Journal of Functional Foods 21 (2016) 10–26

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Antioxidant peptides from marine by-products:

Isolation, identification and application in food
systems. A review

Assaad Sila a,b, Ali Bougatef a,c,*

a
Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Route de Soukra,
3038 Sfax, Tunisia
b
Institut Charles Viollette, équipe ProBioGEM, Polytech’Lille, 59655 Villeneuve d’Ascq Cedex, France
c
Institut Supérieur de Biotechnologie de Sfax, Université de Sfax, Route Sokra Km 4, BP. 1175, 3038 Sfax,
Tunisia

A R T I C L E I N F O A B S T R A C T

Article history: In recent years, several attempts have been made for the utilisation of the protein rich fish
Received 4 April 2015 processing by-product discards and underutilised fish proteins for the production of com-
Received in revised form 27 October mercially valuable food ingredients. There has been an increasing interest in the utilisation
2015 of marine products, and novel bioprocessing technologies are developing for isolation of
Accepted 3 November 2015 some bioactive substances. Antioxidant peptides isolated from marine food products have
Available online 8 December 2015 been used as functional foods and nutraceuticals. Peptides obtained by enzymatic hydro-
lysis of fish proteins exhibit not only nutritional but also biological properties for use in diet
Keywords: or in therapeutic purposes. In this review, we have focused on the enzymatic process for
Antioxidant peptides generating antioxidant peptides from marine by-products as well as on the isolation pro-
Degree of hydrolysis cedures of selected antioxidant peptides.
Identification © 2015 Elsevier Ltd. All rights reserved.
Fish by-products
Marine organisms
Proteases

Contents

1. Introduction ........................................................................................................................................................................................ 11
2. Seafood products and by-products proteins ................................................................................................................................. 11
3. Production of antioxidant peptides by enzymatic hydrolysis .................................................................................................... 13
3.1. Proteases used for marine protein by-products hydrolysis ............................................................................................. 13
3.2. Influence of the degree of hydrolysis on the antioxidant activity .................................................................................. 13
3.3. In vitro tests for antioxidant activity evaluation ................................................................................................................ 14
3.4. In vivo evaluation of antioxidant activity ............................................................................................................................ 18
4. Isolation of antioxidant peptides .................................................................................................................................................... 18
4.1. Purification of antioxidant peptides by chromatographic methods ............................................................................... 18

* Corresponding author. Unité Enzymes et Bioconversion, Ecole Nationale d’Ingénieurs de Sfax, Université de Sfax, Route de Soukra,
3038 Sfax, Tunisia. Tel.: +216 74 674 354; fax: +216 74 674 364.
E-mail addresses: ali.bougatef79@gmail.com; ali.bougatef@isbs.rnu.tn (A. Bougatef).
http://dx.doi.org/10.1016/j.jff.2015.11.007
1756-4646/© 2015 Elsevier Ltd. All rights reserved.
 Journal of Functional Foods 21 (2016) 10–26 11

4.2. Identification of antioxidant peptides ................................................................................................................................. 20

4.3. Structure–activity relationship of antioxidative peptides ................................................................................................ 20
5. Antioxidant peptides in food systems ........................................................................................................................................... 21
6. Conclusion .......................................................................................................................................................................................... 21
Acknowledgement ............................................................................................................................................................................. 22
References ........................................................................................................................................................................................... 22

associated with bioactive peptides encrypted within protein

1. Introduction
Free radical-mediated lipid oxidation, oxidative stress and
antioxidants are widely discussed in many current research
areas. Uncontrolled generation of free radicals that attack
membrane lipids, proteins and DNA is believed to be in-
volved in many health disorders such as diabetes mellitus,
cancer, neurodegenerative and inflammatory diseases
(Butterfield et al., 2002; Pryor & Ann, 1982). In addition,
deterioration of some foods has been identified to be caused
by the oxidation of lipids and formation of secondary lipid
peroxidation products.
Many synthetic antioxidants, such as butylated hydroxy-
anisole (BHA) and butylated hydroxytoluene (BHT), are used
as food additives to prevent deterioration. Although these syn-
thetic antioxidants show stronger antioxidant activities than
those of natural antioxidants, such as α-tocopherol and ascor-
bic acid, the use of these chemical compounds has begun to
be restricted because of their induction of DNA damage and
their toxicity (Ito et al., 1986). Therefore, in recent years, there
is a great interest in finding new and safe antioxidant com-
pounds from natural sources for their use in foods and
medicinal materials to replace synthetic antioxidants. Vitamin
C, α-tocopherol and phenolic compounds, which are natu-
rally present in vegetables, fruits and seeds, possess the ability
to reduce oxidative damage associated with many diseases,
including cancer, cardiovascular diseases, atherosclerosis,
among others (McCall & Frei, 1999).
Different extracts from plants (Afnani & Manaf, 2011; Cui
et al., 2013; Ke et al., 2013; Parejo et al., 2003; Polydoro et al.,
2004), microorganisms (Benedetti et al., 2004; Gantar, Simovic,
Djilas, Gonzalez, & Miksovska, 2012; Mohamed, Osman, Salem,
& Elmalawany, 2014), and foods (Bougatef et al., 2009, 2010;
Saiga, Tanabe, & Nishimura, 2003; Suetsuna, Ukeda, & Ochi,
2000) have been already reported to exhibit antioxidant prop-
erties through their capacity to inhibit lipid peroxidation.
Furthermore, dietary proteins have been found to play a sig-
nificant role in improving human health beyond their well
recognised nutritional value (Hartmann & Meisel, 2007). In ad-
dition, several studies in the past few decades have reported
that protein hydrolysates from various food sources, in addi-
tion to their nutritional properties, exhibited various biological
functions including antioxidant (Bougatef et al., 2009, 2010,
2012), antimicrobial (Salampessy, Phillips, Seneweera, &
Kailasapathy, 2010; Sila et al., 2014a, 2014b), hypotensive (Balti
et al., 2012, 2015; Balti, Nedjar-Arroume, Bougatef, Guillochon,
& Nasri, 2010), anticoagulant (Ren et al., 2014), and cholesterol-
lowering ability (Ben Khaled et al., 2012). These functions are
structure.
Bioactive peptides, which consist of 2 to 20 amino acid resi-
dues, are inactive in the sequence of their parent proteins and
can be released by enzymatic hydrolysis either during gastro-
intestinal digestion in the body or during food processing (e.g.,
cheese ripening and milk fermentation). Once they are liber-
ated in the body, bioactive peptides may act as regulatory
compounds with hormone-like activity. Further, bioactive pep-
tides may also be generated by in vitro hydrolysis of protein
sources using appropriate proteolytic enzymes. The nature of
the protein substrate, the specificity of the enzyme used for
the proteolysis, the conditions used during hydrolysis (time and
temperature) as well as enzyme/substrate ratio greatly influ-
enced the molecular weight and amino acid composition of
bioactive peptides, and thus their biological activities (Van der
Ven, Gruppen, de Bont, & Voragen, 2002).
Antioxidant peptides are among the most studied bioactive
peptides. In particular, fish protein hydrolysates (FPH) with an-
tioxidant properties have become a topic of great interest for
pharmaceutical, health foods, as well as for food processing/
preservation industries (Alasalvar, Shahidi, & Quantick, 2002;
Bougatef et al., 2009, 2010, 2012; Hagen & Sandnes, 2004). The
bioactive molecules in FPH responsible for these properties are
peptides that are released upon hydrolysis of fish proteins by
the enzymes already present in fish mince (endogenous) and/
or by appropriate enzyme(s) added to the fish mince
(exogenous).
This review describes the use of fish by-products as sources
for the production of antioxidant peptides, and points to chro-
matographic methods used for their purification and
identification. Additionally, the structure–activity relation-
ship of antioxidant peptides was presented.
2. Seafood products and by-products proteins
Oceans constitute an extremely diversified alimentary rich-
ness (algae, crustaceans, shellfish, molluscs and fish). According
to the Food and Agricultural Organization (FAO, 2010), more than
145.1 million tons of fish are actually caught or farmed annu-
ally worldwide. In 2010, the quantity of global releases was
estimated at 24 million tons, i.e., about 16.54% of the total
catches. Commercial fish production and seafood processing
generate large amounts of fish waste, which create burden-
some disposal problems and environmental concerns. This
biowaste contains, however, several biomass materials that can
be biotechnologically exploited for the production of useful mar-
ketable products.
12 Journal of Functional Foods 21 (2016) 10–26

Table 1 – Marine by-products used for antioxidant peptides preparation.

