.80 ± 16.26 ␮g/mL for SVAs, 20.07 ± 1.93 ␮g/mL for GSH, and 4.26 ± 0.03 ␮g/mL for ascorbic acid, respectively. This indicated the weak activity of SVAs on scavenging DPPH radical compared to GSH and ascorbic acid. However, SVAs demonstrated stronger activity in quenching DPPH radical than L-carnosine and some reported fish protein hydrolysates, such as alcalase hydrolysates of patin sarcoplasmic protein (IC50 , 1.44 ± 0.08 mg/mL) and papain hydrolysates of patin sarcoplasmic protein (IC50 , 1.48 ± 0.17 mg/mL) [30]. Moreover, SVAs showed higher activity in quenching hydroxyl radical than GSH (IC50 , 0.95 ± 0.03 mg/mL), with the IC50 value of 0.74 ± 0.11 mg/mL (P < 0.05). Hydroxyl radical is considered as the strongest component among oxygen free radicals because it reacts easily with biomolecules (e.g. lipids, proteins and DNA) [31]. To date, some crude and purified peptide fractions derived from aquatic viscera have been shown to possess noteworthy hydroxyl radicalscavenging activities, such as abalone viscera hydrolysates (AVH) [32], and horse mackerel (Magalaspis cordyla) viscera protein hydrolysates and their resulting purified fraction B (by gel filtration chromatography) [33]. The present study suggested that the SVAs could be explored as an efficient hydroxyl radical scavenger. Furthermore, the SVAs displayed stronger reducing power ability than L-carnosine under the same initial concentration of 1.0 mg/mL.

3.2. Amino acid composition of SVAs The features of amino acids in protein hydrolysates plays an important role for the antioxidative activity of hydrolysates and peptides [34,35]. As shown in Table 2, amino acid profiles revealed that the SVAs contained 38.52% hydrophobic amino acids (HAA), which could contribute to form hydrophobic structures. Peptides containing one or more hydrophobic amino acids at the C- or N-terminus of the peptide can help to solubilize peptide at the lipidwater interface, thereby facilitating the detoxification of the free radicals/lipid peroxides produced in the lipid phase [16,36]. Moreover, some of amino acids like Gly, Leu, Asp, Ala, Val, Phe, Pro, and Lys, which are believed to be related with the antioxidative activity of protein hydrolysates and peptides [28,37–39], were abundant in the SVAs. Additionally, the SVAs were found to contain high level of essential amino acids (EAA, 33.02%). Glu and Gly, two amino acids related with typical meaty aroma and sweet taste [10], were prominent in the SVAs. Taken all together, these results indicated that the SVAs, from the standpoint of amino acid profile, could be adequate as a potential flavoring and nutritional antioxidant.

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Table 1 The antioxidative activities of SVAs on scavenging DPPH and hydroxyl radicals and reducing power ability compared with ascorbic acid, GSH and L-carnosine.a Sample

DPPH radicalb (IC50 , ␮g/mL)

Hydroxyl radicalc (IC50 , mg/mL)

Reducing powerd (Absorbance at 700 nm)

SVAs Ascorbic acid GSH L-carnosine

231.80 ± 16.26 4.26 ± 0.03A 20.07 ± 1.93B NDe

0.74 ± 0.11 0.54 ± 0.09A 0.95 ± 0.03C NDf

0.430 ± 0.020B 1.216 ± 0.016D 0.593 ± 0.021C 0.042 ± 0.010A

C

B

The data were presented as the mean ± SD (n = 3). Different capital letters in the same column represented significant difference between samples (P < 0.05). In DPPH radical scavenging activity assay, the initial concentrations of SVAs were ranged from 0 to 1 mg/mL. The initial concentrations of GSH and ascorbic acid were changed from 0 to 50 ␮g/mL. c In hydroxyl radical scavenging activity assay, the initial concentrations of SVAs, GSH and ascorbic acid were ranged from 0 to 2 mg/mL. d The reducing power abilities of samples including SVAs, GSH, ascorbic acid and L- carnosine were determined in the initial concentration of 1 mg/mL. e L- carnosine did not show DPPH radical scavenging activity in the initial concentration of 2 mg/mL. f The hydroxyl radical scavenging activity of L-carnosine was lower than 15% in the initial concentration of 2 mg/mL. a

b

Table 2 Amino acid composition of the SVAs, expressed as mg per g and% of total amino acids. Amino acid

Content/(mg/g)

Relative content%

Amino acid

Content/(mg/g)

Relative content%

Asp Thr Ser Glu Gly Ala Cys Val Met Trp

50.39 ± 1.75 29.68 ± 1.22 20.99 ± 0.91 107.25 ± 3.60 78.65 ± 2.47 47.95 ± 1.65 0.97 ± 0.17 30.20 ± 1.05 5.18 ± 0.28 ND

8.22 ± 0.28 4.48 ± 0.20 3.42 ± 0.15 17.49 ± 0.59 12.83 ± 0.40 7.82 ± 0.27 0.16 ± 0.08 4.92 ± 0.17 0.84 ± 0.04 ND

Ile Leu Tyr Phe Lys His Arg Pro EAA HAA

26.54 ± 0.98 50.04 ± 1.72 14.73 ± 1.44 24.12 ± 0.93 36.67 ± 1.27 12.08 ± 0.77 41.26 ± 1.64 36.46 ± 1.66 202.43 236.19

