Antioxidant and angiotensin I-converting enzyme inhibitory activities of northern shrimp (Pandalus borealis) by-products hydrolysate by enzymatic hydrolysis

P. borealis was purchased from the market of Yang yang-gun, Gangwon-do, Korea, in May, 2012. Alcalase® 2.4 L, papain, trypsin, serine, o-phthaldialdehyde (OPA), pyrogallol, 2, 2′-azino-bis [3-ethylbenzothiazoline-6-sulfonic acid] (ABTS⁺), dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), potassium ferricyanide (K₃Fe(CN)₆), trichloroacetic acid (TCA), hippuryl-l-histidyl-l-leucine (HHL), angiotensin I-converting enzyme (ACE), sodium borate, sodium chloride, hydrochloric acid, pyridine, benzene sulfonyl chloride (BSC), captopril, and l-ascorbic acid were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and iron(III) chloride (FeCl₃) was obtained from Junsei Chemical Co. (Tokyo, Japan). Protamex and flavourzyme were purchased from Novo Co. (Novozyme Laboratories, Copenhagen, Denmark).

The by-products of P. borealis (PBB) were lyophilized and stored at −20 °C until use. The crude protein content of PBB was 44.50 ± 0.35 % by AOAC method (AOAC, 2000) and enzymatic hydrolysis of PBB was performed using five enzymes, alcalase, papain, trypsin, protamex, and flavourzyme, under their optimal conditions (Table 1). A 100-g sample (on the basis of protein weight) and 1 % enzyme were mixed and then the mixture was incubated for 8 h at each optimal temperature with stirring. After incubation, the mixture was heated at 100 °C to inactivate the enzyme. The inactivated mixture was centrifuged at 2000×g for 20 min. After centrifugation, the supernatant was lyophilized and stored at −20 °C until use.

The protamex hydrolysate (38 g) was dissolved in 50 mL deionized water and filtered by two ultrafiltration membranes (Amicon Ultra-filter devices; Millipore, Billerica, MA, USA) with 3 and 10 kDa molecular weight cut-offs (< 3 kDa, 3–10 kDa, and > 10 kDa). The soluble fractions were prepared by centrifuging at 3000×g for 20 min and passed through the membrane sequentially, beginning with the largest molecular weight cut-off membrane cartridge (10 kDa). The retentate and permeate were collected separately, and the retentate was recirculated into the feed until the maximum permeate yield was reached. Permeate from the 10 kDa membrane was then filtered through the 3 kDa membrane with recirculation until the maximum permeate yield was reached.

The DH of enzymatic hydrolysates of PBB was calculated by determining free amino groups with OPA (Nielsen 2001).

The total amino acids composition was determined using an amino acid analyzer (S43000; Sykam, Eresing, Germany). Samples were hydrolyzed in hydrochloric acid (6 N) in vacuum-sealed tubes at 110 °C for 24 h.

Tryptophan is measured after alkaline hydrolysis (Sato et al. 1984). Samples (9–10 mg, 0.1 mg precision) were dissolved in 10 mL of 4.2 N sodium hydroxide and hydrolysed in an oven at 110 °C for 20 h. The hydrolysates were filtered using filter paper (ADVANTEC No. 5B) and mass up 50 mL by 0.2 N sodium citrate buffer (pH 4.2). The 1–5 mL of solution was dried in a waterbath at 70 °C. The pH of the hydrolysates was adjusted to 4.2 with 6 N HCl and 0.2 N sodium citrate buffer and mass up 25 mL. The solutions were filtered through 0.2 μm membrane filter and analyzed by amino acid analyzer (Sykam 4300, Sykam, Germany).

The ABTS⁺ radical scavenging activity was determined using the method of Roberta et al. (1999). The ABTS solution was diluted with water to achieve an absorbance of 0.75 ± 0.03 at 734 nm. Then, 180 μL of ABTS solution was added to 20 μL of different concentrations of samples. The mixture was incubated in the dark for 10 min and measured the absorbance by spectrophotometer (BIO-TEK US/MQX 200, USA) at 734 nm. l-ascorbic acid was used as a positive control and the ABTS⁺ radical scavenging activity of each sample was expressed as IC₅₀ value.

