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Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319

“ST26943”, 2nd International Conference on Agricultural and Food Engineering, CAFEi2014”

Characterization of Fish Protein Hydrolysate from Tilapia

(Oreochromis niloticus) by-Product
Jumardi Roslana, Khairul Faezah Md. Yunosa, Norhafizah Abdullahb, Siti Mazlina
Mustapa Kamala,*
a
Department of Process and Food Engineering,Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor,Malaysia
b
Department of Chemical and Environmental Engineering,Faculty of Engineering, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor,Malaysia

Abstract

Several by-products, such as skin, bones, frames, heads and tails are produced during the processing of tilapia fish (Oreochromis
niloticus) into fillets. These by-products can be converted into tilapia by-producthydrolysate powder(TBHP) with beneficial
functional properties. The TB hydrolysate produced through enzymatic hydrolysis was characterized in term of chemical and
amino acid (AA) compositions, molecular weight distribution and ACE inhibition activity. TBHP has showna good nutritional
value with high protein content (62.71%)and essential amino acids (199.15 mg/g).High value of ACE inhibition activity
(88.26%) in TBHP might be due to the presence of low molecular weight peptides.

© 2014 The Authors. Published

© Published by
by Elsevier
Elsevier B.V.
B.V. This is an open access article under the CC BY-NC-ND license
Peer-review under responsibility of the Scientific Committee of CAFEi2014.
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of the Scientific Committee of CAFEi2014
Keywords:Alcalase; amino acid composition; enzymatic hydrolysis; peptides; tilapia by-product; tilapia by-product hydrolysate
____________________________________________________________________________________________________________________

1. Introduction

Tilapia is a popular freshwater fish due to their nutritional benefits and wide availability. Intensive tilapia
farming in Asia has increased steadily and become an important source of fish within the last few years (Murthy et
al., 2011). The major tilapia species farmed in Malaysia is red tilapia (Oreochromis sp.), accounting for about 81%

* Corresponding author. Tel.: +603-89466294; fax: +603-89464440.

E-mail address: smazlina@upm.edu.my

2210-7843 © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Peer-review under responsibility of the Scientific Committee of CAFEi2014
doi:10.1016/j.aaspro.2014.11.044
 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319 313

(33,260 tons) of total tilapia production (Ng, 2009; Department of Fisheries, 2011). Due to the growing demand for
tilapia fillet in producing fish-based food products, large amounts of by-product have been generated. These by-
products are usually discarded and cause numerous environmental problems (Arvanitoyannis and Kassaveti, 2008)
and contain considerable amounts of proteins that are known to possess high nutritional value with respect to
essential amino acid (AA) composition (Venugopal et al., 2008) and rich protein content (Arnesen and Gildberg,
2006) varying from 15-60% (Valdimarson and James, 2001;Je et al., 2004; Jung et al., 2006, Fahmi et al., 2004;
Sathivel et al. 2004). Recovery of proteins from these by-products and conversion to high value products, such as
bioactive peptides is a very exciting and promising alternative. In recent years, the bioactive peptide production
from fish by-products has received a growing attention due to their physiological functions such as antihypertensive
and antioxidative activities which are suitable for applications in healthcare and pharmaceutical products (Je et al.,
2004; Jung et al., 2006).
In comparison to the chemical hydrolysis, enzymatic hydrolysis is preferable due to several advantages, such as
mild reaction conditions, low undesirable products, and high product quality and yield. Alcalase has been found to
be a highly efficient enzyme in fish protein hydrolysis (Benjakul and Morrissey, 1997; Adler-Nissen, 1986; Aspmo
et al., 2005) due to its ability to attain a high degree of hydrolysis in a relatively short period under mild conditions
(Aspmo et al., 2005) and can produce fish protein hydrolysate with good nutritional and functional properties
(Benjakul and Morrissey, 1997; Amiza et al., 2011; See et al., 2011; Kristinsson and Rasco, 2000; Wasswa et al.,
2007). A few studies have been conducted on the enzymatic hydrolysis of fish by-products using alcalase enzyme
such as pacific whiting solid waste (Benjakul and Morrissey, 1997), catfish frames (Amiza et al., 2011), salmon skin
(See et al., 2011), and Sardinelle by-product (Bougatef et al., 2008). However, there is no reported research on the
nutritional value and size distribution of tilapia by-product (TB) hydrolysate and their potential as antihypertension.
The objective of this study was to investigate the characteristics of TB protein hydrolysate in terms of chemical
and amino acids compositions, molecular weight distribution as well as the angiotensin converting enzyme (ACE)
inhibition activity of the TB hydrolysate. These findings are important in providing useful information regarding TB
protein hydrolysate that can be useful for various applications particularly for food, healthcare, and pharmaceutical
products.