4.33 ± 0.16 8.16 ± 0.28 2.40 ± 0.24 3.93 ± 0.15 5.98 ± 0.21 1.97 ± 0.13 6.73 ± 0.27 5.95 ± 0.21 33.02 38.52

The data were presented as the means ± SD (n = 3). ND: not determined. EAA: total essential amino acids. HAA: total hydrophobic amino acids (Ala, Val, Met, Ile, Leu, Phe, Pro, Cys and Tyr).

3.3. Gel filtration chromatography Protein hydrolysates obtained after enzymatic hydrolysis are generally composed of undigested proteins, long and short chain bioactive peptides and free amino acids [40]. Gel filtration is generally applied to isolate the protein hydrolysates according to their molecular weights. The larger masses are eluted earlier than the smaller ones [41]. At the present study, the SVAs were seperated into four fractions, i.e., SVAs1, SVAs2, SVAs3 and SVAs4, on Sephadex G25 chromatography (Fig. 1a). The fraction SVAs3 showed the strongest hydroxyl radical scavenging activity and reducing power ability, followed by SVAs4, SVAs2 and SVAs1. By comparing the elution profile with molecular weight standards, it was noticed that the rough size distributions of SVAs2, SVAs3 and SVAs4 were below 1500. The firstly eluted fraction SVAs1, with MW close to BSA, had the highest scavenging activity on DPPH radical. The fraction SVAs1, however, showed less hydroxyl radical scavenging activity and reducing power ability compared to other three fractions (Fig. 1b). The fraction SVAs1 might be a complex mixture of large masses, such as undigested proteins, large peptides and/or some exposed antioxidative groups (i.e. phenolic hydroxyl group and indole group), which could be responsible for its high DPPH radical scavenging activity. The lately eluted fraction, SVAs4, indicated its smallest MW compared to other fractions. In a study by Giri et al. who confirmed that antioxidative peptides, especially certain peptides of small MWs, were contributed more remarkably to radical scavenging than heterocyclic volatiles and phenolics in squid fermented paste [42]. However, in our present study, the smallest MW fraction SVAs4 showed less antioxidative activity than SVAs3. Similar to our results, the lowest MW portion (lately eluted) of loach protein hydrolysate separated by Sephadex G-25 did not show the highest antioxidant activity [41]. The less antioxidative activity of lowest MW fraction might be due to its significant amount of free amino acids, which have been reported to be less effective antioxidants

Fig. 1. Elution profile of SVAs on Sephadex G 25 column eluting with distilled water (a) and the antioxidative activities of separated fractions assayed using DPPH and hydroxyl radical scavenging activities and reducing power ability (initial concentration of 1 mg/mL) (b), the results were plotted as the means ± SD (n = 3).

