The document summarizes a study on the enzymatic protein hydrolysis of catfish (Clarias batrachus) muscle using the enzyme subtilisin. The key findings are: 1) Degree of hydrolysis and peptide content of the catfish protein hydrolysates increased with longer hydrolysis time, reaching maximum levels at 180 minutes. 2) Amino acid profiles and proximate compositions were generally not affected by hydrolysis time. The hydrolysates were rich in essential amino acids and high in protein content. 3) Catfish protein hydrolysates produced using subtilisin have potential applications in food, health and other industries due to their high nutrient and bioactive peptide content.

1. Biochemical Properties and
Proximate Composition of Catfish
Enzymatic Protein Hydrolysates
Made Using Subtilisin
Maizatul Sarah Bt Md Seniman
Dr. Salma Mohamad Yusop
Prof. Dr. Abdul Salam Babji
2nd International Conference on Science and Technology (ICST)
June 17, 2014
Hotel Dynasty, Kuala Lumpur

2. Introduction
• Clarias batrachus or Keli is one of the high demand freshwater fishes
▫ because of unique taste and cheaper price
▫ total amount of catfish production in year 2012 was 46,523 metric tons
[ Department of Fisheries Malaysia (2012) ]
▫ sale of catfish produced in Malaysia in year 2012 earned RM 189 million out
of the total aquaculture fish production of RM 992 million
• Though catfish is on a high demand in Malaysia, yet its commercial
value is low.
▫ due to the utilization of cultured catfish is still limited to local consumption.
• Hence, an alternative to enhance the commercial value of catfish is by
generating catfish protein hydrolysates (CPH).
▫ protein hydrolysates can be a source of raw material for many industries.

3. Introduction
• Protein hydrolysis refers to all possible process in which the protein is
broken down to produce lower molecular weight peptides.
• Applications of protein hydrolysates have expanded in a variety of
industries, including pharmaceuticals, nutraceuticals, cosmetics or
animal nutrition.
• Recently, fish protein hydrolysates have become popular in the food
industry due to its:-
▫ high protein content,
▫ dynamic physicochemical properties e.g. emulsifying, foamability
▫ biological active properties e.g. antioxidant, antihypertensive

4. Introduction
• The use of commercial enzyme is preferred in protein hydrolysis
▫ hydrolysis process and the properties of resulting products can be
controlled.
• Different enzymes have been used for hydrolysis of fish:-
▫ plant-derived enzymes e.g. papain
▫ animal-originated enzymes e.g. trypsin
▫ microbial-originated enzymes e.g. subtilisin
• Subtilisin is an alkaline proteinase originating from Bacillus
subtillis.
• From a technical and economic point of view, enzymes from
microbial sources operating at alkaline pH were shown to be one
of the most efficient in the hydrolysis of fish proteins.

5. Objective
This study was designed to facilitate enzymatic protein
hydrolysis of catfish (Clarias batrachus) muscle using
subtilisin in order to characterise its biochemical
properties and proximate composition as affected by
hydrolysis time.

6. Minced fish
muscle
Protein hydrolysate
was stored at -20 °C
Centrifuged
(3000 x g, 20
min)
Enzyme reaction
inactivated at 90°C,
10 min
Hydrolysis
(0 – 180 min)
Adjusted pH and
temperature of sample (pH
8; 55 °C)
Added subtilisin to
sample
(1:100, v/w )
Added dH2O
(2:100, w/v)
Supernatant was
lyophilised.
Materials & Methods
Protein hydrolysate preparation:

7. Materials & Methods
Degree of hydrolysis
DH was analyzed according to percent of trichloroacetic acid
(TCA) method.
- (Hoyle and Merritt, 1994)
The soluble nitrogen was analyzed using Kjeldahl method
(AOAC 2005).
%DH = (10% TCA–soluble N in sample/ total N in sample) x
100
Peptide content quantification
using o-phthaldialdehyde (OPA) spectrophotometric assay.
- (Church et al., 1983)

8. Materials & Methods
Amino acid profile
Amino acid composition was analysed by HPLC.
- Alaiz et al. (1992)
Proximate composition
Moisture, ash, fat and protein content of raw catfish and fish
protein hydrolysates were determined according to AOAC
(2005).
Statistical analysis

9. Results & Discussion
Figure 1: Effect of hydrolysis time on degree of hydrolysis of C.batrachus

10. • DH of C.batrachus depends on the reaction time.
• The highest DH value of 59% was obtained at maximum incubation
time (180 min).
• The DH values were increased with increasing hydrolysis time.
• The result was in agreement with previous studies by Herpandi et al.
(2012) and Norma et al. (2005).
• Hydrolytic curve of C.batrachus exhibited an initial fast reaction
rate.
• Thereafter, the rate of enzymatic hydrolysis decreased and reached a
stationary phase.
• Such shapes of hydrolysis curves were similar to those previously
published for salmon muscle (Kristinsson et al., 2000) and cod
muscle (Ravallec-Plé et al., 2000).
• In the initial period of hydrolysis, a large number of peptide bonds
were cleaved, leading to an increase of soluble peptides in the
reaction mixture.
• Meanwhile in the second stage, the reaction speed decreases. This
may due to some enzyme partial inactivation and/or an increase of
peptides may also act as effective substrate competitors to the
undigested or partially digested proteins .

11. Results & Discussion
Figure 1: Effect of hydrolysis time on peptide content of C.batrachus
hydrolysate

12. • Quantitatively the peptide content of CPH was affected by
hydrolysis time.
• At maximum incubation time (180 min), the peptide content of
subtilisin-treated CPH was 7200 µg/ml.
• The result also indicated that increasing hydrolysis time from 0 min
to 180 min increased the peptide content of CPH.
• From these results, we infer that the peptide content increased with
increasing hydrolysis time as more peptides are released as a result
of proteolytic enzymes activity.

