ACE Inhibitory and Antioxidant Activities of Collagen Hydrolysates from the Ribbon Jellyfi sh ( Chrysaora sp . )

Hypertension is one of the most common lifestyle-related diseases and has become one of the most signifi cant health problems in recent years (1). Angiotensin-I-converting enzyme (ACE; EC 3.4.15.1) is a circulating enzyme that plays an important physiological role in regulating blood pressure by converting angiotensin I (inactive form) to angiotensin II, which is a potential vasoconstrictor, or by inactivating bradykinin, which is a vasodilator (2–5). Therefore, inhibition of ACE can be used to suppress blood pressure elevation and to treat myocardial infarction and other cardio-related diseases (2,4). A negative result of angiotensin II production is the formation of intercellular reactive radicals. Free radicals are the product of normal aerobic reactions in organisms and they play anti-infection roles. However, if their levels exceed normal levels, they can cause diseases such as cancer, arthritis, atherosclerosis, and diabetes (2). Increased concentrations of free radicals in the body can also occur via the consumption of oxidant-containing foods (6). Thus, neutralizing radicals both in foods and in the body can be helpful in the prevention and treatment of diseases.


Introduction
Hypertension is one of the most common lifestyle-related diseases and has become one of the most signifi cant health problems in recent years (1).Angiotensin-I-converting enzyme (ACE; EC 3.4.15.1) is a circulating enzyme that plays an important physiological role in regulating blood pressure by converting angiotensin I (inactive form) to angiotensin II, which is a potential vasoconstrictor, or by inactivating bradykinin, which is a vasodilator (2)(3)(4)(5).Therefore, inhibition of ACE can be used to suppress blood pressure elevation and to treat myocardial infarction and other cardio-related diseases (2,4).
A negative result of angiotensin II production is the formation of intercellular reactive radicals.Free radicals are the product of normal aerobic reactions in organisms and they play anti-infection roles.However, if their levels exceed normal levels, they can cause diseases such as cancer, arthritis, atherosclerosis, and diabetes (2).Increased concentrations of free radicals in the body can also occur via the consumption of oxidant-containing foods (6).Thus, neutralizing radicals both in foods and in the body can be helpful in the prevention and treatment of diseases.
In recent years, there has been growing interest in fi nding safe alternatives to artifi cial antioxidants and syn-thetic antihypertensive (ACE inhibitor) medicines because of their health risks and side eff ects.Biologically active peptides have antioxidant and/or antihypertensive activities and also possess nutritive value; thus they may prove to be useful in the pharmaceutical and food industries (7).Bioactive peptides have been isolated from diff erent proteins, including soy (8), whey (9), and casein (10), and from various marine sources such as fi sh-derived proteins, e.g.sardine, tuna, cod, bonito (3), and waste from fi sh processing facilities (2,11).More and more investigations are being conducted to identify potential sources of bioactive peptides (4,6,7).Although collagen peptides are assumed to exhibit high antiradical and antihypertensive activities due to their high content of hydrophobic amino acids (5,6,12), to date few studies have focused on collagen or gelatin hydrolysates as a source of these peptides.
Proteolytic hydrolysis of proteins is the most eff ective method for the production of bioactive peptides because the process releases bioactive fragments that are inactive within the protein chain (13).Moreover, by controlling the enzymatic hydrolysis process, polypeptides of certain sizes can be obtained, and they can be modifi ed to improve their functional properties (6).The characteristics and bioactivity of protein hydrolysates depend on many factors, including type of protease, enzyme-to-substrate ratio, concentration of the substrate, incubation time, temperature, and pH (7).The eff ects of diff erent enzymes (e.g.trypsin, alcalase, Protamex, pepsin, and others) and variations of other parameters have been evaluated in studies focused on the preparation and optimization of bioactive peptides.However, to identify the optimal conditions for isolating bioactive peptides from any individual source, the eff ects of diff erent proteases and other conditions must be studied individually.
Jellyfi sh, which are o en abundant in coastal waters, have been used as a food source in Asia, particularly China, for more than a thousand years.They have also been used for a traditional treatment of diseases such as hypertension, arthritis, gastric ulcer, and many others.The use of enzymatic hydrolysates of jellyfi sh collagen as antioxidant and/or antihypertensive reagents could add value to this underutilized resource, and the hydrosylation process could provide a multifunctional ingredient with potential applications in the pharmaceutical, nutraceutical, and food and dietary supplement industries.
The ribbon jellyfi sh (Chrysaora sp., class Scyphozoa, order Semaeostomeae) has recently been found in the coastal area around Penang Island, Malaysia, in huge blooms.Its bell is about 8-12 cm in diameter, and its fl at and ribbon-like oral arms are 20-50 cm long (14).In this study we extracted collagen and subsequently produced collagen hydrolysates from Chrysaora sp. using three different enzymes (trypsin, alcalase and Protamex).The ACE inhibitory and antioxidant activities of the diff erent hydrolysates were then measured and compared.Finally, potential bioactive peptide sequences present in highly bioactive (ACE inhibitor or antioxidant) samples were identifi ed.

