Production and identification of angiotensin I-converting enzyme (ACE) inhibitory peptides from Mediterranean fish discards

1 PRODUCTION AND IDENTIFICATION OF ANGIOTENSIN I1 CONVERTING ENZYME (ACE) INHIBITORY PEPTIDES 2 FROM MEDITERRANEAN FISH DISCARDS 3 Pedro J. García-Moreno*, F. Javier Espejo-Carpio, Antonio Guadix, Emilia M. Guadix 4 Department of Chemical Engineering, University of Granada, 18071 Granada, Spain 5 ABSTRACT 6 This work studies the production of peptides exhibiting Angiotensin I-converting enzyme 7 (ACE)-inhibitory activity from discarded Mediterranean fish species such as sardine, horse 8 mackerel, axillary seabream, bogue and small-spotted catshark. The evolution of the ACE9 inhibitory activity with the degree of hydrolysis (DH) of protein hydrolysates was also 10 investigated. Hydrolysates of horse mackerel and small-spotted catshark, both obtained with 11 the simultaneous addition of subtilisin and trypsin, showed the highest antihypertensive 12 activity (IC50 of 279 and 302 g/mL, respectively). For horse mackerel hydrolysate, fraction 13 B (130-2350 Da) exhibited the highest ACE-inhibitory activity (IC50=85 g/mL). In the case 14 of small-spotted catshark hydrolysate, fraction D (<470 Da) presented the lowest IC50 value 15 (27 g/mL). In addition, 14 novel ACE-inhibitory peptides were identified in horse mackerel 16 and small-spotted catshark hydrolysates. The peptide VAMPF, identified in fraction D of 17 small-spotted catshark hyhdrolysate, is one the most promising according to its low IC50 value 18 obtained by the QSAR-model (IC50=0.44 M). 19 20 Keywords: fish discards, enzymatic hydrolysis, SEC fractionation, ACE-inhibitory activity, 21 bioactive peptides 22 23 * Corresponding author: Tel.: +34 958 241329; Fax: +34 958 248992; E-mail: [email protected] 2 1. INTRODUCTION 24 Discards are defined as that part of the catch which is not retained on board and is returned to 25 the sea (Kelleher, 2005). Discarding has a negative impact on fisheries sustainability since it 26 reduces the stock of juveniles and spawning biomass due to the high mortality of discarded 27 fish (Catchpole, Frid, & Gray, 2005). In addition, discard practices produce a significant 28 environmental problem due to alterations on marine trophic chains (Bozzano & Sardà, 2002). 29 As a result, the EU Fisheries Commission has approved a reformed common fisheries policy 30 to gradually eliminate discards in all the European fisheries (EU, 2013). One of the proposed 31 measures, that would be gradually implemented, is the obligation to land all catches. Thus, 32 apart from the implementation of measures destined to reduce unwanted catches, added-value 33 products must be also developed for the up-grading of these landed non-commercial 34 specimens. 35 In the Alboran Sea, portion of Mediterranean Sea lying between the Iberian Peninsula and the 36 north of Africa, discards comprise non-commercial species such as bogue (Boops boops) and 37 small-spotted catshark (Scyliorhinus canicula). Additionally, commercial species such as 38 sardine (Sardina pilchardus), horse mackerel (Trachurus mediterraneus) and axillary 39 seabream (Pagellus acarne) are also discarded in this fishery due to quota restrictions, 40 minimal landing-size requirements or high-grading practices. These species present valuable 41 protein contents, which are practically constant along the year ranging from 17 to 23 %, 42 depending on the species (García-Moreno, Pérez-Gálvez, Morales-Medina, Guadix A, & 43 Guadix EM, 2013a). Therefore, added-value products such as bioactive peptides produced by 44 enzymatic proteolysis can be obtained from the protein fraction of these fish discarded 45 species. 