A review of fish-derived antioxidant and antimicrobial peptides: Their production, assessment, and applications

Peptides 33 (2012) 178–185

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Peptides journal homepage: www.elsevier.com/locate/peptides

Review

A review of fish-derived antioxidant and antimicrobial peptides: Their production, assessment, and applications L. Najafian ∗ , A.S. Babji School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

a r t i c l e

i n f o

Article history: Received 17 October 2011 Received in revised form 12 November 2011 Accepted 14 November 2011 Available online 26 November 2011 Keywords: Fish-derived peptides Antioxidant peptides Antimicrobial peptides Protein hydrolysates

a b s t r a c t Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Technological approaches for the production of bioactivepeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Preparation of fish protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Extraction of sarcoplasmic and myofibrillar proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Extraction of gelatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Enzymatic hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Purification and identification bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological properties of bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Measuring the antioxidant activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

178 179 179 179 179 179 180 180 180 181 182 183 184 184

1. Introduction Fish can serve as a source of functional materials, such as polyunsaturated fatty acids, polysaccharides, minerals and vitamins, antioxidants, enzymes and bioactive peptides [63]. Recently, much attention has been focused on the identification and characterization of the structure, composition and, sequence of bioactive peptides. Biologically active peptides play an important role in metabolic regulation and modulation. These peptides can be used

∗ Corresponding author. Tel.: +60 3 89215988; fax: +60 3 89213232. E-mail address: najafian [email protected] (L. Najafian). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.11.013

as functional food ingredients, or nutraceuticals and pharmaceuticals to improve human health and prevent disease. The importance of fish as a source of novel bioactive substances is growing rapidly [3,8]. Bioactive peptides can be produced by one of three methods: solvent extraction, enzymatic hydrolysis and microbial fermentation of food proteins (Fig. 1). The solvent extraction system mainly use at laboratory-scale. This technique has several drawbacks like low selectivity, low extraction efficiency, solvent residue, and environmental pollution [1]. In addition, the proteolytic systems of lactic acid bacteria (LAB) are implicated in the hydrolysis of proteins during fermentation of foods such as milk and meat products [12].

L. Najafian, A.S. Babji / Peptides 33 (2012) 178–185

Solvent extraction

Proteins

Microbial fermentation

Preferred method

Enzymatic hydrolysis Lack of residual Organic solvent

179

The mixture is centrifuged and then incubated at 4 ◦ C for 30 min. The supernatant is filtered through a double-layered cheesecloth. For precipitating the myofibrillar proteins, the pH of this supernatant is adjusted to 5.5 using 2 M HCl. After the last centrifugation, myofibrillar proteins are recovered as sediment. This sediment, which includes myofibrillar proteins, is used for enzyme hydrolysis.

Lack of toxic Chemical

Bioactive peptides Fig. 1. The generation methods of peptides from food proteins.

However, the enzymatic hydrolysis method is preferred in the food and pharmaceutical industries because the other methods can leave residual organic solvents or toxic chemicals in the products. Bioactive peptides are inactive within the sequences of the parent proteins. They are released by enzymatic hydrolysis and then they may exert various physiological functions [45,54,89]. These peptides generally contain 2–20 amino acid units. The amino acid composition and sequences can affect the activity of biopeptides [61]. Fish-derived bioactive peptides based on their structural propertiesand their amino acid composition and sequences, in addition to nutrient utilization, these peptides may be involved in various biological functions [5,17] including inhibition of angiotensin-I-converting enzyme (ACE) [42,65] and antioxidant [69,75,87], immunomodulatory [79], antimicrobial [9,73,93] and anticoagulant [39] activities. With the increasing knowledge of the functional properties of fish protein hydrolysates, there are many researchers are conducting studies on the developments and applications of fish-derived functional foods and nutraceuticals. This present paper focuses on the antioxidative and antimicrobial bioactive peptides derived from fish. In addition, it presents an overview of the issues relevant to their enzymatic production, isolation and characterization. Finally, we discuss their biological activities and potential applications as ingredients in functional foods, nutraceuticals, and pharmaceuticals. 2. Technological approaches for the production of bioactivepeptides 2.1. Preparation of fish protein 2.1.1. Extraction of sarcoplasmic and myofibrillar proteins There are several methods for the extraction of meatsarcoplasmic and myofibrillar proteins [21,34,55]. Minced fish meat can be used for the production of proteins with biological activities. The separation of meat proteins into different protein fractions can be accomplished using various methods. For example, sarcoplasmic proteins can be extracted by diluting minced meat in phosphate buffer, and after homogenizing and centrifugation, the supernatant is filtered and the resulting pellet is used for myofibrillar extraction according to the method described by Sanz et al. [76]. Then, pellet is homogenized after resuspendingin phosphate buffer, containing Triton X-100. After centrifugation, the pellet is washed three times by resuspending it in the same buffer to remove muscle proteases. The resulting pellet is resuspended in nine volumes of phosphate buffer with KI. The suspension is centrifuged and diluted 10 times to prevent the possible inhibition of bacterial proteases by KI. An alkali solubilization method has recently been developed as a new way to extract of myofibrillar proteins. According to this method [34,65], the minced meat is homogenized with 9 parts of cold deionized water, and then the pH of mixture is adjusted to 11.0 using 2 M sodium hydroxide in order to solubilize themyofibrillar proteins.