Materials
Ribbon jellyfi sh (Chrysaora sp.) were caught along the north coast of Penang Island, Malaysia in March 2011.The umbrella was dissected, washed with deionized water, and stored at -80 °C until use.

Isolation of collagen
Collagen was extracted according to the method of Nagai et al. (15) with slight modifi cation as described by Barzideh et al. (16).All procedures were performed at 4 °C.Jellyfi sh umbrellas were thawed at 4 °C for 4-5 h, cut into small pieces (0.5 cm×0.5 cm), and washed with distilled water.To remove non-collagenous substances, each sample was treated with 0.1 M NaOH at a sample/solution ratio of 1:10 (mass per volume) with gentle stirring for 2 days; the solution was changed once a day.A er centrifugation at 10 000×g for 30 min, the remaining insoluble matter was washed with distilled water until neutral pH was achieved.It was then suspended in 0.5 M acetic acid (10 mL of acetic acid for each gram of collagenous material) containing 10 % (by mass) pepsin with gentle stirring for 3 days (digestion process).The fi nal viscous liquid was centrifuged at 20 000×g and 4 °C for 1 h.Digestion of the remaining pellet was carried out once more, and the supernatants were combined.The supernatant was dialysed against 10 volumes of 0.02 M Na 2 HPO 4 (pH=8.8)for 3 days to inactivate the enzyme.The dialysed sample was centrifuged at 20 000×g and 4 °C for 1 h.The resulting precipitate was dissolved in 0.5 M acetic acid and salted out by adding NaCl to a fi nal concentration of 1 M, followed by centrifugation at 20 000×g and 4 °C for 1 h.The resulting precipitate was dissolved in 0.5 M acetic acid and dialysed against distilled water for 2 days.The sample, called jellyfi sh pepsin-solubilized collagen (JPSC), was lyophilised and stored at -80 °C until further analysis.

Preparation of collagen hydrolysates
Samples (100 mg) of the isolated pepsin-solubilised collagen (JPSC) were suspended in 100 mL of distilled water and digested using one of three diff erent enzymes at an enzyme-to-substrate ratio of 1:50 (by mass).Digestion was carried out for 9 h at 50 °C, and optimum pH values for each enzyme (as suggested by the manufacturer and previous research) were as follows: pH=8 for alcalase and trypsin, and pH=7 for Protamex (17,18).To evaluate the eff ect of the duration of exposure to the enzyme on the degree of hydrolysis and bioactivity (ACE inhibitory and antioxidant activities) of the peptides, samples were taken at 0 (non-hydrolysed collagen), 1, 3, 5, 7, and 9 h.A er 9 h, the hydrolytic reaction was terminated by heating the samples at 95 °C for 10 min.The samples were then cooled to room temperature and centrifuged at 1077×g (3000 rpm) for 30 min.The resulting supernatants were lyophilised as jellyfi sh collagen hydrolysates (JCHs) and stored at -80 °C for further analysis.