46 Peptides containing 2-20 amino acid residues, which were released from fish protein by 47 enzymatic hydrolysis, have been reported to exhibit numerous bioactivities such as 48 3 antihypertensive, antioxidant, anticholesterolemic, antithrombotic and antimicrobial (He, Liu, 49 & Ma, 2013; Harnedy & FitzGerald, 2012). Among them, angiotensin I-converting enzyme 50 (ACE)-inhibitory peptides, which do not exhibit known side effects, have been 51 comprehensively studied as alternative antihypertensive agents (Aluko, 2015). To this regard, 52 the in vitro measurement of ACE-inhibitory activity is a common first approach to identify 53 marine protein derived cardioprotective peptides (Mora & Hayes, 2015). This is due to the 54 fact that ACE (EC 3.4.15.1), a zinc metallopeptidase, plays a crucial role in the regulation of 55 blood pressure. In the renin-angiotensin system, ACE transforms the inactive decapeptide 56 angiotensin I (DRVYIHPFHL) into the potent vasoconstrictor octapeptide, angiotensin II 57 (DRVYIHPF). Besides, in the kallikrein-kinin system, ACE catalyzes the degradation of 58 bradykinin, a vasodilator nonapeptide (Li, Le, Shi, & Shrestha, 2004). Thus, the inhibition of 59 ACE would originate a reduction in blood pressure. 60 A number of previous studies have described the ACE-inhibitory activity of marine protein 61 hydrolysates produced from different species such as yellowfin sole (Limanda aspera) (Jung 62 et al., 2006), pacific hake (Merluccius productus) (Cinq-Mars & Li-Chan, 2007), sardinelle 63 (Sardinella aurita) (Bougatef et al., 2008), cuttlefish (Sepia officinalis) (Balti, Nedjar64 Arroume, Yaba-Adjé, Guillochon, & Nasri, 2010), loach (Migurnus anguillicaudatus) (Li, 65 Zhou, Huang, Sun, & Zeng, 2012) and chum salmon (Oncorhynchus keta) (Lee, Jeon, Byun, 66 2014). However, to the best of the authors’ knowledge, apart from sardine (Sardina 67 pilchardus) which has been extensively studied (Bordenave et al., 2002; García-Moreno, 68 Pérez-Gálvez, Espejo-Carpio, Muñío, Guadix A, & Guadix EM, 2013b; Matsui, Matsufuji, 69 Seki, Osajima, Nakashima, & Osajima, 1993), there is no previous work on the production of 70 fish protein hydrolysates with ACE-inhibitory activity from the chosen discarded species in 71 the Alboran Sea. 72 4 A special attention should be given to the specificity of the enzymes employed since they play 73 an important role on the bioactivity of the hydrolysates produced. Subtilisin and trypsin have 74 been previously reported to yield fish protein hydrolysates exhibiting ACE-inhibitory activity 75 (Bougatef et al., 2008; Matsui et al., 1993). Nevertheless, there is a limited knowledge about 76 the production of ACE-inhibitory hydrolysates from fish protein by combinations of these two 77 enzymes (García-Moreno et al., 2013b). Subtilisin preferentially cleaves at the C-terminal of 78 hydrophobic residues, whereas trypsin permits to release peptides with basic amino acids in 79 the C-terminal (Espejo-Carpio, De Gobba, Guadix A, Guadix EM, & Otte, 2013). Both 80 hydrophobicity and basicity in the C-terminal are desired characteristics for ACE-inhibitory 81 peptides (Li et al., 2004). 82 In the light of the above, this work aimed to investigate the ACE-inhibitory activity of fish 83 protein hydrolysates produced from five discarded fish species (S. pilchardus, H. mackerel, A. 84 Seabream, B. boops and S. canicula). The following specific objectives were pursued: i) to 85 study the influence of the enzymatic treatment (subitlisin and trypsin added sequentially or 86 simultaneously) on ACE inhibition of hydrolysates, ii) to investigate the effect of the degree 87 of hydrolysis of hydrolysates and the molecular weight range of peptides on the ACE88 inhibitory activity, and iii) to identify ACE-inhibitory peptides within the most active 89 fractions. 