2. Materials and methods

Fresh red tilapia (Oreochromis niloticus) was purchased from a local wet market in Selangor, Malaysia and
washed, eviscerated and hand filleted with the muscle and by-product (head, frames, and tail) separated. Tilapia by-
products (TB) was then minced using a blender, packed in polyethylene bags, frozen, and stored at -20qC until
further use.

2.1Proximate analysis
The proximate compositions of TB and tilapia by-product hydrolysate powder (TBHP) were determined
according to the AOAC method (AOAC, 2005). Moisture was determined by drying a sample in an air oven for 5 h
at 105qC. The Kjeldahl method was used to determine the crude protein content using Tecator Kjeltec protein
analyzer (FOSS, Hillerod, Denmark). The fat content of TB and TBHP were determined using the soxhlet method.
Ash was determined by heating the samples in a furnace at 550qC for 8–12 h.

2.2. Enzymatic hydrolysis reaction

Minced TB was thawed overnight in a cold room (4qC), then 15% w/v of minced TB was mixed with 50 mL of
50 mM phosphate buffer solution (PBS, pH 7.5) and the mixture preincubated at 60 oC for 20 min prior to adding the
2.5% w/w alcalase enzyme to initiate the enzymatic hydrolysis reaction. After 120 min of hydrolysis, the reaction
was terminated by heating the mixture in a water bath at 90qC for 15 min with occasional agitation. The mixture was
immediately cooled on ice, sedimented at 10000 rpm for 20 min in a refrigerated high speed centrifuge and the
supernatant was collected and the degree of hydrolysis (DH) was measured using OPA method (Nielsen et al., 2001;
Zarei et al., 2012). TB hydrolysate was then freeze dried and characterized.
314 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319

2.3. Amino acid analysis

The AA compositions of TB and TBHP were identified using a Waters-Pico Tag Amino Acid Analyzer System
(Waters 2690/5,Waters Corp., Milford, MA, USA) (Shamloo et al., 2012). The samples were hydrolysed, then
derivatised prior to the injection for analysis. The hydrolysis was carried out by mixing 0.2 g sample with 5 ml 6 N
HCl at 110°C for 24 h. Upon completion, 4 ml of the internal standard (AABA) was added to the residues and then
made up to 100 ml by deionised water.

2.4. Tricine SDS-PAGE analysis

Tricine-SDS-PAGE was performed according to a method described by Schagger and von Jagow (1987) with a
slight modification. Briefly, 4% of stacking and 16% of separating gels were prepared using gel buffer (3M Tris-
HCl, 0.3% SDS, pH 8.45), acrylamide/bisacrylamide (48% acrylamide, 1.5% bisacrylamide), distilled water,
glycerol, ammonium persulfate and TEMED. Sample solution was mixed with the sample buffer (1M Tris-HCl, pH
6.8, containing 20% SDS, glycerol and Coomassie Brilliant Blue) and heated at 90°C for 10 min before loading. 8 μl
of aliquots were loaded into individual wells. Protein standards (1.06 kDa to 26.6 kDA) were also run on the gels.
After electrophoresis, the gels were mixed with fixing solution (50% methanol, 10% acetic acid), staining solution
(Coomassie Blue, 10% acetic acid) and destaining solution (10% acetic acid) orderly. Once the desired bands were
appeared, then the samples were compared with known bands of protein standards.

2.5. ACE inhibitory activity of TB hydrolysate

ACE inhibition activity was measured by monitoring the release of hippuric acid (HA) from the hydrolysis of the
substrate hippuryl-histidyl-leucine (HHL) using ACE solution according to the method described by Jimsheena and
Gowda (2009) with a slight modification. Firstly, HHL was prepared in 0.05 M of potassium phosphate buffer (pH
8.2) containing of 0.3 M NaCl. About 125 μL of TB hydrolysate was mixed with 50 μL of HHL solution and 25 μL
of ACE solution and then incubated at 37ºC for 30 min. The reaction was terminated by adding 200 μL of 1 M HCl.
Then, 400 μL of pyridine was added followed by 200 μL of BSC. The solution was slowly mixed using a vortex
mixer and cooled on ice. The yellow colour developed was measured at 410 nm using spectrophotometer. The
percentage of ACE inhibition was calculated based on the absorbance value of the samples:

(1)

where A = absorbance of solution without sample, B = absorbance of solution with sample, C = Absorbance of
blank solution (without ACE solution and sample).

