Optimization of enzymatic hydrolysis conditions for yellowfin tuna rest raw materials using alcalase enzyme

MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY MUSIIGE DENIS OPTIMISATION OF ENZYMATIC HYDROLYSIS CONDITIONS FOR YELLOW FIN TUNA REST RAW MATERIALS USING ALCALASE ENZYME MASTER THESIS KHANH HOA - 2020 MINISTRY OF EDUCATION AND TRAINING NHA TRANG UNIVERSITY MUSIIGE DENIS OPTIMISATION OF ENZYMATIC HYDROLYSIS CONDITIONS FOR YELLOW FIN TUNA REST RAW MATERIALS USING ALCALASE ENZYME MASTER THESIS Major: MSc In Food Technology Topic allocation Decision 192/QD-DHNT Decision on establishing the Committee: Defense date: 18/09/2020 Supervisors: Assoc Prof Nguyen Van Minh Dr Pham Duc Hung Chairman: Assoc Prof Trang Si Trung Faculty of Graduate Studies: (Full name) KHANH HOA - 2020 UNDERTAKING I undertake that the thesis entitled: “Optimization of enzymatic hydrolysis conditions for yellow fin tuna rest raw materials using Alcalase enzyme” is my own work The work has not been presented elsewhere for assessment until the time this thesis is submitted Khanh Hoa, Date 25 month 09 year 2020 Musiige Denis iii FUNDING This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.05-2019.46 to Dr Pham Duc Hung iv ACKNOWLEDGEMENT I am extremely honored for the opportunity bestowed upon me to work under the versatile guidance of Assoc Professor Nguyen Van Minh, Faculty of food technology, Nha Trang University for his excellent guidance, continuous support, resourceful advice, encouragement and understanding throughout the experimental period until thesis completion His uncommon scientific knowledge, despite his busy schedule provided timely feedbacks and correction to my thesis, making it a useful library and reference material It is my privilege to record a deep sense of gratitude for the invaluable and constant inspiration, help, kind, constructive criticism, unfailing interest, meticulous planning right from suggesting the problem till the completion of this thesis I appreciate with immense pleasure the support obtained from my second supervisor, Dr Hung, Institute for Aquaculture, Nha Trang University providing all sorts of resources to me for easy completion of my work and for his constant supervision, invaluable guidance and all the facilities extended in the course of this investigation My sincere gratitude goes to the entire VLIR international master’s program Management Board at Nha Trang University with special regards to the vice dean, faculty of food technology, Dr Mai Thi Tuyet Nga and the Graduate Studies Department, Nha Trang University for making my stay in Vietnam a successful one I am also indebted to the Nha Trang University’s entire teaching staff and my classmates for providing me with a good and world-class working environment I am extremely grateful for the love, care and all the support provided by the department of external cooperation, Nha Trang