90 2. MATERIALS AND METHODS 91 2.1 Raw material and separation of protein fraction 92 Raw sardine (Sardine pilchardus), horse mackerel (Trachurus mediterraneus), bogue (Boops 93 boops), axillary seabream (Pagellus acarne) and small-spotted catshark (Scyliorhinus 94 canicula), were provided by the fishing harbour of Motril (Spain). Fish were kept in ice 95 during transportation. In the same day, whole fish, included skin, bones and internal organs, 96 5 was preheated at 40 °C for 30 min (Digiterm 100, Selecta, Barcelona, Spain) and hydraulic 97 pressed according to García-Moreno et al. (2014) in order to obtain a dewatered and defatted 98 protein cake. In the case of small-spotted catshark, muscle was employed as raw material for 99 the enzymatic hydrolysis due to the high resistance of its skin. 100 2.2 Enzymatic hydrolysis 101 Two serine endoprotease enzymes were employed: one of bacterial origin (subtilisin, EC 102 3.4.21.62) and other from an animal source (pancreatic trypsin, EC 3.4.21.4). Both were 103 provided by Novozymes (Denmark) as Alcalase 2.4L and PTN 6.0S, respectively. The 104 following enzymatic patterns were evaluated: (a) 2-hour hydrolysis with subtilisin followed 105 by addition of trypsin until completing 4 h of reaction; (b) 2-hour reaction with trypsin 106 followed by subtilisin until completing 4 h of reaction and; (c) 4-hour hydrolysis with 107 simultaneous addition of both enzymes. 108 For all the experiments, the hydrolysis reaction was carried out at pH 8 and 50 ºC as described 109 previously (García-Moreno et al., 2014). To determine the influence of DH on ACE110 inhibitory activity of hydrolysates, samples were drawn for each experiment at different times 111 of reaction (0, 5, 10, 20, 30, 45, 60, 90, 120, 125, 130, 140, 150, 165, 180, 210, and 240 min). 112 Final hydrolysates obtained after 4 h of reaction were also further analysed. The enzyme was 113 deactivated by heating the samples in a boiling water bath for 15 min. The samples were 114 centrifuged and filtered in order to remove the solids remained. Then, they were kept at -20 115 ºC until analyses were performed. They were also lyophilized and stored at -20 ºC until 116 analyses were carried out. 117 2.3 Fractionation by size-exclusion chromatography (SEC) 118 Selected lyophilized hydrolysates were re-dissolved in distilled water (5 mg of 119 hydrolysate/mL) and were then fractionated by SEC using an FPLC system (AKTA purifier 120 6 UPC 100, GE Healthcare, Uppsala, Sweden) mounted with a Superdex Peptide 10/300 GL 121 column (GE Healthcare, Uppsala, Sweden). Five hundred microliters of the sample solution 122 were injected and eluted with ultrapure water at a flow rate of 0.5 mL/min. The effluent was 123 monitored at 280 nm and the fractions were automatically collected according to slope 124 changes. The area of each fraction was integrated using Unicorn 5.1 software (GE Healthcare, 125 Uppsala, Sweden). Up to five injections were performed for each selected hydrolysate in 126 order to collect enough amount of protein for each fraction. Then, each fraction was 127 concentrated by freeze-drying for the subsequent ACE-inhibitory activity determination and 128 peptides identification. Five standards with different molecular weights, Ribonuclease A 129 (13700 Da), Aprotinin (6511 Da), Vitamin B12 (1355 Da), tri-glycine (189 Da) and glycine 130 (75 Da) (Sigma-Aldrich, St. Louis MO, USA), were analyzed to set a calibration curve which 131 allowed to relate the elution volume with the peptide size. 132 2.4 Protein determination 133 The protein content of the final lyophilized hydrolysates was determined using a FP-528 134 LECO nitrogen analyser (LECO, St Joseph, MI, USA) calibrated with 135 ethylenediaminetetraacetic acid according to the Dumas method (Saint-Denis & Goupy, 136 2004). The protein concentration of the SEC fractions was evaluated using a bicinchoninic 137 acid (BCA) protein assay kit acquired from Sigma-Aldrich Quimica SA (Madrid, Spain). 138 Triplicate measurements were performed. 139 2.5 Determination of ACE-inhibitory activity 140 The ACE inhibitory activity of the hydrolysates and of the SEC fractions was determined in 141 vitro by the methodology described by Shalaby, Zakora, and Otte (2006). This method is 142 based in the hydrolysis of the tripeptide N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl143 glycine (FAPGG, Sigma F7131) with the Angiotensin converting enzyme (ACE) from rabbit 144 7 lung (Sigma A6778). The assay was carried out in 96-well microplate at 37 ºC. Each well 145 contained 10 L of enzyme solution (0.25 U/mL), 10 L of sample, and 150 L of 0.88mM 146 of FAPGG in buffer Tris-HCl 50 mM, pH 7.5 and 0.3 M of NaCl. The wavelength was set at 147 340 nm and the absorbance was monitored during 30 minutes by means of a Multiskan FC 148 microplate photometer (Thermo Scientific, Vantaa, Finland). Each sample was analysed in 149 triplicate. 