2.6. Statistical analysis

Proximate analyses and AA compositions were determined by ANOVA and Duncan’s multiple range test, using
the SAS program (SAS, 1989). Standard deviation was calculated using the same software.

3. Results and discussion

3.1. Chemical composition of TB and TBHP

The chemical composition of food is very important not only from the perspective of human nutritional health,
but also for assessment of the potential development and application of food materials in a food system. Proximate
analyses were conducted on TB and TBHP (Table 1) and the results showed that TB contained 14.60 ± 0.30%crude
protein, 66.57 ± 0.39% moisture, 5.50 ± 0.30% fat, and 8.93 ± 0.46% ash. Several studies have shown that the crude
protein content of fish by-products varies from 8 to 35%, TB was within this range, indicating that it possessed
considerable protein content. The TB moisture content was in agreement with results obtained by several authors for
 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319 315

catfish frame (Amiza et al., 2011), salmon skin (See et al., 2011), catla visceral waste (Bhaskar et al., 2008), and
Alaska pollock (Hou et al., 2011). Relatively low fat content was observed in TB compared with other fish by-
products such as catla visceral waste (Bhaskar et al., 2008) and catfish frame (12.8% and 68.21%, respectively)
(Amiza et al., 2011). TB showed relatively higher ash content when compared with other fish by-products such as
cod head (Arnesen and Gildberg, 2006), catfish frame (Amiza et al., 2011) and catla visceral waste (6.8, 7.08, and
2.50%, respectively) (Bhaskar et al., 2008), but showed slightly lower content compared with Alaska pollock frame
(14.99%) (Hou et al., 2011). High ash content is largely contributed by bones in every part of fish by-product
including head, fin, tail and frame (Batista et al., 2010).
When compared with TB, significant differences in TBHP’s protein, moisture, fat and ash content were observed
(P<0.05), with protein the major component, followed by ash, moisture, and fat (62.71 ± 0.29, 25.34 ± 0.94, 6.48 ±
0.40, 0.08 ± 0.05%, respectively). The high protein content was due to protein solubilization during hydrolysis,
removal of insoluble undigested non-protein substances and partial removal of fats after hydrolysis (Benjakul and
Morrissey, 1997). However, protein content in TBHP was lower compared with hydrolysates from other fish
species’s by-products, such as salmon skin (See et al., 2011), pacific whiting solid waste (Benjakul and Morrissey,
1997), and catfish frame (Amiza et al., 2011) (89.53, 82.25, and 65.05%, respectively). The percentage of
solubilized protein was found to depend on the amount of fats in the raw material, such that raw materials containing
the highest amount of fats produce the lowest percentage of solubilized protein (Slizyte et al., 2005).
Several studies have reported the fat content of various fish protein hydrolysates to be <5% (Benjakul and
Morrissey, 1997; Kristinsson and Rasco, 2000; Wasswa et al., 2007; Bhaskar et al., 2008). Here, the lower TBHP fat
content was due to the removal of the fat layer after hydrolysis. During hydrolysis, muscle cell membranes tend to
round up and form insoluble vesicles, leading to removal of membrane structured lipids during centrifugation to
recover the soluble protein (Sathivel et al., 2005).The ash content of fish protein hydrolysates has been reported in
the range of 0.45% - 27% of total composition (Benjakul and Morrissey, 1997; Bhaskar et al., 2008). The ash
content of TBHP was within the range indicated and this relatively high ash content might have been because of the
addition of sodium phosphate buffer during enzymatic processing (Benjakul and Morrissey, 1997; See et al., 2011).
Most studies have demonstrated that protein hydrolysates from various fish proteins contain moisture <10%
(Kristinsson and Rasco, 2000; Wasswa et al., 2007; Bhaskar et al., 2008; Chalamaiah et al. 2010). Here, the TBHP
moisture content was 6.48 ± 0.40% in agreement with the literature and possibly due to the sample type and
temperatures employed during evaporation, in which the sample lost most of its moisture (Bueno-Solano et al.,
2009).