2.1.2. Extraction of gelatin Although fish filets usually account for less than 50% of the total fish weight, the fish processing industry poorly utilizes other parts such as the viscera, backbone, skin and head [6]. Gelatin is traditionally produced from the bones and skins or bovine by acid or alkaline treatment to give type A or type B gelatins, respectively. Collagen and gelatin are recently used in functional food, and pharmaceutical applications [28]. Gelatin can be extracted from fish skin using various methods [18,44,57]. According to the method described by Montero, the skin is cleaned with 0.8 M sodium hydroxide and water. The skins are then treated with 0.2 M sodium hydroxide and 0.05 M acetic acid. The fish skins are kept in distilled water at 45 ◦ C for 18 h. The literature about fish bone gelatin is somewhat limited [18], but there are also reported methods for the extraction of gelatin from bone [18,57]. Muyonga et al. [57] outlined a method in which the bones are pre-treated by tumbling them in warm (35 ◦ C) water and then demineralized using 3% HCl, at ambient temperature (20–25 ◦ C) until the bones lose their hard cores (9–12 days). The spent solution from deminerization (pre-treated minerals) is covered with warm (∼60 ◦ C) water, and gelatin extracted in water baths for 5 h at different temperatures (50, 60 and 70 ◦ C) and followed by boiling for 5 h [57]. 2.2. Enzymatic hydrolysis Enzymatic hydrolysis of whole protein molecules is the most commonly used method for producing bioactive peptides. The physicochemical conditions of the reaction media, including time, temperature and, pH enzyme/substrate ratio, must be optimized for the activity of the enzyme. The type of enzyme used in enzymatic protein hydrolysis is very important because it dictates the cleavage patterns of the peptide bonds [80]. Many proteases have been used for the generation of bioactive peptides from proteins. Proteinases from animal, plant, and microbial sources have been used for enzymatic protein hydrolysis. Many bioactive peptides have been experimentally generated using various commercial proteases [51,62,80]. Qian et al. [64] used ␣-chymotrypsin, papain, neutrase, and trypsin for the hydrolysis of tuna dark muscle under theoptimal pH and temperature conditions for the respective enzymes (Table 1). Jun et al. [40] investigated protein hydrolysates of yellow fin sole frame protein by alcalase, neutrase, papain, trypsin, pepsin, ␣-chymotrypsin, and pronase E for their antioxidative potential. Ranathunga et al. [70] reported that pepsin hydrolysates, although they had the lowest degree of hydrolysis

Table 1 Optimum conditions for the hydrolysis of tuna dark muscle. Enzyme

Buffer

pH

Temperature (◦ C)

Alcalase ␣-Chymotrypsin Papain Pepsin Neutrase Trypsin

0.1 M PBa 0.1 M PB 0.1 M PB 0.1 M GHBb 0.1 M PB 0.1 M PB

7.0 8.0 6.0 2.0 8.0 8.0

50 37 37 37 50 37

a b

Phosphate buffer. Glycine–HCl buffer.

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and exhibited the highest antioxidative activities, which was possibly due to the smaller size of the peptides produced. After hydrolization, the degree of hydrolysis (DH) in the extracts is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with stacking and resolving gels. Proteins are visualized by staining with Coomassie Brilliant Blue R-250 [25,53].