University which made my stay in Vietnam worthwhile with special consideration to the head, Dr Ngan for the timely assistance as and whenever sought I am as well grateful to my biological and spiritual family for their unending boost, patience and understanding Special thanks goes to my sister Lydia and all my friends for their moral support, and motivation during this research work Musiige Denis September 2020, Nha Trang, Vietnam v TABLE OF CONTENTS UNDERTAKING iii FUNDING iv ACKNOWLEDGEMENT v TABLE OF CONTENTS .vi LIST OF SYMBOLS ix LIST OF ABBREVIATIONS x LIST OF TABLES xi LIST OF FIGURES xiii ABSTRACT xiv Chapter INTRODUCTION 1.1 Problem statement and purpose of study 1.2 Objectives of the study 1.2.1 Main objective 1.2.2 Specific objectives .5 Chapter LITERATURE REVIEW 2.1 Tuna .6 2.1.1 Tuna waste 2.1.2 Applications of tuna waste/by-products (rest raw materials) 2.1.2.1 Pet food sources from Tuna dark muscle 2.1.2.2 Oil from Tuna .9 2.1.2.3 Tuna collagen and gelatin 2.1.2.4 Tuna bone powder 10 2.1.2.5 Tuna digestive enzymes .11 vi 2.2 Fish protein hydrolysates 11 2.3 Recovery methods of fish protein from fish rest raw materials 12 2.3.1 Chemical hydrolysis .12 2.3.1.1 Acid hydrolysis 12 2.3.1.2 Alkaline hydrolysis 12 2.3.2 Fermentation Hydrolysis .13 2.3.3 Isoelectric Solubilization and Precipitation (ISP) .13 2.3.4 Enzymatic hydrolysis 15 2.3.4.1 Enzymes 15 2.3.4.2 Application of enzymes 16 2.3.4.3 Factors that influence enzyme activity during hydrolysis .16 2.3.4.4 Alcalase 18 Chapter MATERIALS AND METHODS 25 3.1 Materials 25 3.1.1 Head and viscera from Yellow fin Tuna 25 3.1.2 Enzyme and chemicals 25 3.2 Experimental design 25 3.2.1 Preparation of protein hydrolysates 25 3.2.2 Experimental design for optimization and analysis of data 26 3.3 Analysis methods 29 3.3.1 Proximate Chemical composition 29 3.3.2 Determination of the degree of hydrolysis 29 3.3.3 Determination of protein solubility 29 3.3.4 Amino acid analysis .30 3.4 Statistical analysis 30 Chapter RESULTS AND DISCUSSION 31 vii 4.1 Proximate composition of the rest raw materials 31 4.2 Optimization of hydrolysis parameters for DH and solubility of viscera 31 4.2.1 Optimal plot for DH and Solubility of viscera .43 4.3 Optimization of hydrolysis parameters for DH and solubility of head .44 4.3.1 Optimal plot for DH and solubility of head 52 4.4 Optimization and validation of the models 53 4.5 Proximate composition of the hydrolysates 55 4.6 Amino acid composition .55 Chapter CONCLUSIONS AND RECOMMENDATIONS 58 5.1 Conclusions 58 5.2 Recommendations 58 REFERENCES 60 APPENDICES I Appendix I Appendix VII Appendix XX viii LIST OF SYMBOLS Time Temperature Enzyme concentration Percentage h Hours Degrees Celsius Axial/ star points Intercept/constant/offset term Regression coefficient for linear effect Regression coefficient for quadratic effect Regression coefficient for interaction effect Broken