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36 Biological Activities and Production of Marine-Derived Peptides

Table 36.2. Source, Amino Acid Sequence and Molecular Weight of Antioxidative Peptides from Some Aquatic
Protein Hydrolysates
Sources

Enzymes

Da
751
1300

Giant squid muscle

Protease XXIII

Pepsin, Mackerel
intestine crude enzyme
Mackerel intestine crude
enzyme
Trypsin

Jumbo squid skin gelatin

Trypsin

880

Tuna cooking juice
Yellowfin sole frame
Alaska Pollack frame

Jao and Ko (2002)
Jun et al. (2004)

Rajapakse et al. (2005b)

938
584
431

Asn–Gly–Leu–Glu–Gly–Leu–Lys
Asn–Ala–Asp–Phe–Gly–Leu–Asn–
Gly–Leu–Glu–Gly–Leu–Ala
Phe–Asp–Ser–Gly–Pro–Ala–Gly–
Val–Leu

Asn–Gly–Pro–Leu–Gln–Ala–Gly–
Gln–Pro–Gly–Glu–Arg
His–Gly–Pro–Leu–Gly–Pro–Leu
Leu–Gly–Leu–Asn–Gly–Asp–Asp–
Val–Asn
Val–Lys–Ala–Gly–Phe–Ala–Trp–
Thr–Ala–Asn–Gln–Gln–Leu–Ser
Glu–Ser–Thr–Val–Pro–Glu–Arg–
Thr–His–Pro–Ala–Cys–Pro–Asp–
Phe–Asn
Leu–Lys–Gln–Glu–Leu–Glu–Asp–
Leu–Leu–Glu–Lys–Gln–Glu
Leu–Asn–Leu–Pro–Thr–Ala–Val–
Tyr–Met–Val–Thr
Pro–Val–Ser–His–Asp–His–Ala–Pro–
Glu–Tyr
Pro–Ser–Asp–His–Asp–His–Glu
Val–His–Asp–Tyr
Leu–His–Tyr

756

Pro–Met–Asp–Tyr–Met–Val–Thr

Hsu (2010)

978

Leu–Pro–Thr–Ser–Glu–Ala–Ala–
Lys–Tyr

Pro–Ser–Tyr–Val

You et al. (2010)

672
747
1307

Hoki skin gelatin
Conger eel

Trypsin
Tryptic enzyme

797
928

Tuna backbone

Pepsin

1519

Hoki frame

Pepsin

1801

Oyster protein


Pepsin

1600

Bigeye tuna dark muscle

Pepsin

1222

Tuna cooking juice

Orientase

1305

Loach muscle

Crude extract from
sardine viscera
Protease XXIII

Papain

References

Pro–His–His–Ala–Asp–Ser
Arg–Pro–Asp–Phe–Asp–Leu–Glu–
Pro–Pro–Tyr

Leu–Pro–His–Ser–Gly–Tyr

1242

Sardinelle (head,
viscera)
Tuna dark muscle
by-product

Peptide Sequence

464

causes of many serious human diseases, such as cardiovascular disease, cancer, and neurological disorders as well as the
aging process (Jittrepotch et al. 2006). To prevent foods from
undergoing oxidative deterioration and to provide protection
against serious diseases, it is important to inhibit the oxidation
of lipids and formation of free radicals occurring in the living
body and foodstuffs. In this regard, several antioxidants have
been used to maintain food quality. Also, there is keen interest
in antioxidants from natural sources for use as food supplements or processing aids. The commonly used antioxidants are
chemically synthesized antioxidants such as butylated hydroxyanisole, butylated hydroxytoluene, tert-butylhydroquinone, etc.
However, these antioxidants are suspected to pose toxicity problems in the long term and their use in foodstuffs is restricted
or prohibited in some countries (Sakanaka et al. 2004, Je et al.
2005a, Pihlanto et al. 2008).

