Initially, some of the dietary components of soft-sheel mud crabs have been analyzed: lipid
content was of 1.2 % and protein content was 30.3 %. The result of this research proves that it is
a source of raw materials for the production of nutrient-rich products.
The process of hydrolysis of lobster crabs by enzyme has been studied the material:water
ratio and E/S ratio which showed the highest productivity at 1:3 and 1 %, respectively. Typical
content of calcium in crab peeling is 1.76 % and accounts for 1.16 % in hydrolysed crab meal,
similar to the content of zinc in extraction of 203.53 mg/kg and 111.03 mg/kg of hydrolyzed
crab powder. Especially, hydrolysate of crab provides high content of essential amino acids such
as arginine, histidine.
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Vietnam Journal of Science and Technology 56 (2A) (2018) 201-208
ESTABLISHING THE HYDROLYSIS PROCEDURE OF
SOFT-SHELL CRAB (Scylla sp.) IN VIET NAM
Nguyen Thi Phuong Lan
1
, Do Thi Thanh Trung
2
, Van Thu Vu
2
,
Dao Thi Dung
2
, Le Tat Thanh
2, *
1
Graduate University of Sciences and Technology, 18 Hoang Quoc Viet, Cau Giay, Ha Noi
2
Institute of Natural Products Chemistry,VAST, 18 Hoang Quoc Viet street, Cau Giay, Ha Noi
*
Email: thanh.biotech@gmail.com
Received: 12 March 2018; Accepted for publication: 12 May 2018
ABSTRACT
The mud crab (Scylla sp.) belonging to Portunidae family has been aquacultured and used
to provide a lot of nutrient foods and materials for supplementary food. Recent researches
showed nutrient content of soft-shell crabs is higher than that of harden crabs. In this study, the
content of total lipid and protein of Scylla sp. are found to be 1.2% and 30.5%, respectively.
Additionally, trace elements contents (calcium and zink) were obtained of high level with 1.76%
and 203.53 mg/kg, respectively. This is a potential source of materials for application in
medicine and pharmaceutical industry in Vietnam. A enzymatic hydrolysis procedure was
established as well in order to keep the high nutritional value of soft-shell crab materials. Based
on these results, this procedure will be optimized as prospective in next step to obtain the
maximized hydrolysate yield.
Keywords: Mud crab, soft-shell crab, enzymatic hydrolysis.
1. INTRODUCTION
The mud crab Scylla belongs to Portunidae, Decapoda, Crustacea class, Arthropoda, and
includes 4 species (S. paramamosain; S. serrata; S. olivacea and S. tranquebarica) [1, 2]. The
mud crab has expanded to aquaculture in recent years due to increasing demands and rising
market value of this commodity [3, 4]. Soft-shell crab is a culinary term for crabs that have
recently molted their old exoskeleton and are still soft [6]. Soft-shells are removed from the
water as soon as they molt to prevent any hardening of their shell. This means that almost the
entire animal can be eaten, rather than having to shell the animal to reach the meat. The
exceptions are the mouthparts, the gills and the abdomen, which, though edible when shells are
very soft, are sometimes discarded [5]. According to M. Vilasoa-Martinez et al. [6], the protein
content of crabs Chionoecetes opilio at soft-shell stage was very high, about 33.0 ± 13.8 g/100 g
in the meat crab. In addition, unsaturated fatty acids in meat (36-39 %) have also been shown to
be higher than in the hepatopancreas (16-18 %) and genital (16-24 %), especially, the level of
Nguyen Thi Phuong Lan, Do Thi Thanh Trung, Van Thu Vu, Dao Thi Dung, Le Tat Thanh
202
docosahexaenoic acid DHA is higher. The amino acids in the different components are quite
similar [7].
Enzymatic hydrolysis of proteins by some commercial enzymes to cleave specific peptide
bonds is widely applied in the food industry [8]. Application of enzyme is high value method
because of their controllability and reproducibility [9]. There are many researchers who have
focused on the enzyme hydrolysis because enzymatic reaction do not involve side reactions and
do not reduce the nutritional value of the protein source [10].