By-products Species References
Viscera Black Pomfret Jai Ganesh, Nazeer, and Sampath kumar (2011)
(Parastromateus niger)
Horse mackerel (Magalaspis cordyla) Sampath Kumar et al. (2011)
Heads Bluefin leatherjacket (Navodon septentrionalis) Chi et al. (2015a)
Sardinelle (Sardinella aurita) Barkia, Bougatef, Khaled, and Nasri (2010)
Tuna (Thunnus thynnus) Bougatef et al. (2012)
Heads and viscera Sardinelle (S. aurita) Bougatef et al. (2010)
Skin Bluefin leatherjacket (Navodon septentrionalis) Chi et al. (2015b)
Alaska pollock Guo et al. (2013)
Alaska pollack (Theraga chalcogramma) Kim et al. (2001)
Jumbo skid (Dosidiuc gigas) Mendis et al. (2005)
Giant kingfish (Caranx ignobilis) Nazeer and Anila Kulandai (2012)
Tilapia Yang, Liang, Chow, and Siebert (2009)
Tilapia (Oreochromis niloticus) Zhuang and Sun (2011)
Blue shark (Prionace glauca) Rodrıguez-Dıaz, Kurozawa, Netto, and
Hubinger (2011)
Skin and viscera Cuttlefish (Sepia officinalis) Ktari et al. (2013)
Viscera and carcass Nile Tilapia (Oreochromis niloticus) Silva, Ribeiro, Silva, Cahú, and Bezerra (2014)
— Tilapia Zhang, Duan, and Zhuang (2012)
Frames Yellow fin sole (Limanda aspera) Jun et al. (2004)
Hoki (Johnius belengrii) Kim et al. (2007)
Alaska pollack (T. chalcogramma) Je et al. (2005)
Backbones Tuna Je et al. (2007)
Cod (Gadus morhua) Slizyte et al. (2009)
Dark muscle Tuna Saidi et al. (2014)
Tuna (Thunnus obesus) Hsu (2010)
Tuna (Thunnus obesus) Je et al. (2008)
Frame, dark, muscle, cutoffs, viscera, silver carp (Hypophthalmichthys molitrix) Zhong et al. (2011)
skin, scales, small bones and fins
Pectoral fin Salmon Ahn et al. (2014)
Heads, cephalothorax, shells and appendix Shrimp (Parapenaeus longirostris) Sila et al. (2014c)

Nowadays, an integrated and sustainable exploitation of fish-
eries resources is obligatory as only 50% of the catch is used
for human consumption. Fish by-products (viscera, heads,
frames, cut-offs, bone, skin) (Table 1) contain both valuable lipid
and protein fractions as well as other interesting and valu-
able compounds.
Seafood products can serve as a source of functional ma-
terials, such as protein (Cahú et al., 2012; Sila, Nasri, & Bougatef,
2012; Sila et al., 2014c), chitin (Cahú et al., 2012; Sila, Mlaik,
Sayari, Balti, & Bougatef, 2014d), carotenoids (Cahú et al., 2012;
Sila, Ayed-Ajmi, Sayari, Nasri, & Bougatef, 2013; Sila et al., 2012),
glycosaminoglycans (Abdelmalek et al., 2015; Cahú et al., 2012;
Krichen et al., 2015), gelatin (Balti et al., 2011; Sila et al., 2015a),
polyunsaturated fatty acids, minerals and vitamins, antioxi-
dants, enzymes (Sila, Haddar, Sayari, Nasri, & Bougatef, 2015b)
and bioactive peptides (Khora, 2013).
Food proteins have long been recognised for their nutri-
tional and functional properties. The nutritional properties of
proteins are associated with their amino acid content in con-
junction with the physiological utilisation of specific amino
acids upon digestion and absorption (Friedman, 1996; Korhonen
& Pihlanto, 2006). On the other hand, the functional proper-
ties of proteins relate to their contribution to the physiochemical
and sensory properties of foods (Vercruysse, van Camp, &
Smagghe, 2005).
Research on marine proteins (Kim & Mendis, 2006;
Thorkelsson, Slizyte, Gildberg, & Kristinsson, 2009; Underland
et al., 2009) has shown that they have bioactive properties that
make them a very interesting alternative for the food indus-
try. In general, the crude protein content of raw fish flesh ranges
between 17 and 22% (w/w). In crustaceans and molluscs, protein
levels can vary from 7 to 23% (w/w) (Murray & Burt, 2001).
Several studies have shown that the crude protein content of
fish by-products varies from 8 to 35% (Roslana, Faezah,
Abdullahb, & Mustapa, 2014). It was reported that fish are a
sources of protein rich in essential amino acids, micro and
macroelements (calcium, phosphorus, fluorine, iodine), fats that
are valuable sources of energy, fat-soluble vitamins, and un-
saturated fatty acids that, among other benefits, have a
hypocholesterolic effect (Ismail, 2005). The nutritive value of
fish proteins is comparatively high because of the favourable
essential amino acid pattern. Fish proteins are rich in all the
essential amino acids (particularly methionine and lysine), in
contrast with most proteins from plant sources, which lack ad-
equate amounts of one or more essential amino acids (Sakaara
& Regenstein, 1990). Sidhu (2003) reported, additionally, that
the nutritional benefits of fish consumption are related to the
exploitation of its protein of high biological quality and the pro-
vision of valuable mineral compounds and vitamins.
Seafood products and by-products represent a vast re-
source for the mining of bioactive peptides. Given their high
structural diversity, marine proteins contain in their primary
structure novel biofunctional peptides that can be used in food
and pharmaceutical applications (Harnedy & FitzGerald, 2014).
 Journal of Functional Foods 21 (2016) 10–26
3. Production of antioxidant peptides by
enzymatic hydrolysis
Solvent extraction, chemical treatment, enzymatic hydroly-
sis and microbial fermentation of food proteins can be used
for bioactive peptides production (Alasalvar, Shahidi, Miyashita,
& Wanasundara, 2010).
Although several protein hydrolysates have been pro-
duced from plant and animal sources using exogenous
proteases, few studies have been conducted on the genera-
tion of biologically active peptides using microbial fermentation.
Proteolytic systems of lactic acid bacteria are used in the hy-
drolysis of proteins during fermentation of foods such as milk
and meat products (Brink & Huis in’t Veld, 1992). These prop-
erties can be attributed to the release of bioactive peptides from
protein substrates by secreted microbial proteases. Rajapakse,
Mendis, Byun, and Kim (2005) purified and identified a radical-
scavenging peptide from fermented mussel sauce. In the same
context, Kleekayai et al. (2015) studied the extraction of anti-
oxidant peptides from Thai traditional fermented shrimp pastes.
However, the enzymatic hydrolysis method is preferred in
the food and pharmaceutical industries because the other
methods can leave residual organic solvents or toxic chemi-
cals in the final products.
3.1. Proteases used for marine protein
by-products hydrolysis
The specificity of the enzyme used for the proteolysis is one
of the most important factors for the production of antioxi-
dant peptides. In fact, treatment of protein substrate with
different proteases, which displayed various spectra of sub-
strate specificity, produces several types of protein hydrolysates.
These hydrolysates exhibited, to a various extent, antioxi-
dant activities against various antioxidant systems in vitro. The
differences between the antioxidant activities of the diverse
hydrolysates are probably due to the fact that peptides are dif-
ferent in terms of chain length and amino acid sequence, and
due to the diversity of mechanisms of antioxidant action. The
process that can be used for the production, purification and
identification of antioxidant peptides from fish waste is shown
in Fig. 1.
Different enzymes have been used for the production of
protein hydrolysates. The use of exogenous enzymes is pre-
ferred in most cases over the autolytic process, due to the
shorter time required to obtain similar degree of hydrolysis (DH)
as well as better control of the hydrolysis to obtain more con-
sistent molecular weight profiles and peptide composition.
Industrial food-grade proteinases such as Alcalase ® ,
Flavourzyme®, and Protamex® derived from microorganisms,
as well as enzymes from plants (e.g. papain) and animal sources
(e.g., pepsin, chymotrypsin and trypsin), have been widely used
for the production of antioxidative peptides.
Proteases such as pepsin, chymotrypsin and trypsin are fre-
quently used to produce antioxidant peptides from marine by-
products (Bougatef et al., 2009; Sampath Kumar, Nazeer, &
Jaiganesh, 2011). Microbial alkaline proteases are also utilised
in the production of antioxidants peptides from marine pro-
teins such as tuna (Thunnus thynnus) heads (Bougatef et al., 2012),
13
tuna backbones (Je, Qian, Byun, & Kim, 2007), sardinelle
(Sardinella aurita) heads and viscera (Bougatef et al., 2010) and
Johnius belengerii frame (Kim, Je, & Kim, 2007). For example,
Bougatef et al. (2010) used alcalase, crude enzyme prepara-
tions from Bacillus licheniformis NH1 and Aspergillus clavatus ES1,
and crude enzyme extract from sardine viscera to produce an-
tioxidant peptides from sardinelle by-products. The results
showed that hydrolysates obtained with visceral serine pro-
teases had the highest antioxidant activity (DPPH radical-
scavenging activity (53.76 ± 1.2% at 2 mg/ml)), indicating that
these proteases are more suitable for marine protein diges-
tion and production of antioxidant peptides. However, higher
DH (11%) obtained with NH1 proteases did not guaranteed a
higher antioxidant activity. This may be due to the fact that
B. licheniformis NH1 extract contains multiple proteases, which
offer the ability to achieve higher DH of sardinella heads and
viscera proteins. Nevertheless, the use of endogenous enzymes
from fish viscera or invertebrate has also been reported in the
literature, especially for the production of various antioxidative
FPH. Bougatef et al. (2009) reported the use of pepsin, alka-
line protease, trypsin-like protease and crude enzyme extract
from Mustelus mustelus intestine for the production of
antioxidative hydrolysates. In the same context, Je, Park, and
Kim (2005) used the crude proteinase from mackerel intes-
tine for antioxidant peptides production.
Ktari et al. (2013) investigated the antioxidant activities of
eight hydrolysates from cuttlefish by-products obtained by treat-
ment with various gastrointestinal proteases (chymotrypsin,
trypsin, and crude alkaline enzyme extracts from cuttlefish and
sardinelle) and bacterial proteases (Alcalase and crude enzymes
from Bacillus pumilus A1, Bacillus mojavensis A21, and Bacillus
cereus BG1). They showed that all hydrolysates showed differ-
ent degrees of hydrolysis (DH) and varying degrees of
antioxidant activity.
Since antioxidant peptides typically consist of 2 to 20 amino
acids, in some cases, specific enzyme combinations, which con-
tained multiple proteases, could be used to enhance protein
hydrolysis and to obtain hydrolysates enriched with low-
molecular-weight peptides and acceptable antioxidant activity.
3.2. Influence of the degree of hydrolysis on the
antioxidant activity
The conditions employed for the enzymatic hydrolysis, in-
cluding process time, temperature and pH for the optimal
activity of the enzyme, as well as enzyme/substrate ratio, greatly
influence the degree of hydrolysis (DH) of the protein sub-
strate. DH affects the size and the amino acid composition of
peptides, which could modulate their biological activity.
The hydrolysis of protein, which is measured in terms of
DH, is an important parameter for the determination of the
functional and biological properties of protein hydrolysate
preparations (Kristinsson & Rasco, 2000). It has been demon-
strated that the antioxidant activities of proteins can be
increased through hydrolysis with certain enzymes, and some
peptides or fractions possess stronger antioxidant potential than
others (Chen, Muramoto, & Yamauchi, 1995). Therefore, DH
should be controlled to obtain reproducible bioactive pep-
tides. In this context, Ktari et al. (2013) reported that antioxidant
14 Journal of Functional Foods 21 (2016) 10–26