Je et al. (2005c)

Mendis et al. (2005b)


Mendis et al. (2005a)
Ranathunga et al. (2006)
Je et al. (2007)
Kim et al. (2007)

Qian et al. (2008b)
Je et al. (2008)
Hsu et al. (2009)

Bougatef et al. (2010)

Recently, a number of studies have demonstrated that peptides derived from different marine animal protein hydrolysates
act as potent antioxidants (Table 36.2). Some of these antioxidant peptides have exhibited varying capacities to scavenge free
radicals. Several studies have indicated that peptides derived
from marine fish proteins have greater antioxidant properties
than α-tocopherol in different oxidative systems (Jun et al. 2004,
Rajapakse et al. 2005b). However, the exact mechanism of action of bioactive peptides as antioxidants is not clearly known.
Some peptides are capable of chelating metal ions, which acts
as the pro-oxidant (Klompong et al. 2007). The antioxidant activity of protein hydrolysates has been attributed to the presence
of certain amino acids in the peptide sequence. High amounts of
histidine and some hydrophobic amino acids are related to the
antioxidant potency. D´avalos et al. (2005) indicated that tryptophan, tyrosine and methionine showed the highest antioxidant


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activity among the amino acids, followed by cysteine, histidine,
and phenylalanine, while histidine and hydrophobic amino acids
were suggested by Pe˜na-Ramos et al. (2004) as the key factors
in delaying lipid oxidation. Mendis et al. (2005b) reported that
the presence of nonaromatic amino acids such as proline, alanine, valine, and leucine in jumbo squid skin hydrolysate contributed to the higher antioxidative activities, and phenylalanine
and leucine residues at N- and C-terminals of peptide could
contribute to the high activity. Wang et al. (2008c) stated that
peptides exhibiting good antioxidative activity usually contain
certain amino acids such as histidine, proline, tyrosine and lysine. Bougatef et al. (2010) indicated that peptides containing
histidine, tryptophan and tyrosine residues possessed antioxidative activity. Guo et al. (2009) concluded that peptides containing tyrosine residues at the C-terminus, lysine or phenylalanine
residues at the N-terminus, and tyrosine residues in their sequences had strong free radical scavenging activity. Moreover,
Suetsuna et al. (2000) indicated that some other amino acids
such as proline, alanine, and leucine contribute to free radical
scavenging activity. Leucine and proline could favor antioxidant
activity when they occur at the C-terminus end of the sequence
(Suetsuna et al. 2000). In general, peptides containing tyrosine
tend to exhibit strong free radical scavenging activity due to
the phenolic hydroxyl groups, which contribute substantially to

scavenging activity toward free radicals via the mechanism of
donating a hydrogen atom from their hydroxyl group (Suetsuna
et al. 2000, Guo et al. 2009). Other aromatic amino acids, tryptophan and phenylalanine, are generally considered as effective
radical scavengers, because they can donate protons easily to
electron deficient radicals while at the same time maintaining
their stability via resonance structures (Rajapakse et al. 2005b,
Zhang et al. 2009). In case of histidine-containing peptides, it is
thought to be connected to hydrogen-donating ability, lipid peroxy radical trapping, and/or the metal ion chelating ability of the
imidazole group (Mendis et al. 2005a, b). Hydrophobic peptides
can help in scavenging of free radicals by keeping close contact
with oxidizing substances leading to the rapid scavenging of
radicals (Mendis et al. 2005a, b). Some aromatic amino acids
and histidine are reported to play a vital role in the observed activity. Gelatin peptides contain mainly hydrophobic amino acids
and abundance of these amino acids favors a higher emulsifying
ability. Hence, marine-gelatin-derived peptides are expected to
exert higher antioxidant effects among other antioxidant peptide sequences (Mendis et al. 2005a). Therefore, marine-derived
bioactive peptides with antioxidative properties may have great
potential for use as nutraceuticals and pharmaceuticals and a
substitute for synthetic antioxidants.

Antimicrobial Activity
Antimicrobial peptides (AMPs) have captured the attention of
researchers in recent years because of their efficiency in inhibiting pathogens, bacteria, fungi, and virus. Adding preservative is
a common way of preventing or slowing microbial growth, the
major reason of food spoilage and poisoning. However, there
is a shortage of efficient and safe preservatives as a result of
appearance of resistant forms of food pathogens in response to

massive use of preservatives. Marine-derived AMPs are found
in a wide range of marine animals. These peptides are naturally

