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MINISTRY OF EDUCATION AND TRAINING VIETNAM ACADEMY OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY - NGUYEN THI PHUONG LAN RESEARCH ON CHEMICAL COMPOSITION AND DEVELOPMENT OF A PRODUCTION PROCESS OF FOOD SUPPLEMENTS FROM SOFT-SHELL CRABS (SCYLLA) Major: Chemical Engineering Code: 9.52.03.01 SUMMARY OF DOCTORAL THESIS IN CHEMISTRY HANOI – 2021 The thesis was completed at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisor 1: Assoc Prof Dr Nguyen Quyet Chien Supervisor 2: Dr Le Tat Thanh Opponent 1: … Opponent 2: … Opponent 3: … The thesis will be defended at an institutional-level Thesis Committee, meeting at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at …… hour……., on day … month … year 2021 The thesis is available at: - Library of the Graduate University of Science and Technology - National Library of Vietnam INTRODUCTION Rationale of the doctoral thesis In our country, the mud crab (Scylla spp.) is a traditional saltwater-brackish aquatic species with important socio-economic values in the coastal fishing community, and a preferred food source of the majority of Vietnamese people Along with traditional crab farming, soft-shell crab farming (raising and harvesting freshly molted crabs) is increasingly developing, helping to increase economic value of crabs and diversify aquaculture Soft-shell crabs are rich in nutrients, but soft-shell crab products can only be used as “instant” food, which means that products are only processed, packed, frozen, then consumed domestically or exported to be used as ingredients for restaurants to process a variety of dishes Researches on chemical composition, biological activity of soft-shell crabs as well as processing technology to increase value and prepare high quality products are still very limited In recent years, the trend of applying enzyme technologies in the food processing field has grown strongly, especially in the field of food supplement production+ from nutrient-rich, biologically active compounds that promote health Marine organisms in general and saltwater-brackish crustaceans in particular have a high protein content that can be used as an ideal source of raw materials to obtain functional compounds with various biological activities by enzyme technology in order to serve development and production of food supplements For the above reasons, in order to affirm values of soft-shell crabs and study the processing technology to create products with nutritional value and health-promoting effects, contributing to promoting stable and sustainable development of soft-shell crab farming, we chose the thesis title “Research on Chemical Composition and Development of a Production Process of Food Supplements from Soft-Shell Crabs (Scylla)” 2 Research objectives of the thesis The objectives of the thesis are to research chemical composition, explore biological activities of soft-shell crabs, and research to optimize enzymatic hydrolysis and spray drying process, thereby developing a production process of food supplements from soft-shell crabs Main research contents of the thesis - Research to determine chemical composition and biological activity of soft-shell crabs - Research to optimize the hydrolysis technology process of soft-shell crabs and the spray drying process to create hydrolyzed soft-shell crab powder with health-promoting effects - Assess the activity of hydrolyzed soft-shell crab products and health protection preparations from hydrolyzed soft-shell crab powder CHAPTER OVERVIEW 1.1 Overview of Scylla spp This section gives a general introduction to crabs and the genus of mud crabs (Scylla spp.) (morphological