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In the present study, we investigated to the antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities of the northern shrimp (Pandalus borealis) by-products (PBB) hydrolysates prepared by enzymatic hydrolysis. The antioxidant and ACE inhibitory activities of five enzymatic hydrolysates (alcalase, protamex, flavourzyme, papain, and trypsin) of PBB were evaluated by the 2, 2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS ) radical scavenging and superoxide dismutase (SOD)-like activities, reducing power and Li’smethod for ACE inhibitory activity. Of these PBB hydrolysates, the protamex hydrolysate exhibited the most potent ACE inhibitory activity with IC value of 0.08 ± 0.00 mg/mL. The PBB protamex hydrolysate was fractionated by two ultrafiltration membranes with 3 and 10 kDa (below 3 kDa, between 3 and 10 kDa, and above 10 kDa). These three fractions were evaluated for the total amino acids composition, antioxidant, and ACE inhibitory activities. Among these fractions, the < 3 kDa and 3–10 kDa fractions showed more potent ABTS radical scavenging activity than that of > 10 kDa fraction, while the > 10 kDa fraction exhibited the significant reducing power than others. In addition, 3–10 kDa and > 10 kDa fractions showed the significant ACE inhibitory activity. These results suggested that the high molecular weight enzymatichydrolysate derivedfromPBB couldbeusedfor control oxidative stress and prevent hypertension. Keywords: Northern shrimp, Enzymatic hydrolysate, Antioxidant, Angiotensin I-converting enzyme Abbreviations: ACE, Angiotensin I converting enzyme; PBB, Pandalus borealis by-products; ABTS ,2,2'-Azino- bis[3-ethylbenzothiazoline-6-sulfonic acid]; SOD, Superoxide dismutase; RAS, Renin angiotensin system; OPA, o-phthaldialdehyde; DMSO, Dimethyl sulfoxide; EDTA, Ethylenediaminetetraacetic acid; K Fe(CN) , Potassium 3 6 ferricyanide; TCA, Trichloroacetic acid; HHL, Hippuryl-L-histidyl-L-leucine; BSC, Benzene sulfonyl chloride; FeCl , Iron (III) chloride; MWCO, Molecular weight cut-offs; ROS, Reactive oxygen species Background main active component of hypertension (Paul et al. 2006; Hypertension is one of the primary causes of cardiovascu- Takahashi et al. 2011). The increased angiotensin II causes lar disease which leads to stroke, coronary artery disease, vasoconstriction along with increased blood volume and and sudden cardiac death (Bhuyan and Mugesh 2011). water retention (Parish and Miller 1992). Several ACE in- The renin-angiotensin system (RAS) plays a key role in hibitors have been tested and developed in order to inhibit regulating blood volume and hypertension (Hall 1991; angiotensin II-mediated hypertension. Most synthetic Hall et al. 1989). Angiotensin I-converting enzyme (ACE) ACE inhibitors such as captopril, enalapril, lisinopril, is important in the functioning of the RAS. Renin converts ramipril, and benzapril on the market have been reported angiotensinogen to angiotensin I, and ACE catalyzes with their side effects including skin rash, loss of taste, cleavage of angiotensin I into angiotensin II which is the and dry cough (Dr and Lisa 2012). Moreover, a recent re- search has found that oxidative stress is a principal factor * Correspondence: whasoo@korea.kr for hypertension (Bagatini et al. 2011). Excess reactive Food Safety and Processing Research Division, National Institute of Fisheries oxygen species affects cellular functions and reduces the Science, Busan 46083, South Korea © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kim et al. Fisheries and Aquatic Sciences (2016) 19:29 Page 2 of 6 bioavailability of endothelial nitric oxide and enhances Table 1 The proximate compositions including moisture, crude fat, ash, and crude protein of PBB low-density lipoprotein oxidation in the