Degree of hydrolysis of collagen hydrolysates
The method by Hoyle and Merri (19) was used to determine the degree of hydrolysis (DH) of all the isolated JCHs.Trichloroacetic acid (TCA, 20 %) was mixed with an equal volume of the hydrolysate solution.TCA-soluble materials (10 %) were collected a er centrifugation at 7000×g for 20 min at 10 °C.The nitrogen content was measured according to the Kjeldahl method (20).The following equation was used to compute the DH:

Amino acid composition of collagen hydrolysates
To determine the amino acid composition of the extracted collagen hydrolysates, 0.1 g of the freeze-dried JCH samples prepared using all of the enzymes were hydrolysed with 6 M HCl (5 mL) at 110 °C for 24 h.Subsequently, 400 µL of 50 µmol/mL of -α-amino-n-butyric acid (AABA) were added (as the internal standard) to the resulting hydrolysates, and distilled deionized water was added to reach a volume of 100 mL.Samples were then fi ltered through Whatman no. 1 fi lter paper, and then through 0.22-µm Millipore fi lter (21).The amino acids of the JPSC hydrolysate were derivatised by incubating 10 µL of the hydrolysed samples with 20 µL of AccQ•Fluor TM reagent (also known as AQC, and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate) (Waters Corporation, Milford, MA, USA) for 1 min at room temperature.Samples were then placed on the 150-µL glass with polyspring insert (Thermo Scientifi c, San Jose, CA, USA), which was equipped with the screw-capped vial, and the vial was heated for 10 min at 55 °C before analysis.
The derivatised samples were analysed using a HPLC (Waters Corporation).For each sample, a 10-µL aliquot was injected into the column, and the solution was eluted at a fl ow rate of 1 mL/min.Determination of amino acids was carried out according to the AccQ•Tag TM method using the Waters HPLC system equipped with a Waters 1525 binary pump, Waters 717 plus autosampler, Waters 2475 Multi λ fl uorescence detector, and Waters AccQ•Tag TM amino acid analysis column (3.9 mm×150 mm; packing material: silica-based bonded with C 18 ).The column was fi xed at 37 °C with fl uorescence detection at 250 nm for excitation and at 395 nm for emission.AccQ•Tag TM eluent A and acetonitrile/water (60:40) were used as eluents, and calibration of the HPLC system was performed using the amino acid standard H (Pierce, Rockford, IL, USA) as the reference.Methionine and cysteine were analysed separately using the performic acid procedure described by Moore (22).Breeze Workstation v. 3.20 (Waters Corporation) was used for data analysis.The area under the peak of each amino acid in the chromatogram was calculated and compared with that of the standard.The analysis was carried out in triplicate, and the results are reported as per 1000 amino acid residues.
In this assay, a 2.7-µL sample of JCHs (an aliquot containing 1 mg of JCH in 1 mL of distilled water) was mixed with 200 µL of prewarmed FRAP reagent at 37 °C and incubated at 37 °C for 1 h.Absorbance was then measured at 593 nm using a spectrophotometer (Spectramax M5, Molecular Devices, Sunnyvale, CA, USA).The antioxidant potential of the samples was determined according to a standard curve generated using a 0-2 mM solution of FeSO 4 •7H 2 O. FRAP values were expressed as mM of FeSO 4 •7H 2 O equivalent per mg of freeze-dried sample.