Atherosclerosis Inflammation

Ischemia: brain, heart

Arthritis Shock

Diabetes Reactive Oxygen Species

Cancer

Frostbite Infection:malaria, entamoeba, AIDS

2.3. Purification and identification bioactive peptides Radiation damage

Enzymatically hydrolyzed fish peptides exhibit different physicochemical properties and biological activities depending on their molecular weight and amino acid sequence. Therefore the molecular weight of the bioactive peptide is one of the most important factors in producing bioactive peptides with the desired biological activities [47,48]. An ultrafiltration membrane system can separate the peptides that have the desired molecular weights and functional properties from fish protein hydrolysates [35,37]. Such a system can also control the molecular weight distribution of the appropriate peptide [15,43]. Small peptides with different bioactivities are concentrated from the higher molecular weight fractions and remaining enzymes using a membrane with a low-molecularmass cutoff such as 500, 1000 or 3000 Da. Nanofiltration, ionexchange membranes, and column chromatography can also be used to [62]. Often the most useful method for peptide separation is HPLC. Commercially available reversed-phase columns allow rapid separation and detection of their hydrophilic and hydrophobic characteristics [80]. Ferreira et al. [23] recommended that peptides with different surface hydrophobicities be separated by reversed-phase columns with a polystyrene–divinylbenzene copolymer-based packing. Choosing the right pore size to achieve optimal separation of peptides is important, as the wrong pore size will result in poor resolution. In addition to pore size, the ligand on the gel also plays an important role in obtaining effective separation. Anather essential factors for effective separation is determining the appropriate hydrophobicity of the gel. The appropriate pore size, hydrophobicity, particle size and column size should be combined to achieve high recovery and resolution in the isolation of peptides and proteins [52]. HPLC is usually used in conjunction with other analyzing equipment including a UV detector or mass spectrometer. Liquid chromatography followed by tandem mass spectrometry detection (LC–MS/MS) is commonly used to identify peptide sequences [60]. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometric analysis is also useful for generating peptide profiles of protein hydrolysates or semipurified fractions. However this method is limited, as peptides with molecular masses below 500 Da are difficult to detect because the ␣-cyano-4-hydroxycinnamic acid matrix molecules used for absorbing energy during the analysis also appear in this region of the chromatogram [24]. In addition, some peptides may not be ionized and “fly” with the matrix molecules and therefore may not be detected. 3. Physiological properties of bioactive peptides 3.1. Antioxidant activity Oxidation is a vital process in aerobic organisms, particularly in vertebrates and humans although it leads to the formation of free radicals. The formation of reactive oxygen species (ROS) including free radicals such as superoxide anion radicals (O2 − ), hydroxyl radicals (OH• ), and non-free radical species such as hydrogen peroxide (H2 O2 ) and singlet oxygen (1 O2 ), is an unavoidable consequence in the body’s normal use of oxygen during respiration [10,26,36]. Oxidation primarily occurs on unsaturated fats by a free

Aging Parkinsonism

Fig. 2. Disease and damage caused by reactive oxygen species: Courtesy of Dr. M.T. Huang.

