ENZYMATIC HYDROLYSIS OF TRASH FISH (HERRING FISH - ARDINELLA GIBBOSA) USING FLAVOURZYME ENZYME

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1 THOÂNG BAÙO KHOA HOÏC ENZYMATIC HYDROLYSIS OF TRASH FISH (HERRING FISH - ARDINELLA GIBBOSA) USING FLAVOURZYME ENZYME NGHIÊN CỨU ỨNG DỤNG ENZYME FLAVOURZYME THỦY PHÂN CÁ TRÍCH (SARDINELLA GIBBOSA) Nguyễn Thị Vân 1, Đỗ Thị Thanh Thủy 2 Ngày nhận bài: 10/3/2014; Ngày phản biện thông qua: 11/3/2014; Ngày duyệt đăng: 02/6/2014 ABSTRACT The hydrolysis of herring for production of protein hydrolysate was studied. The hydrolysis process was carried out using Flavourzyme enzyme at natural ph with a water/material ratio of 1:1. The results show that the appropriate hydrolysis conditions were: temperature 55 0 C, the ratio of enzyme-substrate LAPU/g protein, hydrolysis time is 6 hours. The hydrolysis degree, the nitrogen recovery and total evaporation bases nitrogen obtained in the hydrolysis protein solution are respectively 50.58%, 63.38% and 0.92 g/l. The resulting product contains g/l amino acid nitrogen, have the potential applications in many fields. Keywords: Enzymatic hydrolysis, protein hydrolysis, herring fi sh, Flavourzyme TÓM TẮT Dịch đạm hòa tan đã được nghiên cứu sản xuất từ protein cá trích bằng enzyme Flavourzyme ở ph tự nhiên với tỷ lệ nước/nguyên liệu là 1/1. Kết quả nghiên cứu cho thấy điều kiện thủy phân thích hợp là: nhiệt độ 55 o C, tỷ lệ enzyme - cơ chất LAPU/g protein, thời gian 6 giờ. Độ thủy phân, hiệu suất thu hồi nitơ và tổng nitơ bazơ bay hơi trong dịch thủy phân thu được lần lượt đạt là 50.58%, 63.38% và 0.92 g/l. Sản phẩm thu được chứa hàm lượng nitơ a xít amin g/l, có tiềm năng ứng dụng trong nhiều lĩnh vực. Từ khóa: thủ y phân, dịch đạm thủy phân, cá trích, Flavourzyme I. INTRODUCTION Herring is a species of trash fi sh that caught in large amount - account for about 16.46% of the total trash fi sh exploitation. Currently, herring used only to eat fresh, produced dried products, used in animal feed, a few exported as frozen bait, create low economic effi ciency. Research to create value-added products from plentiful source of herring fi sh to improve the effi ciency of natural resource, bring more income to the people are very necessary. There is a great potential in marine bioprocess industry to convert this kind of trash fi sh into value-added products. Enzymatic hydrolysis is one of the most effi cient methods to recover proteins from trash fi sh and to produce the protein hydrolysates. The research to fi nd out the appropriate mode hydrolysis to produce soluble protein fl uid rich in amino acids from herring is a feasibility idea. The potential use of fi sh protein hydrolysate in food and feed was also reported by several authors (Refstie et al., 2004; Nguyen et al., 2012). Production of protein hydrolysate produced from herring protein could generate signifi cant revenue for fi sh processing industry. II. MATERIALS AND METHODS 1. Materials 1.1. Herring The herring fi sh (Sardinella Gibbosa) are purchased in Hon Ro ports - Nha Trang City. 1 ThS. Nguyễn Thị Vân, 2 ThS. Đỗ Thị Thanh Thủy: Khoa Công nghệ thực phẩm Trường Đại học Nha Trang TRƯỜNG ĐẠI HỌC NHA TRANG 73