In Vietnam, the mud crab farming has been improved using synthetic feed, in plastic tanks
on the shelf, rack, therefore, the quantity of mud crab product was improved remarkablely.
However, there is a large percentage (30%) softshell crabs which lost their pincers,
consequently, their commercial value is very low. The crab product of droping pincers is not
different in terms of nutritional value compared to intact crabs, thus, processing with improved
technique is essential in order to increase the value for mud crab products. In this study, we plan
to establish a hydrolysis procedure applying enzymatic technology and aim to investigate
biochemical compositions of soft-shell crab materials.
2. MATERIALS AND METHODS
2.1. Material
Soft-shell crab (Scylla) was cultured with large scale in South Vietnam. The fresh crab
samples were collected at Nha Trang in 2017, then immediately placed in ice and transported to
the laboratory. Upon arrival, the flesh was separated manually and then washed.
Protease with declared activity 2.4 AU-A/g was purchased from Novozyme (Denmark) in
optimal reaction conditions at pH of 7-9, at a temperature of 30-65°C.
2.2. Methods
The total lipid content (Bligh & Dyer, 1959) [11]
10 g of fresh sample was grinded and soaked with 18 ml of a mixture of Chloroform :
Methanol (1:2, v:v) at room temperature, then filtered after several hours. 10 ml of Chloroform
was added, the mixture was shaked well and added with 10 ml of water. The resulting mixture
was shaked again and stood overnight at 0
o
C, then removed of the lower phase, dried and
evaporated of the solvents under the pressure of the total lipid mixture. The total lipid fraction
was weighted then preserved by dilution in Chloroform and stored at 0 °C. The total lipid
content was dertermined by percentate rate of weight of total lipid fraction per 10 g sample.
Content of total protein [12]
The Bradford assay relies on the binding of the dye Coomassie Blue G250 to protein. The
more anionic blue form of the dye, which binds to protein, has an absorbance maximum at 590
nm. Thus, the quantity of protein can be estimated by determining the amount of dye in the blue
ionic form. The result is usually achieved by measuring the absorbance of the solution at 595 nm
and comparing with standard curve.
Fomula: y = 0.0001x + 0.2457 (R² = 0.9926)
Establishing the hydrolysis procedure of soft-shell crab (Scylla sp.) in Viet Nam
203
Analysis of free amino acid from hydrolysate [13]
An amount of 0.1 grams of soft-shell crab hydrolysate was deproteinized and derivatized
using phenyl isothiocyanate prior to AccQ Tag HPLC analysis. The free amino acid was
analyzed by the AccQTag method using an AccQ Tag column (3.9 × 150 mm) at a flow rate of 1
mL/min equipped with fluorescence detector. The mobile phase used was AccQTaq Eluent A
consists of 100 mL Eluent A consited 1000 mL deionized water while AccQ Tag Eluent B
consisted of 60 % acetonitrile and 40 % deionized water. The total running time per injection
was 50 minutes.
Amount of trace elements, heavy metals by ICP-OES method [14]
Determination of micronutrient content was done by atomic absorption spectrometer and its
components: Thermo Elemental-Model Solaar M6 Dual Zeeman Atomic Absorption
Spectrometer with VP 90 hydride generation system, Halogen light, HCL data Coded.
Preparation of mud crab (Scylla) hydrolysate
An amount of flesh and distilled water was mixed and homogenized in a blender. The
mixture was incubated in a circulated water bath. At the optimal temperature (45 °C) protease
was used at enzyme-substrate ratio of 1 % and the hydrolysis continued for 6 hours. At the end
of hydrolysis, the enzymatic reaction was terminated by placing the samples in a water bath at
80 °C for 10 min. This was followed by centrifugation at 6000 rpm for 10 min. Supernatant
obtained was freeze-dried then the powdered hydrolysate was stored in -20 °C for further
analysis.
3. RESULTS AND DISCUSSION
3.1. Investigation of nutrient contents of the soft-shell mud crab Scylla
The moisture content
The results of moisture content was shown in Table 1. After three experiments, the average
moisture content of soft-shell mud crab was 85.74 %. Comparing with other researches, it is
slightly higher than moisture content of the blue swimmer crab and mud crab with 78.8 % and
79.72 %, respectively [7, 15]. The high moisture can be explained that the crabs need moisture
to maintain their nomal activities of body when molting their old exoskeleton.