DPPH free radical scavenging assay
The antioxidant capacity of JPSC and JCHs was determined using the DPPH radical scavenging assay according to the method of Lee et al. (24).An aliquot of 6.66 µL of sample (1 mg/mL) was mixed with 200 µL of DPPH reagent (0.1 mM in ethanol).The mixture was then mixed and incubated at 30 °C for 30 min in the dark.The absorbance of the sample was measured at 517 nm using a spectrophotometer (Spectramax M5).The control consisted of methanol instead of the sample.Gallic acid at concentrations ranging from 0 to 60 µg/mL was used as the standard.The results are expressed as percentage of inhibition of DPPH radicals using the following equation: where A sample is the absorbance of the samples at diff erent times and A control is the absorbance of DPPH solution without the sample.

Evaluation of ACE inhibitory activity of collagen hydrolysates
The ACE inhibitory activity was measured according to the method of Cushman and Cheung (25) with modifications.For this analysis, a sample (50 µL at a concentration of 1 mg/mL) was mixed with 50 µL of ACE solution (50 mU/mL in sodium borate buff er, pH=8.3) and incubated at 37 °C for 10 min.Next, 150 µL of hippuryl-histidyl-leucine (4.15 mM in borate buff er containing 0.3 M NaCl, pH=8.3) were added to the standard reaction mixture and incubated at 37 °C for 30 min.The reaction was stopped by adding 500 µL of 1 M HCl.To extract the resulting hippuric acid, 1.5 mL of ethyl acetate were added and the mixture was vortexed for 1 min.A er resting for 5 min, 800 µL of the ethyl acetate layer were removed and vacuum dried in a vacuum concentrator (Concentrator 5301, Eppendorf, Hamburg, Germany) at 45 °C for 30 min.The dried sample was dissolved in 1 mL of distilled water, and the absorbance was measured at 228 nm using a spectrophotometer (Spectramax M5).Positive and negative controls consisted of 50 µL of 1 M HCl and 50 µL of distilled water, respectively, instead of the sample.The ACE inhibitory activity was calculated using the equation: where A negative is the absorbance of the negative control, and A sample is the absorbance of the sample at diff erent times.The absorbance of the positive control was used as the reading correction.

Identifi cation of bioactive peptides extracted from collagen hydrolysates
To identify the bioactive peptides in the JCHs, the peptides with the highest DPPH radical scavenging activity, FRAP reducing activity, and ACE inhibitory activity were subjected to peptide sequencing using mass spectrometry.A 200-µL aliquot containing 2 mg/mL of JCH in distilled water was fi ltered through a syringe fi lter (2 µm, Amicon, Merck Millipore, Carrigtwohill, County Cork, Ireland).Mass spectroscopy of the sample was then conducted using a Thermo LTQ/Orbitrap Velos coupled with an EASY-nLC II system (Thermo Scientifi c).
The eluent was sprayed into the mass spectrometer at 2.3 kV (source voltage), and a capillary temperature of 200 °C was used.Peptides were detected by full scan mass analysis from m/z=200-2000 at resolving power of 60 000 (at m/z=400, full width at half maximum (FWHM); 1-s acquisition) with data-dependent MS/MS analyses (ion trap mass spectrometry, ITMS) triggered by the eight most abundant ions from the parent mass list of predicted peptides with the rejection of single or unassigned charge state.The ITMS analysis was performed using the same resolving power (60 000), and collision-induced dissociation (CID) was conducted with isolation width of 2 Da, normalized collision energy of 35, activation q of 0.25, activation time of 50 ms, and charge state of 2 or higher.
Data acquisition was performed using Xcalibur v. 2.1 (Thermo Scientifi c) with a mass tolerance threshold of 5 ppm.Data analysis was performed using PEAKS studio v. 6.0 (Bioinformatic Solutions Inc., Waterloo, Canada) and Peptide Ranker (ShieldsLab, Dublin, Ireland).

Statistical analysis
All experiments were performed at least in triplicate, and data are presented as mean±standard deviation.Analysis of variance (ANOVA) was performed and comparisons of the mean values were conducted using Duncan's multiple range tests.Diff erences were considered signifi cant at the probability value of p<0.05.Analysis was performed using SPSS v. 20 for Mac OS (IBM, Armonk, NY, USA).