Seafood
Endogenous proteases Products

By-products
Stomachs
Skin
Frames
Proteases extraction Viscera Heads Backbone
Intestines Scales

Cooking Exogenous proteases

Mixing
Base

pH adjustment

Enzymatic hydrolysis

Termination of enzymatic
hydrolysis

Centrifugation

Non hydrolysed Supernatant

residue
FPH

Freeze-drying

Gel filtration
Purification RP-HPLC
Ion exchange chromatography
Ultrafiltration membrane

Identification Electrospray mass spectrometry (ESI-MS)

Tandem mass spectrometry (ESI-MS/MS)

Antioxidant
peptides

Fig. 1 – Typical diagram for the production process of antioxidant peptides from marine by-products.

activity in hydrolysates produced from cuttlefish by-products
were positively correlated with the increase of DH.
Bougatef et al. (2010) studied the effect of the DH on the
evolution of antioxidative properties of protein hydrolysates
from sardinelle by-products. In that work, it was reported that
hydrolysates with 6% DH exhibited the highest DPPH (1,1-
diphényl-2-pycrilhdrazil) radical-scavenging activity (87 ± 2.1%).
Same results were reported by Bougatef et al. (2012) when
protein by-products from tuna (T. thynnus) heads were used as
raw materials for antioxidant protein hydrolysates produc-
tion. Wu, Chen, and Shiau (2003) indicated that changes in the
levels and composition of free amino acids and small pep-
tides during hydrolysis were associated with antioxidant
activities of mackerel hydrolysate. Likewise, Kelfala-Foh,
Amadou, Foh, Kamara, and Xia (2010) investigated function-
ality and antioxidant properties of tilapia (Oreochromis niloticus)
skin as influenced by the degree of hydrolysis. Authors re-
ported that different DH led to different peptide chain lengths
that greatly influenced the antioxidant activities of the
hydrolysates.
3.3. In vitro tests for antioxidant activity evaluation
Specific assays have not yet been developed or standardised
to measure the antioxidative capacity of peptides or peptide
mixtures. Therefore, assays that are commonly used for mea-
suring antioxidative capacity of non-peptidic antioxidants have
been used in the literature to measure the antioxidative ca-
pacity of peptides as well (Table 2). In vitro assays based on
chemical reactions are widely used in quantifying antioxidative
effectiveness of whole food, partially purified peptides, and/
or individual peptides. Several studies have indicated that
peptides derived from fish proteins have antioxidative prop-
erties in different oxidative systems (Jeon, Byun, & Kim, 1999;
Rajapakse et al., 2005; Samaranayaka & Li-Chan, 2008; Slizyte
et al., 2009; Yang, Ho, Chu, & Chow, 2008).
 Journal of Functional Foods 21 (2016) 10–26 15

