BLBS102-c36 BLBS102-Simpson March 21, 2012 18:47 Trim: 276mm X 219mm Printer Name: Yet to Come
36 Biological Activities and Production of Marine-Derived Peptides 693et al. 2009). These newly formed peptides can retain the biolog-
ical 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 ra-
tio, 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 concentra-
tion, 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 ob-
tain 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 homogeniza-
tion. The bone, scales, or skin, which may interfere with enzy-
matic hydrolysis, should be removed. Thereafter, the selected
protease is added into the mixture containing the protein sub-
strate, 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 re-
sulting 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 sys-
tem to separate marine-derived bioactive peptides (Jeon et al.
1999, Byun and Kim 2001, Kim and Mendis 2006). This mem-
brane bioreactor technology equipped with ultrafiltration mem-
branes is recently emerging for the development of bioactive
compounds and considered as a potential method to utilize ma-
rine proteins as value added nutraceuticals with beneficial health
effects.Choices of EnzymeDifferent 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 com-
mercial 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.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 sta-
bilities (Diniz and Martin 1997). Generally, Alcalase©R
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 brown-
stripe 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, Phan-
turat et al. (2010) used the pyloric caeca extract from bigeye
snapper (Priacanthus macracanthus) for preparation of gelatin
hydrolysate with antioxidative activity. The antioxidative pep-
tide 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 obtain-
ing the desired peptides by autolysis or the use of endogenous
protease.
The impact of the enzyme’s specificity is a key factor influ-
encing 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 preferen-
tially generated.
Hydrolytic curves reported for enzymatic hydrolysis of differ-
ent 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 sub-
strate 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.