Nghiên cứu chế biến các sản phẩm giàu protein từ cá lóc (channa striata) nuôi tt tiếng anh

MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL THESIS Major: Food Technology Code: 62.54.01.01 VO HOANG NGAN RESEARCHING ON THE PROCESSING OF RICH PROTEIN PRODUCTS FROM CULTURED SNAKEHEAD FISH (Channa striata) Can Tho, 2020 THE PhD THESIS WAS COMPLETED AT CAN THO UNIVERSITY Advisor: Assoc Prof Dr Nguyen Van Muoi The doctoral thesis was evaluated by The Board of Examiners at the basic level Meeting at: Meeting room 2, 2nd floor, Campus II – Control Hall, Can Tho University At: 14 p.m., date 17/11/2018 Reviewer 1: Assoc Prof Dr Dai Thi Xuan Trang Reviewer 2: Prof Dr Dong Thi Anh Dao You can find a copy of this thesis at the library: - Learning Resource Center, Can Tho University - National Library of Vietnam LIST OF PUBLISHED PAPERS Tran, Thanh Truc, Vo, Hoang Ngan, Nguyen, Van Muoi, 2016 Influence of NaCl and additives on gel formation and texture characteristics of frozen fish bologna Journal of Science, Can Tho University, Special issue Agriculture, 1: 122-130 https://doi.org/10.22144/ctu.jsi.2016.030 ISSN 1859-2333 Vo, Hoang Ngan, Tran, Thanh Truc, Nguyen, Van Muoi, 2017 An exploration of the factors affecting the soluble protein extraction from cultured snakehead fish (channa striata) muscle Vietnam Journal of Science and Technology, 55(5A): 74-82 https://doi.org/10.15625/2525-2518/55/5A/12181 ISSN 0866708X Vo, Hoang Ngan., Tran, Thanh Truc, Nguyen, Van Muoi, 2018 Optimization of salt - water suplementation combined with other additives to improve the quality of snakehead fish cake Proceedings in “The 3rd International Conference on Sustainable Global Agriculture and Food (November 2018) HCMC, Vietnam” 296-310 ISBN 978-604-67-1160-5 Nguyen, Van Muoi, Tran, Thanh Truc, Vo, Hoang Ngan, 2019 The influence of additives on frozen snakehead fish surimi and the application of transglutaminase to fish cakes Acta Scientiarum Polonorum Technologia Alimentaria, 18(2): 1-9 http://dx.doi.org/10.17306/J.AFS.2019.0636 pISSN 1644-0730; eISSN 1898-9594 CHAPTER INTRODUCTION 1.1 The rationale of the study In recent years, Vietnam's seafood industry has always experienced strong growth and is one of the leading industries in the country's exports This has contributed to making Vietnam become one of the top ten seafood exporting countries of the world Occupying about 70% of the area and 73% of the country's output, the fisheries industry has become a very important economic strength of Mekong Delta, it has contributed greatly to the domestic consumption and export of our country In particular, snakehead fish farming had a very strong growth rate, snakehead fish production increased rapidly because of diversified farming models and high intensive farming Therefore, snakehead fish farming area was expanding spontaneously, so the supply was larger than the demand, which caused stagnation for the output of snakehead fish material source Besides, many studies have shown that global protein demand will double by 2050 This is not only due to the pressure of population growth but also because of increasing awareness of the importance of protein for human health in general and especially for elderly health Among proteincontaining foods, seafood has been thought to provide up to 17% of the total global protein needs Moreover, snakehead fish has long been known as a good source of protein with great potential for human consumption, especially for people with poor health These values are obtained because snakehead fish protein contains sufficient and balanced of amino acids and essential amino acids In the composition of aquatic proteins, soluble protein had many biological values, especially potential applications in the processing of high value hydrolyzed protein The myofibrillar protein obtaine from the soluble protein extraction process has good structural properties, suitable for the use in snakehead fish surimi processing Fish cake is made from snakehead surimi which will be a rich source of protein for people with diabetes, obesity and those who are allergic to fishy smell In addition, the protein from fish by-product also accounts for a large proportion, the quality of protein is similar in fish fillet but difficult to be used directly for human consumption, so there should be an effective solution to obtain this protein source Therefore, the study of recovering and processing rich protein products from different components of cultured snakehead fish has important economic and scientific significance This helps to increase the economic efficiency of snakehead fish farming and creates high-value protein products to meet human consumption needs 1.2 Objective of the study Optimize the efficiency of recovering different components of the raw materials to process and preserve rich protein products from snakehead fish according to the orientation of building the production process of frozen surimi and fried fish cake, hydrolyzed protein with high bioactivity and recycled protein solution from by-products after processing 1.3 Research contents Identify factors