radical-mediated process. The radicals interact with molecular oxygen to form lipid peroxy radicals. These radicals can abstract a hydrogen atom from adjacent unsaturated fatty acids and produce a hydroperoxide and a new lipid radical, which causes the continuation and acceleration of the chain reaction. Highly reactive molecules generated through these chemical processes are responsible for producing unpleasant and obnoxious odors and flavors in rancid foods and oils. These chemical processes may also destroy nutrients in food. The oxidation of proteins in foods is affected by lipid oxidation because, the products of lipid oxidation react with proteins, which causes their subsequent oxidation [90]. Carbohydrates are less sensitive to oxidation than lipids and proteins [59]. The Formation of ROS has been implicated in many human diseases, including heart disease, stroke, arteriosclerosis, diabetes and cancer (Fig. 2). Hence, lipid peroxidation is a problem not only in the edible oil and the food industry, but also in human health [78]. Antioxidants can interfere with oxidation by donating a hydrogen atom or an electron to radicals formed from unsaturated lipids and can terminate these chain reactions by removing initiators or radical intermediates from the medium either by scavenging singlet oxygen species or through the inactivation of metal catalysts. Thus, antioxidants can increase the stability of food lipids [68]. Antioxidants are widely used in dietary supplements to boost health and reduce the risk of diseases such as cancer and coronary heart disease. In addition, antioxidants have many industrial uses, such as preservatives in food and cosmetics and the prevention of rubber and gasoline degradation. All aerobic organisms have antioxidant defenses, including antioxidant enzymes and antioxidant food constituents, which remove or repair the damaged molecules. Antioxidants can protect the human body from free radicals and ROS effects. They retard the progress of many chronic diseases as well as lipid peroxidation. Moreover, deterioration of some foods has been linked to the oxidation of lipids or rancidity and the formation of undesirable secondary lipid peroxidation products. Therefore, many synthetic antioxidants such as butylatedhydroxytoluene (BHT), butylatedhydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), and propyl gallate (PG) are used in the food and pharmaceutical industries to retard lipid oxidation [10]. However, the use of these synthetic antioxidants, must be strictly controlled due to potential health issues [29]. Hence, it is necessary to identify alternative natural, safe sources of food antioxidants, and the search for natural antioxidants, especially of plant origin, has notably increased in recent years [10]. There is growing interest in the replacement of synthetic antioxidants with natural antioxidants from food sources for their potential health benefits with little or no side effects [7]. Recently, a number of studies have demonstrated that peptides derived from different fish protein hydrolysates act as potential antioxidants. According to the available literature, we list fish with antioxidant peptides, the mane of the organism producing the

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181

Table 2 Antioxidative protein hydrolysates and amino acids derived from fish. Source organism

Enzyme used

Amino acid sequence

References

Sardine (muscle) Mackerel filet Capelin whole fish Conger eel (muscle) Round scad (muscle) Round scad (muscle) Yellow stripe trevally Hydrolysate Royal jelly protein

pepsin Protease N Alcalase® Trypsin Alcalase® 2.4 L and Flavourzyme® 500 L Flavourzyme® Alcalase® 2.4 L and Flavourzyme® 500 L Protease N

[83] [91] [2] [70] [88] [86] [50] [27]

Channel catfish protein isolates Tilapia protein

ProtamexTM Flavourzyme® , Neutrase® , andCryotin-F Alcalase, Pronase E

LQPGQGQQ –a – LGLNGDDVN – – – AL, FK, FR, IR, KF, KL, KY, RY, YD, YY, LDR, KNYP – – GEOGPOGPOGPOGPOG2 GPOGPOGPOGPOG2 RPDFDLEPPY

[46] [41]

HGPLGPL – PLFQDKLAHAK, AEAQKQLR

[56] [58] [74]

Alaska Pollack skin gelatin Yellowfin sole protein (frame) Hoki skin gelatin Ornate threadfin bream (muscle) Pacific hake (muscle) a

Alcalase, Neutrase, Papain, Pepsin, Pronase E, ␣-chymotrypsin Trypsin Pepsin Validase® BNP, Flavourzyme® 500 L

[85] [66]

Amino acid sequence not identified.

peptide, the sequence, and the type of enzyme used for hydrolysis (Table 2). The bioactive antioxidant peptide Leu-Gly-Leu-Asn-Gly-AspAsp-Val-Asn, isolated from conger eel (Conger myriaster), exhibited high levels of antioxidant activity [70]. Nalinanon et al. [58] isolated peptides with antioxidant properties from the muscle of ornate threadfin bream by skipjack tuna pepsin. These authors found that the highest scavenging activities for ABTS and DPPH were obtained whit protein hydrolysates with 20% DH. The inhibition of lipid peroxidation and free radical scavenging by fish bioactive peptides isolated from hoki frame protein has been assayed using an electron spin resonance spin-trapping technique, and its activity was higher than that of ␣-tocopherol. Its amino acid sequence was identified as Glu-Ser-Thr-Val-Pro-Glu-Arg-ThrHis-Pro-Ala-Cys-Pro-Asp-Phe-Asn [49]. Three dipeptides (Lys-Tyr, Arg-Try, and Tyr-Tyr) containing Tyr residues at the C-terminus of royal jelly protein had strong hydroxyl-radical and hydrogenperoxide scavenging activity [27]. In a study by Wu et al. [91] the antioxidant activity of free amino acids and peptides from mackerel (Scomber austriasicus) muscle were investigated. Mackerel meat was hydrolyzed with protease N at a temperature of 50 ◦ C for 0, 5, 10, 15, and 25 h. The levels of free amino acids, anserine and other peptides generated by hydrolysis with protease were much higher than those generated by autolysis. The free amino acids in the hydrolysates and the dipeptides carnosine and anserine were extracted, separated using ion-exchange chromatography and studied during the hydrolysis time points. The antioxidant activity of the hydrolysates was measured in a linoleic acid peroxidation system, the DPPH assay and the reducing power assay. Results showed that the antioxidant activity of the hydrolysates increased from 0 to 10 h, and then it gradually decreased. Three main peptide fractions were separated from the hydrolysate using gel filtration chromatography on a Sephadex G-25 column. Three main fractions of approximately 1400, 900, and 200 Da, were detected and tested for their antioxidant activity. Peptides with a molecular weight of approximately1400 Da possessed stronger in vitro antioxidant activity than that of the 900 and 200 Da peptides [91]. Klompong et al. [50] studied the antioxidant activity and functional properties of yellow stripe trevally (Selaroides leptolepis) muscle hydrolysates by hydrolysis with Flavourzyme® 500 L and Alcalase® 2.4 L. The antioxidant activity of the hydrolysates was determined by the DPPH assay, the metal-chelating activity assay and the reducing power assay. The antioxidant activity of the two hydrolysates was found to depend on the type of enzyme employed