13. Results & Discussion
Table 1: Amino acid profile of C.batrachus protein hydrolysate
Amino acid (%) CPH30 CPH60 CPH120 CPH180
Aspartic acid 9.06 ± 0.01 9.17 ± 0.05 9.41 ± 0.25 9.86 ± 0.01
Serine 5.50 ± 0.05 5.45 ± 0.04 5.51 ± 0.04 4.15 ± 0.10
Glutamic acid 13.66 ± 0.22
13.86 ±
0.22
14.40 ± 0.20 15.66 ± 0.22
Glycine 7.66 ± 0.02 7.65 ± 0.01 6.96 ± 0.11 5.66 ± 0.04
Histidine 2.21 ± 0.06 2.21 ± 0.04 2.21 ± 0.19 2.21 ± 0.01
Arginine 6.42 ± 0.14 6.50 ± 0.05 6.52 ± 0.15 6.61 ± 0.14
Threonine 4.76 ± 0.03 4.71 ± 0.04 4.75 ± 0.04 4.60 ± 0.07
Alanine 6.02 ± 0.15 6.52 ± 0.13 5.62 ± 0.63 5.29 ± 0.05
Proline 5.53 ± 0.07 4.60 ± 0.08 4.12 ± 0.04 4.53 ± 0.97
Tyrosine 3.04 ± 0.00 3.03 ± 0.04 3.53 ± 0.04 4.04 ± 0.14
Valine 6.00 ± 0.08 5.98 ± 0.10 5.77 ± 0.13 5.48 ± 0.08
Amino acid (%) CPH30 CPH60 CPH120 CPH180
Methionine 3.03 ± 0.05 3.03 ± 0.07 3.08 ± 0.13 3.20 ± 0.06
Lysine 10.05 ± 0.04
10.06 ±
0.03
10.26 ± 0.39 10.35 ± 0.09
Isoleucine 4.23 ± 0.14 4.37 ± 0.08 4.47 ± 0.08 4.86 ± 0.23
Leucine 7.08 ± 0.05 7.35 ± 0.23 7.75 ± 0.12 8.08 ± 0.46
Phenylalanine 4.33 ± 0.00 4.25 ± 0.03 4.20 ± 0.01 4.13 ± 0.00
Cysteine 0.37 ± 0.01 0.35 ± 0.00 0.32 ± 0.00 0.17 ± 0.05
Tryptophan 1.05 ± 0.07 1.10 ± 0.09 1.12 ± 0.09 1.11 ± 0.18
TAA 100 100 100 100
TEAA 42.85 43.61 43.92 44.05
TAA, total amino acids; TEAA, total essential amino acids
CPH30, 30min incubated CPH; CPH60, 60min incubated CPH; CPH120, 120min incubated CPH; CPH180, 180min incubated CPH

14. • The catfish hydrolysates were rich in glutamic acid, lysine and
aspartic acid.
• Studies by Ghassem et al. and Hou et al. found glutamic acid and
aspartic acid to be higher compared to other amino acids in the
reported fish protein hydrolysates.
• CPH samples contain the essential and nonessential amino acids.
• Based on the total amino acids, the composition of essential amino
acids are 42.85%, 43.61%, 43.92% and 44.05% of the CPH
incubated for 30 min, 60 min, 120 min and 180 min respectively.
• Several studies have reported high essential amino acids
composition of the fish protein hydrolysate including those of
herring (Liceaga-Gesualdo et al., 1999) and round scad muscle
hydrolysate (Thiansilakul et al., 2007).

15. Results & Discussion
Table 2: Amino acid profile of C.batrachus protein hydrolysate
Hydrolysis time
(min)
Proximate composition (%)
Crude Protein Fat Moisture Ash
Control 20.32 ± 1.42b 4.75 ± 0.61a 76.85 ± 0.17a 1.09 ± 0.04a
30 83.94 ± 0.52a 0.95 ± 0.06b 3.02 ± 0.12b 0.70 ± 0.05b
60 84.69 ± 0.55a 0.97 ± 0.05b 3.09 ± 0.46b 0.71 ± 0.08b
120 85.02 ± 0.24a 1.00 ± 0.08b 3.13 ± 0.12b 0.72 ± 0.06b
180 85.40 ± 0.62a 1.06 ± 0.08b 3.18 ± 0.12b 0.71 ± 0.05b
Control, raw catfish.
a b Means with different superscripts within a column are significantly different (P<0.05)

16. • The protein content of CPH was significantly higher (p < 0.05) than
the raw catfish sample.
• Similar trend was reported in the sardinella byproducts hydrolysate
(Souissi, 2007) and Pacific whiting muscle hydrolysate (Pacheco-
Aguilar et al., 2008).
• The high protein content is due to solubilization of proteins during
hydrolysis and removal of insoluble solid matter by centrifugation.
• There was no significant difference in protein content between all
CPH samples.
• The fat, moisture and ash content of CPH was significantly lower
(p < 0.05) than raw C.batrachus.
• The low fat content of fish protein hydrolysates is due to the
removal of lipids and insoluble protein fractions by centrifugation.
• The low moisture content of protein hydrolysates is related to the
type of sample and the effect of freeze drying. During the process,
the sample loses most of its moisture.

17. Conclusion
• Degree of hydrolysis and peptide content of CPH were
significantly affected by hydrolysis time.
• In contrast, the amino acids profile and proximate composition of
CPH samples were not significantly affected by incubation time.
• Nevertheless C.batrachus protein hydrolysates prepared by
subtilisin demonstrated a high protein content as well as high
peptide and amino acid content.
• Therefore, the CPH may potentially serve as a good source of
useful nutrients and bioactive peptides for application in health,
food and other industries.