characteristics, biological characteristics, distribution) This section also introduces soft-shell crabs, soft-shelled crab farming, use and processing of soft-shell crabs 1.2 Chemical studies of crabs This section reviews international studies on chemical composition and biological activities of sea crabs and mud crabs Scylla 1.3 Introduction of enzymatic hydrolysis technology in food processing This section introduces hydrolysis technology, enzyme technology and enzyme application in seafood processing 1.4 Introduction to experimental planning and optimization of chemical engineering processes This section introduces experimental planning, response surface methodology (RSM) and Design Expert 12.0 software CHAPTER RESEARCH OBJECT AND METHODOLOGY 2.1 Research object The research object is the crab Scylla paramamosain at the molting stage, which was sampled and scientifically named by Dr Nguyen Thi Bich Ngoc – Research Institute for Aquaculture No Raw crabs with a weight of 100-120g/crab and carapace width of 8090 mm were raised separately, each in plastic box (15 cm x 20 cm) in a recirculating aquaculture system using industrial feed at Research Institute for Aquaculture No 2.2 Research methodology 2.2.1 Method for determination of chemical composition * Moisture: TCVN 3700-90 * Ash content: TCVN 5105:2009 * Total protein content: Kjeldahl method (TCVN 3705:1990) * Amino acid content was determined by HPLC method, in which hydrolysis was carried out according to Kang-Lyung Woo (2003), derivatization method and chromatographic analysis were performed according to Agilent and Jajić’s (2013), Zeng (2013) * Total lipids: Method of E.G Bligh and W.J Dyer * The composition and content of lipid classes were analyzed and determined on Sorbfil pre-coated plates according to Imbs A B (2015) * The composition and content of fatty acids were determined by gas chromatography (GC, GC-MS according to the method of Carreau & Dubacq (1978) and ISO/FDIS 5590:1998 * The molecular species of classes of polar lipids was determined according to the method of Imbs (2015) and Sikorskaya (2018) * Vitamins A, D, E: By LC-MS/MS referring to AOAC 992.26, AOAC 2012.10 and AOAC 2001.13 * Vitamin K: By LC-MS/MS referring to TCVN 8974:2011 * Vitamins B2, B3: AOAC 2015.14 * Ca, Mg, Na, K, P, Fe, Zn: AOAC 2011.14 and TCVN 10641:2014 * Cu, Mn: AOAC 2015.01 and AOAC 2015.06 * Cholesterol content: TCVN 12385:2018 and method of Marıa A Paulazo (2020) * Ecdysone content: Method of Soumoff (1985) and Lafont R (1994) 2.2.2 Biological Activity Evaluation Methods 2.2.2.1 In vitro anti-osteoporotic activity from hydrolyzed protein powder and anti-osteoporosis product from soft-shell crabs: According to the method of Alcantara EH (2011) and Lai CH (2014) 2.2.2.2 Anti-inflammatory activity of lipid fractions from soft-shell crabs: Evaluated through the ability to inhibit NO production by the method of Liao H (2014), Tsai PJ (2007) and Cheenpracha S (2010) 2.2.2.3 Antioxidant activity by malondialdehyde assay (MDA assay): According to the method of Jelili A Badmus (2011) and Engoor (2013) 2.2.2.4 Cytotoxic activities against cancer cells: Performed on cancer cell lines according to the method of Monks A (2011) and Tim Mosmann (1983) 2.2.3 Enzymatic hydrolysis method It was carried out according to the method of hydrolysis of mud crabs by protease enzymes of the author Harun (2017) Degree of hydrolysis (DH) was determined according to the method of Nielsen (2001) The peptide content in the hydrolysate liquid and hydrolyzed soft-shell crab powder was also determined by the OPA method and calculated based on the L-glutathione standard curve equation 2.2.5 Method of experimental planning and optimization of