peptide bonds Total number of peptide bonds ⁄ Volume/ weight ⁄ Volume/volume Nanometres Millilitres Microlitres Litres Molarity ix LIST OF ABBREVIATIONS ANOVA Analysis of variance AOAC Association of Analytical Communities AU Anson unit CCD central composite design CPHA Cuttlefish protein hydrolysates Alcalase CPHP Cuttlefish protein hydrolysates protamex CPHS Cuttlefish protein hydrolysates DH Degree of hydrolysis DHA Docosahexaenoic acid DNFB Dinitrofluorobenzene DPPH 2, 2- diphenyl-1-1picryhydrazyl EPA Eicosapentaenoic acid FAD Fish aggregation devices FAO Food and agricultural organization FPH Fish protein Hydrolysate FPI Fish protein isolate ISP Isoelectric solubilization and precipitation KDa kilo Dalton LAB Lactic acid bacteria NRC Nutritional research council pH Potential of hydrogen ions PI Isoelectric point PUFA Polyunsaturated fatty acids RSM Response surface methodology SAS Statistical analysis system WHO World health organization x Appendix Results of proximate analysis, DH and solubility Moisture and ash content in raw materials and hydrolysates Moisture content of head and viscera waste @ 105°C final mass rest raw empty crucible sample moisture replicate (sample material crucible +sample mass content + crucible) Mean STD Mean±STD Head 64.729 68.149 3.42 66.190 57.281 58.645 57.963 0.965 57.96±0.96 Head 59.590 63.877 4.287 61.363 58.645 Viscera 56.103 59.734 3.631 57.076 73.206 73.498 73.352 0.207 73.35±0.21 Viscera 57.827 61.463 3.636 58.791 73.498 Moisture content of head and viscera supernatant @ 105°C final mass rest raw empty crucible sample moisture replicate (sample material crucible +sample mass content + crucible) Mean STD FINAL Head 56.103 59.456 3.353 56.416 90.658 90.540 90.599 0.084 90.6±0.08 Head 58.323 61.506 3.184 58.624 90.540 Viscera 63.569 66.748 3.178 63.931 88.623 88.559 88.591 0.045 88.59±0.04 Viscera 59.994 63.168 3.174 60.357 88.559 Ash content of head and viscera waste @ 600°C final rest raw empty crucible sample mass Ash replicate material crucible +sample mass (sample+ content crucible) Head 42.146 52.544 10.398 42.781 6.107 Head 43.260 54.150 10.890 43.952 6.354 Viscera 34.314 45.165 10.851 34.447 1.230 Viscera 35.095 45.533 10.438 35.262 1.596 VII Mean STD FINAL 6.354 6.231 0.175 6.23±0.17 1.596 1.413 0.259 1.41±0.26 Ash content of head and viscera supernatant @ 600°C final rest raw material replicate mass empty crucible sample (sample crucible +sample mass + Ash Mean content STD FINAL crucible) Head 58.224 62.326 4.102 58.234 0.258 Head 58.224 62.326 4.102 58.234 0.258 Viscera 59.653 65.059 5.406 59.704 0.947 Viscera 64.729 69.994 5.265 64.774 0.860 0.26±0.00 0.258 0.258 0.860 0.904 0.061 0.90±0.06 Lipid content in raw materials and hydrolysates Lipid content of head and viscera waste mass of rest raw material Replicate empty tube (g) volume sample used Final mass content (g) (ml) Lipid Mean STD (Mean±STD) (g) head waste 