synthesized as a part of innate host defense mechanisms (Brown
and Hancock 2006) and may also be generated by enzymatic
hydrolysis of native proteins (Bulet et al. 2004, Liu et al. 2008,
Reddy et al. 2004). Among the marine animals, they are well
described in fish (Kitts and Weiler 2003, Hwang et al. 2010)
and the hemolymph of many marine invertebrates, including
spider crab (Stensvag et al. 2008), American lobster (Battison
et al. 2008), green sea urchin (Li et al. 2008), oysters (Liu et al.
2008), mussels, scallops, venerid clams and abalone (ChengHua et al. 2009), and shrimp (Destoumieux et al. 1997,Bartlett
et al. 2002). For example, the mudfish (Misgurnus anguillicaudatus) produces a strongly basic peptide termed misgurin (Park
et al. 1997). This 2502 Da peptide contains five arginine and four
lysine residues and has antimicrobial activity against a broad
spectrum of microorganisms. The tissues of Atlantic salmon
(Salmo salar) contain an antimicrobial peptide with a molecular
mass of 20,734 Da. This purified peptide has been identified
as a histone protein and is termed histone H1 (Richards et al.
2001). Three antibacterial basic polypeptides, HLP-1, HLP-2,
and HLP-3 have been isolated from acetic extracts of channel
catfish (Ictalurus punctatus) skin (Robinette et al. 1998). The
molecular mass of these peptides were reported to be 15.5 kDa,
30 kDa, and 15.0 kDa, respectively. HLP-1 is the dominant peptide and closely related to histone H2B, with inhibitory effects
against both fish bacterial pathogens and Escherichia coli D31.
Catfish (Parasilurus asotus) also contains a strong antimicrobial
peptide with the molecular mass of 2000 Da, named parasin I
consisting of 19 amino acids that include three arginine and five
lysine residues (Park et al. 1998). Because of high homology
to N-terminal sequence of histone H2A, parasin I may be derived from histone H2A by a specific protease cleavage. These
AMPs have strong activity against gram-negative and grampositive bacteria as well as fungi. Potent antibacterial peptides
originating from pardaxin have been isolated from Moses sole
fish (Pardachirus marmoratus) (Shai 1994). Pardaxin is composed of a helix-hinge-helix structure that is a common motif

found in many antibacterial and cytotoxic peptides. The peptide,
termed pleurocidin, is found specifically in the general epithelial mucous cells of flounder and has an amphipathic α-helical
conformation enabling the binding to anionic phospholipid rich
membranes. In addition, two hydrophobic proteins with molecular masses of 31 kDa and 27 kDa, respectively, have also been
isolated from skin mucous of carp (Cyprinus carpio)(Lemaitre
et al. 1996). In addition, marine-derived AMPs, such as urechistachykinins, piscidins, and arenicin-1 exhibited significant antimicrobial activities against human microbial pathogens without remarkable hemolytic effects against human erythrocytes
(Hwang et al. 2010).
Of marine invertebrates, antibacterial activity has been
reported in the hemolymph of the blue crab, Callinectus
sapidus, and it was highly inhibitory to gram-negative bacteria
(Edward et al. 1996). Although there are several reports on antibacterial activity in seminal plasma, few antibacterial peptides
have been reported in mud crab, Scylla serrata (Jayasankar and
Subramonium 1999). The antimicrobial peptide derived from


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American lobster (Homarus americanus) exhibited bacteriostatic activity against some gram-negative bacteria and both
protozoastatic and protozoacidal activity against two scuticociliate parasites Mesanophrys chesapeakensis and Anophryoides haemophila (Battison et al. 2008). Furthermore, antimicrobial activity and growth inhibition of bacteria such as E. coli,
Pseudomonas aeruginosa, Bacillus subtilis, and fungi such as
Botrytis cinerea, and Penicillium expansum have been reported
by antimicrobial peptide, CgPep33, derived from oyster (Crassostrea gigas) (Liu et al. 2008). In addition, Lee and Susumu
(1998) isolated two peptides, Leu–Leu–Glu–Tyr–Ser–Ile and
Leu–Leu–Glu–Tyr–Ser–Leu, from thermolysin hydrolysate of
oyster, C. gigas that inhibited HIV-1 protease. Moreover, an
active peptide, Arg–Arg–Trp–Trp–Cys–Arg–X (where X is an
amino acid or an amino acid analog) isolated from the enzymatic
hydrolysate of oyster, C. gigas, showed high inhibitory activity
on herpes virus (Zeng et al. 2008). In general, these AMPs are
characterized by relatively short cationic peptides with common
features, including (1) α-helices, (2) ß-sheet and small proteins,
(3) peptides with thio-ether rings, (4) peptides with an overrepresentation of one or two amino acids, (5) lipopeptides, and
(6) macrocyclic cystine knot peptides (Epand and Vogel 1999).
Their modes of action are based on interaction with the microbial cellular lipid bilayer and eventual disintegration of the
cell membrane (Hwang et al. 2010). In addition to antimicrobial
action, AMPs have demonstrated diverse biological effects, all
of which participate in the control of infectious and inflammatory diseases (Kamysz et al. 2003, Guan´ı-Guerraa et al. 2010).
Therefore, there are several potential applications of AMPs to
develop new strategies or substitute the existing ones for more
sustainable and promising future of these peptides.

