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JUMARDI ROSLAN, SITI MAZLINA MUSTAPA KAMAL*, KHAIRUL FAEZAH MD. YUNOS
& NORHAFIZAH ABDULLAH
ABSTRACT
Fish protein hydrolysate was prepared from tilapia muscle using commercial Alcalase enzyme. Optimization of
enzymatic hydrolysis process for preparing tilapia muscle protein hydrolysates (TMPH) was performed by employing
central composite design (CCD) method of response surface methodology (RSM). O-phtaldialdehyde (OPA) method
was employed to calculate the degree of hydrolysis (DH), which is the key parameter for monitoring the reaction of
protein hydrolysis. The suggested model equation was proposed based on the effects of pH, temperature, substrate
concentration and enzyme concentration on the DH. Optimum enzymatic hydrolysis conditions using Alcalase enzyme
were obtained at pH7.5, temperature of 50oC, substrate concentration of 2.5% and enzyme concentration of 4.0%.
Under these conditions, the highest value of the DH was achieved at 25.16% after hydrolysing at 120 min. The TMPH
was further assessed for their nutritional value with respect to chemical and amino acid compositions. Molecular
weight distributions of TMPH were characterized by SDS-PAGE. TMPH contains moderate amount of protein (28.14%)
and good nutritive value with respect to the higher total amino acid composition (267.57 mg/g). Glutamic acid,
aspartic acid and lysine were the most abundant amino acids present in TMPH with values 42.68, 29.16 and 26.21
mg/g, respectively. Protein hydrolysates from tilapia muscle containing a desirable peptide with low molecular weight
which may potentially to be used as functional food products.
Keywords: Alcalase; degree of hydrolysis; enzymatic hydrolysis; response surface methodology; tilapia muscle
ABSTRAK
Protein ikan hidrolisat disediakan daripada otot tilapia dengan menggunakan enzim alkalase. Pengoptimuman proses
hidrolisis berenzim untuk penyediaan protein hidrolisat otot tilapia (TMPH) dilakukan dengan menggunakan reka bentuk
komposit berpusat (CCD) iaitu kaedah sambutan permukaan (RSM). Kaedah O-phtaldialdehid (OPA) telah digunakan untuk
penentuan darjah hidrolisis (DH) yang merupakan kunci utama untuk memantau tindak balas hidrolisis protein. Model
cadangan yang digunakan adalah berasaskan kepada kesan pH, suhu, kepekatan substrat dan kepekatan enzim ke atas
DH. Keadaan hidrolisis optimum bagi enzim alkalase diperoleh pada pH7.5, suhu 50oC, kepekatan substrat 2.5% dan
kepekatan enzim 4.0%. Pada keadaan ini, nilai tertinggi DH dicapai pada 25.16% selepas hidrolisis dijalankan selama
120 min. TMPH seterusnya diuji nilai nutrisinya berdasarkan kepada komposisi kimia dan asid amino. Taburan berat
molekul TMPH dicirikan menggunakan SDS-PAGE. TMPH mengandungi kandungan protein yang sederhana (28.14%) serta
nilai nutrisi yang baik berdasarkan kepada jumlah asid amino yang tinggi (267.57 mg/g). Asid glutamik, asid aspartik
dan lisina merupakan asid amino yang paling banyak terkandung di dalam TMPH dengan nilai masing-masing adalah
42.68, 29.16 dan 26.21 mg/g. Protein hidrolisat daripada otot tilapia mengandungi peptida yang dikehendaki dengan
berat molekul rendah yang berpotensi untuk digunakan sebagai produk makanan berfungsi.
Kata kunci: Alkalase; darjah kehidrolisisan; hidrolisis berenzim; kaedah sambutan permukaan; otot tilapia
INTRODUCTION
Tilapia is abundantly cultured in several parts of the world
due to its special features such as fast-growing, tolerant to
environmental changes and disease, as well as can provide
an excellent source of protein. The composition of amino
acid in the fish muscle is considerably high, particularly an
essential amino acid which is the major factor determining
the nutritional value of food protein (Venugopal et al.