Total lipid and protein content
The lipid content in crab analyzed according to Bligh & Dyer method, and the protein
content determined using Bradford method are indicated in Table 1.
Table 1. Lipid and protein content of mud crab.
Properties Content Zaliha Harun et al. [15] Sarower et al. [16] Ke Chen et al. [17]
Moisture (%) 85.74±0.35 79.72 75.31-78.02 71.61-76.71
Total Lipid (%) 1.2±0.02 0.04±0.03 1.32–1.17 0.49-0.53
Total Protein (%) 30.3±0.06 14.63 16.60–19.38 18.85-19.28
Nguyen Thi Phuong Lan, Do Thi Thanh Trung, Van Thu Vu, Dao Thi Dung, Le Tat Thanh
204
After 3 experiments, we obtained a total lipid content of 1.2 %. This results is in agreement
with previous study by Sarower et al. who reported that wild mud crab contained 1.32–1.17 %
fat [16]. The protein content of soft-shell crab was 30.3 % higher than 16.60–19.38 % of mud
crab [16], 14.63 % of Malaysia mud crab [15] and 18.85-19.28 % of Chinis mitten crab [17].
This suggested that soft-shell crab muscle meat is a good source of protein and beneficial fatty
acids for human health.
3.2. Enzymatic hydrolysis procedure of mud crab Scylla
3.2.1. Investigation of material and water ratio in preparing crab
After preparing, the material was supplemented with water following different ratios. The
ratio of material and water was experimented from 1:1 to 1:10 and the 1:3 ratio showed to be of
the highest product (Figure 1). Thus, 1:3 ratio was chosen for further experiments.
Figure 1: Optimization of material and water ratio.
3.2.2. Investigation of enzyme and substrate ratio (E/S ratio)
When the temperature downed to 40
o
C, enzyme was supplement immediately. Hydrolysis
was performed at temperature of 45-50
o
C, speed of 200 rpm for 6h. The reaction condition was
choosed basing on optimal condition of enzyme following the producer. The enzyme and
substrate (E/S) ratio was investigated from 0.1 % to 2 %. The results showed that 1 % enzyme
had the best activity to the catalysis (Table 2).
Table 2. Comparison of the enzymatic hydrolysis with different E/S ratio.
E/S Ratio (%) 0 0.1 0.5 1.0 1.5 2.0
Protein weight (mg) 1.02 8.98 21.56 38.79 29.05 21.95
3.3.3. The final procedure of crab hydrolysis
After investigation, we established a hydrolysis procedure for mud crab Scylla in order to
obtain the valuable nutient component. The hydrolysis process using enzyme including the
following steps (Figure 2).
Establishing the hydrolysis procedure of soft-shell crab (Scylla sp.) in Viet Nam
205
Figure 2. Hydrolysis procedure of mud crab Scylla
Step 1: Preparing materials: Raw crab materials were washed with acetic acid and salt
water, drained, grinded in the meat grinder, ensure the blender is cleaned and coated before
grinding. Mixture of raw material and water (1:3 ratio g/ml) in the hydrolysis tank was incubated
at a temperature of 80
o
C for 30 minutes then decreased the temperature of the hydrolyzer by
replacing the water tank with hydrolysis and cool water.
Step 2: Hydrolysis: After processing and lowering the temperature of 40
o
C in the
hydrolytic enzyme supplement (E/S ratio at 1%). Hydrolysis was performed at temperature of
45-50
o
C, speed of 200 v/p for 6h. Finally, the enzyme reaction was ended by heating up to
80
o
C for 5 minutes
Step 3: Filtering: Hydrolysate was filtered by sieve then the filtrate was freeze-dried to
obtain hydrolysate powder and stored in the refrigerator at -20
o
C.
Furthermore, optimization experiments will be performed to find out the optimal conditions
for hydrolysis including materials and water ratio, E/S ratio, temperature, pH and time reaction.