affecting on efficiency of extracting soluble protein from snakehead fish fillet; Studying suitable conditions in obtaining hydrolyzed protein with high biological activity; Building the process of surimi processing from snakehead fish paste and application of transglutaminase in processing fried fish cake; Optimizing the efficiency of protein recovery from snakehead fish byproducts by incubation method using Bacillus subtilis 1.4 Scientific and practical significance The research results of the thesis have shown a great potential in the use of snakehead fish in processing rich protein products including surimi, fish cake and hydrolyzed protein Research has great scientific and practical significance in creating food products with high nutritional value, highprotein products by integrating many techniques and technologies in processing processes such as extraction, enzyme technology, microbiological technology and barrier theory in product preservation The results of the study also contribute to solving difficulties in consuming snakehead fish, thereby helping to improve the income of fish farmers as well as build the appearance of new rural areas In addition, the use of byproducts as a source of raw materials for processing rich protein hydrolyzed products has helped take advantage of the raw materials and contribute to solving the environmental pollution problem in seafood processing With the results obtained from the study, it opens up the prospect of a comprehensive use of protein source in snakehead fish for processing into value-added foods as well as other rich protein products for human health 1.5 New contributions of the thesis The thesis has contributed practical and highly effective solutions in recovering and processing rich protein products from snakehead fish The soluble protein extracted from fish fillets after hydrolysis has created a product with high biological activity in terms of antioxidant capacity and in angiotensin converting enzyme inhibition That brings potential applications in the processing of functional foods Fish paste obtained from the extraction process is rich in protein that has been successfully applied in the processing of frozen snakehead surimi with long-term storage capacity Fried fish cake made from frozen surimi with the supporting effect of transglutaminase have good gel structure, both water retention and gel strength Finally, optimizing the incubation process by Bacillus subtilis has helped to obtain quite a thorough amount of protein from by product expressed through high efficiency and quality of recovered protein It can be said that the contributions of the thesis are the overall solution of the process of effectively collecting and processing protein from cultured snakehead fish 1.6 The structure of the thesis The thesis consists of chapters with 146 pages: Chapter 1: Introduction (pages: 1÷3); Chapter 2: Literature review (pages: 4÷33); Chapter 3: Research Methods (pages: 34÷63 with 17 experiments); Chapter 4: Results and discussion (pages: 64÷144) and Chapter 5: Conclusions and suggestions (page 145÷146) The primary content has 37 tables and 31 figures The thesis consists of 323 references (296 English references and 27 Vietnamese references) CHAPTER LITERATURE REVIEW 2.1 Cultured snakehead fish Snakehead fish (Channa striata) is a species of freshwater fish and very typical of Vietnam This fish has high nutritional value, delicious fish meat, popular in the market and is enjoyed by people This is a highly nutritious aquatic species, snakehead fish contains a lot of protein, minerals, low in fat and energy The protein content of snakehead fish is about 16-18%, which is more easily digested than protein from other animal meat The quality of protein is high value because it contains a completed and balanced essential and non-essential amino acids The amino acid content in snakehead fish also has high medical value, playing an important role in pain reduction and wound healing process 2.2 Methods of obtaining and processing protein products from snakehead fish Extraction is the process of separating one or several components of solute inside a liquid or solid with another liquid called solvent Many studies have shown that distilled water with different ionic strength was suitable for extracting soluble protein from aquatic products, and the use of distilled water in extraction helps recover appropriately in food processing Hydrolyzed protein from aquatic species, particularly from fish, is the product of hydrolysis of fish meat or fish by-products by proteases The process of protein hydrolysis creates products that are peptides, free amino acids with nutritional and biological values that meet the physiological functions for humans This is a processing method that brings high protein recovery efficiency and does not greatly affect the final product quality At present, hydrolyzed protein from aquatic species is considered as a functional food source and a very important source of bioactive peptides The fish muscle protein after removing the soluble components will be mixed with cryoprotectant