Calcium Binding Peptides
Calcium is an important mineral for the human body. Generally,
the principal sources of calcium are milk and other diary
products (Anderson and Garner 1996). Casein phosphopeptides
(CPP) obtained after intestinal digestion of casein enhance bone

calcification (Tsuchita et al. 1993). CPP have the capacity of
chelating Ca and preventing the precipitation of Ca phosphate
salts, resulting in the increased availability of soluble Ca for
absorption (Berrocal et al. 1989, Yuan and Kitts 1994). However, some groups of people do not drink milk due to lactose
indigestion and intolerance. Therefore, peptides from other
sources, especially from aquatic source can be an alternative
for use in food to increase Ca solubility and bioavailability.
Bone oligopeptides with high affinity to calcium was prepared
from hoki bone with the aid of tuna intestine crude enzyme,
which was able to degrade hoki bone matrices comprising
collagen, noncollagenous proteins, carbohydrates, and minerals
(Jung et al. 2005). Fish bone phosphopeptides (FBP) containing
23.6% phosphorus and a molecular weight (MW) of 3.5
kDa could solubilize calcium (Jung et al. 2005). Fish bone
peptide II (FBP II) with a high content of phosphopeptide
from hoki bone could inhibit the formation of insoluble Ca
salts. The levels of femoral total Ca, bone mineral density, and
strength were increased in ovariectomized rats fed with FBP

691

II diet (Jung et al. 2006b). Alaska pollack backbone peptide,
Val–Leu–Ser–Gly–Gly–Thr–Thr–Met–Ala–Met–Ala–Met–Tyr
–Thr–Leu–Val with the MW of 1442 Da, prepared using
pepsin hydrolysis showed affinity for calcium ions on the
surface of hydroxyapatite crystals. The peptide could solubilize
calcium at levels similar to that by casein phosphopeptide (Jung
et al. 2006a). Furthermore, calcium binding peptide derived
from pepsinolytic hydrolysate of hoki frame was identified as
Val–Leu–Ser–Gly–Gly–Thr–Thr–Met–Tyr–Ala–Ser–Leu–Tyr–

Ala–Glu with MW of 1561 Da (Jung and Kim 2007). Therefore, fish bone oligophosphopeptide could be used as the
nutraceutical to increase the absorption of calcium.

Anticoagulant Activity
An anticoagulant therapy is a type of medication that may be
used to prevent blood from coagulating or clotting. Blood clotting could be life-threatening for patients with atherosclerosis
and related cardiovascular diseases, the leading cause of heart
attack, stroke, and death (Libby 2002, Levi et al. 2003, Schultz
et al. 2003). The mainstays of clinical anticoagulant treatments
are heparin, which is a cofactor of plasma-derived and naturally
occurring inhibitors of thrombin, and coumarins that antagonize
the biosynthesis of vitamin K-dependent coagulation factors.
Although effective and widely used, heparins and coumarins
have practical limitations because their pharmacokinetics
and anticoagulation effects are unpredictable, with the risk
of many undesirable side effects, such as hemorrhaging and
thrombocytopenia resulting in the need for close monitoring of
their use. More seriously, heparins are involved in many aspects
of cellular physiology (Kakkar 2003), making their long-term
uses as anticoagulants plagued with potential side effects. The
anticoagulant marine-derived bioactive peptides have rarely
been reported, but have been isolated from marine organisms
such as marine echiuroid worm (Jo et al. 2008), starfish
Koyama et al. 1998), and blue mussel (Jung and Kim 2009).
Moreover, marine anticoagulant proteins have been purified
from blood of ark shell (Jung et al. 2001) and yellowfin sole
Rajapakse et al. 2005a). The anticoagulant activity of the above
peptides has been determined by prolongation of activated
partial thromboplastin time (APTT), prothrombin time PT) and
thrombin time assays and the activity was compared with heparin, the commercial anticoagulant. The anticoagulant peptide