Table 1. Proximate analysis of TB and TBHP.

Component Tilapia by-product (TB) Tilapia by-product hydrolysate powder (TBHP)

Moisture content (%) 66.57±0.39a 6.48±0.40b
b
Crude protein (%) 14.60±0.30 62.71±0.29a
a
Fat (%) 5.50±0.30 0.08±0.05b
b
Ash (%) 8.93±0.46 25.34±0.94a
All data expressed as mean ± S.D. (n=3).
Means within each row sharing same upper case were not significantlt different (P>0.05).

3.2. Tricine SDS-PAGE of TBHP

Characterization of the molecular weights of TBHP by SDS-PAGE showed the presence of strong bands ranging
between 3.5 – 26.6 kDa, which indicated that alcalase enzyme was able to produce small-sized peptides in 120 min
(Fig. 1). Several studies have shown alcalase’s ability to produce low molecular weight peptides via a high degree of
hydrolysis (Benjakul and Morrissey, 1997; Liaset et al., 2000; Lalasidis et al., 1978). According to Bhaskar et al.
(2008), fish protein hydrolysate with high nutritional values should be rich in low molecular weight peptides, and
the successful production of such desired peptides from TB indicated its potential application in functional food
products.
316 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319

Fig. 1. SDS PAGE Analysis of TBHP.

3.3. AA compositions of TB and TBHP

The AA compositions of fish protein hydrolysates are important due to their nutritional value and influence on
functional properties (Santos et al., 2011). Tables 2 shows the AA compositions of TB and TBHP. TB was most rich
in glutamic acid followed by glycine, lysine and alanine (18.61 mg/g, 15.43 mg/g, 10.53 mg/g, and 10.38 mg/g,
respectively). The lowest AA concentration in TB was cysteine which was similar to reports by several authors that
cysteine was the lowest AA in fish protein (Benjakul and Morrissey, 1997; See et al., 2011; Bhaskar et al.,
2008).The TBHP AA composition was slightly different from TB such that glutamic acid was the largest component
in TBHP followed by glycine, aspartic acid, and alanine (79.60 mg/g, 67.82 mg/g, 45.85 mg/g, and 45.64 mg/g,
respectively). The TBHP total AA was significantly higher, by 4 fold, than in TB. The total AAs in TBHP was
significantly higher than in TB (199.15 ± 2.29 mg/g and 48.82 ± 0.57 mg/g, respectively), lysine and leucine the
highest essential AA in both samples which indicated that TB and TBHP could contribute significantly to dietary
essential AA content. According to Bhaskar et al. (2008), the nutritive value of any ingredient depends on the
protein’s capacity to meet an organism’s requirements based on the essential AAs. Among all AAs, aspartic and
glutamic acid have been found to be the most abundant AAs in most reported fish protein hydrolysates. Fish protein
hydrolysates have been reported to exhibit variation in their AA composition (Benjakul and Morrissey, 1997;
Wasswa et al., 2007; Bhaskar et al., 2008) due to several factors such as raw material, enzyme source, and
hydrolysis conditions (Klompong et al., 2009). Many researchers have extensively studied peptides derived from
fish proteins as functional foods and particularly in the synthesis of angiotensin converting enzyme (ACE) inhibitory
(Je et al., 2004). There is a relationship between AA sequences and ACE inhibition activity, such that the most
potent and specific peptide inhibitors have similar structures and ACE activity is strongly influenced by the C-
terminal tripeptide sequence. Tripeptides with Trp, Tyr, Phe, Pro and a hydrophobic AA at the C-terminal possess
 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319 317

ACE inhibitory activity because of interaction with three subsites at the ACE active site (Pihlanto, 2000). In the
present study, TBHP was found to contain higher proportions of hydrophobic AAs (278.85 ± 0.17 mg/g), which
might contribute to ACE inhibitory activity.

Table 2. AA compositions in TB and TBHP (in mg/g of protein).