Degree of hydrolysis
Degree of hydrolysis (DH) is an important parameter when monitoring the hydrolysis process and comparing diff erent protein hydrolysates in terms of peptide length, functional and sensory properties, and nutritional value (6,11).DH is directly correlated with the solubility of the protein hydrolysates, which is increased through digestion of the parent protein (11).Fig. 1 shows the DH of the JCHs digested using the three enzymes over the course of the incubation period.The hydrolysis of all enzymes was fast during the initial stage of the process (3-5 h), but then the rate of hydrolysis gradually decreased until it reached a stationary phase, during which no apparent hydrolysis took place.This result, which shows that most peptides were cleaved within the fi rst 3-5 h of hydrolysis, is in agreement with the classic protease-induced hydrolysis reported for other proteins (26).However, there is a diff erence between the patterns of hydrolysis using Protamex and the other two enzymes.Increase of DH was biphasic when using trypsin and alcalase, while it was monophasic when using Protamex.It has been suggested that the activity of alcalase and trypsin starts with hydrolysing some reachable sites (up to 3 h of hydrolysis, phase 1).A er cleavage of the fi rst susceptible bonds and deformation of the polypeptide chain, new susceptible bonds become exposed to the enzyme.Therefore, cleavage goes fast again for another phase (up to 5 h, phase 2) before reaching a stationary phase (biphasic hydrolysis).However, with Protamex, which is a mixture of two enzymes and is specialized in cleaving the peptide bonds at certain sites, cleavage of all target bonds occurs simultaneously.Therefore, the hydrolysis trend using Protamex is monophasic.
During the fi rst 3 h of hydrolysis, exposure of JPSC to Protamex resulted in the highest DH (37 %); values for alcalase and trypsin were 30 and 25 %, respectively.However, a er 9 h of hydrolysis, the highest DH was found in the JCH produced by alcalase (48 %), followed by Protamex (43 %) and trypsin (36 %).This result is in agreement with other studies that reported alcalase to be more efficient than trypsin (26).In our experiment, the parent protein was identical in all three treatments, and hydrolysis conditions were optimized for each enzyme.Thus, the difference in DH among treatments might be due to the ability of the diff erent enzymes to cleave certain hydrolysable bonds (6).
DH is an important factor aff ecting the bioactivity of protein hydrolysates because it infl uences the size of peptides and the exposure of certain amino acids or functional Fig. 1.Enzymatic hydrolysis of jellyfi sh collagen using trypsin, alcalase, and Protamex.DH=degree of hydrolysis groups at the peptide terminals (26).Therefore, to evaluate the eff ect of DH on the bioactivity (ACE inhibitory and antioxidant activities) of hydrolysates, all assays were carried out on JCHs digested using the three diff erent enzymes at every hour of hydrolysis.

Amino acid content
Table 1 shows the amino acid composition of the JCHs expressed per 1000 amino acid residues.The amino acid composition of all JCH samples hydrolysed by three enzymes was similar, and also similar to that of jellyfi sh collagen (16) and to the typical profi le of collagen, with Gly as the most abundant amino acid (32 %) (26) and with Hyp present.
The amino acid profi le revealed that amino acids mainly consisted of hydrophobic residues.Wan Mohtar et al. (27) reported that the presence of a considerable quantity of hydrophobic amino acids in peptide sequences contributes to ACE inhibitory activity and antioxidant activity in several bioactive peptides.Among the hydrophobic amino acids, Gly was dominant with 319 residues.Ala and Pro were the next most abundant hydrophobic amino acids, with 91 and 73 residues, respectively, followed by Leu, Ile, Val, Phe, Met, and Tyr.The JCHs also contained a high amount of the non-hydrophobic amino acids Asp (69 residues) and Glu (82 residues), which have been reported to exhibit high antioxidant activity due to the donation of electrons to free radicals (13,28).
Pro plays an important role in ACE inhibitory and antioxidant activities (27,29).The presence of Tyr, Trp, Met, Lys (2,13,28), Pro, Leu, and Asp has also been reported to contribute to the antioxidant activity (29), and a high content of Gly, Pro, Ala, Glu, and Asp has been found in many other ACE inhibitory peptides (13).Therefore, the amino acid composition of the JCHs indicates that they have antioxidant and/or ACE inhibitory properties.

