* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Khoa hoc - Cong nghe
Paracrine signalling wikipedia , lookup
Gene expression wikipedia , lookup
Ultrasensitivity wikipedia , lookup
Ribosomally synthesized and post-translationally modified peptides wikipedia , lookup
Enzyme inhibitor wikipedia , lookup
Expression vector wikipedia , lookup
G protein–coupled receptor wikipedia , lookup
Genetic code wikipedia , lookup
Ancestral sequence reconstruction wikipedia , lookup
Magnesium transporter wikipedia , lookup
Biochemistry wikipedia , lookup
Point mutation wikipedia , lookup
Interactome wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Amino acid synthesis wikipedia , lookup
Biosynthesis wikipedia , lookup
Metalloprotein wikipedia , lookup
Protein purification wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Western blot wikipedia , lookup
Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 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 Vân1, Đỗ Thị Thanh Thủy2 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 550C, the ratio of enzyme-substrate 38.96 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 11.79 g/l amino acid nitrogen, have the potential applications in many fields. Keywords: Enzymatic hydrolysis, protein hydrolysis, herring fish, 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 55oC, tỷ lệ enzyme - cơ chất 38.96 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 11.79 g/l, có tiềm năng ứng dụng trong nhiều lĩnh vực. Từ khóa: thủy phân, dịch đạm thủy phân, cá trích, Flavourzyme I. INTRODUCTION Herring is a species of trash fish that caught in large amount - account for about 16.46% of the total trash fish exploitation. Currently, herring used only to eat fresh, produced dried products, used in animal feed, a few exported as frozen bait, create low economic efficiency. Research to create value-added products from plentiful source of herring fish to improve the efficiency 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 fish into value-added products. Enzymatic hydrolysis is one of the most efficient methods to recover proteins from trash fish and to produce the protein hydrolysates. 1 The research to find out the appropriate mode hydrolysis to produce soluble protein fluid rich in amino acids from herring is a feasibility idea. The potential use of fish 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 significant revenue for fish processing industry. II. MATERIALS AND METHODS 1. Materials 1.1. Herring The herring fish (Sardinella Gibbosa) are purchased in Hon Ro ports - Nha Trang City. 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 Tạp chí Khoa học - Công nghệ Thủy sản Fish was fresh, bright, no stains lesions, size 19 - 20 units/kg. Fish are washed, removing impurities, storage and transport to the laboratory in insulation foam box at 0 - 400C. At the laboratory, to ensure uniformity, fish were washed, drained, minced, mixed thoroughly, divided, and packaged in vacuum PA bags, frozen and stored at -20 ± 20C 1.2. Flavourzyme Enzyme Flavourzyme protease enzyme was produced by Novozymes A/S (Bagsvaerd Denmark). Flavourzyme Herring fish Hydrolysis Inhibit enzyme Dregs Filter Số 2/2014 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 5.0 - 7.0 and at a temperature of 50 - 550C. 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: 45 ÷ 65oC, δ = 5oC Determine the effect of E/S: 9.74 ÷ 48.7 LAPU/g protein Determine the effect of time: 3 - 7 hours, δ = 1 hour Natural pH of the fish, W/NL =1/1 The filtrate 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 0 - 40C, in 15 hours. - Identify appropriate hydrolysis temperature: set th = 45 - 650C, δ = 50C 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 fish. - Identify the ratio of enzyme-substrate: set E/S = 9.74 - 48.7 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 fish. - 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 fish. - The enzyme was inactivated by heating at 950C for 15 minutes in a water bath. 74 • TRƯỜNG ĐẠI HỌC NHA TRANG 2.2. Chemical analyses Water content: TCVN 3700-90; Ash content: calcined at 6000C methods; lipid content: according to TCVN 3703:2009, the total nitrogen content: TCVN 3705-90; NH3 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., 2002. 2.3. 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 Office Excel 2007 software was used to calculate results. Differences in treatment means were considered significant at p < 0.05. Tạp chí Khoa học - Công nghệ Thủy sản 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 73.87 ± 0.01 Protein 19.25 ± 0.04 Lipid 2.53 ± 0.03 Ash 0.96 ± 0.01 Số 2/2014 Table 1 showed that the herring have high total protein content (19.25%) higher than cuttlefish (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%), classified as lean fish 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 efficiency of the hydrolysis Figure 2. Effect of temperature on the hydrolysis degree (DH) and nitrogen recovery efficiency (NRE) Figure 3. Effect of temperature to the rate of Naa/NT and TVB-N of hydrolysed protein Figures 2 and 3 show that, when the temperature rose from 450C to 550C, then 3 indicators DH, NRE and Naa/NT increased and peaked at 55°C; if the temperature continues to rise to 650C, the measured indices were reduced. But TVB-N concentration decreased continuously as the temperature increases from 450C to 650C. This can be explained as follows: when the temperature increases from 450C to 550C, the activity of Flavourzyme enzyme increased due to the activation energy of the reaction is enhanced, then further increase in temperature up to 65oC, the activity of Flavourzyme enzyme decreased because high temperature inhibits the activity of Flavourzyme enzyme. When the temperature increases from 45 - 650C, the activity of microbial flora reduced because the microbiota in herring active at topt less than 450C, when the temperature increases from 45 - 650C their activity are inhibited, leading to the TVB-N concentration decreases with hydrolysis temperature. At 550C (DH, NRE, Naa/NT 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. 