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than peptides [13]. Taken all together the results of antioxidative activity, the active fraction SVAs3 was collected for further purification. 3.4. Identification of peptides by LC-ESI-Q-TOF-MS/MS The fraction SVAs3 contained a multitude of closely eluting peptides with varying intensity, as can be seen from the peptide profile shown in Fig. 2. With the aim to identify all potential antioxidative peptides, SVAs3 was directly subjected to RP-HPLC separation coupled with an ESI source under positive ionization mode. The molecular weights of peptides were identified according to their proton charged [M+H]+ precursor ions in scan mass spectrum (m/z, 350–1500). The precursor ion in single charged of peptide was collapsed into series of fragments. A single peptide fragment could be matched by de novo peptides sequencing [11]. In this study, nineteen peptides were identified in SVAs3 through de novo peptide automated spectrum processing, with ALC ≥95% and local confidence of each residue in peptide sequence ≥90% (Table 3). The MWs of these peptides ranged from 700 Da to 1300 Da. This result was in accordance with various antioxidative peptides derived from squid hydrolysates, with MWs between 500 and 1500 Da [39,43]. Among these peptides, the interpretations of ion series of six peptides LLAPPER (794.44 Da), FPGLADR (774.41 Da), WVAPLK (712.41 Da), FFNPVH (759.39 Da), FNVVLK (719.37 Da) and LELPLK (711.79 Da), with relative small MWs between 700 and 800 Da, were also manually interpretated (Fig. 3). The physicochemical properties of the six peptides (named as SVAs3-I, SVAs3-II, SVAs3-III, SVAs3-IV, SVAs3V and SVAs3-VI) were summarized in Table 4. The differences between the theoretical mass and the observed mass of the six peptides were all small and acceptable. Moreover, the six peptides had some common features like hydrophobic amino acid residues L (Leu), F (Phe), or W (Trp) at the N-terminal, and basic amino acid residues like R (Arg), K (Lys) or H (His) at the C-terminal. Four peptides (LLAPPER, FPGLADR, WVAPLK and LELPLK) had one or more L (Leu) and P (Pro) residues within the peptides sequence, three peptides (WVAPLK, FFNPVH and FNVVLK) had one or two V (Val) residues in peptides sequence, and three peptides (WVAPLK, FNVVLK and LELPLK) had common residues LK (Leu-Lys) at the Cterminal. These properties of the six peptides are consistent with most of the published studies, which emphasize the presence of hydrophobic amino acid residues at the N- or C-terminus of peptides and specific amino acid residues (e.g. L, P, F, W, K, H, and V) within the peptide sequence in determining the antioxidative activity of peptides [16,27,30,34,44]. Meanwhile, the differences among all six peptides are noticeable. For instance, peptides SVAs3-I, SVAs3-II and SVAs3-IV had relatively large MWs, peptide SVAs3-III had the highest total hydrophobic ratio, and each peptide had different amino acid residues in peptide sequence. These differences could account for the structure-activity relationship, and therefore ultimately lead to different antioxidative activity [30,44]. Hence, the six peptides from SVAs3 were chemically synthesized respectively to determine their antioxidative activities in vitro. 3.5. Antioxidative activity of synthetic peptides The six synthesized peptides scavenged DPPH and hydroxyl radicals in dose-dependent manners under the initial concentrations ranging from 0 to 5 mg/mL. Similar results were found for their reducing power ability (Fig. 4). Peptides SVAs3-III (WVAPLK, 712.89 Da) and SVAs3-V (FNVVLK, 718.90 Da) exhibited stronger DPPH radical-scavenging activities than other peptides in non-linear dose-dependent manners (Fig. 4a). The IC50 values of scavenging DPPH radicals were 0.82 ± 0.08 mg/mL (or 1.14 ± 0.11 mM) for SVAs3-V and 1.05 ± 0.08 mg/mL (or

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1.47 ± 0.11 mM) for SVAs3-III, respectively. Peptides with small MWs could demonstrate greater molecular mobility and diffusivity than larger peptides [31]. The properties of small MWs for peptides SVAs3-III and SVAs3-V should be a positive factor for their high DPPH radical scavenging activities. By comparison, the relatively large MWs could be a negative feature for peptides SVAs3-I (794.95 Da) and SVAs3-II (774.88 Da) in quenching DPPH radicals. Apart from the relatively small MWs, peptides SVAs3-III and SVAs3-V had hydrophobic residues W or F at the N-terminal and the same basic residue K at the C-terminal (Table 4). The hydrophobic residues W and F at the N-terminus of SVAs3-III and SVAs3-V sequences could contribute to the solubility of peptides in DPPH solutions. Furthermore, the indole and benzene ring of W and F residues in sequences had important contribution for radical scavenging because the indole and benzene ring of these amino acids could donate protons to electron deficient radicals, and thereby making reactive oxygen species stable [45]. Chi, Wang, Wang, Zhang and Deng identified a novel antioxidative peptide WEGPK derived from protein hydrolysate of bluefin leatherjacket heads [19], which shares features with peptide SVAs3-III (WVAPLK). Both of them demonstrate the presence of W and K residues at the N- and C-terminal respectively, and a P residue within peptide sequence. Peptide SVAs3-V (FNVVLK) shared common features with a novel antioxidative peptide FRDEHKK from rice endosperm protein hydrolysate, containing F and K residues at the N- and Cterminal [46]. Besides, the amino acid compositions, especially of peculiar amino acid residues locating in peptide sequence, play important roles for the observed antioxidative activity of peptides. For example, P and L in peptide GFGPEL (Gly-Phe-Gly-Pro-GluLeu, 618.89 Da), V and P in peptide VGGRP (Val-Gly-Gly-Arg-Pro, 484.56 Da), and L and A in peptide ACFL (Ala-Cys-Phe-Leu, 518.5 Da) were reported as major reasons for their good antioxidant activities [17,33]. In this study, the sequence properties of peptides SVAs3III (WVAPLK) and SVAs3-V (FNVVLK) were consistent with those published antioxidative peptides. As for scavenging hydroxyl radicals, these six peptides demonstrated activities in linear dose-dependent manners. Moreover, the smaller peptides SVAs3-III (WVAPLK, 712.89 Da) and SVAs3VI (LELPLK, 711.90 Da) showed higher activities than other four peptides (Fig. 4b). The IC50 values were 1.85 ± 0.04 mg/mL (or 2.60 ± 0.06 mM) for SVAs3-III (R2 = 0.9821) and 2.01 ± 0.10 mg/mL (or 2.82 ± 0.14 mM) for SVAs3-VI (R2 = 0.9702), respectively. Similar to our results, in a recent study by Chi et al. (2015), who described the smallest peptide GPP (Gly-Pro-Pro, 269.33 Da) derived from bluefin leatherjacket heads hydrolysates exhibited the highest scavenging activity in DPPH radicals, hydroxyl radicals, and ABTS radicals compared to other two identified peptides WEGPK (Trp-Glu-Gly-Pro-Lys, 615.69 Da) and GVPLT (Gly-Val-ProLeu-Thr, 485.59 Da) [19]. However, another peptide SVAs3-V, which displayed strong DPPH radical scavenging activity (Fig. 4a), showed moderate ability in quenching hydroxyl radical. In addition of similar MW and hydrophobic residues at the N-terminal, peptide SVAs3-V (FNVVLK) had two common residues LK at the C-terminal like peptides SVAs3-III (WVAPLK) and SVAs3-VI (LELPLK) (Table 4). Therefore, the amino acids composition and position within peptides SVAs3-III, SVAs3-V and SVAs3-VI should play core roles for their different hydroxyl radical scavenging activities. Within the sequences, residues VAP, NVV and ELP were located in SVAs3-III (WVAPLK), SVAs3-V (FNVVLK) and SVAs3-VI (LELPLK) sequences, respectively. The hydrophobic residues V, A and L, and acidic residue E were related with the strong hydroxyl radical scavenging activity of peptides [17,30,33,34,47,48]. Furthermore, the P residue had been shown to act positively as direct radical scavenger [39,47]. Therefore, we speculated that the lack of P residue in SVAs3-V might be a key reason for its relatively weak hydroxyl radical scavenging