19.636 19.815 15.813 13.975 14.894 1.299 14.89± 1.30 head waste 21.248 21.406 13.975 Viscera waste 21.262 21.315 4.655 3.419 4.037 0.875 4.04±0.87 Viscera waste 21.113 21.152 3.419 Lipid content of head and viscera supernatant mass supernatant (hydrolysate) of Replicate empty tube (g) volume sample used (ml) Final mass (g) Lipid content Mean STD (Mean±STD) head hydrolysate 11.041 11.042 0.098 0.177 0.138 0.056 0.14± 0.06 head hydrolysate 11.135 11.137 0.177 Viscera hydrolysate 11.211 11.212 0.050 0.130 0.090 0.056 0.09±0.06 Viscera hydrolysate 10.980 10.981 0.130 VIII Protein content in raw materials and hydrolysates Protein content for the rest raw materials replicate initial volume Titre (ml)/mass(g) volume(ml) Blank %Protein head waste 0.510 13.45 0.2 22.715 head waste 0.515 10.35 0.2 17.248 Viscera waste 0.510 14.45 0.2 24.434 Viscera waste 0.509 10.25 0.2 17.266 mean STD Mean±STD 17.248 19.982 3.865 19.98±3.9 17.266 20.850 5.068 20.85±5.1 Protein content for the hydrolysates replicate initial volume Titre (ml)/mass(g) volume(ml) Blank %Protein head hydrolysates 21 0.2 9.100 head hydrolysates 2 21.05 0.2 9.122 Viscera hydrolysates 21.05 0.2 9.122 Viscera hydrolysates 2 21.25 0.2 9.209 IX mean STD Mean±STD 9.122 9.111 0.015 9.11±0.02 9.209 9.166 0.062 9.17±0.06 Degree of hydrolysis viscera Run order Standard order Time Temp Enzyme Abs.1 Abs.2 Abs %DH Viscera %DH Viscera %DH Viscera, actual STD Mean±STD 12 -1 -1 -1 0.525 0.686 0.606 41.60 54.85 48.26 9.37 48.26±9.37 11 -1 -1 0.64 0.801 0.721 51.06 64.32 57.73 9.37 57.73±9.37 -1 -1 0.369 0.601 0.485 28.75 47.85 38.30 1 -1 0.552 0.641 0.597 43.82 51.15 47.52 5 -1 -1 0.547 0.835 0.691 43.41 67.12 55.26 -1 0.779 0.827 0.803 62.51 66.46 64.48 2.79 64.48±2.79 10 -1 1 0.572 0.576 0.574 45.47 45.79 45.63 0.23 45.63±0.23 1 0.62 0.942 0.781 49.42 75.93 62.67 -1.68 0 0.533 0.487 0.51 42.25 38.47 40.36 2.68 40.36±2.68 10 20 1.68 0 0.636 0.789 0.713 50.73 63.33 57.07 8.91 57.07±8.91 11 13 -1.68 0.628 0.778 0.703 50.08 62.43 56.25 8.73 56.25±8.73 12 1.68 0.505 0.509 0.507 39.95 40.28 40.11 0.23 40.11±0.23 13 0 -1.68 0.693 0.697 0.695 55.43 55.76 55.59 0.23 55.59±0.23 14 18 0 1.68 0.86 0.706 0.783 69.18 56.50 62.84 8.97 62.84±8.97 X 13.51 38.30±13.51 5.18 47.52±5.18 16.77 55.26±16.77 18.75 62.67±18.75 Run order Standard order Time Temp Enzyme Abs.1 Abs.2 Abs %DH Viscera %DH Viscera %DH Viscera, actual STD Mean±STD 15 0 0.816 0.822 0.819 65.56 66.05 65.80 0.35 65.80±0.35 16 16 0 0.807 0.811 0.809 64.81 65.14 64.98 0.23 64.98±0.23 17 12 0 0.791 0.797 0.794 63.50 63.99 63.74 0.35 63.74±0.35 18 15 0 0.809 0.813 0.811 64.98 65.31 65.14 0.23 65.14±0.23 19 10 0 0.78 0.786 0.783 62.59 63.09 62.84 0.35 62.84±0.35 20 0 0.789 0.793 0.791 63.33 63.66 63.50 0.23 63.50±0.23 XI Degree of hydrolysis head Run order standard order Time Temp Enzyme Abs Abs Abs %DH, head %DH, head %DH, head STD Mean±STD 12 -1 -1 -1 0.15 0.164 0.158 10.717 