technological processes It was applied according to Box-Wilson quadratic orthogonal plan model Design Expert 12.0 software was used to model and optimize technological parameters of the process CHAPTER EXPERIMENT 3.1 Preliminary processing and treatment of soft-shell crab samples 3.2 Study diagram of soft-shell crab samples 3.3 Analysis of basic chemical components This section describes experimentally how to determine basic chemical composition of soft-shell crabs: moisture content, ash content, protein content, total lipids, vitamins, minerals, cholesterol and ecdysone content 3.4 Analysis of composition and content of amino acids 3.5 Determination of composition and content of fatty acids 3.6 Determination of composition and content of lipid classes in total lipids 3.7 Analysis of molecular species of polar lipids This section presents how to determine each species of substances in polar lipids according to retention time (Table 3.2) and highresolution mass spectrometry (HRMS) data of 58 molecular species of phospholipids including species of PE, 23 species of PC, 15 species of PS, 10 species of PI, species of LPC and species of LPE Table 3.2 Retention time of species of substances Subclass Species Phospholipid PE PC PS PI Retention 4.9-6.0 5.6-10.3 13.3-15.2 15.1-18.1 time (minute) LPC LPE 15.8-18.2 18.4-19.0 3.8 Bioactivity testing This section describes experimentally how to determine the biological activity (cancer cytotoxicity, antioxidant activity by MDA measurement, anti-inflammatory activity) of lipid fractions from soft-shell crabs and anti-osteoporotic activity of hydrolyzed protein powder and anti-osteoporotic products from soft-shell crabs 3.9 Research and optimization of enzymatic hydrolysis of soft-shell crabs 3.9.1 Equipment and materials 3.9.2 General procedure for hydrolysis Raw material soft-shell crab samples were minced Each experiment weighed 50 g and put into the reaction vessel, and then added the required amount of water for each experiment The raw material solution and water were stirred at 200 rpm and inhibited endogenous enzymes at 80oC for 10 minutes After adding the necessary enzymes, hydrolysis was carried out at different temperatures, pH, and time to determine optimal conditions The hydrolysis was terminated by raising temperature to 80oC for 15 minutes to inactivate enzymes The hydrolysate liquid was then centrifuged, taking the liquid for determining degree of hydrolysis (DH) Each experiment was performed times to obtain the mean value 3.9.3 Determination of degree of hydrolysis (OPA method) 3.9.4 Survey of influencing factors 3.9.5 Research on optimization of hydrolysis process The studied technological factors include: pH, temperature (oC), bromelain enzyme/substrate ratio (%), chitinase enzyme/substrate ratio (%), hydrolysis time (hour) Target function Y: Hydrolysis (%) Select the survey model according to Box-Wilson with k = 5, choose the swing arm α = 1.546 and the number of experiments at the center is The total number of experiments of the matrix is 27 3.10 Research and optimization of the spray drying process 3.10.1 Equipment and materials 3.10.2 General Procedure The hydrolysate liquid of soft-shell crabs after filtering through a sieve (3 mm sieve) was put into a 10-liter bucket, gently stirred at 80 rpm and pumped directly into a spray dryer with specified parameters of spray disc speed and temperature The inlet drying gas and the pump speed are determined Hydrolyzed soft-shell crab powder was collected in the drying chamber every 45 minutes for analysis Spray drying results were evaluated through two criteria: protein content and moisture 3.10.3 Survey of influencing factors 3.10.4 Research on