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Fig. 2. RP-HPLC chromatographic profile of active fraction SVAs3 from Sephadex G-25 chromatography, measured at 215 nm (a) and 280 nm (b), respectively.

Table 3 Identification of peptides in the active fraction of SVAs3 derived from Sephadex G-25 chromatography by using LC-ESI-Q-TOF-MS/MS. Peptides

RT/mina

Tag length

ALC/%b

m/z

z

Mass

KLWHHT KN(+0.98)TNELPVYK LLAPPER DM(+15.99)EKLWH MN(+0.98)LEKLK GPGLEPKGV VDLRKN(+0.98)L VN(+0.98)FPEER ETGPLGLAWSP A(+42.01)HTEKPPVL

13.44 19.86 19.89 20.28 20.60 20.83 21.43 22.13 22.93 23.49

6 10 7 7 7 9 7 7 11 9

97 99 97 98 97 97 96 95 97 98

411.22 603.82 398.24 487.72 438.74 427.24 429.76 446.21 564.29 517.29

2 2 2 2 2 2 2 2 2 2

820.43 1205.63 794.47 973.43 875.48 852.47 857.50 890.41 1126.57 1032.56

WLGLENVP FPGLADR WVAPLK TDN(+0.98)GVQLVTVK FFNPVH K(+42.01)VPVKPLQ(+0.98)

23.86 24.18 24.61 25.29 25.73 25.75

8 7 6 11 6 8

95 95 97 97 96 96

464.25 388.21 357.21 587.82 380.69 476.30

2 2 2 2 2 2

926.49 774.40 712.41 1173.62 759.39 950.58

VTVPVVVR FN(+0.98)VVLK LELPLK

26.27 27.82 27.83

8 6 6

96 99 95

434.78 360.69 356.89

2 2 2

867.55 719.37 711.79

a b c

PTMc Deamidation (NQ) Oxidation (M) Deamidation (NQ) Deamidation (NQ) Deamidation (NQ) Acetylation (Protein N-term)

Deamidation (NQ) Acetylation (Protein N-term); Deamidation (NQ) Deamidation (NQ)

local confidence/% 98 99 92 95 99 99 99 100 99 98 100 100 100 100 100 99 99 100 100 99 92 98 92 91 98 100 99 99 100 99 96 99 99 99 98 98 96 94 96 95 98 100 99 99 99 99 95 96 98 92 97 98 99 91 96 99 94 99 98 92 98 95 99 98 99 99 100 99 96 96 97 99 99 98 99 97 99 97 99 99 98 99 95 94 95 91 97 96 98 98 99 99 92 93 90 98 98 99 99 97 95 93 98 100 98 99 98 100 97 98 98 92 98 97 97 96 92 93 95 100 99 100 99 90 97 93

93 97 99 98 98 97 96 89 98 99 99 99 99 99 94 99 100 97 95 90

RT: represented the retention time in LC system connected to ESI-Q-TOF-MS. ALC: represented the average local confidence scores. PTM: was the abbreviation of post-translational modification.

activity. In relation to this observation, several peptides with good antioxidative activity identified from aquatic protein hydrolysates, all of them contained P residue within their peptides sequences,

such as peptide GFGPEL from grass carp skin [17], peptide EWPAQ from monkfish muscle protein [49], and peptide HDHPVC from round scad muscle protein [50].

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Fig. 3. MS/MS spectra of six smaller peptides identified in SVAs3, peptide LLAPPER with m/z 794.44 (a), peptide FPGLADR with m/z 774.41 (b), peptide WVAPLK with m/z 712.41 (c), peptide FFNPVH with m/z 759.39 (d), peptide FNVVLK with m/z 719.37 (e), and peptide LELPLK with m/z 711.79 (f).