11.873 11.38 0.82 11.38±0.82 11 -1 -1 0.273 0.285 0.279 20.842 21.835 21.38 0.70 21.38±0.70 -1 -1 0.145 0.149 0.147 10.306 10.638 10.5 0.23 10.5±0.23 1 -1 0.21 0.216 0.213 15.656 16.154 15.91 0.35 15.91±0.35 5 -1 -1 0.211 0.221 0.216 15.738 16.566 16.19 0.59 16.19±0.59 -1 0.318 0.338 0.328 24.546 26.199 25.38 1.17 25.38±1.17 10 -1 1 0.194 0.202 0.198 14.339 15.001 14.63 0.47 14.63±0.47 1 0.254 0.274 0.264 19.278 20.930 20.03 1.17 20.03±1.17 14 -1.68 0 0.192 0.196 0.194 14.174 14.507 14.34 0.24 14.34±0.24 10 20 1.68 0 0.29 0.292 0.291 22.241 22.412 22.33 0.12 22.33±0.12 11 -1.68 0.291 0.301 0.296 22.323 23.153 22.78 0.59 22.78±0.59 12 18 1.68 0.3 0.1 0.200 23.064 6.603 14.84 11.64 14.84±11.64 13 0 -1.68 0.223 0.243 0.233 16.726 18.377 17.58 1.17 17.58±1.17 14 0 1.68 0.27 0.278 0.274 20.595 21.259 20.95 0.47 20.95±0.47 15 15 0 0.351 0.34 0.346 27.262 26.364 26.86 0.64 26.86±0.64 16 0 0.3 0.355 0.350 23.064 27.599 27.2 3.21 27.2±3.21 XII 17 10 0 0.362 0.4 Run order standard order Time Temp Enzyme Abs Abs 0.366 28.168 31.304 28.53 Abs %DH, head %DH, head %DH, head 2.22 28.53±2.22 STD Mean±STD 18 0 0.325 0.331 0.328 25.122 25.623 25.36 0.35 25.36±0.35 19 0 0.37 0.376 0.373 28.826 29.328 29.03 0.35 29.03±0.35 20 16 0 0.35 0.362 0.356 27.180 28.175 27.69 0.70 27.69±0.70 XIII Solubi-lity (%) Solubi-lity (%) Solubi-lity (%) mass of total protein in sample (mg) mass of total protein in sample (mg) 2593.7 2689.8 9500 9700 9650 28.313 26.739 27.873 27.63 0.812 0.358 0.347 0.358 75.8 73.4 75.8 40 3030.2 2934.2 3030.2 9500 9700 9650 31.897 30.250 31.401 31.19 0.845 0.295 0.341 0.295 62.0 72.0 62.0 40 2480.2 2881.8 2480.2 9500 9700 9650 26.108 29.710 25.702 28.17 2.206 0.389 0.312 0.389 82.5 65.7 82.5 40 3300.9 2628.7 3300.9 9500 9700 9650 34.746 27.099 34.206 32.02 4.267 0.307 0.205 0.307 64.6 42.4 64.6 40 2585.0 1694.5 2585.0 9500 9700 9650 27.211 17.469 26.788 23.83 5.506 0.33 69.6 71.4 69.6 40 2785.8 2855.6 2785.8 9500 9700 9650 29.324 29.440 28.868 29.21 0.302 0.306 0.247 0.306 64.4 51.5 64.4 40 2576.3 2061.2 2576.3 9500 9700 9650 27.119 21.249 26.697 25.01 3.274 0.338 0.357 0.338 71.4 75.5 71.4 40 2855.6 3021.5 2855.6 9500 9700 9650 30.059 31.150 29.592 30.27 0.799 0.33 69.6 69.9 69.6 40 2785.8 2794.5 2785.8 9500 9700 9650 29.324 28.810 28.868 10 0.332 0.267 0.332 70.1 55.9 70.1 40 2803.3 2235.8 2803.3 9500 9700 9650 29.508 23.049 29.049 27.22 3.604 0.33 0.338 0.33 0.331 XIV STD 40 2689.8 Average mass of soluble protein (mg) mass of total protein in sample (mg) mass of soluble protein (mg) 67.2 A3 64.8 A2 67.2 A1 0.319 0.308 0.319 Runs mass of soluble protein (mg) soluble protein conc (mg/mL) soluble protein conc (mg/mL) Volume of supernatant (mL) soluble protein conc (mg/mL) Solubility, head 29 0.282 11 0.356 0.33 0.356 75.3 69.6 75.3 40 3012.8 2785.8 3012.8 9500 9700 9650 31.713 28.720 31.220 30.55 1.605 12 0.365 0.447 0.365 77.3 95.2 77.3 40 3091.3 3807.2 3091.3 9500 