vascular system (Ray et al. 2012; Toeroek 2008; Mattson 2009). Therefore, Moisture (%) Crude fat (%) Ash (%) Crude protein (%) there is a necessity for the development of new ACE in- PBB 21.07 ± 0.22 12.19 ± 0.12 17.18 ± 0.64 44.50 ± 0.35 hibitors with potent oxidative stress inhibition and fewer Protamex 7.05 ± 0.21 1.12 ± 0.15 18.35 ± 0.21 73.92 ± 0.95 side effects from natural resources. < 3 K 14.04 ± 0.13 4.66 ± 0.21 15.85 ± 0.18 62.07 ± 3.23 Northern shrimp (Pandalus borealis), one of the most 3–10 K 7.26 ± 0.17 2.27 ± 0.25 11.75 ± 0.26 69.31 ± 2.11 popular shrimp species in Korea, belongs to family > 10 K 5.48 ± 0.22 2.30 ± 0.17 12.69 ± 0.31 74.02 ± 2.63 Pandalidae and distributes widely in the deep sea at The proximate composition of PBB was measured by AOAC methods depths of 20–1330 m with a temperature of 2–14 °C found in the water around the eastern coast of Korea (Bauer 2004). This is rich in nutrients such as proteins, each optimal temperature with stirring. After incubation, minerals, and vitamins. However, the inedible parts of the mixture was heated at 100 °C to inactivate the enzyme. shrimp by-products including head, shell, and tail portions The inactivated mixture was centrifuged at 2000×g for account for approximately 50 % of the catch and constitute 20 min. After centrifugation, the supernatant was lyophi- valuable and useful bioactive materials, such as caroteno- lized and stored at −20 °C until use. protein, pigments, chitin, and chitosan (Chakrabarti 2002; The protamex hydrolysate (38 g) was dissolved in Babu et al. 2008; Younes et al. 2015). Recently, much 50 mL deionized water and filtered by two ultrafiltration research has been carried out on the utilization of protein- membranes (Amicon Ultra-filter devices; Millipore, Bil- rich fisheries by-products as nutraceuticals and nutritional lerica, MA, USA) with 3 and 10 kDa molecular weight supplements with high nutrient (Chae et al. 1998; Guerard cut-offs (< 3 kDa, 3–10 kDa, and > 10 kDa). The soluble et al. 2001; Arvanitoyannis and Kassaveti 2008). fractions were prepared by centrifuging at 3000×g for The aim of the present study was to determine antioxi- 20 min and passed through the membrane sequentially, dant and ACE inhibitory activities of enzymatic hydroly- beginning with the largest molecular weight cut-off mem- sates and its molecular weight cut-off fractions of the brane cartridge (10 kDa). The retentate and permeate P. borealis by-products for prevention of hypertension. were collected separately, and the retentate was recircu- lated into the feed until the maximum permeate yield was Methods reached. Permeate from the 10 kDa membrane was then Materials filtered through the 3 kDa membrane with recirculation P. borealis was purchased from the market of Yang until the maximum permeate yield was reached. yang-gun, Gangwon-do, Korea, in May, 2012. Alcalase® 2.4 L, papain, trypsin, serine, o-phthaldialdehyde (OPA), Degree of hydrolysis (DH) pyrogallol, 2, 2′-azino-bis [3-ethylbenzothiazoline-6-sul- The DH of enzymatic hydrolysates of PBB was cal- fonic acid] (ABTS ), dimethyl sulfoxide (DMSO), ethylene- culated by determining free amino groups with OPA diaminetetraacetic acid (EDTA), potassium ferricyanide (Nielsen 2001). (K Fe(CN) ), trichloroacetic acid (TCA), hippuryl-L-histi- 3 6 dyl-L-leucine (HHL), angiotensin I-converting enzyme DH ¼ h = h 100 tot (ACE), sodium borate, sodium chloride, hydrochloric acid, pyridine, benzene sulfonyl chloride (BSC), captopril, and where h is the total number of peptide bonds per pro- tot L-ascorbic acid were purchased from Sigma-Aldrich Chem- tein equivalent, and h is the number of hydrolyzed bonds. ical Co.