150 The absorbance decreases linearly with time as ACE hydrolyses the substrate FAPGG. The 151 slope of this descent is commonly used as a measurement of the enzyme activity. Indeed, the 152 numerical value of inhibitory activity of each hydrolysate can be calculated by Eq. 1: 153 ACE inhibition (%)= (1 − 𝜌𝑖 𝜌0)×100 (1) 154 where i was the slope in the presence of inhibitor (hydrolysate) and o the slope obtained in 155 the absence of inhibitor (pure water). These slopes were calculated from the values obtained 156 within the interval of 10 to 25 minutes, where a better linearity was observed. 157 The IC50 value, which is defined as the concentration of hydrolysate needed to inhibit 50 % of 158 ACE activity, was determined experimentally for the final hydrolysates and for the selected 159 SEC fractions. In the case of identified peptides, IC50 values were determined by the 160 quantitative structure-activity relationship (QSAR) model proposed by Pripp, Isaksson, 161 Stepaniak, and Sørhaug (2004). This model (Eq. 2) gives a calculated IC50 value for peptides 162 according to: i) the side-chain hydrophobicity (x1), ii) the positively charged side chain for 163 amino acid in C-terminal position (x2), and iii) the van der Waals volume for the amino acid 164 next to C-terminal position (x3): 165 logIC50% = 1.46−9.29∙10−5𝑥1+0.52𝑥2+3.21∙10−2𝑥3 (2) 166 8 2.6 Identification of ACE-inhibitory peptides 167 The most active fractions collected from SEC were analyzed employing a Waters ACQUITY 168 UHPLC system coupled to a Synapt Mass Quadrupole Time-of-Flight Mass Spectrometer. 169 Samples of 5 L were injected in a Waters ACQUITY BEH C18 column (100 mm × 2.1 mm, 170 1.7 m) and components were eluted using a flow rate of 0.3 mL/min of water– acetic acid 171 (100:0.5, v/v) (buffer A) and methanol (buffer B) as described by Liu et al. (2015). The MS 172 spectra were acquired under the positive electrospray ionization using a capillary energy of 173 3.0 kV and sampling cone of 30 V. The analyses were performed using the standard range 174 from 50-1900 m/z at the normal scan resolution. PepSeq program from BioLynx software was 175 employed for sequencing peptides. 176 2.7 Statistical analysis 177 The Statgraphics software (version 5.1) was used to carry out one-way analysis of variance 178 (ANOVA) on the data. The Tukey’s test was employed for that purpose and differences 179 between means were considered significant at p≤0.05. 180 3. RESULTS AND DISCUSSION 181 3.1 Hydrolysis of press cake 182 The hydrolysis curves of the four press cakes and of the muscle of small-spotted catshark 183 exhibited an initial fast reaction rate after the addition of each enzyme or mixture of enzymes. 184 This initial period was followed by a slowdown which finished in a plateau where no apparent 185 hydrolysis took place. This remarkable and progressive decrease in the reaction rate is mainly 186 due to enzyme inhibition by hydrolysis products (Valencia, Pinto, & Almonacid, 2014). 187 Further, the addition of subtilisin resulted in higher increase of DH when compared with the 188 addition of trypsin, as observed in Fig. 1 for the hydrolytic curves of small-spotted catshark. 189 This fact was attributed to the specificity of trypsin, which only cleaves peptidic bonds 190 9 involving arginine and lysine, while subtilisin is an endoprotease of broad spectrum (Adler191 Nissen, 1986). In addition, an increase of DH was also observed (Fig. 1) after the addition of 192 the second enzyme in the sequential enzymatic treatments. The shape of these hydrolysis 193 curves was similar to those reported by Guerard, Guimas, and Binet (2002) when intermediate 194 addition of fresh enzyme was carried to hydrolyze tuna stomachs. 