9700 9650 32.541 39.250 32.035 34.61 4.028 13 0.287 0.262 0.287 60.3 54.8 60.3 40 2410.4 2192.1 2410.4 9500 9700 9650 25.373 22.599 24.978 24.32 1.500 14 0.381 0.3 0.381 80.8 63.1 80.8 40 3231.0 2523.9 3231.0 9500 9700 9650 34.011 26.019 33.482 15 0.418 0.334 0.418 88.9 70.5 88.9 40 3554.0 2820.7 3554.0 9500 9700 9650 37.411 29.080 36.830 34.44 4.651 16 0.38 80.6 80.6 80.6 40 3222.3 3222.3 3222.3 9500 9700 9650 33.919 33.220 33.392 35.51 0.364 17 0.388 0.455 0.388 82.3 96.9 82.3 40 3292.1 3877.1 3292.1 9500 9700 9650 34.654 39.970 34.115 36.24 3.236 18 0.335 0.438 0.365 70.7 93.2 77.3 40 2829.4 3728.7 3091.3 9500 9700 9650 29.784 38.440 32.035 33.42 4.491 19 0.398 0.376 0.398 84.5 79.7 84.5 40 3379.4 3187.4 3379.4 9500 9700 9650 35.573 32.860 35.020 34.48 1.434 20 0.402 85.4 84.9 85.4 40 3414.4 3396.9 3414.4 9500 9700 9650 35.941 35.020 35.382 35.45 0.464 0.38 0.38 0.4 0.402 XV 31.1 4.469 HS HS 0.366 0.366 0.387 77.5 83.8 82.1 40 3100.1 3353.3 3283.4 9500 9700 9650 32.63 34.57 34.03 33.74 1.00 0.387 77.5 83.8 82.1 30 2325.1 2514.9 2462.6 2730 2730 2730 85.17 92.12 90.20 89.16 3.59 XVI solubility (%) sample (mg) sample (mg) mass of total protein in sample (mg) mass of total protein in mass of total protein in (mg) mass of soluble protein (mg) mass of soluble protein (mg) mass of soluble protein (mL) Volume of supernatant (mg/mL) soluble protein conc (mg/mL) soluble protein conc (mg/mL) soluble protein conc A3 A2 A1 STD 0.395 Average Final solubility solubility (%) 0.395 solubility (%) Validation at optimal conditions of 0.7% enzyme concentration, 6.9 hours at 55°C for solubility of head soluble protein conc (mg/mL) soluble protein conc (mg/mL) mass of soluble protein (mg) mass of soluble protein (mg) mass of soluble protein (mg) mass of total protein in sample (mg) mass of total protein in sample (mg) mass of total protein in sample (mg) solubility (%) solubility (%) solubility (%) 0.277 58.1 55.7 58.1 40 2323.1 2227.1 2323.1 9500 9700 9650 24.5 23.0 24.1 23.83 0.8 0.245 0.278 0.245 51.1 58.3 51.1 40 2043.7 2331.8 2043.7 9500 9700 9650 21.5 24.0 21.2 22.24 1.6 0.274 0.3 0.274 57.4 63.1 57.4 40 2296.9 2523.9 2296.9 9500 9700 9650 24.2 26.0 23.8 22.67 1.2 0.258 0.208 0.258 53.9 43.0 53.9 40 2157.2 1720.7 2157.2 9500 9700 9650 22.7 17.7 22.4 20.95 2.8 0.269 0.36 0.269 56.3 76.2 56.3 40 2253.3 3047.7 2253.3 9500 9700 9650 23.7 31.4 23.3 26.16 4.6 0.259 0.244 0.259 54.1 50.9 54.1 40 2166.0 2035.0 2166.0 9500 9700 9650 22.8 21.0 22.4 22.07 1.0 0.312 0.235 0.312 65.7 48.9 65.7 40 2628.7 1956.4 2628.7 9500 9700 9650 27.7 20.2 27.2 25.03 4.2 0.26 0.331 0.26 54.4 69.9 54.4 40 2174.7 2794.5 2174.7 9500 9700 9650 22.9 28.8 22.5 20.75 3.5 0.299 0.314 0.299 62.9 66.2 62.9 40 2515.2 2646.1 2515.2 9500 9700 9650 26.5 27.3 26.1 26.61 0.6 10 0.21 0.26 0.21 43.5 54.4 43.5 40 1738.2 2174.7 1738.2 9500 9700 9650 18.3 22.4 18.0 19.58 2.5 11 0.263 0.334 0.263 55.0 70.5 55.0 40 2200.9 