Antioxidant activity
Antioxidant activity of proteins and peptides is not a ributed to a single mechanism because proteins contain various amino acids with diff erent antioxidant properties.Some antioxidant components are more eff ective in metal chelating, whereas some others are radical scavengers or lipid peroxidation inhibitors (26,30).Thus, diff erent antioxidant assays are required to evaluate the antioxidant capacity of protein hydrolysates, and none of them can individually be referred to as a standard method.In this study, FRAP and DPPH radical scavenging assays were applied to investigate the antioxidant activity of the JCHs.

Results of FRAP assay
The ability of the sample to reduce the ferric ion is an indicator of potential antioxidant activity.In this process, the free radical is neutralized by receiving an electron donated by the reducing agent (i.e. the sample) and subsequently by acquiring a hydrogen from its environment solution (6).In this assay, the result of the reduction of ferric (Fe 3+ ) to ferrous (Fe +2 ) ion in the FRAP reagent is the formation of Fe 2+ -TPTZ, which is a blue-coloured complex with the highest absorbance at 593 nm (30).Fig. 2 shows the ferric reducing capacity of non-hydrolysed JPSC and the JCHs.The JCH samples had higher values than the non-hydrolysed collagen, probably due to the release of protons and electrons (hydrogen ions) during hydrolysis (31).The ability of all three JCHs to scavenge ferric ion increased as incubation time increased, with the highest activity at 7 and 9 h, with no signifi cant diff erence for each individual JCH (p>0.05).Among the three enzymes tested, digestion with Protamex and trypsin resulted in the highest FRAP activities, which might be due to the release of peptides with certain sequences as well as exposure of some amino acid residues (Tyr, Lys, and Met) and side chains that exhibit higher antioxidant activity (6,13).
The correlation between the DH and reducing capacity is not always clear.For example, the alcalase-induced hydrolysate with the highest DH had the lowest reducing power, and the reducing power of the Protamex-induced hydrolysate and the trypsin-induced hydrolysate did not diff er signifi cantly even though the former had a higher DH than the la er.This might be due to the position of specifi c amino acids within the peptide sequences that confer antioxidant activity to the purifi ed peptide (29).

Results of DPPH free radical scavenging activity assay
DPPH is an organic nitrogen radical.The percentage of DPPH radical inhibition by the sample within a certain period of time is an indicator of the antioxidant capacity of the sample (13).JPSC and JCHs obtained using diff erent enzymes for various periods of incubation were evaluated for their radical scavenging activity.Although DPPH radical scavenging activity was observed in both non-hydrolysed collagen and collagen hydrolysates, the la er showed higher values (Fig. 3).This result shows that enzymatic hydrolysis improved the antioxidant activity via the release of low molecular mass peptides and by increasing the exposure of hydrogen-donating amino acids (13,30).
For all of the JHCs, the highest value of DPPH scavenging activity was observed within the fi rst 1 to 3 h of hydrolysis, with no signifi cant diff erences among enzyme treatments (p<0.05).However, as the duration of hydrolysis increased, DPPH scavenging activity decreased.This result emphasizes the premise that the size of the peptide plays an important role in the bioactivity of the peptide.Excess hydrolysis might have a reverse eff ect by producing very short peptides or free amino acids that do not exhibit radical scavenging activity (13).
The DPPH scavenging activity of the trypsin-induced JCH was more than two times higher than that of the non--hydrolysed collagen, and the values of Protamex and alcalase were following the trypsin-induced one.Several factors, including amino acid composition, DH, peptide size, peptide sequence, and type of the applied enzyme, can infl uence the antioxidant capacity of the collagen hydrolysate.For the JCHs in this study, the parent protein used in all treatments was the same, so the observed differences cannot be due to amino acid composition; however, they might be due to various amino acid sequences within the peptides (particularly at N-and C-terminals) or various peptide lengths (2).