to create a product called surimi This is a semi product that is usually frozen in block, stored in frozen form, and used as a raw material to research and process gel protein products such as fish cake with high nutritional and sensory value In addition, it is thought that it is possible to dissolve protein components from aquatic by-products to create products that are free peptides and amino acids, microorganisms and their enzymes are a useful biological tool to collect nutrition components in aquatic by-products The collection of protein from by-products is very important, helping to increase the efficiency of aquatic processing while also reducing the impact of the by-products on the environment 2.3 Domestic and foreign research on snakehead fish protein There have been many international publishments affirming the nutritional and medical value of snakehead fish The study of Gam et al (2005, 2006) and Haniffa et al (2014) on snakehead protein analysis has shown that this protein component is rich in amino acids with a high and balanced content, playing a very important role in nutrition as well as medicine Firlianty et al (2013) also showed that snakehead fish is a high protein content fish Besides being a high-value food source, snakehead fish also has medicinal properties that are appreciated as shown in the studies of Jais (2007) and Ma and Mj (2012) Buhari (2015) also suggested that snakehead fish extract could be applied in cancer treatment Evaluation of the self-transformation processes of snakehead fish after freezing in cold conditions was also conducted by Varghese and Mathew in 2016 By 2018, Rosmawati et al has initially announced the determination of chemical composition, amino acids and collagen of snakehead skin and bones The results of the above studies have shown that snakehead fish is a source of valuable nutritional and medical materials that need to be exploited Despite being a potential source of nutritional and medical materials, there are still limited domestic and foreign announcements about the collection and processing of protein components from snakeheads fish In 2012, Marimuthu et al conducted research on the impact of different cooking methods on the quality of snakehead fish fillet The study of Mustafa et al (2012, 2013) have shown that extracts from snakehead fish are rich in albumin and Zinc, these components have a great role for human health and especially the healing of post operative wounds In 2014, in a hydrolysis study to collect peptides inhibiting angiotensin-converting enzyme from snakehead fish protein, Ghassem et al has shown that alcalase has the highest hydrolytic efficiency with the highest inhibition of angiotensin-converting enzyme Lestari and Nanisa (2014) conducted a study on the effects of different types of chitosan on the quality of minced snakehead fish at cold storage Tan and Azhar (2014) also have researched to create snakehead fish powder from snakehead fish fillet with a high content of some amino acids In 2015, Wiranata et al used ethanol as a coagulating agent to recover albumin from the wash water in snakehead surimi processing Romadhoni et al (2016) studied the extraction of albumin from snakehead fish to make a rich albumin powder Research by Rahayu et al (2016) demonstrated that biologically active peptides derived from snakehead fish protein are a potent natural compound that has the potential to accelerate wound healing under cases such as postoperative, postpartum, and it also helps increase albumin In the 2017 study, Syukroni et al created albumin tablet from snakehead fish surimi wash water that meets Indonesia's National standards In 2018, Fahrizal et al conducted a comparison study of surimi quality from tilapia and snakehead fish (Channa striatus) and concluded that the addition of 5% sucrose, 5% sorbitol and 0.4% STPP created surimi with highest quality In 2018, Zakaria and Sarbon used hydrolyzed protein from snakehead fish to limit the lipid oxidation that occurs on refrigerated sausages Ikasari and Donny (2018) also studied the use of acids and bases in making protein products from snakehead extract byproducts In addition to the above-stated international publications, domestic research on cultured snakehead fish is also very limited Truong et al (2015) used raw bromelain to shorten fermentation time of snakehead fish with high efficiency Tran Thanh Truc et al (2016) investigated the effects of additives to increase the quality of snakehead fish floss In 2017, Tran et al used proteases collected from snakehead fish viscera to improve the physical and chemical properties of snakehead fish sausages All the above publications shows that snakehead protein has very high nutritional and medical value but has not been exploited effectively, especially the abundant source of snakehead fish in the Mekong Delta This will help open up great potential for the research on collecting and processing products from snakehead fish protein sources At the same time, the above problems also indicate that the need for a solution for thorough collection and processing of