The results will be reported in other publications.
3.3. Comparison of content of composition before and after hydrolysis
Amount of trace elements, heavy metals
The result of minerals in the crab specimens was showed in Table 3 and 4.
From Table 3, the content of essential minerals in peeling is very high such as calcium is
1.76 %, phosphorus is 1.11 %, in addition, the content of zinc in 1kg peeled to 203.53 mg.
Similarly in the hydrolysed crab meal mineral content also accounted for quite as high as 1.16 %
calcium, zinc accounted for 11.03 mg/kg. Whereas, with light increasing after hydrolysis,
content of heavy metal has still maintained in the safety limits of heavy metals (according to
QCVN 8-2:2011/BYT).
Nguyen Thi Phuong Lan, Do Thi Thanh Trung, Van Thu Vu, Dao Thi Dung, Le Tat Thanh
206
Table 3. Trace elements in crab produces.
N0 Trace elements Powder of dried crab Powder of crab hydrolysate
1 Calcium (Ca) 1.76 % 1.16 %
2 Magnesium (Mg) 0.54 % 0.31 %
3 Sodium (Na) 4.39 % 2.50 %
4 Potasium (K) 1.58 % 1.01 %
5 Phosphorus (P) 1.13 % 0.86 %
6 Copper (Cu) 67.23 mg/kg 80.84 mg/kg
7 Zinc (Zn) 203.53 mg/kg 111.03 mg/kg
8 Iron (Fe) 78.18 mg/kg 39.17 mg/kg
9 Manganese (Mn) 9.19 mg/kg 11.05 mg/kg
Table 4. Heavy metals in crab produces.
N0 Heavy metal Powder of dried crab Powder of crab hydrolysate
10 Cadmium (Cd) 0.90 mg/kg 1.18 mg/kg
11 Lead (pb) 4.52 mg/kg 5.44 mg/kg
12 Mercury (Hg) 0.058 mg/kg 0.138 mg/kg
Content of free amino acid after hydrolysis
By the method of determining free amino acids, we measured the content of dissolved
amino acids in hydrolysed crab powder as shown in the Table 5 below.
Table 5. Free amino acid after hydrolysis.
N0 Amino acid Content (µg/100g)
1 Aspatic acid 28383.2
2 Threonin 35701.7
3 Serine 31231.3
4 Glutamic acid 31878.6
5 Proline 58109.5
6 Glycine 22438.1
7 Alanine 10187.9
8 Valine 27196.8
9 Methionin 60697.7
10 Isoleucine 44190.5
11 Leucine 66420.2
12 Tyrosine 967517.0
13 Phenylalanine 994285.0
14 Lysine 360776.0
15 Histidine 436891.0
16 Arginin 368859.0
Total content 3544762.0
Establishing the hydrolysis procedure of soft-shell crab (Scylla sp.) in Viet Nam
207
The result indicated that high levels of precious amino acids such as lysine, proline,
arginine, histidine, glutamic acid, thionine, leucine, isoleucine, aspartic acid, tyrosine were
obtained after hydrolysis. Comparing with the case of before hydrolysis, most content of free
amino acids seemed to increase by a number of times after hydrolysis such as arginin, tyrosine
[18].
4. CONCLUSION
Initially, some of the dietary components of soft-sheel mud crabs have been analyzed: lipid
content was of 1.2 % and protein content was 30.3 %. The result of this research proves that it is
a source of raw materials for the production of nutrient-rich products.
The process of hydrolysis of lobster crabs by enzyme has been studied the material:water
ratio and E/S ratio which showed the highest productivity at 1:3 and 1 %, respectively. Typical
content of calcium in crab peeling is 1.76 % and accounts for 1.16 % in hydrolysed crab meal,
similar to the content of zinc in extraction of 203.53 mg/kg and 111.03 mg/kg of hydrolyzed
crab powder. Especially, hydrolysate of crab provides high content of essential amino acids such
as arginine, histidine.
Acknowledgements. This work was financial supported by The Ministry of Agriculture and Rural
Development (MARD) with the code contract 16/2017 belonging to the Agriculture and Aquaculture
Biotechnology Program.
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