Antioxidant Activity and Amino Acid Profiling of Protein Hydrolysates from the Skin of Sphyraena barracuda and Lepturacanthus savala

Abstract

The antioxidant activity of protein hydrolysates prepared from the skin of Sphyraena barracuda (Seela fish) and Lepturacanthus savala (Ribbon fish), using the commercial enzymes pepsin, trypsin, and papain were determined. The protein hydrolysate showed high antioxidant activity in which trypsin hydrolysate of the skin of both seela and ribbon fish proved good DPPH scavenging activity with 66 and 60% (p < 0.05), respectively. These active hydrolysates were purified using fast protein liquid chromatography on ion exchange and gel filtration chromatography procedure and the active purified fractions were determined using electron spin resonance spectrophotometer against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. Further the purified fractions were analyzed for amino acid composition using high performance liquid chromatography and it consisted of antioxidant inducing amino acids along with a considerable quantity of essential amino acids.

Keywords:

• Antioxidant
• Sphyraena barracuda
• Lepturacanthus savala
• ESR spectrophotometer
• FPLC

INTRODUCTION

Bioactive peptides act as potential physiological modulators of metabolism during intestinal digestion of nutrients.[1] These peptides are liberated depending on their structural, compositional, and sequential properties, and may exhibit various bioactivities, such as antioxidants,[1, 2] antihypertensive,[3, 4] and immunomodulatory effects.[5] These peptides can be released from their precursor proteins by digestive enzymes during gastrointestinal digestion or by in vitro proteolytic processes with exogenous proteases. Bioactive peptides generally contain 3–20 amino acid units, but in some cases this range may be extended.[6] Current, state-of-the-art antioxidants have a major role in maintaining the homeostasis in human health because one-electron reduction of oxygen leads to the formation of reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, and related species.^[1] These radicals are very unstable and react rapidly with other groups or substances in the body, leading to cell or tissue injury. Moreover, free radicals mediate modifications in DNA, proteins, lipids, and small cellular molecules, which are associated with a number of pathological processes, including atherosclerosis, arthritis, diabetes, cataractogenesis, muscular dystrophy, pulmonary dysfunction, inflammatory disorders, ischemiareperfusion tissue damage, and neurological disorders such as Alzheimer's disease.[7]

Lipid oxidation by reactive oxygen species (ROS), is the predominant cause of qualitative decay of foods, which leads to rancidity, toxicity, and destruction of biochemical components that are important in physiologic metabolisms.[2] Especially, lipid peroxidation in foods affects the nutritive value and may cause disease conditions following consumption of potentially toxic reaction products. Antioxidants are used to preserve food products to retard discoloration and deterioration that occur as a result of oxidation. However, the use of synthetic antioxidants, such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and propyl gallate (PG), is under strict regulation because of the potential health hazards.[8] Therefore, during the last few decade researchers focused on an antioxidant derived from a food ingredient that could act against reactive oxygen species. Among various marine bio-resources, antioxidant activities have not been studied adequately in Sphyraena barracuda (Seela)- and Lepturacanthus savala (Ribbon fish)-derived peptides. The current article deals with the purification and characterization of antioxidant peptides derived from the skin of seela and ribbon fish. For this purpose, the extracted fish skin protein was enzymatically hydrolyzed to obtain antioxidant peptides and was assessed for free radical scavenging effects.

MATERIALS AND METHODS

Collection of Fish

Two marine fish, Sphyraena barracuda (Seela fish) and Lepturacanthus savala (Ribbon fish), were collected from the Royapuram sea coast, Bay of Bengal (13° 6′ 26′′ N, 80° 17′ 43′′ E) during October, 2008. The fish were dissected and the skin was collected separately from each fish, wiped with the blotting paper, and weighed. Samples were then cut into small pieces (0.2 × 0.2 cm) and stored at −20°C until used.

Enzymatic Hydrolysis of Fish Skin

The skin of Seela and Ribbon fish were submitted to enzymatic hydrolysis by pepsin, trypsin, and papain. Hydrolysis was carried out in controlled conditions (pH, temperature, enzyme concentration, and stirring speed). Both fish's skin protein were dissolved (4% w/w) in 10 mM glycin-Hcl buffer (pH 2 for pepsin, animal origin, 4.63 AU/g proteins) and 10 mM phosphate buffer (pH 6 for papain, plant origin, 1.0 AU/g protein and pH 8 for trypsin, animal origin, 0.2 AU/g protein) and submitted to enzymatic hydrolysis at 37°C for 6 h with an enzyme/substrate ratio of 1:100 (w:w) at a stirring speed of 200 rpm. The pH of the reaction was kept constant by continuously adding 1 N HCl and NaOH to the reaction medium. Afterwards, the enzyme was inactivated by heating the mixture at 100°C for 10 min and then centrifuged at 3800 rpm for 15 min at 4°C. The supernatants, corresponding to hydrolysates, were freeze-dried and stored at −80°C. The yield of protein can be calculated by:

Antioxidant Assays

DPPH radical scavenging activity was determined according to the method of Yen and Hsieh.[9] An aliquot of the sample (1 mg/ml) was mixed with 3 ml of methanol and then added to 1 ml of 0.15 mM DPPH in methanol. The mixture was then mixed vigorously and allowed to stand at room temperature in the dark for 30 min. The absorbance of the mixture was measured at 517 nm using a UV-Vis spectrophotometer. The control was conducted in the same manner but methanol was used instead of the sample. DPPH radical scavenging activity was calculated as follows:

where the sample is the absorbance of the sample and control is the absorbance of the control at 517 nm. The reducing power assay was as follows: The ability of the hydrolysate to reduce iron (III) was determined according to the method of Yen and Chen.[10] An aliquot of 1 mg/ml sample of each hydrolysate was mixed with 2.5 ml of 0.2 M phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The mixture was incubated at 50°C for 30 min, followed by the addition of 2.5 ml of 10% (w/v) trichloroacetic acid. The mixture was then centrifuged at 1,650× g for 10 min. Finally, 2.5 ml of the supernatant solution was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) ferric chloride. After 10 min, reaction the absorbance of the resulting solution was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

Measurement of the Antioxidant Activity in Linoleic Acid Model System

The antioxidant activity was measured in a linoleic acid model system according to the methods of Osawa and Namiki.[11] Briefly, a sample (1 mg) was dissolved in 10 ml of 50 mM phosphate buffer (pH 7.0) and added to a solution containing 0.13 ml of linoleic acid and 10 ml of 99.5% ethanol. Then the total volume was adjusted to 25 ml with distilled water. The mixture was incubated in a brown bottle at 40 ± 1°C in a dark room and the degree of oxidation was evaluated by measuring the ferric thiocyanate values. The ferric thiocyanate value was measured according to the standard method.[12] The reaction solution (100 μl) incubated in the linoleic acid model system was mixed with 4.7 ml of 75% ethanol, 0.1 ml of 30% ammonium thiocyanate, and 0.1 ml of 2 mM ferrous chloride solution in 3.5% HCl. After 3 min, the thiocyanate value was measured by reading the absorbance at 500 nm following color development with FeCl₂ and thiocyanate at different intervals during the incubation period at 40 ± 1°C.

Purification of the Antioxidant Peptide

Ion exchange chromatography

The lyophilized skin protein hydrolysates of S. barracuda and L. savala (20 mg/ml) was dissolved in 20 mM sodium acetate buffer (pH 4.0) and loaded onto fast protein liquid chromatography (FPLC) on a XK26 DEAE anion exchange column (GE Healthcare, Stockholm, Sweden) equilibrated with 20 mM sodium acetate buffer (pH 4.0) and eluted with a linear gradient of NaCl (0–1.5 M) in the same buffer at a flow rate of 60 ml/h. Each fraction collected at a volume of 5 ml was monitored at 280 nm, pooled fractions were then concentrated using a rotary evaporator; and antioxidant activities were investigated. The fraction having strong antioxidant properties was lyophilized and subjected to next separation.

Gel filtration chromatography

The antioxidant active fractions of both the fish hydrolysates were dissolved in distilled water and loaded onto a Sephadex G-25 gel filtration column (2.5 × 70 cm; GE Healthcare, Stockholm, Sweden), which was previously equilibrated with distilled water. The column was then eluted with the distilled water at a flow rate of 60 ml/h. Each fraction collected at a volume of 2 ml was monitored at 280 nm, pooled fractions were then concentrated using a rotary evaporator; and antioxidant activities were investigated. Fractions showing antioxidant activity were pooled and lyophilized.