different protein components of snakehead fish is necessary and highly feasible CHAPTER RESEARCH METHODS 3.1 Materials - Time: from 6/2015 to 6/2019 - The research has used the means, tools and equipment at the Laboratory of Department of Food Technology, College of Agriculture, Can Tho University - Chemicals: Commercial protease (Alcalase 800 U/mL, Flavourzyme 700 U/mg, Papain (750 U/mg), Neutrase (400 U/mg), Novozymes, Denmark; Transglutaminase 40 U/g ( Activa TG-SR-MH), Malaysia; ACE (Angiotensin converting enzyme) (400 U/L, extracted from crossed rabbit lungs); Analytical chemicals of suitable purity are imported and distributed by CEMACO Vietnam Company Limited Branch in Can Tho - The necessary additives for research - Main research subject: Cultured snakehead fish (with average weight from 400÷700 g) was purchased directly from the farming area in Tam Binh district, Vinh Long province, Vietnam After being collected early in the morning, the fish was transported live (in a bucket of water) to the laboratory, which took about hour At the laboratory, the live fish was kept stable in the water tank for at least h before further processing 3.2 Research Methods 3.2.1 Analytical methods Table 3.1: The criteria and analytical methods No 10 11 Criteria Total nitrogen content (%) Solube protein content (%) Lipid content in solid (%) Lipid content in liquid (%) Total volatile base nitrogen, TVBN (%) Total Aerobic Microbial Count (CFU/g) Water holding capacity, WHC (%) pH value Moisture (%) Whiteness (L* - 3.b*) Gel strength, GS (g x mm) Methods Kjeldahl, TCVN 8125:2009 Lowry et al (1951) Soxhlet, TCVN 8125:2009 Adam-Rose-Gottlieb TCVN 9215-2012 TCVN 4884:2005 Grau and Hamm (1957; cited by Honikel and Hamm, 1994) ISO 2917:1999(E) NMKL 57-1994 Park (1994) GS = Breaking force x Deformation (Nowsad et al., 2000) No 12 Criteria Hydrolysis degree, DH (%) 13 Protein recovery efficiency, (%) 14 15 16 α-amino nitrogen content (%) Ammonia nitrogen content (%) Amino nitrogen content (%) 17 18 19 Angiotensin converting enzyme inhibitor capacity, ACEI (%) DPPH scavenging activity (%) Molecular weight 20 Microstructure image 21 Amino acid compositions 22 Total protein extraction efficiency (%) 23 Soluble protein extraction efficiency (%) 24 Lipid extract content (%) Methods DH% = α-amino nitrogen content / total nitrogen content (Liu et al., 2013) Protein recovery efficiency = (Total protein content in product x volume of product) x 100%/ (Total protein content in raw material x amount of raw material) Formol titration (Liu et al., 2013) TCVN 3706 – 90 Amino nitrogen content = α-amino nitrogen content – Ammonia nitrogen content Restrepo et al (2013) Vo et al (2016) SDS-PAGES, analyzed at the Biotechnology Research and Development Institute, CTU SEM, analyzed at the Intensive Laboratory, CTU TCVN 8764:2012, analyzed at NAFIQAD-6 Total protein extraction efficiency = (Total protein content in product x volume of product) x 100%/ (Total protein content in raw material x weight of raw material) Soluble protein extraction efficiency = (soluble protein content in product x volume of product) x 100%/ (Total protein content in raw material x weight of raw material) Lipid extract content = Lipid content in extract x 100% / Total protein content in extract 3.2.2 Methods of collecting and processing results The experiments were randomized with replicates The results of the experiments were analyzed and statistical using the Statgraphics Centurion XVII program and Excel software Methods of analysis of variance (ANOVA) and LSD test, Duncan test are used to conclude about the difference between the treatments Optimal experiments are designed showed that fillet has low lipid content (2.63±0.11%) while in by-products is 9.76±0.37% The water holding capacity of fillet meat is quite high (67.95±0.28%), the pH of fillet and by-products is around pH6.77±0.06 and pH6.45±0.08 Besides, the efficiency of recovering fillets and by-products is 47.12±1.48% and 46.87±1.21%, respectively 4.2 Extraction of soluble protein from cultured snakehead fish fillet 4.2.1 The appropriate ratio of fish muscle and water to extract soluble proteins from snakehead fish Survey results show that the use of different fish muscle and water ratios has an effect on the efficiency of soluble protein extraction and the further research processes Table 4.1 showed that the use of a ratio of 1:2 between the fish muscle and water helped to highly dissolve the soluble protein, creating a protein extract with a high content of soluble protein (10.11±0.2 mg/mL) and low lipid content (3.84±0.08%) The efficiency of soluble protein extraction and total protein extraction efficiency were 10.62±0.21% and 21.47±0.21%, respectively Table 4.1: Influence of the ratio of fish muscle and water on soluble protein extraction from snakehead fish Fish muscle: Soluble Protein extraction Total protein extraction Extracted lipid water (w:v) efficiency (%) efficiency (%) content (%) 1:1 8.87±0.29a 19.76±0.38a 3.67±0.11a b bc 1:2 10.62±0.21 21.47±0.21 3.84±0.08ab b b 1:3 10.69±0.20 21.07±0.19 4.30±0.09c b c 1:4 10.60±0.25 