(St.Louis,MO, USA) and iron(III) chloride (FeCl ) The factor h is dependent on the amino acids compos- 3 tot was obtained from Junsei Chemical Co. (Tokyo, Japan). ition of the raw material (Adler-Nissen 1986). Protamex and flavourzyme were purchased from Novo Co. (Novozyme Laboratories, Copenhagen, Denmark). Total amino acids contents The total amino acids composition was determined Enzymatic hydrolysis and fractionation of shrimp by-product using an amino acid analyzer (S43000; Sykam, Eresing, The by-products of P. borealis (PBB) were lyophilized and Germany). Samples were hydrolyzed in hydrochloric acid stored at −20 °C until use. The crude protein content of (6 N) in vacuum-sealed tubes at 110 °C for 24 h. PBB was 44.50 ± 0.35 % by AOAC method (AOAC, 2000) Tryptophan is measured after alkaline hydrolysis (Sato and enzymatic hydrolysis of PBB was performed using five et al. 1984). Samples (9–10 mg, 0.1 mg precision) were enzymes, alcalase, papain, trypsin, protamex, and flavour- dissolved in 10 mL of 4.2 N sodium hydroxide and hy- zyme, under their optimal conditions (Table 1). A 100-g drolysed in an oven at 110 °C for 20 h. The hydrolysates sample (on the basis of protein weight) and 1 % enzyme were filtered using filter paper (ADVANTEC No. 5B) were mixed and then the mixture was incubated for 8 h at and mass up 50 mL by 0.2 N sodium citrate buffer (pH Kim et al. Fisheries and Aquatic Sciences (2016) 19:29 Page 3 of 6 4.2). The 1–5 mL of solution was dried in a waterbath at by the addition of 10 μL of ACE solution (100 mU/mL), 70 °C. The pH of the hydrolysates was adjusted to 4.2 and the mixture was incubated at 37 °C for 30 min. The with 6 N HCl and 0.2 N sodium citrate buffer and mass reaction was stopped by adding 100 μL of 1 M HCl, and up 25 mL. The solutions were filtered through 0.2 μm then sodium borate buffer (320 μL), pyridine (600 μL), membrane filter and analyzed by amino acid analyzer and BSC (200 μL) were added to the reaction mixture. (Sykam 4300, Sykam, Germany). After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 492 nm ABTS radical scavenging activity and the captopril was used as a positive control. The ABTS radical scavenging activity was determined using the method of Roberta et al. (1999). The ABTS so- Statistical analysis lution was diluted with water to achieve an absorbance The data were analyzed using analysis of variance of 0.75 ± 0.03 at 734 nm. Then, 180 μL of ABTS solution through the general linear model procedure (SAS Insti- was added to 20 μL of different concentrations of sam- tute, Cary, NC, USA). Duncan’s multiple range test was ples. The mixture was incubated in the dark for 10 min applied to determine the significance of the differences and measured the absorbance by spectrophotometer between means (P < 0.05). (BIO-TEK US/MQX 200, USA) at 734 nm. L-ascorbic acid was used as a positive control and the ABTS rad- Results and discussion ical scavenging activity of each sample was expressed as Enzymatic hydrolysis and fractionation IC value. Recently, the enzymatic hydrolysates have been studied and utilized as nutraceutical resources. In particular, Superoxide dismutase (SOD)-like activity the interest of many researchers on the enzymatic hy- The SOD-like activity of the sample was evaluated ac- drolysates derived from fish and shrimp processing by- cording to the procedure of Marklund and Marklund products and the various biological activities such as (1974) with a slight modification. One hundred microli- antioxidant, antibacterial, antiobesity, and antihyperten- ters of sample solutions were mixed with 100 μLof sive activities is growing (Sila et al. 2015; Benoit et al. pyrogallol (7.2 mM) and 100 μLof50mMTris-HCl 2008; Cancre et al. 1999). buffer at pH 8.5 containing 0.2 mM EDTA. After In the present study, PBB was hydrolyzed by five spe- 10 min, 50 μL of 1 N HCl was added to the mixture to cific enzymes including alcalase, papain, trypsin, prota- stop the reaction and measured