Table 2 – Proteases and antioxidative tests used for antioxidant protein hydrolysates (FPH) preparation from marine
by-products.
Fish species By-products Proteases used for Antioxidant activity tests IC50 or scavenging Reference
protein hydrolysis activity (%)
Limanda Frame Mackerel intestine Linoleic acid autoxidation — Jun et al.
aspera proteins crude enzyme, Alcalase, inhibition activity (2004)
α – chymotrypsin,
papain, pepsin, Pronase
E, Neutrase and trypsin
Theragra Frame Crude proteinase from Linoleic acid peroxidation — Je et al. (2005)
chalcogramma proteins mackerel intestines inhibition activity
Tuna Backbone Pepsin DPPH radical scavenging DPPH (35.82% Je et al. (2007)
activity, hydroxyl radical at 1.3 mg/ml)
scavenging activity, superoxide Hydroxyl
radical scavenging activity and (80.91% at
lipid peroxydation inhibition 1.3 mg/ml)
activity
Johnius Frame Pepsin DPPH radical scavenging DPPH (83.93% Kim et al.
belengerii activity, hydroxyl radical at 0.5 mg/ml) (2007)
scavenging activity, peroxyl Hydroxyl
radical scavenging activity, (84.94% at
superoxide radical scavenging 0.5 mg/ml)
activity and lipid peroxydation
inhibition activity
Salmon Protamine, Pancreatin DPPH radical scavenging DPPH (66.61% Wang, Zhu,
derived from activity, hydroxyl radical at 0.5 mg/ml) Han, and
fish milt scavenging activity and Hydroxyl Wang (2008)
superoxide anion radical (31.45% at
scavenging activity 5 mg/ml)
Rachycentron Skin Trypsin DPPH radical scavenging DPPH (60% at Yang et al.
canadum activity, linoleic acid 10 mg/ml) (2008)
autoxidation inhibition activity
Sole Skin gelatin Alcalase Fe2+ chelating activity, ferric ion ABTS (16 mg Gimenez,
reducing capacity, ABTS radical vitamin C Aleman,
(2,2-azinobis-(3- equivalent Montero, and
ethylbenzothiazoline-6- antioxidant Gomez-Guillen
sulphonic acid)) scavenging capacity (2009)
capacity (VCEAC)/g
Tuna Liver Neutrase DPPH radical scavenging DPPH (90% at Je et al. (2009)
(katsuwonus activity, hydroxyl radical 5 mg/ml)
pelamis) scavenging activity, hydroxyl Hydroxyl
radical scavenging, hydrogen (58% at 2 mg/
peroxide scavenging activity ml)
and ferrous ion chelating
activity
Tuna Cooking juice Orientase DPPH radical scavenging DPPH (80% at Hsu, Lu, and
(Thunnus activity and linoleic acid 5 mg/ml) Jao (2009)
tonggol autoxidation inhibition activity
Gadus morhua Backbones Protamex DPPH radical scavenging DPPH (50% at Slizyte et al.
activity and iron mediated 2.5 mg/ml) (2009)
liposomes oxidation reducing
activity
Tilapia Skin gelatin Thermal hydrolysis DPPH radical scavenging DPPH (79.4% Yang et al.
activity and linoleic acid at 20 mg/ml) (2009)
autoxidation inhibition activity
Theragra Skin collagen Trypsin and DPPH radical scavenging DPPH (72.5% (Zhuang et al.
chalcogramma flavourzyme activity, Superoxide anion at 2 mg/ml) 2009a, b)
radical scavenging activity, Hydroxyl
hydroxyl radical scavenging (77.88% at
activity and hydrogen peroxide 1 mg/ml)
scavenging
Tuna Liver Alcalase and DPPH radical scavenging DPPH (70% at Ahn, Lee, and
flavourzymes activity, peroxide and hydroxyl 5 mg/ml) Je (2010)
radical scavenging activity and
reducing power
(continued on next page)
16 Journal of Functional Foods 21 (2016) 10–26

Table 2 – (continued)
Fish species By-products Proteases used for Antioxidant activity tests IC50 or scavenging Reference
protein hydrolysis activity (%)
Aphanopus By products Protamex DPPH radical scavenging — Batista,
carbo activity, Reducing activity, Ramos,
hydroxyl radical scavenging Coutinho,
activity Bandarra,
and Nunes
(2010)
Sardinella Heads and Smooth hound digestive DPPH radical scavenging DPPH (66% at Barkia et al.
aurita viscera proteases activity ferric (Fe3+) reducing 300 µg/ml) (2010)
antioxidant activity, β-carotene
bleaching inhibition activity
Sardinalla Heads and Enzyme Sardina DPPH radical scavenging DPPH (53.76% Bougatef
aurita viscera pilchardus activity, linoleic acid at 2 mg/ml) et al. (2010)
proteins autoxidation activity and
reducing power activity
Thunnus Dark muscle Orientase DPPH radical scavenging DPPH (41% at Hsu (2010)
tonggol activity and linoleic acid 3 mg/ml)
autoxidation inhibition activity
Oreochromis Scale gelatin Alcalase DPPH radical scavenging DPPH Ngo et al.
niloticus activity, hydroxyl radical (IC50 = 600 µg/ml) (2010)
scavenging activity, hydroxyl Hydroxyl
radical scavenging activity and (IC50 = 263 µg/
superoxide radical anion ml)
scavenging activity
Priacanthus Skin gelatin Neutrase DPPH radical scavenging 13 µmol Phanturat
macracanthus activity, ABTS radical Trolox et al. (2010)
scavenging activity, Ferric equivalents
reducing antioxidant power (TE)/mg
(FRAP) and linoleic acid
autoxidation inhibition activity
and lecithin liposome
oxidation preventing activity
North Skin Bacterial endopeptidase DPPH radical scavenging DPPH Picot et al.
Atlantic activity and beta-carotene- (IC50 = 24.7 mg/ml) (2010)
lean fish linoleate oxidation preventing
activity
Theragra Skin Protamex DPPH radical scavenging DPPH Jia et al.
chalcogramma activity and Ferric (Fe3+) (IC50 = 2.5 mg/ (2010)
reducing antioxidant power ml)
Tuna and Skin gelatin Alcalase Ferric reducing antioxidant Tuna : ABTS Aleman et al.
Halibut power (FRAP), and ABTS radical (16 mg (2011)
scavenging activity (VCEAC)/g
Halibut :
ABTS(13 mg
(VCEAC)/g
Silver carp Processing Pepsin DPPH radical scavenging DPPH (68.4% Zhong et al.
(Hypophthalmichthys by-product activity, hydroxyl radical at 5 mg/ml) (2011)
molitrix) scavenging activity, superoxide
anion radical scavenging
activity and linoleic acid
autoxidation inhibition activity
Parastromateus Viscera Pepsin, trypsin, and DPPH radical scavenging DPPH (54% at Jai Ganesh
niger α-chymotrypsin activity, Fe2+ chelating activity 1 mg/ml) et al. (2011)
and ferric (Fe3+) reducing
antioxidant power
horse Skin Pepsin,trypsin and Ferric (Fe3+)reducing — Sampath
mackerel α-chymotrypsin antioxidant activity and Fe2+ Kumar,
(Magalaspis chelating activity Nazeer, and
cordyla) and Jaiganesh
croaker (2012)
(Otolithes
ruber)
(continued on next page)
 Journal of Functional Foods 21 (2016) 10–26 17