and the DH, in accordance with previous research. The Alcalase® 2.4 L hydrolysates showed high DPPH radical-scavenging activity that decreased with an increase of the DH. No differences in the antioxidant activity were observed in the Flavourzyme® 500 L hydrolysates with different levels of DH. As DH levels increased, so did the metal-chelating activity of both hydrolysates [50]. In a study carried out on Alaska Pollack, two antioxidant peptides were characterized [46]. Gelatin extracted from Alaska Pollackskin was hydrolyzed with Alcalase, Pronase E, and Collagenase using a three-step recycling membrane reactor. The hydrolysates were fractionated on the basis of their molecular weight by ultrafiltration (10, 5, and 1 kDa) using HPLC on a GPC column. The antioxidant activity of the hydrolysates was assessed in a linoleic acid peroxidation system using the TBARS method. The sequences of the two-antioxidant peptides were G-E-Hyp-G-P-Hyp-G-P-HypG-P-Hyp-G-P-Hyp-G and G-P-Hyp-G-P-Hyp-G-P-Hyp-G-P-Hyp-G. although the exact mechanism of action through which the peptides exert their antioxidants effects is not known, some aromatic amino acids as well as histidine have been reported to play a vital role in this process. 3.1.1. Measuring the antioxidant activity The identification and quantification of individual antioxidants present in a desired food complex is important, but separating the antioxidative compounds from these mixtures and studying each one individually is expensive and inefficient. Therefore, it is necessary to have a convenient method to quantify the antioxidative effectiveness of a whole food or partially purified antioxidant peptides. On the basis of the chemical reactions involved, assays to assess antioxidant capacity are classified into two groups: methods based on hydrogen atom transfer (HAT) reactions [32] and methods based on electron transfer (ET) reactions [32]. The HAT-based assays quantify the ability of an antioxidant to donate a hydrogen atom. Most HAT-based assays monitor competitive reaction kinetics, and the quantization is obtained from the kinetic curves. The ET-based assays measure the reducing capacity and often rely on a color change as a readout [32]. The ORAC assay is an example of a HAT-based assay in which an antioxidant and a substrate (fluorescein probe) compete kinetically for hydroxyl radicals [38]. In this assay, as the reaction progresses, the antioxidant compound present limits the decrease in fluorescence, which is a meature of the extent of damage to the fluorescein probe. The inhibition of linoleic acid autoxidation by antioxidants is another example of a HAT reaction. Fig. 3 shows the major steps involved

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Initiation

R2N2 R˙ + O2 ROO˙ + LH

2R˙ + N2 ROO˙ ROOH + L

Propagation L˙ + O2

LOO˙

LOO˙ + LH

LOOH + L˙

LOO˙ + LA

LOOH + A˙

Inhibition

Termination A˙ + A˙

A2 (nonradical products)

LOO˙ + A˙

LOOA ( nonradical products)

R2N2 = azo compound, LH = substrate, AH = antioxidant Fig. 3. Steps of the autoxidation process and the action of antioxidants.