195 Different DH values were obtained after 4 h of hydrolysis depending on the species and the 196 enzymatic treatment employed (Table 1). The sequential enzymatic treatment subtilisin and 197 trypsin resulted in the highest DH for all species with exception to horse mackerel where the 198 highest DH was achieved with the simultaneous addition of subtilisin and trypsin. It should be 199 also noted that horse mackerel protein presented the best degradability by the enzymes used 200 which is in accordance with previous studies (García-Moreno et al., 2013b). 201 3.2 Influence of DH on ACE-inhibitory activity 202 The DH of a hydrolysate, which indicates the extent of the protein degradation, together with 203 the characteristics of the raw material, the specificity of enzymes and the hydrolysis 204 conditions play an important role on its ACE-inhibitory activity (pH, temperature and 205 enzyme/substrate ratio) (Balti, Nedjar-Arroume, Yaba-Adjé, Guillochon, & Nasri, 2010). 206 Undigested press cakes from bogue and small-spotted catshark did not present ACE207 inhibitory activity, whereas medium ACE inhibition values were found for press cakes from 208 axillary seabream and horse mackerel (Fig. 2a). In the case of sardine, a higher ACE209 inhibitory activity was observed for the no hydrolysed press cake. According to Kristinsson 210 (2006), intact fish proteins may also exhibit high ACE inactivation activity. However, intact 211 proteins would not play a role directly in ACE regulation in vivo, as they would be 212 hydrolyzed in the digestive system. In addition, the presence of non peptide but biologically 213 active materials (e.g. phenolic compounds) can also contribute to the ACE-inhibiting activity 214 of non hydrolysed samples (Aluko, 2015). 215 16 peptides derived from marine sources has been tested using a set of previously known ACE 365 inhibitory peptides (He et al., 2013). As a result of this assessment, the model was able to 366 predict IC50 values of 70% of the peptides with an error lower than 85 M. Moreover, when 367 the model was used just for distinguishing active peptides (IC50 < 500 M) from peptides with 368 low activity (IC50 > 500 M), its efficacy rose to 90%. However, the predictability of the 369 method decreased considerably when amino acid with a positively charged side group was in 370 the C-terminal position. 371 According to these results, this QSAR-model is an adequate method to predict the ACE 372 inhibitory capacity of identified peptides and thus, it was used in this work (Table 3). 373 Nevertheless, because of the limitations of the model employed, ACE-inhibitory activity of 374 peptides with positive charge in the C-terminal position was not evaluated. Although the good 375 predictive results of the QSAR-model suggested that most of the identified peptides are good 376 inhibitors, none of them has been previously identified as ACE-inhibitory peptide. 377 Particularly interesting seems the peptide VAMPF identified in fraction D of small-spotted 378 catshark (Fig. 4). The high ACE-inhibitory activity of this peptide may be explained by its C379 terminal tripeptide sequence “MPF”. This sequence was also found in the potent ACE 380 inhibitory peptide DPALATEPDPMPF, which was identified in the subtilisin hydrolysate of 381 Nile tilapia gelatin (Vo et al., 2011). The final dipeptide of the peptide LQPY have also been 382 previously identified in ACE inhibitors such as YRPY (320M) and LPYPY (28.9M) 383 derived from bonito bowels (Meisel et al., 2006) and ovine caseins (Gómez-Ruiz et al., 2007), 384 respectively. It should be also noted that the peptides VAMPF and LQPY, identified in small385 spotted catshark hydrolysate, contain proline residue at the C-terminal which may contribute 386 to their ACE-inhibitory activity. 