Table 2 – (continued)
Fish species By-products Proteases used for Antioxidant activity tests IC50 or scavenging Reference
protein hydrolysis activity (%)
Seela Backbones Trypsin and pepsin DPPH radical scavenging Seela : DPPH Nazeer,
(Sphyraena activity, Ferric (Fe3+) reducing (39% at Deeptha,
barracuda) antioxidant power and lipid 3 mg/ml) Jaiganesh,
and Ribbon peroxidation inhibition activity Ribbon fish : Sampath
fish DPPH (38% at kumar, and
(Lepturacanthus 3 mg/ml) Naqash
savala) (2011)
Exocoetus Backbone Papain, pepsin and DPPH radical scavenging DPPH (40.1% Naqash and
volitans trypsin activity, Lipid peroxidation at 1.5 mg/ml) Nazeer (2011)
inhibition activity superoxide Hydroxyl
anion radical scavenging (44.6 at
activity and hydroxyl radical 1.5 mg/ml)
scavenging activity
Catla (Catla Visceral Fungal proteases DPPH radical scavenging DPPH Hathwar
catla) and waste activity and total antioxidant (IC50 = 3.47 mg/ml) et al. (2011)
Rohu (Labeo activity
rohita)
Tilapia Skin gelatin Properase E Scavenging abilities on Hydroxyl 60% Zhuang and
(Oreochromis °O2, H2O2, ONOO°, and NO° (at 2 mg/ml) Sun (2011)
niloticus)
Horse Viscera Pepsin, trypsin and Reducing power assay; DPPH DPPH (61.1% Sampath
mackerel chymotrypsin radical scavenging activity and at 1 mg/ml) Kumar et al.
(Magalaspis Hydroxyl radicals scavenging Hydroxyl (2011)
cordyla) activity; (43.1% at
1 mg/ml)
Alaska Skin collagen Typsin and flourzyme Iron-chelating activity ; — Guo et al.
pollock (2013)
Salmon Pectoral fin Pepsin DPPH radical scavenging DPPH Ahn et al.
activity; ABTS+ radical (IC50 = 1.63 mg/ml) (2014)
scavenging activity; Ferric
reducing power
Bluefin Heads Papain Radical scavenging activity and DPPH (20% at Chi et al.
leatherjacket Lipid peroxidation inhibition 10 mg/ml) (2015b)
(Navodon assay
septentrionalis)
Bluefin Skin Trypsin, flavourzyme, DPPH radical scavenging DPPH Chi et al.
leatherjacket neutrase, papain, activity; Hydroxyl radicals (IC50 = 5.22 mg/ml) (2015b)
(Navodon alcalase, and pepsin scavenging activity and Lipid Hydroxyl
septentrionalis) peroxidation inhibition assay (IC50 = 1.04 mg/ml)