Assays conducted using electron spin resonance (ESR) spectroscopy

Hydroxyl radicals were generated by iron-catalyzed Fenton Haber–Weiss reaction and the generated hydroxyl radicals were rapidly reacted with nitrone spin trap DMPO solution.[13] The resultant DMPO-OH adducts was detected with an ESR spectrometer. The peptide solution (0.2 ml) was mixed with DMPO (0.3 M, 20 μl), FeSO4 (10 mM, 20 μl), and H₂O₂ (10 mM, 20 μl) in a phosphate buffer solution (pH 7.4) and then transferred into a 100-μl quartz capillary tube. After 2.5 min, the ESR spectrum was recorded using an ESR spectrometer. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 200 sweep time, 4 min. The DPPH scavenging activity was determined using ESR spectrometer using the methods of Nanjo et al.[14] A 500-μl peptide solution was added to 500 μl of DPPH in an ethanol solution. After mixing vigorously for 10 s, the solution was transferred into a capillary tube, and the scavenging activity of peptide on DPPH radical was measured using an ESR spectrometer. The spin adduct was measured on an ESR spectrometer exactly 2 min later. The experimental conditions employed were as follows: magnetic field, 336.5 ± 5 mT; power, 1 mW; modulation frequency, 9.41 GHz; amplitude, 1 × 1000 sweep time, 2 min.

Amino Acid Composition

The amino acid composition was identified by using the methods of Li et al.[15] with small modifications. The lyophilized hydrolysate fractions were digested with HCl (6 M) at 110°C for 24 h. High performance liquid chromatography (HPLC) analysis was carried out in an Agilent 1100 assembly system (Hewlett-Packard, Boeblingen, Germany) after precolumn derivatization with o-phthaldialdehyde (OPA). Each sample (1 μl) was injected on a Zorbax 80 A C₁₈ column at 40°C with detection at 338 and 262 nm. Mobile phase A was 7.35 mmol/l sodium acetate/triethylamine (500:0.12, v/v), adjusted to pH 7.2 with acetic acid, while mobile phase B (pH 7.2) was 7.35 mmol/l sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed as (w/w%) of protein.

Statistical Analysis

Experimental results were expressed as mean of triplicate ± standard deviation. Statistical analysis of the result was performed using SPSS 10.1 for Windows (SPSS, Chicago, IL, USA).

RESULTS

Fish Protein Hydrolysate

The yield of protein hydrolysate using different proteolytic enzymes is given in Table 1. Although many factors affect the yield of hydrolysis, the type of enzyme used had a marked effect on the yield and properties of the final product.[16] At the same temperature, enzyme concentration, and stirring speed during a 6 h hydrolysis period, the yield of protein recovery of trypsin hydrolysate (55%) was higher than the remaining enzymatic hydrolysates in ribbon fish and pepsin hydrolysate (62%) was recovered better than other hydrolysates of seela fish.

Table 1  The percentage of skin protein hydrolysates obtained after proteolysis

S. No.	Hydrolysate	Seela fish (SF)	Ribbon fish (RF)
1.	Trypsin	18.2 ± 1.3	27.6 ± 1.1
2.	Pepsin	29.1 ± 1.9	23.4 ± 1.6
3.	Papain	20.6 ± 2.1	18.7 ± 0.9
All the results are means of triplicates ± SD, n = 3.

Antioxidant Activity

The antioxidant activities of seela and ribbon fish skin protein hydrolysate were determined through the DPPH radical-scavenging activity and reducing power, with the remarkable percentage of radical scavenging of hydrolysate as shown in Tables 2 and 3. The protein hydrolysate prevented the DPPH radical-scavenging activity and showed a remarkable reducing ability. As shown in Tables 2 and 3, the antioxidant assays have proved that trypsin hydrolysate of seela and ribbon fish were higher active than the remaining hydrolysates.

Table 2  The DPPH radical scavenging activity of skin protein hydrolysates

Conc. (mg/ml)	BHT	Tocopherol	SF Trypsin	SF Pepsin	SF Papain	RF Trypsin	RF Pepsin	RF Papain
0.5	45.9 ± 1.7^a	37.6 ± 3.1^a	34.3 ± 1.8^b	23.3 ± 1.3^b	25.7 ± 0.8^c	33.1 ± 0.7^c	29.3 ± 2.6^d	27.5 ± 1.2^d
1.0	59.4 ± 1.6^a	45.8 ± 2.7^a	41.6 ± 0.9^b	29.3 ± 2.8^b	31.4 ± 1.5^c	40.5 ± 1.6^c	36.8 ± 3.1^d	31.6 ± 1.9^d
1.5	60.2 ± 2.1^a	52.8 ± 1.9^a	50.2 ± 2.7^b	35.3 ± 3.6^b	39.77 ± 3.6^c	49.4 ± 1.1^c	42.0 ± 3.3^d	35.71 ± 2.1^d
2.0	68.5 ± 2.5^a	59.9 ± 2.6^a	59.9 ± 1.7^b	41.5 ± 3.9^b	46.1 ± 3.1^c	55.5 ± 3.5^c	49.3 ± 3.1^d	41.3 ± 2.3^d
2.5	76.2 ± 2.9^a	67.1 ± 3.5^a	66.9 ± 2.4^b	47.0 ± 2.6^b	51.9 ± 4.1^c	60.0 ± 4.2^c	55.1 ± 2.7^d	45.0 ± 3.4^d
All the results are means of triplicates ± SD, n = 3. Different superscripts indicated significant difference (p < 0.05).
BHT: butylated hydroxytoluene; SF: Seela Fish; RF: Ribbon Fish.