387 Nevertheless, although promising results were obtained by using the QSAR-model and some 388 of the subsequences of the identified peptides have been previously reported in literature, 389 17 further studies are required in order to evaluate in vitro the ACE-inhibitory capacity of these 390 peptides. 391 4. CONCLUSIONS 392 The simultaneous addition of subtilisine and trypsine led to the final hydrolysates with the 393 highest ACE-inhibitory activity: horse mackerel hydrolysate (IC50=279 g/mL) and small394 spotted catshark hydrolysate (IC50=302 g/mL). For the horse mackerel hydrolysate, a 395 fraction containing peptides in the range 130-2350 Da exhibited de highest antihypertensive 396 activity (IC50=85 g/mL). For the small-spotted catshark hydrolysate, a purified fraction 397 (<470 Da) showed the highest ACE-inhibitory activity with an IC50 of 27 g/mL. Fourteen 398 novel ACE-inhibitory peptides have been identified in horse mackerel and small-spotted 399 catshark hydrolysates. The peptide VAMPF, identified in small-spotted catshark hydrolysate, 400 is one of the most promising when considering its tripeptide C-terminal sequence and its IC50 401 value predicted by the QSAR-model (IC50=0.44 M). 402 These findings denote that it is feasible to obtain short chain length peptides from fish 403 discards in the Mediterranean Sea exhibiting a high inhibition of ACE. Nevertheless, future 404 studies are required on the bioavailability of the hydrolysates (e.g. gastrointestinal digestion 405 and/or studies with spontaneously hypertensive rats) in order to confirm their ACE-inhibitory 406 activity in vivo. 407 5. ACKNOWLEDGEMENTS 408 This work was supported by the Spanish National Plan I+D+i (CTQ2011-23009) and by the 409 Andalusian Government (project P12-AGR-1993). P.J. García-Moreno also acknowledges a 410 postdoctoral contract from the University of Granada. 411 18 6. REFERENCES 412 Adler-Nissen, J. (1986). Enzymic hydrolysis of food proteins. London: Elsevier Applied 413 Science Publishers. 414 Aluko, R. E. (2015). Antihypertensive peptides from food proteins. Annual Review of Food 415 Science and Technology, 6, 235-262. 416 Balti, R., Nedjar-Arroume, N., Yaba-Adjé, E., Guillochon, D., & Nasri, M. (2010). Analysis 417 of novel angiotensin I-converting enzyme inhibitory peptides from enzymatic hydrolysates 418 of cuttlefish (Sepia officinalis) muscle proteins. Journal of Agriculture Food Chemistry, 419 58, 3840-3846. 420 Bordenave, S., Fruitier, I., Ballandier, I., Sannier, F., Gildberg, A., Batista, I., & Piot, J. 421 (2002). HPLC preparation of fish waste hydrolysate fractions. Effect on guinea pig ileum 422 and ace activity. Preparative Biochemistry and Biotechnology, 32, 65-77. 423 Bougatef, A., Nedjar-Arroume, N., Ravallec-Plé, R., Leroy, Y., Guillochon, D., Barkia, A., & 424 Nasri, M. (2008). Angiotensin I-converting enzyme (ACE) inhibitory activities of 425 sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with 426 microbial and visceral fish serine proteases. Food Chemistry, 111, 350-356. 427 Bozzano, A., & Sardà, F. (2002). Fishery discards consumption rate and scavenging activity 428 in the northwestern Mediterranean Sea. ICES Journal of Marine Science, 59, 15-28. 429 Byun, H., & Kim S. (2001). Purification and characterization of angiotensin I converting 430 enzyme (ACE) inhibitory peptides from Alaska pollack (Theragra chalcogramma) skin. 431 Process Biochemistry, 36, 1155-1162. 432 Catchpole, T. L., Frid, C. L. J., & Gray, T. S. (2005). Discards in North Sea fisheries: causes, 433 consequences and solutions. Marine Policy, 29, 421-430. 434 19 Cinq-Mars, C. D., & Li-Chan, E. C. Y. (2007). Optimizing angiotensin I-converting enzyme 435 inhibitory activity of pacific hake (Merluccius productus) fillet hydrolysate using response 436 surface methodology and ultrafiltration. Journal of Agriculture and Food Chemistry, 55, 437 9380-9388. 438 Espejo-Carpio, F. J., De Gobba, C., Guadix, A., Guadix, E. M., & Otte, J. (2013). Angiotensin 439 I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein 440 fractions. International Dairy Journal, 32, 175-183. 