2 Fish was fresh, bright, no stains lesions, size units/kg. Fish are washed, removing impurities, storage and transport to the laboratory in insulation foam box at C. At the laboratory, to ensure uniformity, fi sh were washed, drained, minced, mixed thoroughly, divided, and packaged in vacuum PA bags, frozen and stored at -20 ± 2 0 C 1.2. Flavourzyme Enzyme Flavourzyme protease enzyme was produced by Novozymes A/S (Bagsvaerd Denmark). Flavourzyme Herring fi sh Hydrolysis including the activity of endopeptidase and exopeptidase but mostly exopeptidase, derived from Aspergillus oryzae (Kamnerdpetch et al., 2007). The optimal working conditions for Flavourzyme are reported to be at ph of and at a temperature of C. Flavourzyme has a declared activity of 500 LAPU (Leucine aminopeptidase Units)/g. 2. Research Methodologies 2.1. Process diagram and experimental design Hydrolysis conditions of Flavourzyme enzyme Determine the effect of temperature: o C, δ = 5 o C Dregs Inhibit enzyme Filter Determine the effect of E/S: LAPU/g protein Determine the effect of time: 3-7 hours, δ = 1 hour Natural ph of the fi sh, W/NL =1/1 The fi ltrate Lipid Suspended solids Centrifugal Dregs Hydrolysis solution Figure 1. Process diagram and experimental design - Materials: the samples were collected and processed as Section 1.1, defrost at C, in 15 hours. - Identify appropriate hydrolysis temperature: set t h = C, δ = 5 0 C and the ratio of enzyme-substrate (E/S) = 9.74 LAPU/g protein, hydrolysis time is 3 hours, W/NL = 1/1, the natural ph of the fi sh. - Identify the ratio of enzyme-substrate: set E/S = LAPU/g protein, δ = 9.74 LAPU/g protein, the temperature found in previous experiments, the time 3 hours, W/NL = 1/1, the natural ph of the fi sh. - Identify suitable hydrolysis time: set T= 3-7 hours, δ = 1 hour; temperature, E/S found in two previous experiments, W/NL = 1/1, the natural ph of the fi sh. - The enzyme was inactivated by heating at 95 0 C for 15 minutes in a water bath Chemical analyses Water content: TCVN ; Ash content: calcined at C methods; lipid content: according to TCVN 3703:2009, the total nitrogen content: TCVN ; NH 3 and TVB-N content: TCVN 9215:2012; amino acid nitrogen: formol method; amino acid composition: HPLC, hydrolyzsis degree DH: DNFB method; The nitrogen recovery in protein hydrolysate was calculated according to Liaset et al., Statistical analyses Each experiment was performed three times in parallel, each time test three samples. Data were processed with SPSS 16.0 statistical software, the Microsoft Offi ce Excel 2007 software was used to calculate results. Differences in treatment means were considered signifi cant at p < TRƯỜNG ĐẠI HỌC NHA TRANG

3 III. RESULTS AND DISCUSSION 1. Chemical compositions of herring Chemical compositions of herring are shown in table 1. Table 1. Chemical compositions of herring Component Percentage (%) compared to wet weight Water ± 0.01 Protein ± 0.04 Lipid 2.53 ± 0.03 Ash 0.96 ± 0.01 Table 1 showed that the herring have high total protein content (19.25%) higher than cuttlefi sh (17-21%), much higher than some other species such as oysters (8-9%), squil (13-16%) and sea snail (11-12%), low lipid content (2.53%), classifi ed as lean fi sh and are suitable for the production of hydrolysed protein solution. 2. Determine the appropriate parameters for the hydrolysis of herring protein by Flavouyme enzyme 2.1. Affect of the temperature to effi ciency of the hydrolysis Figure 2. Effect of temperature on the hydrolysis degree (DH) and nitrogen recovery efficiency (NRE) Figures 2 and 3 show that, when the temperature rose from 45 0 C to 55 0 C, then 3 indicators DH, NRE and N aa increased and peaked at 55 C; if the temperature continues to rise to 65 0 C, the measured indices were reduced. But TVB-N concentration decreased continuously as the temperature increases from 45 0 C to 65 0 C. This can be explained as follows: when the temperature increases from 45 0 C to 55 0 C, the activity of Flavourzyme enzyme increased due to the activation energy of the reaction is enhanced, then further increase in temperature up to 65 o C, the activity of Flavourzyme enzyme decreased because high temperature inhibits the activity of Flavourzyme Figure 3. Effect of temperature to the rate of N aa and TVB-N of hydrolysed protein enzyme. When the temperature increases from C, the activity of microbial flora reduced because the microbiota in herring active at t opt less than 45 0 C, when the temperature increases from C their activity are inhibited, leading to the TVB-N concentration decreases with hydrolysis temperature. At 55 0 C (DH, NRE, N aa and TVB-N corresponding in the order of 32.08%, 51.00%, 44.94% and 0.53 g/l) achieved the best results in experimental conditions and should be selected as appropriate temperature to proteolytic herring Affect of the ratio of enzyme-substrate (E/S) to hydrolysis effi ciency of Flavourzyme enzyme Figure 4. Affect of the rate of enzyme - substrate to the hydrolysis degree (DH) and nitrogen recovery efficiency (NRE) Figure 5. Affect of the rate of enzyme - substrate to the ratio of N aa and TVB-N of hydrolysed protein TRƯỜNG ĐẠI HỌC NHA TRANG 75