22.21±0.84 4.01±0.13b b b 1:5 10.50±0.13 21.05±0.13 3.70±0.17a (Different letters in the same column signify the significant difference of survey treatments at 95% confidence) 4.2.2 Effect of salt concentration on the efficiency of soluble protein extraction The salt concentration of the solvent has greatly influenced the efficiency of extracting soluble proteins from cultured snakehead fish The addition of salt with the concentration of 0.15M gave the highest efficiency of soluble protein extraction, reaching 11.7±0.24% of the total protein content of the raw material Too high or too low salt concentration reduced the efficiency of soluble protein extraction 4.2.3 Conditions of temperature and time to extract soluble protein from snakehead fish a Effects of temperature on the extraction of soluble proteins Temperature is one of the factors that greatly affects the soluble protein extraction process When the temperature is too high or too low, there is an 11 effect of reducing extraction efficiency This result help to identify the extraction at room temperature (30C), which has high efficiency of soluble protein extraction while saving energy and controlling the process easily b Effects of time on the extraction of soluble proteins Time is the decisive factor for the efficiency of extraction process, too short time is not enough for the diffusion process of dissolved components to solvent making the extraction efficiency low However, too long extraction time not only does not bring higher efficiency but also affects the quality of extracted products The results of the study showed that the implementation of the 20-minute extraction process yielded the highest protein extraction efficiency with a value of 12.80±0.14% soluble protein and 23.57±0.39% for total protein The lipid content in the extract at this time was also low, only at 3.74±0.12% c Optimizing temperature and time extraction Based on the results of determining the impact of each factor of temperature and time on the efficiency of extracting soluble protein from snakehead fish muscle, the correlative regression effect of the two factors on the response functions of the process of extracting soluble protein from snakehead fish (Y1 - Soluble protein extraction efficiency, %; Y2 - Total protein extraction efficiency, % and Y3 - Extracted lipid content, %) determined by Response Surface Methodology using Central Composite Design was established The regression equations that show the correlation between temperature and extraction time to the response functions of soluble protein extraction are shown as follows Y1 = 0.6 + 0.59X1 + 0.35X2 - 0.008X12 - 0.005X1X2 - 0.006X22 R2 = 96.40% and R2adjusted = 95.80% Y2 = -16.36 + 2.08 X1 + 0.88X2 - 0.031X12 - 0.013X1X2 - 0.011X22 R2 = 99.23% and R2adjusted = 99.11% Y3 = 5.056 - 0.069X1 - 0.125X2 + 0.001X12 + 0.001X1X2 + 0.003X22 R2 = 95.12% and R2adjusted = 94.30% The regression equations set up with quite high correlation coefficients and the lack of fit tests always have P> 0.05 has proved the suitability of the regression equations to the experimental results The equations can explain the correlation of temperature and time of extraction to the soluble protein extraction efficiency, total protein extraction efficiency and the extracted lipid content at 96.40%, 99.23% and 95.12% The relationship between temperature and time in soluble protein extraction is shown by Figure 4.1 and Figure 4.2 12 Figure 4.1: Impact of temperature and time interaction on soluble protein extraction Figure 4.2: Impact of temperature and time interaction on the response functions of soluble protein extraction The results of multiple responses optimization have helped to determine optimum conditions of temperature and time extraction of 34.98°C and 13.76 minutes The optimal value of each response function is shown in Table 4.2 The desired value achieved by optimizing multiple responses is 99% Table 4.2: The result of optimizing multiple responses in soluble protein extraction Level Optimal Y1max, Y2min, Y3min, Factors level % % % Low High Temperature, °C 20 40 34.98 13.59 22.00 3.29 Time, minute 10 30 13.76 To verify optimal conditions, controlled experiments at adjusted optimum conditions (35°C and 14 minutes) were conducted and obtained values of soluble protein extraction efficiency, total protein extraction efficiency and extracted lipid content were respectively 13.47±0.20%, 25.40±0.43% and 3.33±0.11% 4.2.4 Impact of solvent pH on soluble protein extraction In protein extraction, adjusting pH is also an effective solution for dissolving protein in raw materials into solvent, thereby increasing protein recovery efficiency Survey results of extraction solvent pH changed from pH2 to pH12 (Table 4.3) showed that pH10 is suitable for extracting soluble protein from snakehead fish muscle When extraction was performed at pH10, the efficiency of soluble protein extraction, total protein extraction and extracted lipid content were 14.39±0.18%, 25.45±0.52% and 3.50±0.10% respectively The extract had a soluble protein content of 13.69±0.17 mg/mL, and the proteins in