optimization of spray drying process The surveyed technological factors include: spray disc speed (rpm), inlet drying air temperature (oC), and hydrolysate liquid injection speed (mL/min) The target functions are Y1: Protein content in the obtained product (g/100g), and Y2: Product moisture (%) Select the survey model according to Box-Wilson with k = 3, swing arm α = 1.215 and the number of experiments at the center is The total number of experiments of the matrix is 15 experiments 3.11 Production of hydrolyzed soft-shell crab powder on pilot scale 3.12 Research orientation diagram of the technological process to create food supplement products on laboratory scale Raw materials of soft-shell crabs were preliminarily processed, minced and hydrolyzed by the method of applying enzyme technology The conditions of pH, temperature, enzyme/substrate ratio, hydrolysis time were surveyed and assessed, and the hydrolysis process was optimized to achieve the highest degree of hydrolysis The hydrolysate liquid obtained after passing through a 0.3 mm sieve was spray-dried to obtain hydrolyzed soft-shell crab powder The spray drying process was also optimized for the speed of the spray disc, the temperature of the inlet drying air, and the hydrolysate liquid injection speed Hydrolyzed soft-shell crab powder was evaluated for quality criteria To produce food supplement products, some additional ingredients and excipients were added to soft-shell crab powder, and then mixed, homogenized and packaged CHAPTER RESULTS AND DISCUSSION 4.1 Chemical composition of soft-shell crab Scylla paramamosain 4.1.1 Basic chemical composition 4.1.1.1 Moisture and ash content of soft-shell crabs: Presented in Table 4.1 Table 4.1 Moisture and ash content of soft-shell crabs Soft-shell crabs Composition Unit name Carapace Whole Hepatopancreas Meat and soft-shell Moisture content %/fresh sample 85.74 ± 2.06 82.50 ± 1.98 85.70 ± 2.06 81.9 ± 1.97 %/fresh sample 0.42 ± 0.03 0.39 ± 0.03 0.30 ± 0.02 0.56 ± 0.04 %/dry sample 2.98 ± 0.22 2.24 ± 0.17 2.12 ± 0.16 3.09 ± 0.23 Ash content 4.1.1.2 Protein content: Protein content in whole crab, hepatopancreas and meat reached 4.56%, 7.98% and 10.6%, respectively (based on fresh weight) 4.1.1.3 Lipid content: Lipid content in whole crab and meat was almost similar and reached 1.62% and 1.53%, respectively, while hepatopancreas was the richest in lipids, reaching 4.39% (based on fresh weight) 4.1.1.4 Vitamin content: vitamins were identified: A, E, K, B2, PP, in which vitamin E is the highest among vitamins with values from 804.4-2708 µg/100g 4.1.1.5 Mineral content: minerals were identified, including macro minerals Ca, P, Mg, Na, K and trace minerals Fe, Zn, Cu, Mn Ca and P contents in soft-shell crab samples ranged from 0.55-2.29 g/100g and 1.06-1.41g/100g dry weight, respectively Zn was found in high concentrations in meat and whole soft-shell crab samples (203.53 and 267.83mg/kg, respectively) 4.1.2 Composition and content of amino acids 17 amino acids were identified from soft-shell crab Scylla paramamosain Similar to the protein content, the total amino acid (TAA) content of soft-shell crab meat was times higher than that of whole soft-shell crab with values of 10.6 and 4.53 g/100g fresh weight, 11 29 22:4n-6 - 0.24±0.01 0.28±0.01 - - 30 22:5n-6 0.78±0.02 0.45±0.02 0.29±0.01 - - 31 22:5n-3 1.67±0.05 1.13±0.04 1.09±0.03 - - 32 22:6n-3 11.37±0.51 7.29±0.32 7.19±0.34 18.33±0.86 6.19±0.31 ∑PUFA Others 32.46±1.52 32.71±1.44 32.90±1.38 50.55±2.12 9.96±0.45 8.50±0.32 3.44±0.11 3.25±0.10 7.01±0.24 2.03±0.09 Omega3 (n-3) 28.65±1.22 14.65±0.67 14.57±0.57 23.45±0.76 6.19±0.29 Omega (n-6) 3.81±0.12 18.06±0.87 18.33±0.83 27.10±1.23 3.77±0.13 PUFA/SFA 0.86 