The radical-quenching activities of food antioxidants are due
to the ability of the antioxidants to participate in single elec-
tron transfer reaction (Huang, Ou, & Prior, 2005). Trolox
equivalent antioxidant capacity (TEAC) assay, oxygen radical
absorbance capacity (ORAC) assay, and the total radical-
trapping antioxidant parameter (TRAP) assay have been widely
reported in the literature for measuring antioxidative capac-
ity of food and biological samples (Cao & Prior, 1998; Ghiselli,
Serafini, Natella, & Scaccini, 2000; Re et al., 1999).
Due to the complexity of oxidative processes occurring in
food or in biological systems as well as the different
antioxidative mechanisms by which various compounds may
act, finding one method that can characterise the overall
antioxidative potential of food is not an easy task. Bougatef
et al. (2009) reported the antioxidant activities of different
smooth hound protein hydrolysates using various in vitro an-
tioxidant assays, such as DPPH radical-scavenging activity,
reducing power, total antioxidant capacity, lipid peroxidation
inhibition in rat liver homogenate and β-carotene bleaching
assay. Sila et al. (2014c) studied the antioxidant activities of
shrimp by-products peptides at different concentrations using
various in vitro antioxidant assays, including the DPPH radical
method, reducing power, chelating effects assay and β-carotene
bleaching. In the same context, Saidi, Deratani, Belleville, and
Ben Amar (2014) used superoxide radical scavenging assay, hy-
droxyl radical scavenging activity assay, reducing power assay,
ferrous ion chelating activity and inhibition of linoleic acid au-
toxidation for the evaluation of antioxidant properties of
peptide fractions from tuna dark muscle protein by-product
hydrolysate.
Protein hydrolysate obtained from tuna backbone with
pepsin show the strong antioxidative activity about 35.82 and
80.91% at a concentration of 1.3 mg/ml for DPPH and hy-
droxyl scavenging activities, respectively (Je et al., 2007). Je, Lee,
Lee, and Ahn (2009) produced protein hydrolysate from tuna
liver with Neutrase exhibiting antioxidative activity with re-
spective scavenging activities on DPPH (90% at 5 mg/ml) and
hydroxyl (58% at 2 mg/ml).
18 Journal of Functional Foods 21 (2016) 10–26
Ngo, Qian, Ryu, Park, and Kim (2010) also elaborated protein
hydrolysate from scale gelatin with Alcalase. According to their
results, the produced hydrolysate showed a good scavenging
activity with IC50 values of 600 and 263 µg/ml for DPPH and hy-
droxyl, respectively. In the same context, Picot et al. (2010),
Hathwar, Bijinu, Rai, and Narayan (2011) and Ahn, Kim, and
Je (2014) reported IC50 valued for DPPH radical scavenging ac-
tivity of 24.7, 3.47 and 1.63 mg/ml for protein hydrolysates from
North Atlantic lean fish skin, catla visceral waste and salmon
pectoral fin, respectively.
The cellular antioxidant assays are more biologically rel-
evant than the chemical assays because it takes into account
some aspects of cell uptake, distribution, and metabolism of
antioxidant compounds. In this context, antioxidant activi-
ties of peptide isolated from Nile tilapia (O. niloticus) scale gelatin
were evaluated. This peptide showed protective effect on DNA
damage caused by hydroxyl radicals and showed no cyto-
toxic effect on mouse macrophages (RAW 264.7) and human
lung fibroblasts (MRC-5) (Ngo et al., 2010).
Je et al. (2007) studied the cytotoxic effects of antioxidant
peptide obtained from tuna backbone protein on human lung
fibroblast and human endothelial cell line, and the results
showed that this peptide did not show any cytotoxic effects
on MRC-5 and ECV304 cells.
The antioxidative activities of the purified peptides from
gelatin hydrolysate of Alaska pollock skin were measured using
the thiobarbituric acid method, and the cell viability was mea-
sured with MTT assay. Moreover, the cell viability of cultured
liver cells was significantly enhanced by addition of the peptide
(Kim et al., 2001).
3.4. In vivo evaluation of antioxidant activity
Antioxidants can scavenge reactive oxygen species (ROS) to
inhibit lipid peroxidation. Antioxydation maintain the cellu-
lar redox homeostasis and protect skin cells from UV radiation
(Murakami et al., 2009). ROS, induced by UVA and UVB irra-
diation, played a substantial role in collagen oxidation and
degradation (Wlaschek et al., 2001).
To date, only a small number of studies have evaluated
the efficacy of antioxidant peptides from marine by-products
using animal models. Sun, Zhang, and Zhuang (2013) evalu-
ated protective effects of tilapia skin gelatin peptide on mice
skin photoageing induced by UV irradiation. The IC50 value of
the purified peptide on hydroxyl radical scavenging activity
was 22.47 µg/ml. They also showed that skin gelatin peptide
was useful to protect the collagen fibres, and this protective
effect was in dose-dependent manners. Zhuang, Hou, Zhao,
Zhang, and Li (2009a) reported the production of collagen
hydrolysate from jellyfish, and then they evaluated their
protective effects on mice skin photoageing induced by UV
irradiation.
Kamoun et al. (2012) studied the possible protective effects
in vivo of sardinelle by-products protein hydrolysates on the
ethanol-induced cardiotoxicity in adult rats (7.27 mg of SPH/
kg body weight). They concluded from biochemical analysis that
peptides offer good curative efficacy against ethanol-induced
cardiotoxicity of lipid peroxidation and protein carbonyl and
by enhancing the activities of enzymatic and the levels of non-
enzymatic antioxidants.
4. Isolation of antioxidant peptides
4.1. Purification of antioxidant peptides by
chromatographic methods
Several studies have looked at the contribution of molecular
size and structural characteristics of peptide mixtures in protein
hydrolysates to their bioactivity. These studies showed that low
molecular weight fractions (1 to 5 kDa) in general contained
more potent antioxidative peptides (Aleman, Gimenez, Montero,
& Gomez-Guillen, 2011; Je et al., 2005; Mendis, Rajapakse, Byun,
& Kim, 2005; Nalinanon, Benjakul, Kishimura, & Shahidi, 2011.
Protein hydrolysates produced by treatment of protein sub-
strate by appropriate proteolytic enzyme contained a complex
mixture of active and inactive peptides having various sizes
and different amino acid composition. Furthermore, bioactive
peptides, based on their amino acid composition and se-
quences, exhibited multifunctional activities. Antioxidant
peptides are present at low concentrations. Therefore, several
enrichment methods need to be applied to produce fractions
with high concentration of antioxidant peptides.
Chromatographic techniques have been used for partition-
ing protein and peptides mixtures depending on their affinity
to either mobile or stationary phases. Gel filtration separates
proteins and peptides according to their size as they pass
through a gel medium in a packed column. Peptides sepa-
rated by gel filtration do not bind to the medium and therefore
a buffer does not directly affect resolution or their biological
activity. Chromatography using a gel filtration column has been
used to fractionate and concentrate biopeptides in protein hy-
drolysates (Bougatef et al., 2010; Byun, Lee, Park, Jeon, & Kim,
2009; Rajapakse et al., 2005) and is often combined with other
separation techniques based on differences in acidity, basic-
ity, charge, hydrophobic interaction, metal chelating, and
adsorption affinities (Table 3).
As an example, a typical procedure of a bioassay guided iso-
lation applied to the purification of antioxidant peptides.
Bougatef et al. (2010) produced hydrolysates with different
degrees of hydrolysis from sardinelle protein by-products with
crude enzyme extract from sardine (Sardina pilchardus). Hydro-
lysate with 6% DH exhibited the highest DPPH radical-
scavenging activity (87 ± 2.1%, at 2 mg/ml). This hydrolysate was
fractionated by size exclusion chromatography on a Sephadex
G-25 column into eight major fractions (P1–P8). Fraction P4, which
exhibited the highest DPPH scavenging activity (48%, at 1 mg/
ml), was then fractionated by reversed-phase high performance
liquid chromatography (RP-HPLC). Seven antioxidant pep-
tides were isolated from that fraction. The molecular masses
and amino acids sequences of the purified peptides were de-
termined using electrospray mass spectrometry (ESI-MS) and
tandem ESI-MS (ESI-MS/MS), respectively.
The membrane separation is a useful technology for frac-
tionation of molecules and offers a good alternative separation
for achieving an environmental friendly and cost-effective
process (Drioli, Stankiewicz, & Macedonio, 2011). Ultrafiltra-
tion membrane systems using different cut-off sizes (100, 20,
10, 5, 3, and 1 kDa) were employed to separate different pep-
tides with the desired molecular weights and functional
properties from FPH (Jeon et al., 1999). This system can also
Table 3 – Sequences and chromatographic methods employed for antioxidant peptide production.
Fish species By-products Methods used for purification Isolated peptides sequence and molecular weight IC50 or scavenging Reference
and identification activity (%)
Limanda aspera Frame proteins C25; G75; RP-HPLC; Protein Arg—Pro—Asp—Phe—Asp—Leu—Glu—Pro—Pro—Tyr — Jun et al. (2004)
sequencer
Theragra Frame proteins G-25; RP-HPLC Leu—Pro—His—Ser—Gly—Tyr (672 Da) — Je et al. (2005)
chalcogramma Protein sequencer
Jumbo skid (Dosidiuc Skin SP-Sephadex C-25 ; His—Gly—Pro—Leu—Gly—Pro—Leu (797 Da) — Mendis et al. (2005)
gigas) Sephadex G-25; RP-HPLC;
Tuna Backbone FPLC; RP-HPLC Val—Lys—Ala—Gly—Phe—Ala—Trp—Thr—Ala— DPPH (80% at 1.2 mg/ml) Je et al. (2007)
Q-TOF ESI/MS/MS Asn—Gln—Gln—Leu—Ser (1519 Da) Hydroxyl (70% at 1.2 mg/ml)
Johnius belengerii Frame FPLC; RP-HPLC; Q-TOF ESI/ Glu—Ser—Thr—Val—Pro—Glu—Arg—Thr—His— DPPH (IC50 = 41.37 µM) Kim et al. (2007)
MS/MS Pro—Ala—Cys—Pro—Asp—Phe—Asn (1801 Da) Hydroxyl radical
(IC50 = 17.77 µM)
Salmon Protamine, derived G35; Macro-Prep High Q; Pro—Arg (271.3 Da) — Wang et al. (2008)
from fish milt YMC-Pack Protein-RP; LC–
MS
Thunnus tonggol Cooking juice G25; RP-HPLC, Pro—Val—Ser—His—Asp—His—Ala—Pro—Glu— — Hsu et al. (2009)
Q-TOF ESI/MS/MS tyr (1305 Da);
Pro—Ser—Asp—His—Asp—His—Glu (938 Da), and
Val—His—Asp—Tyr (584 Da)
Sardinalla aurita Heads and viscera G-25;RP-HPLC Leu—His—Tyr; Leu—Ala—Arg—Leu; Gly—Gly—Glu; DPPH (63% at 150 µg/ml) Bougatef et al. (2010)
proteins ESI/MS/MS Gly—Ala—His; Gly—Ala—Trp—Ala; Pro—His—Tyr— for Leu-His-Tyr
Leu and Gly—Ala—Leu—Ala—Ala—His
Thunnus tonggol Dark muscle G25; RP-HPLC; Leu—Pro—Thr—Ser—Glu—Ala—Ala—Lys—Tyr (978 Da) DPPH (79 % at 100 µg/ml) Hsu (2010)
Q-TOF ESI/MS/MS Pro—Met—Asp—Tyr—Met—Val—Thr (756 Da) DPPH (85.2 % at 100 µg/ml)
Oreochromis niloticus Scale FPLC; RP-HPLC, Q-TOF ESI/ Asp—Pro—Ala—Leu—Ala—Thr—Glu—Pro—Asp—Pro— DPPH (IC50 = 8.82 µM) Ngo et al. (2010)
gelatin MS/MS Met—Pro—Phe (1382.57 Da) Hydroxyl radical
(IC50 = 7.56 µM)
Parastromateus niger Viscera DEAE; G25; ESI/MS/MS Ala—Met—Thr6Gly—Leu—Glu—Ala (701.9 Da) — Jai Ganesh et al. (2011)
Magalaspis cordyla Skin IEC; G25; ESI-MS/MS Asn—His—Arg—Tyr—Asp—Arg (856 Da); Gly—Asn— — Sampath Kumar
and Otolithes ruber Arg—Gly—Phe—Ala—Cys—Arg—His—Ala (1101.5 Da) et al. (2011)
Tilapia (Oreochromis Skin gelatin SP Sephadex C-25 ; G25 ; Leu—Ser—Gly—Tyr—Gly—Pro (592.26 Da) — Zhuang and Sun (2011)
niloticus) RP-HPLC; LC-ESI-Q-TOF MS/
MS
Journal of Functional Foods 21 (2016) 10–26