ACE inhibitory activity of collagen hydrolysates
The JCHs were subjected to the ACE inhibitory activity assay to identify the hydrolysates with the highest activities.All sample types, including the non-hydrolysed collagen, showed ACE inhibition activity to some extent, but treatment with the three enzymes resulted in significantly increased ACE inhibitory activity (Fig. 4).Therefore, enzymatic hydrolysis seems essential to cleave the A direct correlation between the incubation time and ACE inhibition activity was observed only up to 3 h in case of all enzymes; a er 3 h the activity decreased.This result again emphasizes the important role of peptide size in defi ning its bioactivity.However, diff erent enzymes with almost the same DH did not show the same ACE inhibitory potency.For example, the hydrolysates produced by exposure to alcalase for 1 h and trypsin for 3 h had similar DH values (26 and 25 %, respectively), but diff erent ACE inhibitory values (59 and 89 %, respectively).The hydrolysates generated by incubation with trypsin for 5 h and Protamex for 1 h both had 33 % DH, but their ACE inhibitory values were 34 and 51 %, respectively.On the other hand, some of the peptides with diff erent DH (hydrolysed by diff erent enzymes) showed similar ACE inhibitory activities.For example, the hydrolysates produced by exposure to alcalase for 1 h (59 % DH) and Protamex for 3 h (26 % DH) had similar ACE inhibitory values.These observations suggest that instead of the size of the peptide or the DH, the amino acid sequence of the collagen hydrolysates might be important element in determining the bioactivity of a peptide (3).
The trypsin-induced hydrolysates showed the highest ACE inhibitory activity, followed by alcalase and Protamex.In studies of marine protein sources and corn gluten (7,32,33), Protamex was reported to be more eff ective than alcalase, whereas Alemán et al. (26) found that they were equally eff ective.In some studies, trypsin was more eff ective in producing ACE inhibitors than alcalase (3), but in other cases alcalase was more eff ective (18,34).These diff erent actions of the same enzyme on diff erent substrates or diff erent enzymes on the same substrate (as in our study) indicate that along with DH and other parameters, the protein sequence of the substrate or the effectiveness of a protease on a certain substrate (i.e.substrate specifi city) is a very important factor (7). Therefore, it is crucial to select an appropriate protease to produce specifi c bioactive peptide sequences, and this choice depends on the raw material proteins and the expected bioactivity.