1.12 3.15 0.18 n-3/n-6 7.52 0.81 1.15 0.79 0.87 1.64 4.1.5 Determination of molecular species of phospholipids Structural formula of species of phospholipids in soft-shell crabs is as follows: Phosphatidyletanolamin (PE) Phosphatidylcholine (PC) O O R2 R1 O O R2 O O O P HO R1 NH2 O OH O O O O O P HO OH OH O HO O OH OH Phosphatidylserine (PS) Phosphatidylinositol (PI) Lysophosphatidylethanolamine (LPE) Lysophosphatidylcholine (LPC) By HPLC-HRMS method, 58 molecular species of phospholipids were identified, including species of PE, 23 species of PC, 15 species of PS, 10 species of PI, species of LPC and species of LPE PE 38:5 38:6 Symbol % PE 18:0p/20:5 16:0/22:6 39,12 30,03 PS 38:5 38:4 Symbol 18:0/20:5 18:1/20:4 18:0/20:4 % PS 31,23 28,08 12 40:6 34:1 PC 34:1 38:6 38:5 18:0p/22:6 16:0/18:1 Symbol 16:0/18:1 16:0/22:6 18:0a/20:5 28,39 2,46 % PC 10,59 9,78 7,03 36:1 40:6 38:6 36:4 38:5 36:5 38:4 18:0a/20:4 6,85 39:4 38:6 18:0a/22:6 18:1a/20:5 16:0a/22:6 16:0/16:1 14:0/18:1 37//5 36//2 36//4 36//1 36//5 30//0 38//5 36//3 36//1 40//6 40//7 36//2 40//7 35//8 Symbol /20:5 /22:6 /20:4 6,04 38:6 32:1 37:5 36:2 36:4 36:1 36:5 30:0 38:5 36:3 36:1 40:6 40:7 36:2 40:7 35:8 LPE 20:5 22:6 20:4 8,08 7,64 6,34 4,11 3,73 2,89 37:5 18:0/18:1 18:0/22:6 16:0/22:6 16:0/20:4 18:0/20:5 16:0/20:5 18:0/21:4 19:0/20:4 17:0/20:5 5,28 34:1 16:0/18:1 1,52 3,27 38:2 18:0/20:2 1,20 5,94 5,95 5,68 5,02 4,52 4,48 4,00 3,82 3,64 2,76 2,13 1,74 1,17 0,31 % LPE 50,89 32,84 16,28 40:7 PI 38:5 38:4 39:4 38:6 36:5 38:4 36:4 36:4 38:7 39:4 18:1/22:6 Symbol 18:0/20:5 18:0/20:4 19:0/20:4 18:1/20:5 16:0/20:5 18:0p/20:4 16:0/20:4 16:0p/20:4 18:2/20:5 18:0/21:4 1,03 % PI 27,94 26,24 18,40 11,89 9,86 1,47 1,22 1,03 0,99 0,98 LPC 20:5 22:6 20:4 Symbol /20:5 /22:6 /20:4 % LPC 43,36 38,00 18,64 2,17 1,98 * Determination of molecular species of PE On the negative ion spectrum, [M-H]- ion has the strongest signal at m/z value 748.5128 (accounting for 39.12%) The molecular formula determined is C43H76NO7P (different 0.01536, number of double bonds: 8) and is an alkenyl acylglycerophosphoethnanolamine On the MS2spectrum, the signals are obtained simultaneously: at m/z 301.2141 corresponding to anion 20:5 [C20H29O2]-, at m/z 283.2101 corresponding to the ionic fragment of fatty acid 20:5 which losses molecule H2O [C20H27O]- and m/z 257.2249 corresponding to the ion fragment of fatty acid 20:5 which losses molecule CO2 [C19H29]- The fragment with signal at m/z 464.3115 corresponds to [C43H75NO7P]- which losses ketene molecule of fatty acid with m/z 284.2013 [C20H28O] (fatty acid 13 20:5 loses water molecule) The fragment with signal at m/z 329.2454 was obtained by the [M-H]- ion losing C3H6NO4P molecule and C18H36O molecule The fragment m/z 403.2567 is a signal when the [MH]- ion simultaneously loses ethanolamine [C2H5N] and a fatty acid molecule 20:5 [C20H30O2] Thus, the mass spectrometry data proved that the considered molecular species of PE are alkenyl acyl PE 18:0p/20:5 By GC, GC-MS method, fatty acid composition in the polar lipid fraction of soft-shell crabs is PE 18:0p/20:5n-3 By the same method, we have identified phospholipid molecular species of soft-shell crabs Thus, for soft-shell crabs, there is a wide range of species of phospholipids (6 species of substances have been identified) with many molecular species containing polyunsaturated fatty acids such as C20:4n-6 (AA), C20: 5n-3 (EPA) and C22:6n-3 (DHA) which have been determined by analysis using high-resolution mass spectrometry (HRMS) and analysis of fatty acid composition in polar lipid fractionation by GC-MS, which would have the potential to exhibit interesting biological activities 4.1.6 Cholesterol and ecdysone content during