Table 4 Physiochemical properties of the six synthesized peptides identified in the fraction of SVAs3.a Peptides

Amino acid sequence

Observed MW/theoretical MW(Da)

Total hydrophobic ratio/%b

Iso-eletric point

Net charge at pH 7.0

SVAs3-I SVAs3-II SVAs3-III SVAs3-IV SVAs3-V SVAs3-VI

LLAPPER FPGLADR WVAPLK FFNPVH FNVVLK LELPLK

794.44/794.95 774.41/774.88 712.41/712.89 759.39/759.86 719.37/718.90 711.79/711.90

71.43 57.14 83.33 66.67 66.67 66.67

6.97 6.78 10.1 7.85 10.1 6.94

0 0 1 0.1 1 0

a b

Physiochemical properties of peptides were predicted using web-based peptide property calculator tool (http://www.innovagen.se/custom-peptide-synthesis/). Total hydrophobic ratio was calculated according to the percentage of hydrophobic residues (I, V, L, F, C, M, A, W and P) in the peptide sequence.

Fig. 4. DPPH radical (a), hydroxyl radical (b) scavenging activities, and reducing power ability (c) of six synthesized peptides SVAs3-I (LLAPPER), SVAs3-II (FPGLADR), SVAs3-III (WVAPLK), SVAs3-IV (FFNPVH), SVAs3-V (FNVVLK) and SVAs3-VI (LELPLK). The results were plotted as the means ± SD (n = 3).

In the reducing power ability assay, the ferric irons were converted into ferrous, indicating the antioxidant nature of hydrolysates or peptides [33]. The basic and acidic residues in pep-

tides could act as hydrogen donors and metal ion chelators using the carboxyl and amino groups [51]. As shown in Fig. 4c, all synthesized peptides demonstrated no differences in reducing power

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abilities when their concentrations were at 1 mg/mL and 3 mg/mL respectively. However, the significantly increased reducing activity of SVAs3-III was observed at higher initial concentration of 5 mg/mL (or 7.02 mM) (P < 0.05). This suggested that SVAs3-III could demonstrate effective electron donating capacity to ferric ions and formed stable ferrous at the optimal concentrations. Based on the results of Table 1 and Fig. 4, we found peptide SVAs3-III, one of the most active peptides among these six synthesized peptides, demonstrated lower DPPH radical scavenging activity than SVAs according to their IC50 values. Similar results were also observed for scavenging hydroxyl radical and its ferric ion reducing ability. Actually, several researchers proposed that antioxidant peptides or pepitde fractions in hydrolysates might play roles as synergistic effects, and in many cases, the crude hydrolysates would show stronger activity than the individual peptide or peptide fraction [18,38,52]. The mixture of various peptides with other compounds like glycoproteins and phenolic compunds could contribute to the strong antioxidative activity for crude hydrolysates due to the synergistic mechanisms including free radical scavenging and metal ion chelating [17,34,44]. We recently reported peptides NKVKGELD, EMSAGLHE and WRKKDPLND derived from peptic hydrolysates of half-fin anchovy, displayed synergistic effects on scavenging DPPH radical and reducing power ability [26]. Similarly, peptides RPNYTDA and TRTGDPFF identified from rice residue protein hydrolysates demonstrated a synergistic antioxidant effect compared to the single peptide [53]. However, it should be mentioned that several antioxidative peptides fractions or purified pepitdes showed stronger activities than their original hydrolysates. For example, one peptide fraction (SI 3) displayed DPPH radical scavenging activity by 1.83-fold higher than its original sarcoplasmic protein Alcalase hydrolysates [30]. Peptides VPKNYFHDIV, LVMFLDNQHRVIRH and FVNQPYLLYSVHMK from patin myobrillar protein hydrolysate (MPHs) had the IC50 values ranging from 0.268 mg/mL to 0.443 mg/mL in quenching DPPH radicals. These values were much lower than that of the MPHs (IC50 , 1.120 mg/mL) [16]. The different characterics of purified peptides or peptide fractions on antioxidative activity might be correlated to different hydrolytic conditions, which could result in various physiochemical properties for pepides or peptide fractions. Apparently, once peptides own much more favorable structure properties for quenching free radicals or chelating metal ions, they usually demonstrate higher antioxidative activity. However, the structural-activity relationship of antioxidative peptides purified from natural sources needs to be further investigated.