Table 3  The reducing power ability of skin protein hydrolysates

Conc. (mg/ml)	BHT	Tocopherol	SF Trypsin	SF Pepsin	SF Papain	RF Trypsin	RF Pepsin	RF Papain
0.5	0.17 ± 0.01	0.13 ± 0.02	0.07 ± 0.01	0.02 ± 0.009	0.09 ± 0.02	0.05 ± 0.02	0.01 ± 0.007	0.03 ± 0.01
1.0	0.23 ± 0.05	0.19 ± 0.06	0.13 ± 0.04	0.08 ± 0.02	0.14 ± 0.01	0.12 ± 0.01	0.06 ± 0.02	0.09 ± 0.04
1.5	0.31 ± 0.06	0.22 ± 0.03	0.21 ± 0.09	0.13 ± 0.01	0.22 ± 0.04	0.19 ± 0.07	0.14 ± 0.01	0.17 ± 0.01
2.0	0.46 ± 0.09	0.31 ± 0.07	0.29 ± 0.01	0.19 ± 0.06	0.27 ± 0.02	0.26 ± 0.01	0.21 ± 0.06	0.23 ± 0.05
2.5	0.51 ± 0.1	0.45 ± 0.01	0.36 ± 0.06	0.24 ± 0.04	0.33 ± 0.01	0.31 ± 0.02	0.27 ± 0.03	0.29 ± 0.02
All the results are means of triplicates ± SD, n = 3.
BHT: Butylated hydroxytoluene; SF: Seela fish; RF: Ribbon fish.

Purification of the Antioxidant Peptide

The lyophilized active protein hydrolysate was dissolved in 20 mM sodium acetate buffer (pH 4.0), and loaded onto a XK26 DEAE anion exchange column with the linear gradient of NaCl (0–1.5 M). Elution peaks were monitored at 280 nm, and each fraction was collected as 4 ml and fractionated into one non-adsorptive portion and two adsorptive portions for seela fish (Fig. 1a) and for ribbon fish two non-adsorptive portions and two adsorptive portions (Fig. 2a) and labeled as a, b, c, d. Each fraction was pooled, lyophilized, and measured for antioxidant activity using an ESR spectrophotometer. The DPPH and hydroxyl radical scavenging potencies were estimated as shown in Table 4. Based on the obtained results, fraction ‘b’ of seela fish and fraction ‘d’ of ribbon fish exhibited the highest antioxidant potential as well as exhibited substantial scavenging potencies on both DPPH and hydroxyl radicals. The lyophilized active fractions were further separated by Sephadex G-25 (Figs. 1 and 2b) where, the ‘b’ fraction of seela fish further separated into four fractions and fraction ‘d’ of ribbon fish into three fractions when equilibrated and eluted with water, the eluted peaks were labeled as 1, 2, 3, 4. The antioxidant activities of the purified peptide of seela (b-3) and ribbon fish (d-1) were investigated concerning both free radical scavenging effects on DPPH and hydroxyl radicals. As shown in Fig. 3, these purified peptides effectively inhibited lipid peroxidation in the linoleic acid emulsion system. The activity of the purified peptide for lipid peroxidation inhibition was higher than that of α-tocopherol, the positive control used in this experiment after 6 days. Tohma and Gulcin[17] reported 80.1% lipid peroxidation of Turkish liquorices in linoleic acid emulsion, which was more than tocopherol (68%).