Gly–Glu–Leu–Thr–Pro–Glu–Ser–Gly–Pro–Asp–Leu–Phe–Val–
His–Phe–Leu–Asp–Gly–Asn–Pro–Ser–Tyr–Ser–Leu–Tyr–Ala–
Asp–Ala–Val–Pro–Arg, isolated from marine echiuroid worm,
effectively prolonged the normal clotting time on APTT
from 32.3 ± 0.9 to 192.2 ± 2.1 seconds in a dosedependent manner (Jo et al. 2008). This peptide binds
specifically with clotting factor FIXa, a major component
of the intrinsic tenase complex. An anticoagulant peptide
Glu–Ala–Asp–Ile–Asp–Gly–Asp–Gly–Gln–Val–Asn–Tyr–Glu
–Glu–Phe–Val–Ala–Met–Met–Thr–Ser–Lys, derived from blue
mussel, showed prolongation of 321 ± 2.1 seconds clotting
time on APTT (from 35.3 ± 0.5 seconds of control) and 81.3 ±
0.8 seconds clotting time on TT (from 11.6 ± 0.4 seconds
of control), respectively (Jung and Kim 2009). In addition, a


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protein derived from the blood of arch shell, prolonged the
APTT from a 32 seconds control clotting time to 325 seconds.
(Jung et al. 2001). These marine-derived anticoagulant peptides
have potential for use as functional ingredients in nutraceuticals
or pharmaceuticals.

Immunomodulatory Activity
There has been increased interest in the study of the relationship between nutrition and immunity due to the hypothesis
that consumption of specific foods may reduce susceptibility
for the development and/or progression of immunological diseases. Many immunomodulatory peptides are derived from milk
proteins. Some of these peptides have been found to be immunoinhibitory, while others are immunostimulatory in action.
Furthermore, it has been found that oral administration of the
peptides is a highly effective way to induce the desired immunomodulatory effect, even in the absence of any transport
agents such as delivery vehicles. In addition, it has also been
found that the amount of peptide required to produce the therapeutic effect by oral delivery can be significantly lower than
that required to produce a similar effect when the peptide is delivered by parenteral injection. The immunomodulatory effects
of marine-derived bioactive peptides have rarely been reported.
Gildberg et al. (1996) reported that four acid peptide fractions
from cod stomach hydrolysate were shown to possess an ability
to stimulate leucocyte superoxide anion production in Atlantic
salmon (S. salar). Increasing the production of reactive oxygen metabolites or promoting the phagocytosis and pinocytosis
in macrophages can enhance the nonspecific immune defense
system. It has been suggested that acid-derived cod protein hydrolysate peptide fractions might be useful as an adjuvant in fish
vaccine and as an immune stimulant in fish feed. Immunomodulatory effects of marine oligopeptide preparation (MOP) from
Chum Salmon (Oncorhynchus keta) were also reported by Yang
et al. (2009b). Female ICR mice (6–8 weeks old) were administered the MOP for 4 weeks with the dose of 0, 0.22, 0.45, and
1.35 g/kg/body weight. In comparison with the control group,

MOP could significantly enhance the capacity of lymphocyte
proliferation induced by the mitogen concanavalin A, the number of plaque-forming cells, natural killer (NK) cell activity, the
percentage of CD4+ T helper (Th) cells in spleen and the secretion of Th1 (interleukin (IL)-2, IFN-γ ) and Th2 (IL-5, IL-6)
type cell cytokines. Nevertheless, no significant differences in
weight gain, lymphoid organ indices, and phagocytosis capacity
were observed. In irradiation-treated mice (Yang et al. 2010),
MOP significantly increased the survival rate and prolonged the
survival times for 30 days after irradiation, and lessened the
radiation-induced suppression of T- or B-lymphocyte proliferation, resulting in the recovery of cell-mediated and humoral
immune functions. This effect may be produced by augmentation of the relative numbers of radioresistant CD4+ T cells,
enhancement of the level of immunostimulatory cytokine, IL12, reduction of the level of total cellular NF-κB through the
induction of IκB in spleen, and inhibition of the apoptosis of
splenocytes. In addition, it is proposed that MOP can be used as