4 Effect of the rate of E/S to the effi ciency of the hydrolysis shows in fi gures 4 and 5, the indicators DH, NRE and N aa of hydrolysed protein solution increased when the rate of E/S increases from LAPU/g protein, continue to increase E/S to LAPU/g protein, the indicators did not increase signifi cantly, according to the statistical analysis, the difference was not signifi cant (p > 0.05). For TVB-N indicator, E/S increases from LAPU/g protein, the TVB-N increased strongly, then did not increase signifi cantly when further increasing E/S to LAPU/g protein. The previous study also showed that the soluble nitrogen under the action of enzymes in the hydrolysis and nitrogen recovery rate of hydrolysis products, as well as the severing of peptide links increases when the enzyme concentr tion increase (Wachirattanapongmetee et al., 2009; Motamedzadegan et al., 2010). This can be explained: When increasing E/S from LAPU/g protein, protein hydrolysed (disconnected polypeptide) occurs strongly by the excess substrate, leading to increasing DH, pulling NRE and N aa increased. Then continue to increase E/S, the velocity of hydrolysis is little changed since the concentration of enzyme saturation with substrate concentration. Ratio E/S = LAPU/g protein (corresponding to DH = 60.17%; NRE = 60.17%; N aa = 54.65% and TVB-N = 0.69 g/l) gave the most satisfying results of the conditions set out so should be selected as appropriate rate for the hydrolysis of herring protein Effect of time to the effi ciency of the hydrolysis by the Flavourzyme enzyme Figure 6. Effect of the time to the hydrolysis degree (DH) and nitrogen recovery efficiency (NRE) Figure 7. Effect of the time to the rate of N aa and TVB-N of hydrolysed protein Figure 6 and 7 showed that, when the hydrolysis time increased from 3 to 7 hours, all indicators DH, NRE, N aa, TVB-N increased. However, after 6 hours, the mentioned indicators did not increase signifi cantly (p < 0.05). For TVB-N, this indicator increased with hydrolysis time. The previous study also showed that DH increased over time hydrolyzed (Souissi et al., 2007; Chun et al., 2006; Amiza et al., 2012; Wachirattanapongmetee et al., 2009; Ovissipour et al., 2010; Shamloo et al., 2012). This is explained as follows: Time hydrolytic should be able to ensure for enzyme cut the link in the substrate, creating the desired end products met the target to the research. Prolonged duration of action, the enzyme hydrolyzed herring protein thoroughly But, if prolonged hydrolysis process will facilitate the microbial activities that cause rotten produce more low-end products such as NH 3, H 2 S, indole, scaptol... that affecting product quality. 6 hours of hydrolysis time shows best results, satisfy the set conditions so should be chosen as the appropriate time to proteolytic herring by Flavourzyme enzyme, corresponding to DH = 50.57%; NRE = 62.32%; N aa = 69.55% and TVB-N = 0.90 g/l. 76 TRƯỜNG ĐẠI HỌC NHA TRANG

5 3. Determine the effectiveness of the hydrolysis mode Apply appropriate hydrolysis mode, results are shown in table 2 and 3. Table 2. Evaluation the quality of hydrolysed protein of herring No. Indicators Results 1 Sensory Colors Odor Taste Status N aa N T Pale yellow Characteristic odor of hydrolyzed protein solution, pleasant Slightly bitter Pure fl uid g/l g/l 2 Chemistry DH 50.58% NRE 63.38% TVB-N 0.92 g/l Table 2 shows that, the color of protein hydrolysate solution is pale yellow, paler than that of fi sh sauce produced under traditional methods, pleasant smell, TVB-N (5.46%). Compared with the Vietnamese standards 5107:2003 of special fi sh sauce product about ammonia nitrogen content and total soluble protein, the hydrolysed protein can fully studied and put to use in the food sector. Table 3. Amino acid composition of the protein hydrolysate solution Amino acids Content (g/l) Amino acids Content (g/l) Aspartic 1.13 Tyrosine 0.44 Serine 0.91 Valine* 0.76 Glutamine 0.35 Methionine* 0.65 Glycine 0.26 Lysine* 1.38 Histidine* 0.82 Isoleucine* 0.12 Arginine 1.20 Leucine* 0.71 Threonine* 0.73 Phenylalanine* 0.75 Alanine 0.56 TAA Proline 0.11 TEAA 5.71 Cysteine 0.14 TEAA/TAA (*) Essential amino acids, TAA (Total amino acids), TEAA (Total essential amino acids) Results of amino acid composition analysis in Table 3 show that herring protein hydrolysates solution have high nutritional value, rich in essential amino acids (51.81%). The amino acid accounts for high content in herring protein hydrolysates solution is: Lysine (1.38 g/l), Arginine (1.20 g/l), Aspartic (1.13 g/l), this result coincides with research of scad hydrolysis by Flavourzyme (Chun et al., 2006). A number of other studies on hydrolyzed tuna head (Nguyen et al., 2012) and hydrolysis salmon head (Sathivel et al., 2005) also showed that essential amino acids content in hydrolyzed protein obtained from these studied were quite high. IV. CONCLUSION Herring fi sh could be hydrolysed by Flavourzyme enzyme at appropriate conditions: hydrolysis temperature (55 0 C); ratio of E/S LAPU/g protein; and hydrolysis time is 6 hours. Enzymatic hydrolysis of herring fi sh is suitable for production of protein hydrolysate that can be used for different applications. The resulting product can be well applied in the food industry to produce more type sauce and nutritional supplements for a variety of other foods. TRƯỜNG ĐẠI HỌC NHA TRANG 77

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