4. Conclusions Squid viscera autolysates (SVAs) demonstrated strong in vitro antioxidative activities including scavenging DPPH and hydroxyl radicals, and moderate reducing power ability as well. Nineteen peptides were identifed in the active peptide fraction of SVAs3. The antioxidative activities of six peptides, SVAs3-I (LLAPPER), SVAs3II (FPGLADR), SVAs3-III (WVAPLK), SVAs3-IV (FFNPVH), SVAs3-V (FNVVLK) and SVAs3-VI (LELPLK), with relatively small MWs, were determined. The hydrophobic amino acid residues including W, F, or L, at the N-terminal, basic amino acids residues like K, R or H at the C-terminus of peptides, and specific amino acid residues like V, A, L, E and P within peptides sequence could be responsible for the antioxidative activities of these peptides. However, the contribution of specific amino acid residues in the position of peptide sequence and synergistic effects among peptides need to be further investigated, as well as for the study of the antioxidative abilities of SVAs or purified peptides in vivo.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (No. 31301568), Natural Science Foundation of Zhejiang Province (LY15C200018), China, Zhoushan Science and Technology Bureau Project (2014C41005), China, and the Youth Staff Training Program of Zhejiang Ocean University for Dr. Ru Song.

References [1] J.H. Park, H. Lee, G.Y. Lee, H.R. Kim, Y.W. Kim, Development of refined cuttlefish (Todarodes pacificus) oil and its use as functional ingredients, Food Sci. Biotechnol. 20 (2) (2011) 389–394. [2] P.Z. Lian, C.M. Lee, E. Park, Characterization of squid-processing byproduct hydrolysate and its potential as aquaculture feed ingredient, J. Agric. Food Chem. 53 (2005) 5587–5592. [3] R.H. Kim, A.K.M. Asaduzzaman, C.H. You, B.S. Chun, Stability of antioxidant properties and essential amino acids in squid viscera hydrolysate produced using subcritical water, Fish Aquat. Sci. 16 (2) (2013) 71–78. [4] M.S. Uddin, H.M. Ahn, H. Kishimura, B.S. Chun, Comparative study of digestive enzymes of squid (Todarodes pacificus) viscera after supercritical carbon dioxide and organic solvent extraction, Biotechnol. Bioproc. Eng. 14 (3) (2009) 338–344. [5] H. Korhonen, A. Pihlanto, Food-derived bioactive peptides-opportunities for designing future foods, Curr. Pharm. Design 9 (2003) 1297–1308. [6] P.A. Harnedy, R.J. FitzGerald, Bioactive peptides from marine processing waste and shellfish: a review, J. Funct. Foods 4 (2012) 6–24. [7] E.S. Kechaou, J. Dumay, C. Donnay-Moreno, P. Jaouen, J.P. Gouygou, J.P. Bergé, R.B. Amar, Enzymatic hydrolysis of cuttlefish (Sepia officinalis) and sardine (Sardina pilchardus) viscera using commercial proteases: effects on lipid distribution and amino acid composition, J. Biosci. Bioeng. 107 (2) (2009) 158–164. [8] H.G. Kristinsson, B.A. Rasco, Fish protein hydrolysates: production, biochemical, and functional properties, Crit. Rev. Food Sci. Nutr. 40 (1) (2000) 43–81. [9] R. Morales-Medina, F. Tamm, A.M. Guadix, E.M. Guadix, S. Drusch, Functional and antioxidant properties of hydrolysates of sardine (S: pilchardus) and horse mackerel (T. mediterraneus) for the microencapsulation of fish oil by spray-drying, Food Chem. 194 (2016) 1208–1216. [10] W. Xu, G. Yu, C.H. Xue, Y. Xue, Y. Ren, Biochemical changes associated with fast fermentation of squid processing by-products for low salt fish sauce, Food Chem. 107 (4) (2008) 1597–1604. [11] L. Lin, B.F. Li, Radical scavenging properties of protein hydrolysates from Jumbo flying squid (Dosidicus eschrichitii Steenstrup) skin gelatin, J. Sci. Food Agric. 86 (2006) 2290–2295. [12] M. Robert, C. Zatylny-Gaudin, V. Fournier, E. Corre, G.L. Corguillé, B. Bernay, J. Henry, Molecular characterization of peptide fractions of a Tilapia (Oreochromis niloticus) by-product hydrolysate and in vitro evaluation of antibacterial activity, Process Biochem. 50 (3) (2015) 487–492. [13] A.G.P. Samaranayaka, E.C.Y. Li-Chan, Food-derived peptidic antioxidants: a review of their production, assessment, and potential applications, J. Funct. Foods 3 (4) (2011) 229–254. [14] D.Y. Zhou, B.W. Zhu, L. Qiao, H.T. Wu, D.F. Li, J.F. Yang, Y. Murata, In vitro antioxidant activity of enzymatic hydrolysates prepared from abalone (Haliotis discus hannai Ino) viscera, Food Bioprod. Process. 90 (2) (2012) 148–154. [15] C. Wiriyaphan, B. Chitsomboon, S. Roytrakul, J. Yongsawadigul, Isolation and identification of antioxidative peptides from hydrolysate of threadfin bream surimi processing byproduct, J. Funct. Foods 5 (2013) 1654–1664. [16] L. Najafian, A.S. Babji, Isolation, purification and identification of three novel antioxidative peptides from patin (Pangasius sutchi) myofibrillar protein hydrolysates, LWT Food Sci. Technol. 60 (1) (2015) 452–461. [17] L. Cai, X. Wu, Y. Zhang, X. Li, S. Ma, J. Li, Purification and characterization of three antioxidant peptides from protein hydrolysate of grass carp (Ctenopharyngodon idella) skin, J. Funct. Foods 16 (2015) 234–242. [18] S. Saidi, A. Deratani, M.P. Belleville, R.B. Amar, Antioxidant properties of peptide fractions from tuna dark muscle protein by-product hydrolysate produced by membrane fractionation process, Food Res. Int. 65 (2014) 329–336 (Part C). [19] C.F. Chi, B. Wang, Y.M. Wang, B. Zhang, S.G. Deng, Isolation and characterization of three antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) heads, J. Funct. Foods 12 (2015) 1–10. [20] S. Klomklao, S. Benjakul, B.K. Simpson, Seafood enzymes: biochemical properties and their impact on quality, in: Food Biochemistry and Food Processing, second edition, Wiley-Blackwell, 2012, pp. 271–273. [21] A.G.P. Samaranayaka, E.C.Y. Li-Chan, Autolysis-assisted production of fish protein hydrolysates with antioxidant properties from Pacific hake (Merluccius productus), Food Chem. 107 (2) (2008) 768–776.