441 EU, 2013. Regulation (EU) No 1380/2013 of the European Parliament and of the Council of 442 11 December 2013 on the common fisheries policy. 443 Fahmi, A., Morimura, S., Guo, H.C., Shigematsu, T., Kida, K., & Uemura, Y. (2004). 444 Production of angiotensin I converting enzyme inhibitory peptides from sea bream scales. 445 Process Biochemistry, 39, 1195-1200. 446 García-Moreno, P. J., Pérez-Gálvez, R., Morales-Medina, R., Guadix, A., & Guadix, E. M. 447 (2013a). Discarded species in the West Mediterranean Sea as sources of Omega-3 PUFAs. 448 European Journal of Lipid Science and Technology, 115, 982-989. 449 García-Moreno, P. J., Pérez-Gálvez, R., Espejo-Carpio, F. J., Muñío, M. M., Guadix, A., & 450 Guadix, E. M. (2013b). Lipid Characterization and Properties of Protein Hydrolysates 451 Obtained From Discarded Mediterranean Fish Species. Journal of the Science of Food and 452 Agriculture, 93, 3777-3784. 453 García-Moreno, P. J., Batista, I., Pires, C., Bandarra, N. M., Espejo-Carpio, F. J., Guadix, A., 454 & Guadix, E.M. (2014). Antioxidant activity of protein hydrolysates obtained from 455 discarded Mediterranean fish species. Food Research International, 65, 469-476. 456 Geirsdottir, M., Sigurgisladottir, S., Hamaguchi, P. Y., Thorkelsson, G., Johannsson, R., 457 Kristinsson, H. G., & Kristjansson, M. M. (2011). Enzymatic hydrolysis of blue whiting 458 20 (Micromesistius poutassou); functional and bioactive properties. Journal of Food Science, 459 76, C14-C20. 460 Gómez-Ruiz, J. Á., Ramos, M., & Recio, I. (2007). Identification of novel angiotensin461 converting enzyme-inhibitory peptides from ovine milk proteins by CE-MS and 462 chromatographic techniques. Electrophoresis, 28, 4202-4211. 463 Guerard, F., Guimas, L., & Binet, A. (2002). Production of tuna waste hydrolysates by a 464 commercial neutral protease preparation. Journal of Molecular Catalysis B-Enzymatic, 19, 465 489-498. 466 Harnedy, P. A., & FitzGerald, R. J. (2012). Bioactive peptides from marine processing waste 467 and shellfish: A review. Journal of Functional Foods, 4, 6-24. 468 He, H., Liu, D., & Ma, C. (2013). Review on the angiotensin-I-converting enzyme (ACE) 469 inhibitor peptides from marine proteins. Applied Biochemistry and Biotechnology, 169, 470 738-749. 471 Je, J., Park, P., Kwon, J. Y., & Kim, S. (2004). A novel angiotensin I converting enzyme 472 inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein 473 hydrolysate. Journal of Agriculture and Food Chemistry, 52, 7842-7845. 474 Jung, W., Mendis, E., Je, J., Park, P., Byeng, W. S., Hyoung, C. K., Yang, K. C., & Kim, S. 475 (2006). Angiotensin I-converting enzyme inhibitory peptide from yellowfin sole (Limanda 476 aspera) frame protein and its antihypertensive effect in spontaneously hypertensive rats. 477 Food Chemistry, 94, 26-32. 478 Kelleher, K. (2005). Discards in the world’s marine fisheries. Rome: FAO Fisheries 479 Department. 480 21 Kristinsson, H. G. (2006). Aquatic food protein hydrolysates. In F. Shahidi (Ed.), Maximising 481 the value of marine by-products, (pp. 229-248). Cambridge: Woodhead Publishing 482 Limited. 483 Lee, J. K., Jeon, J., & Byun, H. (2014). Antihypertensive effect of novel angiotensin I 484 converting enzyme inhibitory peptide from chum salmon (Oncorhynchus keta) skin in 485 spontaneously hypertensive rats. Journal of Functional Foods, 7, 381-389. 486 Li, G., Le, G., Shi, Y., & Shrestha, S. (2004). Angiotensin I-converting enzyme inhibitory 487 peptides derived from food proteins and their physiological and pharmacological effects. 488 Nutrition Research, 24, 469-486. 489 Li, Y., Zhou, J., Huang, K., Sun, Y., & Zeng, X. (2012). Purification of a novel angiotensin I490 converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach 491 (Misgurnus anguillicaudatus). Journal of Agriculture and Food Chemistry, 60, 1320-1325. 492 Meisel H., Walsh D. J., Murray B., & FitzGerald R. J. (2006). ACE inhibitory peptides. In Y. 493 Mine, & F. Shahidi (Eds.), Nutraceutical proteins and peptides in health and disease (pp. 494 269-315). Boca Raton: CRC Taylor & Francis Group. 