the product have molecular weight mostly below 100 kDa Protein components with a molecular weight of about 40 kDa account for the highest proportion 13 Table 4.3: Influence of pH on soluble protein extraction from snakehead fish Soluble protein Total protein extraction Extracted lipid pH extraction efficiency %) efficiency (%) content (%) 10.70±0.26a 24.27±0.43cd 4.76±0.16d e cde 14.48±0.35 24.51±0.52 4.69±0.28d d c 14.00±0.29 23.98±0.34 6.03±0.13f b b 12.83±0.24 22.23±0.27 3.93±0.10c bc b 13.08±0.27 22.25±0.30 5.40±0.23e c def 13.32±0.27 25.07±0.60 3.24±0.18ab c f 7.4 (C) 13.47±0.20 25.40±0.43 3.33±0.11ab c def 13.43±0.12 24.96±0.79 3.14±0.37a d ef 13.97±0.13 25.16±0.52 3.20±0.12ab de f 10 14.39±0.18 25.45±0.52 3.50±0.10b c ef 11 13.49±0.16 25.33±0.42 4.57±0.08d a a 12 10.85±0.23 19.37±0.06 6.89±0.10g (Different letters in the same column signify the significant difference of survey treatments at 95% confidence) In short, extracting soluble protein from the snakehead fish fillet with a 0.15 M NaCl, pH10, 35°C, for a period of 14 minutes, using a 1:2 ratio of fish muscle and solvent (w/v) obtained the extract with an average soluble protein content of 13.69±0.17 mg/mL At optimum extraction conditions, the efficiency of soluble protein extraction and total protein extraction calculated on the total protein of the raw materials were respectively 14.39±0.18%, 25.45±0.52%, the extracted lipid content was 3.50±0.10% In addition to the soluble protein product, the extraction process also yielded 70.76% fish paste with moisture content of 78.47% and 16.75% of the protein used as a raw material in the study of processing surimi and fried fish cake 4.3 Obtaining hydrolyzed protein with high biological activity 4.3.1 Physicochemical compositions of protein extract The analysis of the basic components of the protein extract showed that the total protein content in 100 mL of the extract was 2.32±0.06% The very low lipid content of 0.08±0.01% and the pH6.46±0.27 indicated that the protein extract was suitable for application in hydrolysis by protease to create products with high biological activity 4.3.2 Effects of enzymes on ACE inhibition ability and antioxidant activity of hydrolyzed product The results of the hydrolytic efficiency survey of proteases (alcalase, bromelain, flavourzyme, neutrase, and papain) showed that alcalase and flavourzyme have better hydrolysis efficiency with high hydrolysis degree of 27.93±0.41% and 32.30±0.23% respectively ACE inhibition ability and DPPH scavenging activity of hydrolyzed product were respectively 14 38.85±1.01%, 71.63±0.76% for alcalase and 39.17±0.68%, 72.95±1.29% for flavourzyme In addition, in terms of hydrolysis mechanism, flavourzyme can both be used as an endoprotease and an exoprotease along with alcalase as an endoprotease with high hydrolysis degree Therefore, the combination of these two enzymes in the hydrolysis of protein will have the high feasibility of inhibiting angiotensin-converting enzyme and antioxidant 4.3.3 Effect of combination ratio of enzymes on ACE inhibition ability and antioxidant activity of hydrolyzed product The results of the study showed that the different combination ratio of alcalase and flavourzyme will give different hydrolysis effects, the combination showed a higher hydrolytic efficiency than the use of each enzyme individually The combination ratio of 25% alcalase and 75% flavourzyme gave the highest efficiency in the hydrolysis of protein extract from snakehead fish muscle At this combination ratio, the hydrolytic degree reached 38.38±0.43%, the angiotensin-converting enzyme inhibition ability and DPPH scavenging activity of hydrolyzed product were 40.49±0.43% and 75.02±0.91% respectively 4.3.4 Effect of temperature and pH on angiotensin-converting enzyme inhibition ability and antioxidant activity of hydrolyzed product Temperature and pH had a great influence on hydrolytic efficiency, so these were the main factors that determine the ability of hydrolytic enzyme to inhibit angiotensin-converting enzyme and scavenge DPPH The results also helped determine whether the temperature of 50°C and pH7 were suitable for hydrolysis of alcalase and flavourzyme The hydrolysis process had the highest hydrolytic degree, the ability to inhibit angiotensinconverting enzyme and the ability to scavenge DPPH, respectively 39.89±0.36%, 41.14±0.41% and 74.80±1.30% 4.3.5 Effect of enzyme activity and hydrolysis time on ACE inhibition and antioxidant activity of hydrolyzed product The research results showed that, when enzyme activity and hydrolysis time increased, hydrolytic efficiency increased However, the biological activity of hydrolyzed products was high when the hydrolysis process reached a certain level Survey results also showed that, with a mixture activity of 600 U/gprotein and hours of hydrolysis time, the product had a high biological activity both in the ability to inhibit the angiotensin converting enzyme and the ability to scavenge DPPH respectively 44.96±0.78% and 78.84±1.37% At this condition, the hydrolysis efficiency of the enzyme mixture reached 50.58±0.66% 15 4.3.6 