molting of softshell crabs Cholesterol and ecdysone are two compounds present in the lipid composition of soft-shell crabs and play an important role in the molting process of crabs During the culture of soft-shell crabs, the cholesterol content gradually increased from 2.541-33.249 mg/mL, reaching the highest in the sample hours after molting, however, the ecdysone content increased gradually from raising and peaked at the beginning of molting (sample Cho5) (from 8.1*10-3-35.8*10-3 mg/mL), and hours after molting, the ecdysone content dropped sharply to only 19.9*10-3 mg/mL) 4.2 Results of assessment of biological activities of lipid classes of softshell crabs 4.2.1 Assessment results of anti-inflammatory activity 14 Table 4.14 Ability to inhibit NO production of the studied samples Total lipidLiver NL- Liver PoL- Liver L-NMMA Concentration % % % % (µg/ml) % NO % NO % NO % NO living living living living inhibition inhibition inhibition inhibition cells cells cells cells 100 58.56 104.21 35.25 97.45 62.88 92.12 92.12 20 25.76 101.11 11.15 104.11 23.60 104.41 75.64 75.64 11.08 0.36 8.06 30.36 0.8 0.29 -1.44 2.88 14.57 IC50 71.52±4.41 Concentration (µg/ml) 100 20 0.8 IC50 >100 95.86 68.60±3.65 7.66±0.57 Total lipid-Meat NL-Meat PoL-Meat % NO % living inhibition cells 52.52 84.22 14.17 97.99 2.86 -1.63 93.82±5.23 % NO % living inhibition cells 27.05 99.30 3.07 106.10 1.58 -7.48 >100 % NO % living inhibition cells 38.71 100.37 1.01 101.54 -0.14 -1.87 >100 Table 4.14 showed that total lipids of soft-shell crabs inhibited NO production with IC50 of 71.5 µg/mL Notably, the polar lipid fraction exhibited strong NO inhibitory activity with an IC50 of 68.6 µg/mL The L-NMMA positive control had an IC50 value of 7.66 µg/mL Specifically, in the positive control sample, viable cells were 75.64% while with lipid samples, viable cells were higher than 97.99% - 106.1% at the equivalent concentration of 20 µg/ml This suggests that cell viability was not affected by lipid samples The anti-inflammatory activity of lipid fractions can be explained by the high content of omega3 (n-3) fatty acids in soft-shell crab sample, especially in the polar lipid fraction 4.2.2 Assessment results of antioxidant activity The studied samples did not show the ability to inhibit lipid peroxidation at concentrations below 500 µg/ml Trolox positive control was stable in the experiment This demonstrates the oxidative stability of lipid fractions in soft-shell crabs 15 4.2.3 Research on cytotoxic activity of cancer cells The lipid fractions inhibited cancer cell lines with IC50 values ranging from 85.45 to 97.34 µg/ml Total lipids exhibited cytotoxic activity against the lung cancer cell lines SK-LU-1 and breast cancer MCF7 with IC50 values of 89.71 and 97.77 µg/ml, respectively Notably, polar lipids showed high cytotoxicity against the five cell lines studied with IC50 values ranging from 85.4 to 95.8 µg/ml Ellipticine positive control was stable in the experiment 4.3 Development and optimization of enzymatic hydrolysis of soft-shell crabs 4.3.1 Research on selection of enzymes for hydrolysis After the survey, we chose to combine chitinase enzyme and bromelain enzyme in the process of hydrolyzing soft-shell crabs to collect the product 4.3.2 Research on influence of single factors on the target function of the process Survey results on the influence of technological factors on the target function Degree of Hydrolysis (DH) of the hydrolysis process were determined as follows: pH 6.0, temperature 50oC, water/material ratio of 3/1, enzyme/substrate ratio of 1%, time of hours 4.3.3 Research on optimization of hydrolysis by response surface methodology From the research results on the influence