Potential bioactive peptides from collagen hydrolysates
A er identifying the samples with the highest bioactivity (FRAP, DPPH and ACE inhibition), the relevant hydrolysates were analysed to determine their amino acid sequence.Using mass spectroscopy and PEAKS studio so ware v. 6.0, peptide sequences present in the samples were identifi ed.Next, by applying the results to Peptide-Ranker so ware (screening of peptides with the certainty of more than 50 %) (35), peptide sequences that may have high antioxidant and ACE inhibitory were identifi ed (Table 2).
All of these peptides contain 7 to 16 amino acid residues, which is the reported range for bioactive peptides (2-20 residues) (36).The molecular mass of these peptides ranged from 677 to 1351 Da.Many of the ACE inhibitory peptides have been reported to be shorter than 1500 Da (18).Low molecular mass peptides are able to pass through the intestine and be absorbed easily (29), which makes these peptides useful as an ingredient in functional foods.
In all of the peptide sequences, the majority of amino acid residues are hydrophobic.He et al. (32) suggested that a high quantity of hydrophobic amino acids (branched side chain or aromatic), such as Pro, Glu, Val, Phe and Tyr, particularly at the C-terminal end of the peptide, enhances the ACE inhibitory capacity of the hydrolysate (32).Therefore, the high ACE inhibitory activity of separated hydrolysates might lie in the high content of these amino acids in the peptide sequence.However, as Li et al. (37) suggested, the presence of hydrophilic amino acids within the peptide sequence at certain intervals between hydrophobic amino acids plays an important role in ACE inhibitory activity too.
It has been reported that the presence of repeating dior tripeptides within the peptide sequence enhances the bioactivity of the peptide as compared to the same individual amino acids (13).In our study, the presence of repeating amino acids, such as Ala-Ala, Pro-Pro and Leu--Leu, was observed within the identifi ed peptide sequences.
In addition, the identifi ed peptides contain shorter fragments (i.e.certain dipeptides, tripeptides, etc.) such as Leu-Gly, Gly-Pro, Gly-Pro-Ala, Leu-Gly-Pro-Val and many others within their sequence, which have been confi rmed as ACE inhibitors and/or antioxidants (29,38).In order to fi nd these short peptides, the identifi ed bioactive peptides were investigated using two bioactive peptide databases, BIOPEP (39) and EROP-Moscow (40).Some of the identifi ed subsequences are summarized in Table 3.However, it should be considered that this study provides only some of the potential bioactive subsequences and there might be more unknown components that play an important role in exhibiting ACE inhibitory and antioxidant activities of the isolated peptides.
Many factors concurrently aff ect the bioactivity of peptides, and it is not easy to identify the best peptide from one type of protein to compare with similar peptides from other proteins.Although the amino acid sequence of a bioactive peptide is an important parameter in conferring high bioactivity (ACE inhibitory and antioxidant activities), amino acid composition and the sequence of the rest of the peptides, which depend on the parent protein, can be important parameters as well, and there is an assumption that the presence of some other fractions can have a synergistic eff ect on the bioactivity of the detected bioactive ones (4).In addition, other factors such as concentration of the hydrolysate, pH, and temperature can be the reasons for diff erences in reports about same proteins.

Conclusion
The ACE inhibitory activity and antioxidant activity of jellyfi sh collagen hydrolysates obtained using three different enzymes were studied.The isolated peptides showed considerable antioxidant and ACE inhibitory activities.Therefore, this underutilized resource may prove useful as a food additive, ingredient for functional foods, and nu- LGPV GSLGPVGDPGQVGR, LGPVGDCKGPPK, LGPVMMLGHGR LGPVGDCKGPPK, GSLGPVGDPGGSVGR LGPVGDPGQVGR, GSLGPVGDPGQVGR SLGPVGDPGQVGR ACE inhibitory1 GDP GSLGPVGDPGQVGR, ALGPSGAAGPAGDPGR GCGLGDPPGHGK, GSLGPVGDPGGSVGR GSLGPVGDPGQVGR, SLGPVGDPGQVGR LGPVGDPGQVGR Antioxidant activity 1  traceutical and pharmaceutical agent.The results of this study also suggest that exposure to diff erent proteases and diff erent degrees of hydrolysis aff ects the bioactivity potency of the hydrolysate from the same parent protein.

Fig. 2 .Fig. 3 .Fig. 4 .
Fig. 2. Results of the FRAP assay for hydrolysates produced using: a) trypsin, b) alcalase and c) Protamex.Diff erent le ers (a, b, c and d) in each graph (within the same hydrolysate) indicate signifi cant diff erences (p<0.05).Data are the mean values±standard deviation from at least three repetitions

Table 2 .
Identifi cation of peptide sequences with the highest bioactivity from three diff erent hydrolysates (35) score was obtained from PeptideRanker(35)

Table 3 .
Bioactive subsequences within the identifi ed potentially bioactive peptides from three diff erent hydrolysates