an ideal adjuvant therapy to alleviate radiation-induced injuries
in cancer patients.
The oligopeptide-enriched hydrolysates from oyster (C. gigas), produced using the protease from Bacillus sp. SM98011
possessed antitumor activity and immunostimulating effects of
the oyster hydrolysates in BALB/c mice (Wang et al. 2010). The
growth of transplantable sarcoma-S180 was obviously inhibited
in a dose-dependent manner in BALB/c mice given the oyster hydrolysates. Mice receiving 0.25, 0.5, and 1 mg/g of body
weight by oral gavage had 6.8%, 30.6%, and 48% less tumor
growth, respectively. Concurrently, the weight coefficients of the
thymus and the spleen, the activity of NK cells, the spleen proliferation of lymphocytes and the phagocytic rate of macrophages
in S180-bearing mice significantly increased after administration of the oyster hydrolysates. These results demonstrated that
oyster hydrolysates produced strong immunostimulating effects
in mice, which might result in its antitumor activity. The antitumor and immunostimulating effects of oyster hydrolysates
prepared in this study reveal its potential for tumor therapy and
as a dietary supplement with immunostimulatory activity.


Gastrin/CCK-Like Activity
Different authors have reported the presence of gastrin/CCKlike molecules in protein hydrolysates from fish by-products
(Cancre et al. 1999, Ravallec-Ple and Van Wormhoudt 2003).
These molecules are the only known members of the gastrin
family in humans, and could have a positive effect on food intake
in humans and fish species in aquaculture. Gastrin is a gastric
hormone that stimulates postprandial gastric acid secretion and
epithelial cell proliferation. In humans, there are two different
gastrins, one with 17 and one with 34 amino acids residues.
CCK is a group of peptides that controls the emptying of the
gallbladder, as well as pancreatic enzyme secretion. It is also a
growth factor, and regulates intestinal motility, satiety signaling,
and inhibition of gastric acid secretion (Rehfeld et al. 2001).
Both gastrin and CCK inhibit food intake and share a common
COOH-terminal pentapeptide amide that also includes the sequences essential for biological activity. The incorporation of
protein hydrolysates including gastrin/CCK-like molecules in
functional foods could be of interest for controlling appetite,
food intake, and obesity. The production of protein hydrolysates
including CCK-like molecules could also be useful in the treatment of paralytic ileus, for the removal of small concrements
from the common bile duct and also for the improvement of
pancreatic insufficiency caused by long-term parenteral nutrition
or chronic pancreatitis (Rehfeld 2004). Above and beyond the
stimulation of satiety through bioactive peptides, lipid-lowering
effects as well as increasing metabolic rate are also beneficial in
fighting obesity.

PRODUCTION OF BIOACTIVE PEPTIDES
Enzyme Hydrolysis
Marine bioactive peptides have widely been produced by enzymatic hydrolysis of proteins derived from marine animals
ˇ zyt˙e

(Zhao et al. 2007, Je et al. 2008, Sheih et al. 2009, Sliˇ


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36 Biological Activities and Production of Marine-Derived Peptides

et al. 2009). These newly formed peptides can retain the biological properties of the native protein or can show new properties.
Using appropriate proteolytic enzymes through the control of
process parameters such as pH, time, and enzyme/substrate ratio, it is possible to produce hydrolysates whose components
may present some interesting biological properties. Especially
for food and pharmaceutical industries, the enzymatic hydrolysis
method is preferred because of lack of residual organic solvents
or toxic chemicals in the products. Hydrolysis can be generally
carried out in a thermostatically stirred-batch reactor in which
the hydrolysis conditions (pH, temperature, enzyme concentration, and stirring speed) are adjusted in order to optimize the
activity of the enzyme used. An initial mixing is usually done
to adjust the pH and temperature to the desired values. To obtain the hydrolysate with high stability with less pro-oxidants,