Amino acid Tilapia by-product (TB) Tilapia by-product hydrolysate protein (TBHP)
Aspartic acid 9.90 ± 0.27 45.85 ± 0.50
Glutamic acid 18.61 ± 0.05 79.60 ± 0.25
Serine 5.74 ± 0.11 23.31 ± 0.53
Glycine 15.43 ± 0.16 67.82 ± 0.96
Alanine 10.38 ± 0.04 45.64 ± 0.27
Cysteine 0.09 ± 0.06 0.86 ± 0.54
Tyrosine 4.07 ± 0.11 15.86 ± 0.28
Arginine 9.98 ± 0.13 32.22 ± 0.04
Proline 9.61 ± 0.05 41.84 ± 0.48
Valine* 6.07 ± 0.04 24.64 ± 0.33
Methionine* 3.37 ± 0.08 14.84 ± 0.05
Isoleucine* 5.27 ± 0.13 21.47 ± 0.03
Leucine* 9.41 ± 0.19 39.22 ± 0.97
Phenylalanine* 5.51 ± 0.05 22.37 ± 0.55
Histidine* 2.95 ± 0.08 11.68 ± 0.69
Lysine* 10.53 ± 0.17 40.47 ± 0.47
Threonine* 5.37 ± 0.01 24.48 ± 0.73
Total AAs 132.61 ± 0.07a 552.13 ± 0.41b
a
Essential AAs 48.82 ± 0.57 199.15 ± 2.29b
a
Hydrophobic AAs 65.74 ± 0.28 278.85 ± 0.17b
All data expressed as mean ± S. D (n=2).
*Essential amino acid.
Means within each row sharing same upper case were not significantlt different (P>0.05).

3.4. ACE inhibition of TBHP

The enzymatic hydrolysis reaction using alcalase enzyme on TB was monitored by the OPA method and 20.20%
of DH was achieved after hydrolyzing for 120 min. This has been shown that hydrolysis reaction using alcalase
enzyme can achieve high DH value in a short time with various peptides sizes. Tricine SDS-PAGE (Fig. 1) analysis
revealed that smaller peptides (1 – 26.6 kDa) were successfully produced and has a great potential to be used as an
antihypertensive. Hypertension is a condition in which the arteries have persistently elevated the blood pressure. It is
caused by the Angiotensin I-converting enzyme (ACE) that plays an important role in regulating blood pressure.
One of the ways to control hypertension is to incorporate antihypertensive peptides in hypertension treatments (Kim
et al., 2001). Several studies have demonstrated that fish protein hydrolysates have the ability to inhibit the ACE
activity (Je et al., 2004; Jung et al., 2006; Kim et al., 2001). In this study, TB hydrolysate produced was evaluated
for their effect on ACE activity. The TB hydrolysate was found to demonstrate a high ACE inhibition activity with
value of 88.26 ± 0.83% and 93.99 mg/ml of soluble peptide. The high ACE inhibition activity in TB hydrolysate
may be attributed to the low molecular weight peptides produced during hydrolysis. Generally, the ACE inhibition
activity is higher for smaller peptide fractions. This is an agreement with study reported by Kim et al. (2001) who
found that a low molecular weight fraction (<1 kDa) of bovine skin gelatin hydrolysates had higher ACE inhibition
activity than the higher-molecular-weight fraction. In addition, protein hydrolysate with molecular weights below 5
kDa in yellowfin sole had higher ACE inhibition activity than the higher-molecular-weight fractions (Jung et al.,
2006). This result indicated that enzymatic hydrolysis using alcalase enzyme on TB in a short duration was
sufficient to obtain very high ACE inhibition activity.
318 Jumardi Roslan et al. / Agriculture and Agricultural Science Procedia 2 (2014) 312 – 319

4. Conclusions

The chemical and amino acid composition of tilapia by-product hydrolysate powderindicate it has good
nutritional value with respect to the high protein content (62.71%) and essential AA composition (199.15 mg/g).
High ACE inhibitory activity (88.26%)in TBHP may be attributed tothe existence of low molecular weight peptides
particularly peptides with molecular weight below than 5 kDa.This study shows the potential applications of TBHP
in preventing hypertension.

Acknowledgements

The authors acknowledge the financial support for this study by the Research University Grant Scheme
(Universiti Putra Malaysia) and Science Fund Research Grant from Ministry of Science, Technology and Innovation
(MOSTI), Malaysia.

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Accepted for poster presentation in CAFEi2014 (December 1-3, 2014 – Kuala Lumpur, Malaysia) as paper 267.