Horse mackerel Viscera FPLC; DEAE; G25; ESI-MS/ Ala—Cys—Phe—Leu (518.5 Da) — Sampath Kumar
(Magalaspis cordyla) MS et al. (2011)
Alaska pollock Skin collagen Affinity chromatography; Ser—Cys—His (345 Da) — Guo et al. (2013)
RP-HPLC ; ESILC-MS/MS
Salmon Pectoral fin DEAE; RP-HPLC; LC/MS/ MS Phe—Leu—Asn—Glu—Phe—Leu—HisVal (1018.48 Da) DPPH (IC50 = 486 µM) Ahn et al. (2014)
ABTS (IC50 = 152 µM)
Bluefin leatherjacket Heads Ultrafiltration; DEAE; G15; Trp—Glu—Gly—ProLys; DPPH (IC50 = 4.43 mg/ml) Chi et al. (2015a)
(Navodon RP-HPLC; protein/peptide Gly—Pro—Pro; DPPH (IC50 = 1.92 mg/ml)
septentrionalis) sequencer and ESI–MS Gly—Val—Pro—Leu—Thr DPPH (IC50 = 4.54 mg/ml)
Bluefin leatherjacket Skin DEAE; G15; RP-HPLC; Gly—Ser—Gly—Gly—Leu; DPPH (IC50 = 0.4 mg/ml) Chi et al. (2015b)
(Navodon protein/peptide sequencer Gly—Pro—GlyGly—Phe—Ile; DPPH (IC50 = 0.19 mg/ml)
septentrionalis) and ESI–MS Phe—Ile—Gly—Pro DPPH (IC50 = 0.11 mg/ml)
G25: Gel filtration chromatography on Sephadex G25; G35: Gel filtration chromatography on Sephadex G35; G75: Gel filtration chromatography on Sephadex G75; RP-HPLC: Reverse-Phase High Per-
formance Liquid Chromatography; FPLC: Fast Performance Liquid Chromatography; Macro-Prep High Q: Macro-Prep® High Q Support, ion exchange; YMC-Pack PROTEIN-RP: Reversed-phase column
utilising a silica gel base; IEC: Ion Exchange Chromatography; Q-TOF ESI/MS/MS: Maldi-Q-TOF Electrospray tandem mass spectrometry; DEAE: Diethylaminoethyl Cellulose.
19
20 Journal of Functional Foods 21 (2016) 10–26
control the molecular weight distribution of the appropriate
peptide (Cheryan & Mehaia, 1990; Kim, Byun, Kang, & Song,
1993).
Saidi et al. (2014) studied the performance of the com-
bined process associating ultrafiltration and nanofiltration
membranes to produce and isolate fractions enriched in pep-
tides of specific molecular weight (MW).
Optimisation of ultrafiltration and nanofiltration steps for
the fractionation of a protein hydrolysate produced by enzy-
matic hydrolysis of tuna dark muscle was investigated through
the studied impact of operating parameters including pres-
sure, peptide concentration and pH of the solution on
performance of two selected membranes (Saidi, Deratani, Ben
Amar, & Belleville, 2013). Bourseau et al. (2009) discussed the
impact of a two-step ultrafiltration and nanofiltration process
producing four different fractions on two industrial fish protein
hydrolysates with different hydrolysis degrees. In addition, an
electromembrane filtration process called electrodialysis with
ultrafiltration membrane was developed and patented by
Bazinet, Amiot, Poulin, Tremblay, and Labbé (2005). This process
has been successfully used to separate bioactive peptides from
various food protein hydrolysates. Suwal et al. (2014) studied
the optimisation of electrodialysis with ultrafiltration mem-
brane process for the separation of bioactive peptides from snow
crab by product hydrolysate. In the same context, Suwal, Roblet,
Amiot, and Bazinet (2015) reported the use of ion-exchange
membranes and ultrafiltration membranes staked in an elec-
trodialysis with filtration membrane system for the production
of peptide fractionations from snow crab by product hydrolysate.
4.2. Identification of antioxidant peptides
In some cases, extensive enzymatic hydrolysis of proteins
results in decreased antioxidant activity due to free amino acids.
Protein hydrolysates (peptides) are more potent antioxidants
than free amino acids, due to their chemical composition and
physical properties (Elias, Kellerby, & Decker, 2008). Liquid chro-
matography followed by MS/MS (LC–MS/MS) is commonly used
to identify peptide sequences (Perkins, Pappin, Creasy, & Cottrell,
1999).
Table 3 shows the antioxidant activities of isolated pep-
tides derived from different fish by-products protein sources.
The first known scientific study reported that the antioxi-
dant activity of FPH was by Shahidi, Han, and Synowiecki (1995);
since then, a number of studies reported the antioxidative ac-
tivities for various FPH (Phanturat, Benjakul, Visessanguan, &
Roytrakul, 2010; Yang et al., 2008; Zhong, Ma, Lin, & Luo, 2011).
Antioxidant peptides have been isolated from several hydro-
lysed fish proteins (Table 3). Several antioxidative peptides
isolated from fish proteins have been reported to contain 2–16
amino acid residues (Bougatef et al., 2010; Je et al., 2005; Je et al.,
2007; Jun, Park, Jung, & Kim, 2004).
In a study Jun et al. (2004) isolated an antioxidative peptide
composed of 10 amino acid residues, Arg—Pro—Asp—Phe—
Asp—Leu—Glu—Pro—Pro—Tyr, from yellowfin sole (Limanda
aspera) frame proteins. In another study, Je et al. (2005) iden-
tified an antioxidative peptide, Leu—Pro—His—Ser—Gly—Tyr
(672 Da), from Alaska pollack (Theragra chalcogramma) frame
protein hydrolysate that was prepared with mackerel intes-
tine crude enzyme. The authors reported that the isolated
peptide showed 35% scavenging activity on hydroxyl radicals
at 53.6 l µM concentration. Kim et al. (2007) described the pro-
duction of antioxidative peptides from hoki (J. belengerii) frame
proteins. The antioxidant peptide isolated from peptic hydro-
lysate having the highest antioxidant activity has the following
sequence: Glu—Ser—Thr—Val—Pro—Glu—Arg—Thr—His—
Pro—Ala—Cys—Pro—Asp—Phe—Asn. This purified peptide was
a potent free radical scavenger with 35.82 and 80.91% scav-
enging activities at a concentration of 1.3 mg/ml for DPPH and
hydroxyl, respectively.
Je et al. (2007) identified antioxidant peptide,
Val—Lys—Ala—Gly—Phe—Ala—Trp—Thr—Ala—Asn—Gln—
Gln—Leu—Ser (1519 Da) from peptic hydrolysate of tuna back-
bone protein and reported that the isolated peptide showed
significant inhibition of lipid peroxidation in a linoleic acid
emulsion system and quenching of free radicals (DPPH (80%
at 1.2 mg/ml)) and hydroxyl (70% at 1.2 mg/ml)). In the same
context, Bougatef et al. (2010) described the isolation of seven
antioxidant peptides from sardinelle by-products. The mo-
lecular masses and amino acids sequences of the purified
peptides were determined using ESI-MS and ESIMS/MS. Their
structures were identified as Leu—His—Tyr, Leu—Ala—Arg—Leu,
Gly—Gly—Glu, Gly—Ala—His, Gly—Ala—Trp—Ala, Pro—His—
Tyr—Leu and Gly—Ala—Leu—Ala—Ala—His. The first peptide
displayed the highest DPPH radical-scavenging activity
(63 ± 1.57%; at 150 µg/ml).
Antioxidant peptide (515.5 Da) from horse mackerel viscera
protein acted as a strong radical scavenger. This peptide per-
formed better as an antioxidant against polyunsaturated fatty
acid (PUFA) peroxidation than that of natural antioxidant,
α-tocopherol (Sampath Kumar et al., 2011).
Three antioxidant peptides (Trp—Glu—Gly—ProLy;
Gly—Pro—Pro; and Gly—Val—Pro-Leu—Thr) have been iso-
lated from the bluefin leatherjacket heads. These peptides
significantly quenched DPPH radical at the IC50 values of 4.43,
1.92 and 4.54 mg/ml, respectively (Chi et al., 2015a). Peptides
purified from bluefin leatherjacket skins were examined for
antioxidative activities. The three peptides, (Gly—Ser—Gly—
Gly—Leu; Gly—Pro—GlyGly—Phe—Ile; and Phe—Ile—Gly—Pro)
were potent scavengers of free radicals with IC50 values of 0.4,
0.19 and 0.11 mg/ml, respectively (Chi, Wang, Wang, Zhang, &
Deng, 2015b). Antioxidant peptide (1382.57 Da) isolated from
Nile tilapia (O. niloticus) scale gelatin bullfrog skin efficiently
quenched different sources of free radicals with IC50 values on
DPPH (8.82 µM) and •OH radical (7.56 µM) (Ngo et al., 2010).
4.3. Structure–activity relationship of
antioxidative peptides
The structural characteristics will provide guides for the evalu-