Please cite this article in press as: R. Song, et al., In vitro antioxidative activities of squid (Ommastrephes bartrami) viscera autolysates and identification of active peptides, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.06.015

G Model PRBI-10719; No. of Pages 9

ARTICLE IN PRESS R. Song et al. / Process Biochemistry xxx (2016) xxx–xxx

˜ M.P. Sánchez-Saavedr, F.J. Márquez-Roch, Characterisation [22] A.B. Castro-Cesen, and partial purification of proteolytic enzymes from sardine by-products to obtain concentrated hydrolysates, Food Chem. 135 (2) (2012) 583–589. [23] A. Vannabun, S. Ketnawa, S. Phongthai, S. Benjakul, S. Rawdkuen, Characterization of acid and alkaline proteases from viscera of farmed giant catfish, Food Biosci. 6 (2014) 9–16. [24] J.L. Arias-Moscoso, A. Maldonado-Arce, O. Rouzaud-Sandez, E. Márquez-Ríos, W. Torres-Arreola, H. Santacruz-Ortega, M.G. Gaxiola-Cortés, J.M. Ezquerra-Brauer, Physicochemical characterization of protein hydrolysates produced by autolysis of Jumbo squid (Dosidicus gigas) byproducts, Food Biophys. 10 (2) (2015) 145–154. [25] AOAC, Official Methods of Analysis of the AOAC, Association of Official Analytical Chemists, Washington DC, 2006. [26] R. Song, R.B. Wei, G.Q. Ruan, H.Y. Luo, Isolation and identification of antioxidative peptides from peptic hydrolysates of half-fin anchovy (Setipinna taty), LWT Food Sci. Technol. 60 (1) (2015) 221–229. [27] I.G.J. de Avellar, M.M.M. Magalhães, A.B. Silva, L.L. Souza, A.C. Leitão, M. Hermes-Lima, Reevaluating the role of 1,10-phenanthroline in oxidative reactions involving ferrous ions and DNA damage, Biochim. Biophys. Acta 1675 (1-3) (2004) 46–53 (BBA—Gen Subjects). [28] B. Ma, K.Z. Zhang, C. Hendrie, C.Z. Liang, M. Li, A. Doherty-Kirty, G. Lajoie, PEAKS: Powerful software for peptide de novo sequencing by tandem mass spectrometry, Rapid Commun. Mass Spectrom. 17 (2003) 2337–2342. [29] A.I. Nesvizhskii, O. Vitek, R. Aebersold, Analysis and validation of proteomic data generated by tandem mass spectrometry, Nat. Methods 4 (2007) 787–797. [30] L. Najafian, A.S. Babji, Production of bioactive peptides using enzymatic hydrolysis and identification antioxidative peptides from patin (Pangasius sutchi) sarcoplasmic protein hydolysate, J. Funct. Foods 9 (2014) 280–289. [31] M.A. Cacciuttolo, L. Trinh, J.A. Lumpkin, G. Rao, Hyperoxia induces DNA damage in mammalian cells, Free Radical Biol. Med. 14 (1993) 267–276. [32] J.Y. Je, S.Y. Park, J.Y. Hwang, C.B. Ahn, Amino acid composition and in vitro antioxidant and cytoprotective activity of abalone viscera hydrolysate, J. Funct. Foods 16 (2015) 94–103. [33] N.S. Sampath Kumar, R.A. Nazeer, R. Jaiganesh, Purification and biochemical characterization of antioxidant peptide from horse mackerel (Magalaspis cordyla) viscera protein, Peptides 32 (7) (2011) 1496–1501. [34] R.J. Elias, S.S. Kellerby, E.A. Decker, Antioxidant activity of proteins and peptides, Crit. Rev. Food Sci. Nutr. 48 (2008) 430–441. [35] J.Y. Je, Z. Qian, H. Byun, S. Kim, Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis, Process Biochem. 42 (2007) 840–846. [36] P. Puchalska, M.L. Marina, M.C. Garcia, Isolation and identification of antioxidant peptides from commercial soybean-based infant formulas, Food Chem. 148 (2014) 147–154. [37] A. Sila, A. Bougatef, Antioxidant peptides from marine by-products: isolation, identification and application in food systems. A review, J. Funct. Foods 21 (2016) 10–26. [38] B.H. Sarmadi, A. Ismail, Antioxidative peptides from food proteins: a review, Peptides 31 (10) (2010) 1949–1956. [39] N. Rajapakse, E. Mendis, H.G. Byun, S.K. Kim, Purification and in vitro antioxidative effects of giant squid muscle peptides on free radical-mediated oxidative systems, J. Nutr. Biochem. 16 (9) (2005) 562–569.