the absorbance at mex, and flavourzyme for 8 h, respectively, and DH 420 nm. L-ascorbic acid was used as a positive control, values of their hydrolysates were given in Table 2. The and the SOD-like activity of the PBB was expressed as cleavage of peptide bonds by protease leads to decom- IC value. position of protein tertiary structure and reduction of the molecular weight of proteins (Adler-Nissen 1986). Reducing power This reaction also increases in the concentration of free The reducing power of shrimp shell extracts were amino and carboxyl groups and its functional proper- measured by the method of Oyaizu (1986). Different ties of proteins (Kristinsson and Rasco 2000). The DH concentrations of samples in 10 % DMSO were mixed values of PBB hydrolysates were as follows: protamex with 50 μL of 0.2 M sodium phosphate buffer (pH 6.6) (59.85 ± 0.09 %) > papain (58.86 ± 0.08 %) > trypsin and 50 μL of potassium ferricyanide (10 mg/mL). The (58.31 ± 0.08 %) > alcalase (55.96 ± 0.04 %) > flavour- mixtures were incubated at 50 °C for 20 min. Then, zyme (55.68 ± 0.08 %). Among the enzymatic hydroly- 50 μL of TCA (100 mg/mL) was added and centrifuged sates, protamex hydrolysate of PBB showed the highest at 2000×g for 10 min. After centrifugation, 100 μLof DH value. the supernatant was mixed with 20 μL of iron(III) Table 2 The conditions of enzymatic hydrolysis and degree of chloride (1 mg/mL) and the mixture was measured at hydrolysis of the enzymatic hydrolysates of PBB 700 nm. Reducing power was expressed as the 0.5 of Enzyme Crude protein (%) pH Temperature (°C) DH (%) absorbance (EC )and L-ascorbic acid was used as a 1,d Alcalase 70.57 ± 2.05 8.0 50 55.96 ± 0.04 positive control. Protamex 73.92 ± 0.95 8.0 45 59.85 ± 0.09 ACE inhibitory activity Flavourzyme 67.40 ± 0.16 7.0 50 55.68 ± 0.08 The inhibitory activity of ACE was monitored according b Papain 69.45 ± 1.33 6.0 37 58.86 ± 0.08 to the method of Li et al. (2005). A 20 μL of sample, Trypsin 66.62 ± 0.66 8.0 37 58.31 ± 0.08 50 μL of 5 mM HHL, and 100 mM of sodium borate Enzymatic hydrolysates were obtained from 8 h under the optical conditions. buffer (pH 8.3) containing 300 mM NaCl were pre- 1 Means within the same row with different superscripts are significantly incubated at 37 °C for 5 min. The reaction was initiated different by Duncan’s multiple range test (P < 0.05) Kim et al. Fisheries and Aquatic Sciences (2016) 19:29 Page 4 of 6 Table 3 Total amino acids composition for the molecular Antioxidant activity weight cut-off fractions of protamex hydrolysate (g/100 g) Reactive oxygen species (ROS), containing superoxide − − Amino acids < 3 kDa 3–10 kDa > 10 kDa (O ), hydrogen peroxide (H O ), hydroxyl radical (OH ), 2 2 2 and singlet oxygen (O ), can cause the oxidative damage Asp 3.66 4.77 3.65 to the important components such as protein, lipid, nu- Thr 1.96 2.21 1.94 cleic acids and have been associated with the occurrence Ser 1.91 2.18 1.10 of various diseases including hypertension (Ngo et al. Glu 6.14 7.16 6.11 2011a, b). Pro 2.95 0.34 3.22 The antioxidant activities of the five PBB enzymatic Gly 5.06 4.72 5.15 hydrolysates were measured by the scavenging activity on ABTS radicals, SOD-like activity, and reducing Ala 4.44 4.10 4.43 power (Table 4). The alcalase and protamex hydroly- Cys 0.08 0.18 0.17 sates of PBB exhibited noticeable ABTS radical Val 2.53 2.62 2.11 scavenging activity with IC value of 0.16 ± 0.02 and Met 1.44 1.24 1.74 0.17 ± 0.00 mg/mL, respectively. However, all hydro- Ile 2.21 2.54 1.88 lysates showed lower ABTS radical scavenging ac- Leu 3.80 3.68 3.73 tivity than that of L-ascorbic acid. SOD, an important antioxidant defense enzyme, cata- Tyr 1.51 1.43 1.50 lyzes the dismutation of the superoxide anion (O ) into Phe 2.24 2.27 2.25 oxygen (O ) and hydrogen peroxide (H O ). The SOD- 2 2 2 His 