1996). Beside of the basic function of fish protein, it can
1716
DH =
h/htot 100
(1)
1717
EXPERIMENTAL DESIGN FOR OPTIMIZATION
(2)
1. Independent variables and their coded and actual levels used in the RSM studies
for optimizing the hydrolysis conditions of tilapia muscle using Alcalase
Factor
pH
Temperature
Substrate concentration
Enzyme concentration
Unit
Symbol
C
%
%
A
B
C
D
Coded level
-2
-1
6.5
40
1.5
3.0
7.0
45
2.0
3.5
7.5
50
2.5
4.0
8.0
55
3.0
4.5
8.5
60
3.5
5.0
1718
2. The actual levels of independent variables used in optimizing the hydrolysis condition of
tilapia muscle using Alcalase with the observed values for the degree of hydrolysis (DH)
TABLE
Run
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
pH(A)
8.0
7.5
7.0
7.0
7.0
8.0
8.0
7.0
8.0
7.5
7.0
7.5
7.0
8.0
8.0
7.5
8.0
7.0
7.0
8.0
6.5
7.5
7.5
7.5
8.5
7.5
7.5
7.5
7.5
7.5
Temperature(B)
55
50
55
45
55
45
45
45
55
50
55
50
55
45
55
50
55
45
45
45
50
50
60
50
50
50
50
40
50
50
Substrate(C)
2.0
2.5
3.0
2.0
2.0
2.0
3.0
3.0
3.0
2.5
2.0
2.5
3.0
2.0
2.0
2.5
3.0
2.0
3.0
3.0
2.5
2.5
2.5
2.5
2.5
2.5
3.5
2.5
1.5
2.5
Enzyme(D)
4.5
4.0
3.5
3.5
3.5
3.5
4.5
4.5
4.5
4.0
4.5
4.0
4.5
4.5
3.5
4.0
3.5
4.5
3.5
3.5
4.0
4.0
4.0
3.0
4.0
4.0
4.0
4.0
4.0
5.0
DH
25.93
24.25
23.10
20.28
21.31
23.11
26.25
24.75
26.83
25.45
22.98
24.47
25.47
24.12
25.13
24.17
24.02
24.14
21.92
23.68
22.13
25.75
25.45
24.27
25.96
26.89
26.37
25.20
26.19
27.26
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TABLE
Source
Model
A
B
C
D
AB
AC
AD
BC
BD
CD
A2
B2
C2
D2
Residual
Lack of fit
Pure error
Cor. Total
Sum of square
62.84
21.62
2.05
3.67
23.80
0.56
1.02
0.78
0.048
0.43
0.66
7.29
1.05
0.052
0.20
7.94
6.24
1.70
85.36
Degree of freedom
14
1
1
1
1
1
1
1
1
1
1
1
1
1
1
14
10
4
29
Mean square
4.49
21.62
2.05
3.67
23.80
0.56
1.02
0.78
0.048
0.43
0.66
7.29
1.05
0.052
0.20
0.57
0.62
0.42
F value
0.0002
<0.0001
0.0778
0.0235
<0.0001
0.3393
0.2012
0.2595
0.7745
0.3991
0.3003
0.0030
0.1956
0.7676
0.5620
1.47
0.3779
R2 = 0.8878, A=pH, B=Temperature (oC), C=Substrate concentration (%) and D=Enzyme concentration (%)
FIGURE
P value
7.91
38.12
3.62
6.46
41.97
0.98
1.80
1.38
0.085
0.76
1.16
12.85
1.85
0.091
0.35
1720
FIGURE
FIGURE 3.
1721
FIGURE 4.
TABLE
Run
4. Degree of hydrolysis (DH) observed during validation experiments using Alcalase and the
corresponding predicted values for the DH
A
7.5
50.0
2.5
4.0
Desirability DH-Predicted
0.929
DH-Experimental
A: pH, B: Temperature, C: Substrate concentration, D: Enzyme concentration and DH: Degree of hydrolysis
All data are expressed as means S. D (n=5)
CONCLUSION
In this study, the production of fish protein hydrolysate
from tilapia muscle was successfully carried out through
an enzymatic hydrolysis using Alcalase enzyme. The
1722
TABLE 5.
Nutritional composition
9.54 0.13
28.14 0.44
0.07 0.03
52.09 0.49
19.71 0.15
29.49 0.63
7.74 0.02
8.64 0.07
11.85 0.13
0.38 0.01
7.87 0.08
12.04 0.08
6.75 0.08
11.30 0.10
6.75 0.04
10.33 0.00
16.44 0.06
8.79 0.01
7.58 0.24
19.74 0.01
8.06 0.01
193.41 0.33a
88.97 0.11a
81.95 0.07b
FIGURE
REFERENCES
1723