The SOD-like activity of the sample was evaluated according to the procedure of Marklund and Marklund (1974) with a slight modification. One hundred microliters of sample solutions were mixed with 100 μL of pyrogallol (7.2 mM) and 100 μL of 50 mM Tris-HCl buffer at pH 8.5 containing 0.2 mM EDTA. After 10 min, 50 μL of 1 N HCl was added to the mixture to stop the reaction and measured the absorbance at 420 nm. l-ascorbic acid was used as a positive control, and the SOD-like activity of the PBB was expressed as IC₅₀ value.

The reducing power of shrimp shell extracts were measured by the method of Oyaizu (1986). Different concentrations of samples in 10 % DMSO were mixed with 50 μL of 0.2 M sodium phosphate buffer (pH 6.6) and 50 μL of potassium ferricyanide (10 mg/mL). The mixtures were incubated at 50 °C for 20 min. Then, 50 μL of TCA (100 mg/mL) was added and centrifuged at 2000×g for 10 min. After centrifugation, 100 μL of the supernatant was mixed with 20 μL of iron(III) chloride (1 mg/mL) and the mixture was measured at 700 nm. Reducing power was expressed as the 0.5 of absorbance (EC₅₀) and l-ascorbic acid was used as a positive control.

The inhibitory activity of ACE was monitored according to the method of Li et al. (2005). A 20 μL of sample, 50 μL of 5 mM HHL, and 100 mM of sodium borate buffer (pH 8.3) containing 300 mM NaCl were pre-incubated at 37 °C for 5 min. The reaction was initiated by the addition of 10 μL of ACE solution (100 mU/mL), and the mixture was incubated at 37 °C for 30 min. The reaction was stopped by adding 100 μL of 1 M HCl, and then sodium borate buffer (320 μL), pyridine (600 μL), and BSC (200 μL) were added to the reaction mixture. After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 492 nm and the captopril was used as a positive control.

The data were analyzed using analysis of variance through the general linear model procedure (SAS Institute, Cary, NC, USA). Duncan’s multiple range test was applied to determine the significance of the differences between means (P < 0.05).

Recently, the enzymatic hydrolysates have been studied and utilized as nutraceutical resources. In particular, the interest of many researchers on the enzymatic hydrolysates derived from fish and shrimp processing by-products and the various biological activities such as antioxidant, antibacterial, antiobesity, and antihypertensive activities is growing (Sila et al. 2015; Benoit et al. 2008; Cancre et al. 1999).

In the present study, PBB was hydrolyzed by five specific enzymes including alcalase, papain, trypsin, protamex, and flavourzyme for 8 h, respectively, and DH values of their hydrolysates were given in Table 2. The cleavage of peptide bonds by protease leads to decomposition of protein tertiary structure and reduction of the molecular weight of proteins (Adler-Nissen 1986). This reaction also increases in the concentration of free amino and carboxyl groups and its functional properties of proteins (Kristinsson and Rasco 2000). The DH values of PBB hydrolysates were as follows: protamex (59.85 ± 0.09 %) > papain (58.86 ± 0.08 %) > trypsin (58.31 ± 0.08 %) > alcalase (55.96 ± 0.04 %) > flavourzyme (55.68 ± 0.08 %). Among the enzymatic hydrolysates, protamex hydrolysate of PBB showed the highest DH value.

Total amino acid composition of the three MWCO fractions of protamex hydrolysate was presented in Table 3. As shown in Table 3, the total amino acid contents of three MWCO fractions were 47.44 g/100 g (< 3 kDa), 48.17 g/100 g (3–10 kDa), and 47.48 g/100 g (>10 kDa), respectively. The total amino acid compositions of three MWCO fractions were similar to each other. All MWCO fractions were rich in Glu, Gly, Asp, Ala, Leu, and Lys, while these fractions contained low levels of Cys and Trp.

Reactive oxygen species (ROS), containing superoxide (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (OH⁻), and singlet oxygen (O₂^·), can cause the oxidative damage to the important components such as protein, lipid, nucleic acids and have been associated with the occurrence of various diseases including hypertension (Ngo et al. 2011a, b).