of univariate technological factors on the target functions, we chose the research model according to the Box-Wilson quadratic model The base levels (or baseline levels) of the factors and the coefficients α = 1,546 (with k = 5) are shown in Table 4.18 Table 4.18 Values at levels of influencing factors Factor pH Temperature (oC) Enzyme bromelain/substrate ratio (%) Chitinase enzyme/substrate ratio (%) Hydrolysis time (hour) +α 6.773 57.73 Encryption x1 x2 -α 5.227 42.27 -1 5.5 45 6.0 50 +1 6.5 55 x3 0.6135 0.75 1.0 1.25 1.3865 x4 0.6135 0.75 1.0 1.25 1.3865 x5 4.454 7.546 Design expert software 12.0 was used to build an experimental plan matrix with 27 experiments and evaluate the convergence of the model through analysis of variance (ANOVA) The analysis results show that this model is completely compatible with the experiment After removing the non-significant variables (p > 0.05), the target 16 function Y of the model is determined and represented by the quadratic regression equation as follows: Y = 44.75 + 2.67x1 + 5.84x2 + 4.19x3 + 4.23x4 - 0.94x1x2 - 0.96x1x5 1.22x2x3 - 0.94x2x4 + 1.47x3x4 - 4.22x1² - 4.09x2² - 3.19x3² 2.41x4² - 1.26x5² The hydrolysis process is optimized so that the hydrolysis of softshell crabs reaches the target function Y (degree of hydrolysis) with the maximum value with the importance of the target function Y being and the value of the variable in the range -1.546 ≤ x1, x2, x3, x4, x5 ≤ 1.546 The optimal solution is selected as shown in Table 4.21 In terms of technological parameters, the predicted value of the target function is Y = 41.87% Table 4.21 Result of optimization of technological variables Factors Encoding variable x1 x2 x3 x4 x5 Actual variable Optimal values of the factors Encoding Actual variable variable 5.6 53 1.2 0.8 hours 0.067 minutes Experimental results at the optimal conditions achieved hydrolysis of 39.12%, which is close to the predicted values of the target function (41.87%) Thus, the optimal computational model is in line with the experiment 4.4 Development and optimization of the spray drying process of hydrolyzed products of soft-shell crabs 4.4.1 Survey of factors affecting product quality of spray drying process The results of surveying the influence of technological factors on two target functions, namely Y1 being the protein content in the obtained product and Y2 being the moisture content of the product, were determined at the following basic levels: spray disc speed 9000 rpm, inlet drying air temperature 140oC, hydrolysate liquid injection speed 45 mL/min pH Temperature (oC) Bromelain/substrate ratio (%) Chitinase/substrate ratio (%) Time (hours) 0.832 0.503 0.640 -0.823 17 4.4.2 Research on optimization of spray drying process by response surface methodology From the results of studying the influence of univariate technological factors on the target functions Y1 and Y2, we chose the research model according to the Box-Wilson quadratic model The base levels (or baseline levels) of the factors and the coefficients α = 1.215 (with k = 3) are shown in Table 4.26 Table 4.26 Experimental levels of technological variables Actual variable Z1: Spray disc speed (rpm) Z2: Inlet drying air temperature (0C) Z3: Hydrolysate liquid injection speed (mL/min) Research level Encoding variable Variation range (Δ) -α -1 A 2000 6570 7000 9000 11000 11430 B 20 115.7 120 140 160 164.3 C 39 40 45 50 51 +α Design Expert 12.0 software was used to build an experimental plan matrix with 15 experiments and evaluate the model’s convergence through analysis of variance (ANOVA) The analysis results show that this model is completely compatible with the experiment After removing the non-significant variables (p > 