2.2. Affect of the ratio of enzyme-substrate (E/S) to hydrolysis efficiency 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 Naa/NT and TVB-N of hydrolysed protein TRƯỜNG ĐẠI HỌC NHA TRANG • 75 Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 Effect of the rate of E/S to the efficiency of the Motamedzadegan et al., 2010). This can be hydrolysis shows in figures 4 and 5, the indicators explained: When increasing E/S from 0 - 38.96 DH, NRE and Naa/NT of hydrolysed protein LAPU/g protein, protein hydrolysed (disconnected solution increased when the rate of E/S increases from 0 - 38.96 LAPU/g protein, continue to increase E/S to 48.70 LAPU/g protein, the indicators did not increase significantly, according to the statistical analysis, the difference was not significant (p > 0.05). For TVB-N indicator, E/S increases from 0-29.22 LAPU/g protein, the TVB-N increased strongly, then did not increase significantly when further increasing E/S to 48.70 LAPU/g protein. The previous study also showed that the soluble nitrogen under the action polypeptide) occurs strongly by the excess substrate, leading to increasing DH, pulling NRE and Naa/NT 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 = 38.96 LAPU/g protein (corresponding to DH = 60.17%; NRE = 60.17%; Naa/NT = 54.65% and TVB-N = 0.69 g/l) gave the most satisfying results of the conditions set out of enzymes in the hydrolysis and nitrogen recovery so should be selected as appropriate rate for the rate of hydrolysis products, as well as the severing of hydrolysis of herring protein. peptide links increases when the enzyme concentr 2.3. Effect of time to the efficiency of the hydrolysis tion increase (Wachirattanapongmetee et al., 2009; 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 Naa/NT 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, Naa/NT, TVB-N increased. However, after 6 hours, the mentioned indicators did not increase significantly (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 NH3, H2S, 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%; Naa/NT = 69.55% and TVB-N = 0.90 g/l. 76 • TRƯỜNG ĐẠI HỌC NHA TRANG Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 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. 1 2 Indicators Sensory Chemistry Results Colors Pale yellow Odor Characteristic odor of hydrolyzed protein solution, pleasant Taste Slightly bitter Status Pure fluid Naa 11.79 g/l NT 16.84 g/l 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 fish sauce produced under traditional methods, pleasant smell, TVB-N/NT (5.46%). Compared with the Vietnamese standards 5107:2003 of special fish 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 11.02 Proline 0.11 TEAA 5.71 Cysteine 0.14 TEAA/TAA 51.81 (*) 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 fish could be hydrolysed by Flavourzyme enzyme at appropriate conditions: hydrolysis temperature (550C); ratio of E/S 38.96 LAPU/g protein; and hydrolysis time is 6 hours. Enzymatic hydrolysis of herring fish 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 Tạp chí Khoa học - Công nghệ Thủy sản Số 2/2014 REFERENCES 1. Amiza, M.A., Kong, Y.L., Faazaz, A.L., 2012. Effects of degree of hydrolysis on physicochemical properties of Cobia (Rachycentron canadum) frame hydrolysate. International Food Research Journal, 19 (1): 199-206. 2. Chun, C., Mouming, Z., Xiaofang, Z., Jiaoyan, R., 2006. Protein degradation of extensive enzymatic hydrolysis of decapterus maruadsi. Transactions of the CSAE, Vol.22, No.1. 3. Liaset, B., Nortvedt, R., Lied, E., Espe, M., 2002. Studies on the nitrogen recovery in enzymatic hydrolysis of Atlantic salmon (Salmo salar, L.) frames by ProtamexTM protease. Process Biochemistry, 37: 1263-1269. 4. Motamedzadegan, A., Davarniam, B., Asadi, G., Abedian, A., 2010. Optimization of enzymatic hydrolysis of yellowfin tuna Thunnus albacares viscera using Neutrase. Int Aquat Res, 2: 173-181. 5. Nguyen, H.T.M., Sylla, K.S.B., Randriamahatody, Z., Donnay-Moreno, C., Moreau, J., Tran, L.T., Bergé, J.P., 2012. Enzymatic hydrolysis of yellowfin tuna (Thunnus albacares) by-products using Protamex protease. Food Technology and Biotechnology, 49 (1): 48 - 55. 6. Ovissipour, M., Benjakul, S., Safari, R., Motamedzadegan, A., 2010. Fish protein hydrolysates production from yellowfin tuna Thunnus albacares head using Alcalase and Protamex. Int Aquat Res, 2: 87-95. 7. Shamloo, M., Bakar, J., Mat Hashim, D. and Khatib, A., 2012. Biochemical properties of red tilapia (Oreochromis niloticus) protein Hydrolysates. International Food Research Journal, 19 (1): 183-188. 8. Santhivel, S., Smiley, S., Prinyawiwatkul, W., Bechtel, P.J., 2005. Functional and Nutritional Properties of Red Salmon (Oncorhynchus nerka) Enzymatic Hydrolysates. Journal of Food Science, 70 (6): 401-406. 9. Souissi, N., Bougatef, A., Triki-Ellouz, Y., Nasri, M., 2007. Biochemical and Functional Properties of Sardinella (Sardinella aurita) By-Product Hydrolysates. Food Technol. Biotechnol. 45 (2): 187-194. 10. Wachirattanapongmetee, K., Wachirattanapongmetee, K., Thawornchinsombut, S., Pitirit, T., Yongsawatdigul, J., Park, J.W., 2009. Functional Properties of Protein Hydrolysates Prepared from Alkali-Aided Protein Extraction of Hybrid Catfish Frame. Trends Research in Science and Technology, (1): 71-81. 78 • TRƯỜNG ĐẠI HỌC NHA TRANG