The antioxidant activities of the five PBB enzymatic hydrolysates were measured by the scavenging activity on ABTS⁺ radicals, SOD-like activity, and reducing power (Table 4). The alcalase and protamex hydrolysates of PBB exhibited noticeable ABTS⁺ radical scavenging activity with IC₅₀ value of 0.16 ± 0.02 and 0.17 ± 0.00 mg/mL, respectively. However, all hydrolysates showed lower ABTS⁺ radical scavenging activity than that of l-ascorbic acid.

SOD, an important antioxidant defense enzyme, catalyzes the dismutation of the superoxide anion (O₂⁻) into oxygen (O₂) and hydrogen peroxide (H₂O₂). The SOD-like activity is widely used for assay to measure the inhibition of pyrogallol autoxidation. The results of SOD-like activity of the five enzymatic hydrolysates were shown in Table 4. The protamex hydrolysate exerted the greatest SOD-like activity (2.04 ± 0.15 mg/mL) among hydrolysates, while the alcalase hydrolysate exhibited moderate activity.

The reducing power is the ability to donate an electron or hydrogen (Dorman et al. 2003). The electron donation capacity of the five PBB enzymatic hydrolysates were evaluated and showed in Table 4. All the PBB hydrolysates exhibited moderate reducing power.

The antioxidant activities of the three MWCO fractions of protamex hydrolysate were evaluated and showed in Table 5. The < 3 kDa and 3–10 kDa fractions showed potent ABTS⁺ radical scavenging activity (IC₅₀ = 0.22 ± 0.01 mg/mL and 0.22 ± 0.00 mg/mL), while the > 10 kDa fraction was exhibiting stronger reducing power with EC₅₀ value of 7.04 ± 0.83 mg/mL than those of the < 3 kDa and 3–10 kDa fractions. However, the three MWCO fractions showed not high antioxidant activities as much as protamex enzymatic hydrolysate.

The inhibition of ACE, a key enzyme regulating the blood pressure, has been recognized as the most effective therapy for the treatment of hypertension. However, many synthetic ACE inhibitors including captopril, enalapril, alacepril, fosinopril, and lisinopril cause side effects such as cough, taste alterations, skin rashes, and angioneurotic edema (Alderman 1996; Cicoira et al. 2001; Vyssoulis et al. 2001). Therefore, it is necessary to develop safe and effective ACE inhibitors from natural products.

The ACE inhibitory activity of the enzymatic hydrolysates of PBB was shown in Table 4. Among the five PBB hydrolysates, the protamex hydrolysate exhibited the most potent ACE inhibitory activity with IC₅₀ value of 0.08 ± 0.00 mg/mL, followed by flavourzyme (IC₅₀ = 0.11 ± 0.00 mg/mL) > alcalase (IC₅₀ = 0.11 ± 0.01 mg/mL) > papain (IC₅₀ = 0.11 ± 0.00 mg/mL) > trypsin (IC₅₀ = 0.13 ± 0.00 mg/mL).

The ACE inhibitory activity of the three MWCO fractions of protamex hydrolysate was measured and shown in Table 5. Among the MWCO fractions, the > 3 kDa fraction including 3–10 kDa and > 10 kDa fractions showed significant ACE inhibitory activity with IC₅₀ value of 0.03 ± 0.00 mg/mL. This result also indicated that high molecular weight fraction, 3–10 kDa and > 10 kDa, exhibited potent ACE inhibitory activity compared with protamex enzymatic hydrolysate.

Recently, many researchers have reported that various bioactive peptides derived from fisheries processing by-products including crab shell (Yoon et al. 2013), Pacific cod skin (Ngo et al. 2011a, b), squid skin and muscle (Mendis et al. 2005; Rajapakse et al. 2005), and tuna (Qian et al. 2007; Je et al. 2005; Lee et al. 2010). In the present study, PBB protamex hydrolysate showed notable ABTS⁺ radical scavenging and ACE inhibitory activities. In addition, its MWCO fractions exerted potent ACE inhibitory activities. Therefore, more detailed investigations are necessary to isolate and identify the peptides from active enzymatic hydrolysate and to clarify the mechanism of active peptides.