9

[40] M. Chalamaiah, T. Jyothirmayi, K. Bhaskarachary, A. Vajreswari, R. Hemalatha, B. Dinesh Kumar, Chemical composition, molecular mass distribution and antioxidant capacity of rohu (Labeo rohita) roe (egg) protein hydrolysates prepared by gastrointestinal proteases, Food Res. Int. 52 (2013) 221–229. [41] L.J. You, M.M. Zhao, J.M. Regenstein, J. Ren, Purification and identification of antioxidative peptides from loach (Misgurnus anguillicaudatus) protein hydrolysate by consecutive chromatography and electrospray ionization-mass spectrometry, Food Res. Int. 43 (2010) 1167–1173. [42] A. Giri, K. Osako, A. Okamoto, E. Okazaki, T. Ohshima, Antioxidative properties of aqueous and aroma extracts of squid miso prepared with Aspergillus oryzae-inoculated koji, Food Res. Int. 44 (1) (2011) 317–325. [43] S.K. Chang, A. Ismail, N.M. YanagitaT. Esa, M.T.H. Baharuldin, Antioxidant peptides purified and identified from the oil palm (Elaeis guineensis Jacq.) kernel protein hydrolysate, J. Funct. Foods 14 (2015) 63–75. [44] E. Mendis, N. Rajapakse, H.G. Byun, S.K. Kim, Investigation of jumbo squid (Dosidicus gigas) skin gelatin peptides for their in vitro antioxidant effects, Life Sci. 77 (17) (2005) 2166–2178. [45] B. Hernández-Ledesma, B. Miralles, L. Amigo, Identification of antioxidant an ACE-inhibitory peptides in fermented milk, J. Sci. Food Agric. 85 (2005) 1041–1048. [46] J. Zhang, H. Zhang, L. Wang, X. Guo, X. Wang, H. Yao, Isolation and identification of antioxidative peptide from rice endosperm protein enzymatic hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS/MS, Food Chem. 119 (2010) 226–234. [47] O.K. Chang, G.E. Ha, G.S. Han, K.H. Seol, H.W. Kim, S.G. Jeong, M.H. Oh, B.Y. Park, J.S. Ham, Novel antioxidant peptide derived from the ultrafiltrate of ovomucin hydrolysate, J. Agric. Food Chem. 61 (2013) 7294–7300. [48] S. Sudhakar, R.A. Nazeer, Structural characterization of an Indian squid antioxidant peptide and its protective effect against cellular reactive oxygen species, J. Funct. Foods 14 (2015) 502–512. [49] C.F. Chi, B. Wang, Y.Y. Deng, Y.M. Wang, S.G. Deng, J.M. Ma, Isolation and characterization of three antioxidant pentapeptides from protein hydrolysate of monkfish (Lophius litulon) muscle, Food Res. Int. 55 (2014) 222–228. [50] H.P. Jiang, T.Z. Tong, J.H. Sun, Y.J. Xu, Z.X. Zhao, D.K. Liao, Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate, Food Chem. 154 (2014) 158–163. [51] J. Qian, Q. Tang, B. Cronin, R. Markovich, A. Rustum, Development of a high performance size exclusion chromatography method to determine the stability of human serum albumin in a lyophilized formulation of interferon alfa-2b, J. Chromatogr. A 1194 (2008) 48–56. [52] M. Gu, J.Y. Ren, W.Z. Sun, L.J. You, B. Yang, M.M. Zhao, Isolation and identification of antioxidative peptides from frog (Hylarana guentheri) protein hydrolysate by consecutive chromatography and electrospray ionization mass spectrometry, Appl. Biochem. Biotechnol. 173 (5) (2014) 1169–1182. [53] Q.J. Yan, L.H. Huang, Q. Sun, Z.Q. Jiang, X. Wu, Isolation, identification and synthesis of four novel antioxidant peptides from rice residue protein hydrolyzed by multiple proteases, Food Chem. 179 (2015) 290–295.

Please cite this article in press as: R. Song, et al., In vitro antioxidative activities of squid (Ommastrephes bartrami) viscera autolysates and identification of active peptides, Process Biochem (2016), http://dx.doi.org/10.1016/j.procbio.2016.06.015