the raw materials can be washed to remove heme protein and
lipids (Kristinsson 2007). In addition, antioxidants can be added
before hydrolysis. To enhance the hydrolysis process, the raw
materials may be subjected to size reduction by homogenization. The bone, scales, or skin, which may interfere with enzymatic hydrolysis, should be removed. Thereafter, the selected
protease is added into the mixture containing the protein substrate, which is previously adjusted to pH to the desired value.
After hydrolysis, the reaction is terminated by inactivation of
protease by heat treatment or pH adjustment. The combination
of pH and temperature to denature the protease used is another
approach to avoid the harsh condition, which may affect the resulting hydrolysate or peptides. The reaction mixture containing
the peptides as well as the unhydrolyzed debris is centrifuged
or filtered. The supernatant or filtrate is concentrated or dried.
The hydrolysate can be an excellent source of peptides with
functionalities and bioactivities, which are determined by the
types of protease, pretreatment of raw materials, the condition
of hydrolysis, and so on. In order to enhance the bioactivity of
peptides produced, several approaches have been implemented
such as the use of multistep hydrolysis with different proteases
(Phanturat et al. 2010). Moreover, it is possible to obtain serial
enzymatic digestions in a system using a multistep recycling
membrane reactor combined with ultrafiltration membrane system to separate marine-derived bioactive peptides (Jeon et al.
1999, Byun and Kim 2001, Kim and Mendis 2006). This membrane bioreactor technology equipped with ultrafiltration membranes is recently emerging for the development of bioactive
compounds and considered as a potential method to utilize marine proteins as value added nutraceuticals with beneficial health
effects.

Choices of Enzyme
Different proteases exhibit varying specificities and reaction
rates in the hydrolysis of polypeptide chains. Many authors,
in view of the economic interest in the recovering of protein
from poorly studied species of fish, have compared some commercial proteases in order to test the most suitable one for the
substrate employed. The most common commercial proteases

reported are both from plant source such as papain (Quaglia
and Orban 1987a, 1987b, Hoyle and Merritt 1994, Shahidi et al.

693

1995) or from animal origin such as pepsin (Viera et al. 1995)
and chymotrypsin or trypsin (Simpson et al. 1998). Enzymes of
microbial origin have been also applied to the hydrolysis of fish.
In comparison to animal- or plant-derived enzymes, microbial
enzymes offer several advantages including a wide variety of
available catalytic activities, greater pH, and temperature staR
bilities (Diniz and Martin 1997). Generally, Alcalase 2.4 L
have been repeatedly favored for fish hydrolysis due to the high
degree of hydrolysis (DH) that can be achieved in a relatively
short time under moderate conditions compared to neutral or
acidic enzymes (Hoyle and Merritt 1994, Shahidi et al. 1994,
Diniz and Martin 1996, Martin and Porter 1995, Benjakul and
Morrissey 1997).
Alternatively, endogenous proteases in the muscle or internal
organs can serve as an important source of proteases, which can
be used for production of peptides. Khantaphant and Benjakul
(2008) used the ammonium sulphate fraction of fish pyloric
caeca extract for preparation of gelatin hydrolysate from brownstripe red snapper skin with 2,2-diphenyl-1-picrylhydrazyl
(DPPH) and 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic
acid) (ABTS) radical scavenging activities. In addition, Phanturat et al. (2010) used the pyloric caeca extract from bigeye
snapper (Priacanthus macracanthus) for preparation of gelatin
hydrolysate with antioxidative activity. The antioxidative peptide of gelatin hydrolysate produced had MW of 1.7 kDa. To
maximize autolysis or hydrolysis, optimal pH and temperature
are implemented, thereby enhancing the cleavage of peptides.
Nevertheless, it could be difficult to control the DH or obtaining the desired peptides by autolysis or the use of endogenous

protease.
The impact of the enzyme’s specificity is a key factor influencing both the characteristics of hydrolysates and the nature
and composition of peptides produced. Proteolysis can operate
either sequentially, releasing one peptide at a time, or through the
formation of intermediates that are further hydrolyzed to smaller
peptides as proteolysis progresses, which is often termed “the
zipper mechanisms” (Panyam and Kilara 1996). Depending on
the specificity of the enzyme, environmental conditions and the
extent of hydrolysis, a wide variety of peptides can be preferentially generated.
Hydrolytic curves reported for enzymatic hydrolysis of different protein substrates by protease generally exhibited an initial
fast reaction followed by a slowdown. With regards to the effect
of the enzyme concentration, it was found that the DH increased
with higher enzyme concentrations. Less important increases
were found with enzyme treatment at higher concentration. The
exact concentration of enzymes required to hydrolyze the substrate at a required DH could be calculated when log10 (enzyme
concentration) versus DH were plotted (Benjakul and Morrissey
1997). Diniz and Martin (1996) used response surface analysis
to study the effects of pH, temperature, and enzyme/substrate
ratio on the DH of Dogfish proteins. The polynomial model they
proposed was well adjusted to the experimental data and was
sufficiently accurate for predicting the DH for any combination
of independent variables within the ranges studied. However,
it should be realized that the higher DH could also negatively
affect the biological activity of peptide.