in lipid autoxidation that are initiated by an azo compound, the action of inhibition and termination of antioxidants in the radical generating process. During the assay, the degree of oxidation can be measured by using thiobarbituric acid (TBA) and ferric thiocyanate values. During the incubation period at 40 ± 1 ◦ C, the reaction of FeCl2 and thiocyanate leads to the development of a red color, and the peroxide value (PV) is measure by reading the absorbance at 500 nm. PV assays meature the total peroxide and hydroperoxideoxygen content of lipids. When lipid hydroperoxides are broken down, secondary lipid oxidation products, including malondialdehyde (MDA), hexanal, and other carbonyl compounds, are formed, and these can also be measured by assessing the extent of lipid oxidation in the linoleic acid model system. The thiobarbituric acid reactive substance (TBARS) measures the concentration of MDA, hexanal, and other secondary lipid oxidation products in a sample [4]. The trolox equivalent antioxidant capacity (TEAC) assay is an ET reaction. It is based on the scavenging of 2,2 -azinobis-(3ethylbenzothiazoline-6-sulfonate) radicals (ABTS• ) and converting them into a colorless product through the reaction between ABTS and potassium persulphate. In the presence of an antioxidant, a preformed radical cation is reduced to ABTS, changing the reaction solution from blue to colorless. The extent of the color change at 734 nm as a function of antioxidant concentration is determined and the TEAC value is calculated by measuring the concentration of a sample that gives the same percent inhibition of absorbance of the ABTS radical cation as 1 mM of trolox [71]. This assay can be used in measure the antioxidative capacity of both water-soluble and lipid-soluble pure compounds as well as food extracts. Because the TEAC method is an end-point assay, the reaction rate differences between antioxidants and oxidants are not reflected [32]. The FRAP assay is also based on ET reaction and measures the ability of an antioxidant to reduce a ferric 2,4,6-tripyridyl-s-triazine salt (Fe3+ –TPTZ) to the blue-colored ferrous complex (Fe2+ –TPTZ) at low pH [32]. There is not a significant difference between the TEAC assay and the FRAP assay except the FRAP assay is conducted at a much lower pH (pH 3.6) than the TEAC assay (pH 7.4). In addition to the above mentioned assays, there are other chemical assays, that measure the scavenging capacity of individual ROS, such as superoxide anion, singlet oxygen, hydrogen peroxide, hydroxyl radical, and peroxynitrite [32]. Another method that can be a powerful tool for monitoring radical–antioxidant interactions and measuring the free-radical scavenging capacities of antioxidants is electron spin resonance (ESR) spectrometry. This analytical

method measures the radical-scavenging activity of antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, the superoxide anion radical (O2 ), the hydroxyl radical (OH), or the peroxyl radical (ROO). This trapping technique measures the interaction between unpaired electrons like free radicals and transition metal ions with an applied magnetic field and chemical species [36,77,92]. Dietary antioxidants can also act as a metal ion chelators, depending on their nature. Reductions in the amount of oxidation due to chelation of transition-metalions by antioxidants can be assayed using a spectrophotometric method where in the reaction of the antioxidant with a mixture containing free Fe2+ ions can be determined by formation of a colored complex with ferrozine [19,81]. An antioxidative compound can yield different results depending on the assay system used because of differences in the mechanism of antioxidative action being measured or the reaction conditions used in the various assays. For example, a good ion chelator may not detect any activity in other radical scavenging assays. In addition, the pH of the reaction media and the solubility of the antioxidant in the reaction media also play important roles in determining a compound’s antioxidative capacity. For these reasons, more than one assay is often used to measure the antioxidative capacity of a food, andoxidant-specific terms, such as “peroxyl-radical-scavenging capacity”, “super-oxide-scavenging capacity”, “ferric-ion-reducing capacity,” are more appropriate to describe the results from specific assays than “total antioxidant capacity” [33,74]. Chemical assays for measuring the antioxidative capacity of protein hydrolysates and peptides from fish are shown in Table 3. 3.2. Antimicrobial activity Antimicrobial peptides usually have less than 50 amino acids, of which nearly 50% are hydrophobic and have a molecular weight below 10 kDa. These peptides, can be generated in vitro by enzymatic hydrolysis [13,72,80]. Research regarding the isolation and identification of antimicrobial peptides derived from animal muscle has not been as extensive as for antioxidant peptides from animal muscle. Fish are a major component of the aquatic fauna. Like other organisms, fish exude different types of antimicrobial peptides, which are positively charged short amino-acid-chain molecules involved in host defense mechanisms. Antimicrobial peptides play key roles in native immunity by interacting directly with bacteria and killing them [93]. A wide variety of organisms produce antimicrobial peptides as a primary innate immune strategy. In medicinal applications, antimicrobial peptides are some times preferred to conventional bactericidal antibiotics because they kill bacteria faster and are unaffected by antibiotic-resistance mechanisms [80]. The methods described here will be useful for the identification of novel peptides with good antimicrobial activities. Several methods for testing the antimicrobial activity of hydrolysates or peptides have been used. The agar diffusion assay (or inhibition zone assay) is a common method used to test the antimicrobial activity of peptidichydrolysates and peptides [30]. This method quantifies the ability of antibiotics to inhibit bacterial growth. The agar diffusion technique is usually used for determining the minimum inhibitory concentration (MIC) in solid media. In a MIC assay, different concentrations of antibiotic solutions are placed in cups, wells or paper discs, and either placed on the surface of or punched into agar plates seeded with the test bacterial strain. Antibiotic diffusion from these sources into the agaros medium leads inhibition of bacterial growth in the vicinity of the source and the formation of clear zones without a bacterial lawn. The diameter of these zones increases with antibiotic concentration [11]. A novel polypeptide antimicrobial activity was isolated and characterized from loach (Misgurnus anguillicaudatus) using Sephadex G-50 gel