495 Liu, R., Zheng, W., Li, J., Wang, L., Wu, H., Wang, X., & Shi, L. (2015). Rapid identification 496 of bioactive peptides with antioxidant activity from the enzymatic hydrolysate of mactra 497 veneriformis by UHPLC-Q-TOF mass spectrometry. Food Chemistry, 167, 484-489. 498 Matsui, T., Matsufuji, H., Seki, E., Osajima, K., Nakashima, M., & Osajima, Y. (1993). 499 Inhibition of angiotensin I-converting enzyme by Bacillus licheniformis alkaline protease 500 hydrolyzates derived from sardine muscle. Bioscience, Biotechnology, and Biochemistry, 501 57, 922-925. 502 22 Mora, L., & Hayes, M. (2015). Cardioprotective cryptides derived from fish and other food 503 sources: Generation, application, and future markets. Journal of Agricultural and Food 504 Chemistry, 63, 1319-1331. 505 Ono, S., Hosokawa, M., Miyashita, K., & Takahashi, K. (2006). Inhibition properties of 506 dipeptides from salmon muscle hydrolysate on angiotensin I-converting enzyme. 507 International Journal of Food Science and Technology, 41, 383-386. 508 Pripp, A. H., Isaksson, T., Stepaniak, L., & Sørhaug, T. (2004). Quantitative structure-activity 509 relationship modelling of ACE-inhibitory peptides derived from milk proteins. European 510 Food Research and Technology, 219, 579-583. 511 Raghavan, S., Kristinsson, H. G. (2009). ACE-inhibitory activity of tilapia protein 512 hydrolysates. Food Chemistry, 117, 582-588. 513 Roberts, P. R., Burney, J. D., Black, K. W., & Zaloga, G. P. (1999). Effect of chain length on 514 absorption of biologically active peptides from the gastrointestinal tract. Digestion, 60, 515 332-337. 516 Saint-Denis, T., & Goupy, J. (2004). Optimization of a nitrogen analyser based on the Dumas 517 method. Analytica Chimica Acta, 515, 191–198. 518 Shalaby, S. M., Zakora, M., & Otte, J. (2006). Performance of two commonly used 519 angiotensin converting enzyme inhibition assays using FA-PGG and HHL as substrates. 520 Journal of Dairy Research, 73, 178-186. 521 Theodore, A. E., & Kristinsson, H. G. (2007). Angiotensin converting enzyme inhibition of 522 fish protein hydrolysates prepared from alkaline-aided channel catfish protein isolate. 523 Journal of the Science of Food and Agriculture, 87, 2353-2357. 524 Valencia, P., Pinto, M., & Almonacid, S. (2014). Identification of the key mechanisms 525 involved in the hydrolysis of fish protein by alcalase. Process Biochemistry, 49, 258–264. 526 23 Vo, T., Ngo, D., Kim, J., Ryu, B., & Kim, S. (2011). An antihypertensive peptide from tilapia 527 gelatin diminishes free radical formation in murine microglial cells. Journal of 528 Agricultural and Food Chemistry, 59, 12193-12197 529 Yokoyama, K., Chiba, H., & Yoshikawa, M. (1992). Peptide inhibitors for Angiotensin I530 converting enzyme from thermolysin digest of dried bonito. Bioscience, Biotechnology, 531 and Biochemistry, 56, 1541-1545. 532 533 24 FIGURE AND TABLE CAPTIONS 534  Figure 1. Hydrolysis curves for small-spotted catshark 535  Figure 2. (a) ACE-inhibitory activity of the hydrolysates obtained with the enzymatic 536 pattern subtilisin(2h)+trypsin(2h) for the five species studied, (b) ACE-inhibitory 537 activity of small-spotted catshark hydrolysates at different time of hydrolysis 538  Figure 3. SEC profile of hydrolysates obtained with the enzymatic pattern 539 subtilisin(2h)+trypsin(2h): (a) horse mackerel, (b) small-spotted catshark 540  Figure 4. Sequence profile of one of the peptides identified from fraction D of small541 spotted catshark 542  Table 1. Degree of hydrolysis and IC50 value of the final hydrolysates 543  Table 2. Characteristics of the SEC fractions for the selected hydrolysates 544  Table 3. Peptide sequences identified by UHPLC–MS/MS in the most active SEC 545 fractions of horse mackerel and small-spotted catshark 546 547 25 548 Fig. 1. Hydrolysis curves for small-spotted catshark 549 550 551 0 4 8 12 16 20 050 100 150 200 250 DH (%) Time (min) Subtilisin(2h) + Trypsin(2h) Trypsin(2h) + Subtilisin(2h) Subtilisin + Trypsin (simultaneous)