ation of food proteins as potential precursors of antioxidative
peptides, and predict the possible release of bioactive pep-
tides from various proteins using appropriate proteolytic
enzymes.
To date, there is still lack of evidence to explain the rela-
tionship between structural characteristics of peptides, such
as molecular size, hydrophobicity, amino acid composition and
sequence, and their antioxidative property specific to each
mechanistic action. Size, especially in the range of 0.5–3 kDa,
has been suggested to be a crucial factor affecting the anti-
 Journal of Functional Foods 21 (2016) 10–26
oxidant activity of protein hydrolysates (Je et al., 2005; Kim et al.,
2007; Nalinanon et al., 2011; Phanturat et al., 2010;
Samaranayaka & Li-Chan, 2011). Another approach is to develop
statistical models that predict the relationship between struc-
ture (amino acid sequence) and activity.
The antioxidative activity of histidine-containing pep-
tides has been reported (Murase, Nagao, & Terao, 1993;
Uchida & Kawakishi, 1992). This activity may be attributed to
the chelating and lipid radical-trapping ability of the imidaz-
ole ring. Chen, Muramoto, Yamavchi, and Nokihara (1996)
reported that peptides with a Pro—His—His sequence showed
the greatest antioxidant activity among many tested pep-
tides. The most reactive amino acids include the nucleophilic
sulphur-containing amino acids Cys and Met, the aromatic
amino acids Trp, Tyr, and Phe. Kawashima, Itoh, Miyoshi, and
Chibata (1979) investigated the effects of many synthetic
peptides on lipid oxidation and found that some peptides
having branched-chain amino acids (Val, Leu, Ile) showed
antioxidant properties. Those dipeptides consisting of Ala,
Tyr, His, and Met at the N-terminal showed higher antioxi-
dant activities than the constituent amino acid mixtures in
an aqueous system. On the correlation between structure
and activity of the antioxidant peptides, Chen, Muramoto,
Yamauchi, Fujimoto, and Nokihara (1998) reported that pep-
tides with highly potent inhibitory activity have hydrophobic
amino acids, Val or Leu, at the N-terminal position, and Pro,
His, or Tyr in the sequences. Yamaguchi, Yokoo, and Fujimaki
(1975) reported that the dipeptides consisting of Tyr and Trp
at the amino terminus, and His and Met at the carboxyl
terminus showed stronger antioxidant activity than the con-
stituent amino acid mixtures in an aqueous system.
Samaranayaka and Li-Chan (2011) reported that lower mo-
lecular weight peptides in the 1000–3000 Da range would be
more effective at interacting with radicals for the termina-
tion of propagation cycles of lipid peroxidation. In the same
context, Chi et al. (2015a) studied the antioxidant activities of
three isolated peptides. They showed that the smaller mo-
lecular size (389.41, 546.63, and 432.52 Da) and hydrophobic and/
or aromatic amino acids in their sequences could contribute
to the antioxidant activities. Peptide with molecular weight
432.52 Da exhibited the highest scavenging activities on DPPH
(IC50 0.118 mg/ml), HO•(IC50 0.073 mg/ml), and O2−• (IC50 0.311 mg/
ml) among the three peptides.
5. Antioxidant peptides in food systems
Lipid oxidation is of great concern to the food industry and con-
sumers because it leads to the development of undesirable off-
flavours, odours and potentially toxic reaction products (Lin
& Liang, 2002). To prevent foods from undergoing such dete-
rioration and to provide protection against serious diseases,
it is very important to inhibit lipid peroxidation occurring in
foodstuffs and in the living body. Antioxidants are used to pre-
serve food products by retarding discoloration and deterioration
as a result of oxidation (Decker, Warner, Richards, & Shahidi,
2005). Antioxidants for use in food processing must be inex-
pensive, nontoxic, effective at low concentrations (0.001–
0.02%), capable of surviving processing (carry-through), stable
21
in the finished products, and devoid of undesirable colour,
flavour and odour effects. Antioxidants in food products are
normally added either as direct additives or indirectly through
diffusion from packaging material (van Aardt et al., 2004). Some
peptides with antioxidant activity occur naturally in food.
Both glutathione (γ-Glu-Cys-Gly) and carnosine (β-alanyl-
L-histidine) are antioxidants that are naturally present in muscle
tissues. They have been found to scavenge hydroxyl radicals
and quench singlet oxygen and inhibit lipid peroxidation
(Samaranayaka & Li-Chan, 2011). In addition to the antioxi-
dants that are naturally present, peptides from hydrolysed
dietary proteins have been reported to have antioxidant ca-
pabilities similar to or better than synthetic antioxidants, such
as BHT, making them safe for food applications. In this context,
Nikoo et al. (2014) reported the scavenging activity against DPPH,
ABTS and hydroxyl radicals of the peptide Pro—Ala—Gly—Tyr
isolated from Amur sturgeon skin gelatin. At a level of 25 ppm,
the peptide prevents lipid oxidation in minced fish, but at higher
levels it was ineffective. Shahidi et al. (1995) indicated that in-
corporation of capelin protein hydrolysate in meat model
system at the 0.5–3.0% level inhibited the formation of TBARS
by 17.7–60.4%. Kittiphattanabawon, Benjakul, Visessanguan, and
Shahidi (2012) monitored lipid oxidation in cooked commi-
nuted pork containing gelatin hydrolysate of 40% DH at levels
of 100, 500 and 1000 ppm and BHA (100 ppm) during storage
at 4 °C for 14 days. They showed that gelatin hydrolysate at a
level of 500 and 1000 ppm could inhibit lipid oxidation in both
β-carotene linoleate and cooked comminuted pork model
systems. Antioxidant peptides with the ability to inhibit lipid
oxidation could be useful agents for maintaining the quality
and freshness of foods by preventing oxidative rancidity, which
leads to rancid flavour and odours (Mills, Stanton, Hill, & Ross,
2011). Gelatin hydrolysates from cobia (Rachycentron canadum)
skin slowed down lipid peroxidation in a linoleic acid model
system. The 8 and 10 mg/ml of cobia gelatin hydrolysate had
a greater inhibition effect on lipid peroxidation, similar to that
of 10 mg/l of BHA (Yang et al., 2008). Cai et al. (2015) showed
that peptides isolated from protein hydrolysate of grass carp
(Ctenopharyngodon idella) skin also effectively inhibited the
peroxidation in linoleic acid model system. Mendis et al. (2005)
reported that peptide (His—Gly—Pro—Leu—Gly—Pro—Leu) pu-
rified from Hoki (J. belengerii) skin gelatin could act as an
antioxidant against linoleic acid peroxidation, and the activ-
ity was closer to the highly active synthetic antioxidant
butylated hydroxytoluene (BHT). Many marine peptides are
available. However, before their utilisation as functional food
ingredients, compatibility with different food matrices, gas-
trointestinal stability, bioavailability and long term stability need
to be studied.
6. Conclusion
Fish-derived bioactive peptides with antioxidative properties
may be a potential substitute for synthetic antioxidants for ex-
ploitation for human nutrition as functional foods. Moreover,
the safety of these peptide-based products should also be evalu-
ated prior to commercialisation especially after extensive food
processing that may affect the natural integrity and quality of
the constituent peptides.
22 Journal of Functional Foods 21 (2016) 10–26
Acknowledgement
This work was funded by the “Ministry of Higher Education,
Scientific Research and Technology, Tunisia.”
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