1.29 1.73 1.24 like activity is widely used for assay to measure the in- Lys 3.06 3.49 3.89 hibition of pyrogallol autoxidation. The results of SOD- Arg 2.94 3.36 3.07 like activity of the five enzymatic hydrolysates were Trp 0.22 0.15 0.30 shown in Table 4. The protamex hydrolysate exerted the greatest SOD-like activity (2.04 ± 0.15 mg/mL) among Total 47.44 48.17 47.48 hydrolysates, while the alcalase hydrolysate exhibited moderate activity. Total amino acids contents The reducing power is the ability to donate an Total amino acid composition of the three MWCO frac- electron or hydrogen (Dorman et al. 2003). The elec- tions of protamex hydrolysate was presented in Table 3. tron donation capacity of the five PBB enzymatic hy- As shown in Table 3, the total amino acid contents of drolysates were evaluated and showed in Table 4. All three MWCO fractions were 47.44 g/100 g (< 3 kDa), the PBB hydrolysates exhibited moderate reducing 48.17 g/100 g (3–10 kDa), and 47.48 g/100 g (>10 kDa), power. respectively. The total amino acid compositions of three The antioxidant activities of the three MWCO frac- MWCO fractions were similar to each other. All tions of protamex hydrolysate were evaluated and MWCO fractions were rich in Glu, Gly, Asp, Ala, Leu, showed in Table 5. The < 3 kDa and 3–10 kDa frac- and Lys, while these fractions contained low levels of tions showed potent ABTS radical scavenging activity Cys and Trp. (IC = 0.22 ± 0.01 mg/mL and 0.22 ± 0.00 mg/mL), Table 4 Antioxidant and ACE inhibitory activities for the enzymatic hydrolysates of PBB Sample ABTS scavenging activity SOD-like activity Reducing power ACE inhibitory activity 1 2 IC (mg/mL) IC (mg/mL) EC (mg/mL) IC (mg/mL) 50 50 50 50 3, c a a b Alcalase 0.16 ± 0.02 2.82 ± 0.72 9.42 ± 0.82 0.11 ± 0.01 b, c a b c Protamex 0.17 ± 0.00 2.04 ± 0.15 6.75 ± 0.94 0.08 ± 0.00 a, b a c b Flavourzyme 0.20 ± 0.00 3.13 ± 0.51 4.63 ± 0.15 0.11 ± 0.00 a a c b Papain 0.21 ± 0.03 2.59 ± 0.48 4.46 ± 0.31 0.11 ± 0.01 a a b a Trypsin 0.22 ± 0.02 2.44 ± 0.13 6.62 ± 0.06 0.13 ± 0.00 A d b d L-Ascorbic acid 0.004 ± 0.000 0.02 ± 0.00 0.04 ± 0.00 B d Captopril 0.00002 ± 0.00000 1 + IC (50 % inhibitory concentration) values of ABTS scavenging, SOD-like, and ACE inhibitory activities were expressed as a mean ± SD The reducing power was expressed as an EC (concentration of the 0.5 absorbance) value Means within the same row with different superscripts are significantly different by Duncan’s multiple range test (P < 0.05) A + L-ascorbic acid was used as a positive control of ABTS radical scavenging and SOD-like activities and reducing power Captopril was used as a positive control of ACE inhibitory activity Kim et al. Fisheries and Aquatic Sciences (2016) 19:29 Page 5 of 6 Table 5 Antioxidant and ACE inhibitory activities for the molecular weight cut-off fractions of the protamex hydrolysate Sample ABTS scavenging activity SOD-like activity Reducing power ACE inhibitory activity 1 2 IC (mg/mL) IC (mg/mL) EC (mg/mL) IC (mg/mL) 50 50 50 50 3, b a a < 3 K 0.22 ± 0.01 >10 20.14 ± 0.39 0.06 ± 0.00 b b b 3–10 K 0.22 ± 0.00 >10 13.84 ± 0.16 0.03 ± 0.00 a c b > 10 K 0.24 ± 0.01 >10 7.04 ± 0.83 0.03 ± 0.00 A c d d L-Ascorbic acid 0.005 ± 0.000 0.07 ± 0.00 0.04 ± 0.00 B c Captopril 0.00001 ± 0.0000 Antioxidant and ACE inhibitory activities were measured using the molecular weight cut-off fractions of the protamex hydrolysate 1 + IC values of ABTS scavenging, SOD-like, and ACE inhibitory activities were expressed as a mean ± SD The reducing power was expressed as an EC value Means within the same row with different superscripts are significantly different by Duncan’s multiple range test (P < 0.05) A + L-ascorbic acid was used as a positive control of ABTS radical scavenging and SOD-like activities, and reducing power Captopril was used as a positive control