2820.7 2200.9 9500 9700 9650 23.2 29.1 22.8 25.02 3.5 12 0.246 0.27 0.246 51.3 56.5 51.3 40 2052.5 2262.0 2052.5 9500 9700 9650 21.6 23.3 21.3 22.06 1.1 XVII STD A3 0.266 Average A2 0.277 Volume of supernatant (mL) A1 soluble protein conc (mg/mL) Runs Solubility, viscera 13 0.243 0.232 0.243 50.7 48.3 50.7 40 2026.3 1930.2 2026.3 9500 9700 9650 21.3 19.9 21.0 20.74 0.7 14 0.287 0.326 0.287 60.3 68.8 60.3 40 2410.4 2750.9 2410.4 9500 9700 9650 25.4 28.4 25.0 26.24 1.8 15 0.296 0.329 0.296 62.2 69.4 62.2 40 2489.0 2777.1 2489.0 9500 9700 9650 26.2 28.6 25.8 26.88 1.5 16 0.294 0.359 0.294 61.8 76.0 61.8 40 2471.5 3039.0 2471.5 9500 9700 9650 26.0 31.3 25.6 27.7 3.2 17 0.298 0.332 0.298 62.7 70.1 62.7 40 2506.4 2803.3 2506.4 9500 9700 9650 26.4 28.9 26.0 27.07 1.6 18 0.292 0.325 0.292 61.4 68.6 61.4 40 2454.0 2742.1 2454.0 9500 9700 9650 25.8 28.3 25.4 26.52 1.5 19 0.301 0.358 0.301 63.3 75.8 63.3 40 2532.6 3030.2 2532.6 9500 9700 9650 26.7 31.2 26.2 28.07 2.8 20 0.298 0.313 0.298 62.7 65.9 62.7 40 2506.4 2637.4 2506.4 9500 9700 9650 26.4 27.2 26.0 26.52 0.6 XVIII VS VS 0.314 0.314 STD 0.314 66.2 62.9 66.2 40 2646.1 2515.2 2646.1 9500 9700 9650 27.85 25.93 27.42 27.07 1.01 0.314 66.2 62.9 66.2 30 1984.6 1886.4 1984.6 2751 2751 2751 72.14 68.57 72.14 70.95 2.06 XIX sample (mg) sample (mg) mass of total protein in sample (mg) mass of total protein in mass of total protein in (mg) mass of soluble protein (mg) mass of soluble protein (mg) mass of soluble protein (mL) Volume of supernatant (mg/mL) soluble protein conc (mg/mL) soluble protein conc (mg/mL) soluble protein conc A3 A2 A1 Average 0.299 solubility (%) Final solubility solubility (%) 0.299 solubility (%) Validation at optimal conditions of 0.8% enzyme concentration, 6.6 hours at 54°C for solubility of viscera Appendix Some of the photos during the experiment Minced viscera, vacuum packaged and Samples mixed with buffer prepared for stored in the freezer hydrolysis Samples after hydrolysis Samples after centrifugation Head samples for hydrolysis During filtration XX Samples for determination of DH after Samples for determination of Solubility addition of DNFB after addition of Biuret’s reagent Samples during filtration Terminating the reactions by heating the solution at 90 °C for 15 minutes, to inactivate enzymes XXI ... Preparation of protein hydrolysates To establish the effect of enzymatic hydrolysis conditions of yellow fin tuna rest raw materials i.e visceral and head rest raw materials by using Alcalase enzyme. .. Optimization of hydrolysis parameters for DH and solubility of viscera The influence of , and on the Degree of hydrolysis and solubility for visceral rest raw materials by a protease enzyme Alcalase. .. influence of enzymatic hydrolysis conditions on the degree of hydrolysis of visceral and head waste proteins of tuna for yield optimization as well as the solubility of the Hydrolysate for their