L. Najafian, A.S. Babji / Peptides 33 (2012) 178–185 Table 3 In vitro chemical assays for measuring the antioxidative capacity of protein hydrolysates and peptides. Source of peptides or hydrolysates

In vitro method(s) used in measuring antioxidative capacity

References

Sardine (muscle)

Superoxide and hydroxyl radical scavenging activity using electron spin resonance (ESR) analysis DPPH assay, reducing power assay, linoleic acid peroxidation system Linoleic acid peroxidation system, hydroxyl-, and carbon-centered radical scavenging activity (ESR) DPPH radical scavenging activity, metal chelation, reducing power DPPH radical scavenging capacity, reducing power, ferrous ion chelation, linoleic acid peroxidation system, lecithin liposome system DPPH radical scavenging capacity, reducing power, ferrous ion chelation Isoluminol enhanced chemiluminescent assay in the presence of a) hydrogen peroxide or b) mononuclear cells isolated from human blood. Ferric reducing antioxidant power (FRAP) assay, TEAC assay Metal chelating ability, DPPH radical scavenging ability, FRAP, ORAC, and the ability to inhibit the formation of thiobarbituric acid reactive substances (TBARS) in washed tilapia muscle containing tilapia hemolysate DPPH radical scavenging capacity, ferric ion reducing antioxidative capacity, TEAC assay, ORAC assay, iron chelation, linoleic acid peroxidation system Linoleic acid peroxidation system

[83]

Mackerel filet Conger eel (muscle) Yellow stripe trevally Round scad (muscle)

Round scad (muscle) Tilapia protein

Channel catfish protein isolates

Pacific hake

Alaska pollack skin gelatin Hoki frame protein

Hoki skin gelatin

Tuna back bone

Skipjack tuna

DPPH, alkyl, hydroxyl-, and superoxide-radical scavenging abilities (ESR) Carbon-centered, and superoxide radical scavenging activity (ESR), DPPH assay (ESR), Linoleic acid peroxidation system DPPH, peroxy- and superoxide radical scavenging (ESR), Linoleic acid peroxidation system DPPH, iron chelation, ABTS radical-scavenging

[91] [70]

[50] [88]

[86] [66]

[85]

[75]

[46] [35]

[56]

[36]

[58]

Adapted from Samaranayaka [74].