Characteristics of hydrolyzed product from protein extract Table 4.4: Amino acid compositions of hydrolyzed product (concentrated 10 times) Amino acid Content (mg/mL) Amino acid Content (mg/mL) Valine 1.2 Glycine 1.47 Tyrosine 0.26 Glutamic acid 0.69 Threonine 1.08 Cystine 0.047 Serine 0.0004 Aspartic acid 0.045 Proline 0.2 Arginine ND Phenylalanine 1.5 Alanine 1.26 Methionine 0.63 Lysine 0.015 Leucine 1.7 Isoleucine 1.1 Histidine 0.8 Figure 4.3: Molecular weight of protein in the extract (4) and in hydrolyzed product (10) The analysis results showed that hydrolyzed product had 0.04±0.002% lipid, 1.94±0.06% protein including amino acids and short-chain peptides with low molecular weight, distributed around 14.4 kDa (Figure 4.3), protein recovery efficiency reached 82.20±0.95% The results of amino acid analysis showed that the product was a good quality protein solution because it contained all essential amino acids (Table 4.4) The analysis of biological activities showed that hydrolyzed product could inhibit 50% of angiotensin-converting enzyme activity and scavenge 50% of DPPH free radicals at the concentration of about 4.55 mgN/mL and 1.55 mgN/mL In summary, from the result of the above study, the hydrolysis of protein extract by different enzymes and hydrolysis conditions has helped improve the biological activity of the extract The combined ratio of 25%: 75% of alcalase and flavourzyme gave high hydrolysis efficiency The appropriate of hydrolysis was determined at condition of hydrolysis was 16 determined at 50°C and pH7 for hours using enzyme mixtures with the activity of 600 U/gprotein The hydrolysis process recovers 82.20% of protein, the product of hydrolysis is a high quality protein solution thanks to its full essential amino acid composition The molecular weight of the product was low, distributed around 14.4 kDa and had high biological activity The hydrolyzed product had a 50% inhibition of angiotensinconverting enzyme activity of 4.55 mgN/mL and a concentration of 50% DPPH scavenging activity was 1.55 mgN/mL Because of its biological activity and high nutritional quality, hydrolyzed product will have the potential to be used in the development of functional food products 4.4 Processing snakehead fish surimi and application of transglutaminase in the production of fried snakehead fish cake 4.4.1 Physicochemical compositions of snakehead fish paste after extraction The recovery rate of fish paste after the extraction process was quite high (70.76±1.59%), with the water content of 78.47±0.31%, higher than fish fillet, so the protein content of the paste only 16.75±0.49% Analytical results also showed that, lipid content in fish paste was also low (2.02±0.13%) In addition, due to its high pH and water holding capacity, respectively, pH6.83±0.06 and 70.89±0.87%, snakehead fish paste is suitable for processing surimi and surimi based products 4.4.2 Effect of cryoprotectant (sucrose and sorbitol in 1: ratio) and sodium tripolyphosphate on the quality of snakehead fish surimi In frozen surimi processing technology, cryoprotectant and sodium tripolyphosphate played a key role in helping maintain product quality through protecting structure and function of protein The research results showed that the addition of 3% cryoprotectant and 0.2% sodium tripolyphosphate was suitable for processing frozen snakehead fish surimi With this addition, fish cake processed from frozen snakehead fish surimi had water holding capacity, gel strength and whiteness were improved corresponding to values of 93.2±0.69%; 4.40±0.11 kgfxmm; 43.50±0.58 4.4.3 Frozen storage capacity of snakehead surimi Frozen snakehead fish surimi with the addition of 3% cryoprotectant and 0.2% sodium tripolyphosphate could be stored in frozen condition (18±2°C) for up to months that physicochemical properties still maintain, especially gel strength and water holding capacity By the 5th month of storage, total volatile base nitrogen was still at 15.86±0.56 mg/100g surimi, gel strength and water holding capacity were not different from before storage, respectively, 92.45±0.43% and 4.06±0.17 kgfxmm 17 4.4.4 Effects of transglutaminase and incubation time on gel characteristics of snakehead fish cake Transglutaminase has been widely used to improve the gel properties of gel protein products by effectively creating bonds within and between proteins In the study results, the combination between the 4-hour incubation period and the ratio of additional transglutaminase 0.7% was the most suitable condition in processing fish cake from frozen snakehead fish surimi Figure 4.4: Changes in molecular weight of protein in snakehead fish cake by the effect of transglutaminase (2- Marker; 7- Fillet; 8- Surimi; 9- surimi with 0.7% transglutaminase and hours incubation) The results of molecular weight analysis also showed that the effect of transglutaminase created links of proteins that help form proteins with a molecular weight of 70 kDa (Figure 4.4), thereby increasing water holding capacity and improving gel strength Because of this combination, water holding capacity and gel strength