Establishing the hydrolysis procedure of Soft-shell crab (Scylla sp.) in Viet Nam

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. REFERENCES 1. 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