filtration, DEAE-52 cellulose ion-exchange chromatography and an improved polyacrylamide gel electrophoresis together with electroelution [20]. Zhang et al. [93] found an antimicrobial component in the skin homogenate of Epinephelus fario using a trypsin digest. The antimicrobial protein was purified by ion-exchange and gelfiltration chromatography. The bacterial growth was monitored by measuring the absorbance of the culture with a microplate reader at 600 nm, and the MIC was determined. MIC was expressed as a range of the highest concentration of Efapat which the bacteria were able to grow and the lowest concentration at which the bacterial growth was completely inhibited. After screening the skin homogenate of E. fario, which showed activity against Gram-positive bacteria (Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio fluvialis, Pasteurella multocida, Escherichia coli, Aeromonas hydrophila and Pesudomonas aeruginosa), the homogenate was treated with trypsin for 90 min at 37 ◦ C, and its antimicrobial activity against E. coli completely stopped. A proteinaceous antibiotic was isolated from the skin homogenate and purified using a DEAE-Sephadex A-50 column (pH 7.8). The purified protein, named Efap, was a 41 kD protein. Zhang

183

reported that Efap has higher antimicrobial activity against Gramnegative bacteria (V. parahaemolyticus, V. alginolyticus, V. fluvialis, P. multocida, and A. hydrophila) than it does against Gram-positive bacteria (S. aureus). Salampessy et al. [73] isolated two antibacterial peptide fractions (fractions 9 and 12) from bromelainhydrolysate derived from leatherjacket (Meuchenia sp.) insoluble muscle proteins. An assay for antimicrobial activity showed that fraction 12 had a MICagainst Bacillus cereus and S. aureus, while fraction 9 only showed some activity against B. cereus. Additional fractionation, on an analytical C-18 column, indicated that many other peptides, such as the cationic antibiotic polymyxin, could account for the high MIC value. Fish antimicrobial peptides can be used as antibacterial, antiviral, antifungal, immunomodulatory, and antitumor agents [67]. Researchers have reported that almost allfish antimicrobial peptides have antibacterial or bacteriostatic functions against several Gram-negative and -positive strains. Su [82] isolated and identified a novel 20-residue antimicrobial peptide, pelteobagrin, from the skin mucus of yellow catfish with the amino acid sequence GKLNLFLSRLEILKLFVGAL. Pelteobagrin exerted broad-spectrum activity against a wide range of bacteria without hemolytic activity. A study on tilapia hepcidin discovered three hepcidin-like antimicrobial peptides (named TH1–5, TH2–2, and TH2–3) [33]. Huang et al. [33] showed that tilapia TH2–3 is similar to the Japanese flounder (Paralichthys olivaceus) 26-amino acid (aa) peptide JF2, tilapia TH2–2 is similar to Japanese flounder 19-aa JF1 [31], and tilapia TH1–5 is similar to sea bream (Chrysophrys major) hepcidin. Falco et al. [22] demonstrated the antiviral activity of the recombinant trout beta-defensin-like peptide in vitro against viral hemorrhagic septicaemia rhabdo virus (VHSV). Piscidin 2, a 22residue cationic peptide isolated from the mast cells of hybrid striped bass, exhibited potent antifungal activity against human pathogenic fungi [84]. Cleavage of histone H2A from the skin mucus of catfish generated a 19-residue antimicrobial peptide, parasin I, that had immunomodulatory properties [16]. Hepcidin TH1–5, an antimicrobial peptide, synthesized from tilapia, showed antitumor activity against several tumor cell lines. It was shown that TH1–5 inhibited the proliferation of tumor cells and reduced colony formation in a soft agar assay [14]. Shabeena and Nazeer [77] tested the purified fraction for cell cytotoxicity on Vero and Hep G2 cell lines. It did not have a cytotoxic effect on Vero cells but exerted a significant antiproliferative effect on Hep G2 cells. Thus, it should be possible to exploit antimicrobial peptides with desirable characteristics in the near future. These findings draw attention to the fact that these peptides have useful antimicrobial properties that can be used in the food industry as well as in the pharmaceutical industry.

4. Conclusions Recent studies have shown that fish-derived bioactive peptides play a vital role in human health and nutrition, and they can be a part of the human diet for several years. More studies should be conducted to further explore the physiological effects of these peptides in humans. Fish-derived bioactive peptides with antioxidative properties may have great potential for use as nutraceuticals and pharmaceuticals and, and they may be a better substitute for synthetic antioxidants. In addition, antimicrobial peptides from fish will provide a new source for the development of measurement novel antimicrobial drugs in the future. These antimicrobial peptides can serve as vaccines in the future to inactive specific pathogens, and they can also be used in food preservatives, and supplements. It is important to discover new antimicrobial substances because of the rise of pathogenic bacteria that are resistant to conventional antibiotics. Based on the evidence

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