of 94.7±0.36% and 5.87±0.27 kgfxmm respectively, the whiteness of the product was also improved (43.06±0.54) A B C Figure 4.5: Scanning electronic micrographs of fried fish cakes processed from different materials (A – fillet, B – surimi, C – surimi with 0.7% transglutaminase and h incubation) 18 The results of principal component analysis (Figure 4.6) show that the sensory attributes that made up the quality of fish cake processed from frozen snakehead fish surimi that had the supporting effects of transglutaminase were mainly gel structure These attributes include crunchy, chewy, white and elastic Figure 4.6: Distribution of sensory attributes by the principal component F1 and the principal component F2 In addition, sensory analysis also helped to confirm that fish cake processed from frozen fish snakehead surimi with the supporting of transglutaminase had good sensory value and achieved higher consumer tastes (Figure 4.7 and Figure 4.8) Figure 4.7: Consumer sensory about attributes of products 19 Figure 4.8: Frequency of sensory points of products on a 9-step Hedonic scale 4.4.5 The effect of setting condition on the change of gel characteristics of fried snakehead fish cake In the processing of fish cake, setting (pre-heat treatment) helps form the gel protein system is one of the most important and indispensable stages, which is decisive for the sensory value as well as the quality of the product The research results show that the appropriate condition in setting fish cake processed from frozen snakehead fish surimi was 55°C for 40 minutes In this condition, fried snakehead fish cake had the best water holding capacity and gel strength, respectively, 95.54±0.36% and 6.07±0.11 kgfxmm, and the product moisture was also maintained (73.73±0.58%) 4.4.6 Influence of the deep fried condition on the quality of fish cake processed from frozen snakehead fish surimi In food processing, frying is a process of heating that helps to increase the structure, as well as creating colors, specific flavors for the product, which will make the product better quality Research results indicated that performing oil deep frying at 175°C for 2.5 minutes was an appropriate parameter to help snakehead fish cake with good gel properties (74.60±0.53% moisture, water holding capacity of 95.40±0.61% and gel strength of 6.21±0.11 kgfxmm), microbiological density was safely controlled (0.05 has proved that the selected design was suitable with experimental data The results of multiple responses optimization was shown in Table 4.5 with desired value of 83.54% Table 4.5: The result of optimizing multiple responses in the process of recovering protein from by-products of snakehead fish Level Optimal Y4max, Y5max, Y6min, Factors level % % % Low High pH 6.72 Salt, % 7.74 57.68 22.53 3.71 Bacillus subtilis, % 1,5 2,5 2.5 Time, day 25 35 28.89 Verification of the suitability of optimum values obtained from the optimization results was also performed, three test samples were conducted repeatedly independently based on value of the optimal conditions such as 22 Table 4.5 (hydrolysis time is adjusted to 29 days) The hydrolyzed product obtained had a protein content of 13.54±0.51%, the criteria of protein recovery efficiency, amino nitrogen content, ammonia nitrogen content, respectively, reached 58.73±0.45%, 22.08±0.43% and 3.96±0.28% The results obtained were not significantly different from those obtained in Table 4.5, this has contributed to the affirmation, the study evaluating the simultaneous impact of factors on the incubation process to find the optimal solution which was very important and highly feasible Table 4.6: Amino acid compositions of hydrolyzed product from snakehead fish by products Amino acid Content (mg/mL) Amino acid Content (mg/mL) Valine 0.23 Glycine 0.22 Tyrosine 0.03 Glutamic acid 0.57 Threonine 0.1 Cystine 0.0022 Serine 0.13 Aspartic acid 0.31 Proline 0.15 Arginine 0.01 Phenylalanine 0.14 Alanine 0.27 Methionine 0.15 Lysine 0.45 Leucine 0.17 Isoleucine 0.1 Histidine 0.09 Results of amino acid analysis showed that hydrolyzed product had the full presence of essential amino acid components (Table 4.6) Hydrolyzed products have an essential amino acid to total amino acid ratio (TEAA/TAA) of 37.71%, this value was very important to express the quality characteristics of hydrolyzed product The results of electrophoresis analysis also showed that hydrolyzed product had very low molecular weight, concentrated mainly at less than 14.4 kDa In conclusion, from the results of the study, it is shown that recovering of protein from snakehead fish byproducts using Bacillus subtilis has been implemented to effectively take advantage of the protein component from cultured snakehead fish Optimizing multiple responses helped determine the incubation conditions corresponding to 2.5% of Bacillus subtilis with 7.74% NaCl at pH 6.72 and carried out in 28.89 days In that condition, the protein recovery efficiency was 57.68%, the amino nitrogen content reached 22.53% and the ammonia nitrogen content was 3.71% Recovered protein products have very low molecular weight (