Table 4  Free radical scavenging activity of FPLC fractions with ESR spectrometer

	Seela fish		Ribbon fish
	DPPH radical	Hydroxyl radical	DPPH radical	Hydroxyl radical
Fraction a	21.0 ± 1.77	17.9 ± 2.21	11.0 ± 1.36	9.6 ± 1.31
Fraction b	59.3 ± 2.19	36.2 ± 2.04	43.3 ± 1.18	16.0 ± 1.60
Fraction c	36.1 ± 1.39	21.8 ± 1.13	25.1 ± 2.19	23.7 ± 2.18
Fraction d	—	—	55.9 ± 3.12	31.5 ± 1.17
Peak 1	35.8 ± 2.19	17.8 ± 1.83	73.4 ± 2.15	49.6 ± 1.85
Peak 2	81.4 ± 1.31	56.3 ± 1.55	59.7 ± 1.89	26.2 ± 1.97
Peak 3	59.7 ± 1.89	31.2 ± 2.90	11.1 ± 1.34	11.3 ± 2.40
Peak 4	11.1 ± 1.34	9.2 ± 1.34	—	—
All the results are means of triplicates ± SD, n = 3.

Table 5  Amino acid composition (% w/w) of the active fractions of purified protein hydrolysates

Amino acid	Seela fish	Ribbon fish
Glycine	1.43	0.60
Histidine	7.55	7.85
Glutamic acid	5.61	4.56
Threonine*	3.47	3.09
Arginine	11.6	13.38
Aspartic acid	1.99	2.79
Valine*	2.81	2.81
Leucine*	3.8	1.57
Alanine	1.25	1.06
Lysine*	12.59	13.51
Serine	2.63	0.94
Tyrosine	9.53	16.09
Phenylalanine*	7.41	15.36
Proline	5.87	5.01
Isoleucine*	9.71	3.01
Methionine*	5.22	4.32
Cysteine	4.96	2.96
Tryptophan*	2.57	1.09
*Essential amino acids.

Amino Acid Composition

To identify the presence of amino acids in the purified peptides, the samples were loaded into HPLC and the results consisted of both essential and non-essential amino acids. Moreover, antioxidant activity of peptide or protein is dependent on molecular size and chemical properties, such as hydrophobicity and electron transferring ability of amino acid residues in the sequence. The purified peptide of both seela and ribbon fish protein hydrolysates were shown in Table 5.

DISCUSSION

DPPH is a stable free radical that shows maximum absorbance at 517 nm in ethanol. When DPPH encounters a proton-donating substance, the radical is scavenged by changing color from purple to yellow and the absorbance is reduced.[18] Trypsin hydrolysates of both the fish had shown comparatively higher than the remaining hydrolysates, whereas the hydrolysate of bullfrog and smooth hound also showed a high scavenging effect of trypsin hydrolysate.[2, 19] For the reducing power assay, the presence of reductants (antioxidants) in tested samples result in reducing Fe3⁺/ferricyanide complex to the ferrous form. The Fe2⁺ can, therefore, be monitored by measuring the formation of Perl's Prussian blue at 700 nm.[20] The results suggested that seela fish and ribbon fish skin protein hydrolysates possibly contained amino acids or peptides, which functioned as electron donors and could react with free radicals to form more stable products. Thus, protein hydrolysate prevented the DPPH radical-scavenging activity and showed a remarkable reducing ability.

Bioactive peptides usually contain 2–20 amino acid residues per molecule,[6] and the lower the molecular weight, the higher their chance to cross the intestinal barrier and exert biological effects.[21] A moderate percentage of alanine (1.25 and 1.06%), valine (2.81 and 2.81%), and leucine (3.8 and 1.57%), which has a non-polar aliphatic group and shows high reactivity to hydrophobic PUFAs, may be the reason for high lipid peroxidation inhibition exhibited by the purified peptides. Also, the presence of tyrosine (9.53 and 16.09%) and histidine (7.55 and 7.85%) can make reactive oxygen species (ROS) stable through direct electron transfer. Moreover, amino acids, such as tyrosine, methionine, histidine, lysine, and tryptophan, were generally accepted as antioxidants.[22] This information seems to be related to the antioxidant activity of the purified fraction of skin protein hydrolysates.

CONCLUSION

Based on the current results, it is suggested that purified peptides from trypsin hydrolysates of the skin of seela and ribbon fish effectively scavenged DPPH and hydroxyl radicals. Moreover, both the fish were potent to show good reducing power and inhibit lipid peroxidation. Further, amino acid composition also proved the presence of histidine, methionine, and tyrosine-like antioxidant amino acids in the active peptide along with other amino acids. Therefore, it could be concluded that peptides derived from both the fish are a good source of nutrients and protect against oxidative damage in living systems in relation to aging and carcinogenesis.

ACKNOWLEDGMENT

The authors gratefully acknowledge Dr. K. Ramasamy, Dean, School of Bioengineering, SRM University, for his support throughout the work, and also the management of SRM University for providing the facilities.