0.05), the target functions Y1 and Y2 of the model are determined and represented by the quadratic regression equation as follows: Y1 = 66.26 + 4.63A+ 2.72B + 1.89C + 1.25 A*B – 7.21A² - 4.31B² 2.74C² (1) Y2 = 4.98 - 0.94A - 0.45B - 0.46C + 0.31AC - 0.32BC + 0.98A2 + 0.47B² + 0.27C² (2) The spray drying process is optimized so that the target function Y1 (protein content) reaches the maximum value and the target function Y2 (product moisture content) reaches the minimum value The optimal solution is selected as in Table 4.29 At the condition of technological parameters, the predicted values of the target functions are Y1 = 67.72 (g/100g) and Y2 = 4.57 (%) Table 4.29 Result of optimization of technological variables Encoding variable Actual variable Hydrolysate Spray disc Inlet drying air A B C liquid injection speed (rpm) temperature ( C) speed (mL/min) 0.411 0.247 0.337 9.822 145 46.7 18 Experimental results at optimal conditions are close to the predicted values of the target functions Therefore, the optimal computational model is in line with the experiment 4.5 Production of hydrolyzed soft-shell crab powder on pilot scale 4.5.1 Production process: Figure 4.25 Production process of hydrolyzed soft-shell crab powder by enzyme 19 4.5.2 Chemical composition of hydrolysis products of soft-shell crabs 4.5.2.1 Main composition and nutritional content The moisture content in hydrolyzed soft-shell crab powder reached 5.07% Protein content in hydrolyzed soft-shell crab powder is 65.67%, more than times higher than that of whole soft-shell crab (31.98% dry weight) The peptide content reached 61.2% The lipid content reached 8.06% dry weight The percentage of total amino acids in hydrolyzed soft-shell crab powder by enzyme increased significantly, from 31.77 g/100g dry weight in whole crab to 65.58 g/100g dry weight (Table 4.31) This can be explained because the hydrolyzed soft-shell crab powder obtained removed the soft chitin shell, gills, thorax and non-resolvable components after hydrolysis Table 4.31 Composition and content of amino acids in hydrolyzed soft-shell crab powder No Criteria Content (%) No Criteria Content (%) I Threonine 2,08 ± 0,01 Proline 8,74 ± 0,03 Leucine 4,28 ± 0,03 10 Serine 3,45± 0,01 Phenylalanine 1,20 ± 0,05 11 Glycine 2,31 ± 0,03 Isoleucine 2,71 ± 0,03 12 Cysteine 5,52 ± 0,04 Lysine 3,45 ± 0,03 13 Aspartic Acid 4,31 ± 0,06 Valine 6,56 ± 0,02 14 Arginine 2,92 ± 0,03 Methionine 4,35 ± 0,04 15 Tyrosine 4,72 ± 0,01 Histidine 3,90 ± 0,02 16 Alanine 3,45 ± 0,04 17 Glutamic Acid 1,63 ± 0,02 EAA 28,53 ± 0,04 NEAA 37,05 ± 0,02 TAA 65,58 ± 0,03 II Protein 65,67 ± 0,03 III Peptide 61,2 ± 0,02 IV Lipid 8,06 ± 0,12 V Moisture 5,07 ± 0,12 20 4.5.2.2 Composition and mineral content The analysis results of mineral compositions in hydrolyzed soft-shell crab powder also identified minerals similar to those in a whole soft-shell crab, including Ca, P, Mg, Na, K, Fe, Zn, Cu, Mn (Table 4.32) Notably, Ca and P content in hydrolyzed soft-shell crab powder reached 1.16 and 0.86 g/100g dry weight, but were not much lower than in a soft-shell crab (1.76 and 1.13g/100g dry weight), which proves that hydrolysis has obtained minerals with high efficiency (with Ca of 65.9% and P of 76.1%) 4.5.2 Anti-osteoporosis activity of hydrolysis products of soft-shell crabs and Boness food supplement from hydrolyzed soft-shell crab powder The activity study results showed that the hydrolyzed soft-shell crab powder and Boness food supplement exhibited good osteoblast differentiation and anti-osteoporosis at concentrations of 20 and µg/ml for indexes of mineralization, collagen production and enhanced ALP activity (P
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