of ACE inhibitory activity while the > 10 kDa fraction was exhibiting stronger re- skin and muscle (Mendis et al. 2005; Rajapakse et al. ducing power with EC value of 7.04 ± 0.83 mg/mL 2005), and tuna (Qian et al. 2007; Je et al. 2005; Lee than those of the < 3 kDa and 3–10 kDa fractions. et al. 2010). In the present study, PBB protamex hy- However, the three MWCO fractions showed not high drolysate showed notable ABTS radical scavenging antioxidant activities as much as protamex enzymatic and ACE inhibitory activities. In addition, its MWCO hydrolysate. fractions exerted potent ACE inhibitory activities. Therefore, more detailed investigations are necessary ACE inhibitory activity to isolate and identify the peptides from active en- The inhibition of ACE, a key enzyme regulating the zymatic hydrolysate and to clarify the mechanism of blood pressure, has been recognized as the most effect- active peptides. ive therapy for the treatment of hypertension. However, many synthetic ACE inhibitors including captopril, Conclusions enalapril, alacepril, fosinopril, and lisinopril cause side In this study, the five enzymatic hydrolysates of PBB de- effects such as cough, taste alterations, skin rashes, and rived from fisheries processing by-products were investi- angioneurotic edema (Alderman 1996; Cicoira et al. gated on the antioxidant and ACE inhibitory activities. 2001; Vyssoulis et al. 2001). Therefore, it is necessary The PBB Protamex hydrolysate which showed the most to develop safe and effective ACE inhibitors from nat- potent ACE inhibitory activity than other fractions was ural products. fractionated as the below 3 kDa, between 3 and 10 kDa, The ACE inhibitory activity of the enzymatic hy- and above 10 kDa fractions respectively to isolate the ac- drolysates of PBB was shown in Table 4. Among the tive materials. But these fractions showed lower antioxi- five PBB hydrolysates, the protamex hydrolysate ex- dant activities than those of enzymatic hydrolysate, while hibited the most potent ACE inhibitory activity with these fractions showed the significant ACE inhibitory IC value of 0.08 ± 0.00 mg/mL, followed by flavour- 50 activity. In addition, 3-10 kDa and >10 kDa fractions zyme (IC = 0.11 ± 0.00 mg/mL) > alcalase (IC = 50 50 showed the better ACE inhibitory activity than <3 kDa 0.11 ± 0.01 mg/mL) > papain (IC = 0.11 ± 0.00 mg/mL) > 50 fraction. These results suggested that the high molecular trypsin (IC = 0.13 ± 0.00 mg/mL). 50 weight enzymatic hydrolysate derived from PBB could The ACE inhibitory activity of the three MWCO frac- be used for prevent hypertension. tions of protamex hydrolysate was measured and shown Acknowledgements in Table 5. Among the MWCO fractions, the > 3 kDa This work was supported by a grant from the National Institute of Fisheries fraction including 3–10 kDa and > 10 kDa fractions Science (NIFS), (R2016064/RP-2016-EC-002). showed significant ACE inhibitory activity with IC value of 0.03 ± 0.00 mg/mL. This result also indicated that Authors’ contributions SB carried out the anti-oxidant and ACE inhibitory activities assay. NY high molecular weight fraction, 3–10 kDa and > 10 kDa, performed the enzymatic hydrolysis and fractionation. KB analyzed the total exhibited potent ACE inhibitory activity compared with amino acids composition. CW participated in the design of the study and protamex enzymatic hydrolysate. performed the statistical analysis. All authors read and approved the final manuscript. Recently, many researchers have reported that vari- ous bioactive peptides derived from fisheries process- Competing interests ing by-products including crab shell (Yoon et al. 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Fisheries and Aquatic Sciences – Springer Journals
Published: Sep 9, 2016
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