AN ABSTRACT OF THE THESIS OF Soottawat Benjakul for the degree of Doctor of Philosophy in Food Science and Technology presented on April 17, 1997. Title: Utilization of Wastes from Pacific Whiting Surimi Manufacturing: Proteinases and Protein Hydrolvsate. Abstract approved: ^___ Michael T. Morml Both liquid and solid wastes from Pacific whiting surimi manufacturing were characterized and value-added products were recovered. A proteinase in surimi wash water (SWW) was determined to be cathepsin L with Mr 54,200 on SDS-substrate gel. Heat treatment and acidification shifted the activity zone to Mr 39,500. No evidence of calpain, cathepsin B or H activity was found. Cathepsin L from SWW was recovered by ohmic heating (550C for 3 min), ultrafiltration, and freeze-drying with overall yield of 0.83 g protein/L SWW and 78% recovery of activity. A 5.9 purification fold was achieved by these processes. The recovered enzyme had an optimum activity at pH 4.0 and showed preferable hydrolytic activity towards casein, acid-denatured hemoglobin and myofibrils. p-Mercaptoethanol, dithiothreitol and urea enhanced the enzyme activity. The recovered proteinase showed 18.5% residual activity after 7 wk storage at 4°C. Proteolytic activity in solid waste and digestive organs from Pacific whiting was investigated. Pepsin-like proteinase predominated in solid waste, while trypsin-like proteinase was predominant in viscera. Carboxypeptidase b was found in both viscera and solid waste. Protein hydrolysate was produced from Pacific whitng solid waste (PWSW) using commercial proteinase, Alcalase, under optimum hydrolysis conditions. Enzyme concentration, reaction time and waste/buffer ratio affected the hydrolysis and nitrogen recovery (NR). Correlation between the degree of hydrolysis (DH) and NR was high (R2=0.978). Freeze-dried hydrolysate contained 79.97% protein and showed similar amino acid composition to PWSW and Pacific whiting muscle but tryptophan was reduced. With different DH (20, 30, 40, 50, 60%), surface hydrophobicity, total and surface sulfhydryl content decreased as the DH increased. The hydrolysate showed a high solubility over a wide pH range. Fat adsorption and fat binding capacity were reduced, while foam expansion was enhanced with an increased DH. Hydrolysate with DH of 30% showed highest emulsifying activity. Low emulsion stability and high foam stability were obtained in all hydrolysates tested. Hydrolysate showed antioxidant activity, but no obvious differences in activity were found with varying DH and hydrolysate concentrations. ^Copyright by Soottawat Benjakul April 17, 1997 All Rights Reserved Utilization of Wastes from Pacific Whiting Surimi Manufacturing: Proteinases and Protein Hydrolysate by Soottawat Benjakul A THESIS submitted to Oregon State University in partial fulfillment of the requirement for the degree of Doctor of Philosophy Presented April 17, 1997 Commencement June 1997 Doctor of Philosophy thesis of Soottawat Benjakul presented on April 17, 1997 APPROVED: Major Professor, representing Food Sciertee and Technology ~T~ {Chaihair of Department of Food Science and Technology Dean of Graduateirate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. -#- Soottawat Benjakul, Author ACKNOWLEDGEMENTS I would like to thank the U.S. Information Agency (USIA) for the Fulbright scholarship and the Institute of International Education (HE) for the continuous support. I would like to express my gratitude to Dr. Michael T. Morrissey, Dr. Thomas A. Seymour, and Dr. Haejung An for their invaluable advice. My appreciation is also expressed to the members of my thesis committee, Dr. R.E. Wroldstad, Dr. W. Gamble and Dr. E.M. Hansen for their beneficial guidance. Special thanks to Nancy Chamberlain for editing my thesis. I would like to express my deepest gratitude to my parents who always support and encourage me. This work is dedicated to them. Last but not least, I would like to thank Wonnqp for his unconditional friendship and help. CONTRIBUTION OF AUTHORS Dr. M.T. Morrissey, Dr. T.A. Seymour and Dr. H. An were involved in the experimental design, analysis and preparing the manuscript. TABLE OF CONTENTS Page 1. INTRODUCTION 1 General considerations 1 Fish proteinase 2 Muscle proteinase 3 Digestive proteinase 5 Protein hydrolysates 7 Physicochemical and functional properties of protein hydrolysate 10 Protein solubility 10 Interfacial properties 11 Antioxidant activity 15 Research objectives 16 References 17 2. PROTEINASE IN PACIFIC WHITING SURIMI WASH WATER: 27 IDENTIFICATION AND CHARACTERIZATION Abstract 28 Introduction 29 Materials and Methods 31 Chemical 31 SWW collection and preparation 31 Enzyme assay 32 Inhibitor study 33 Size exclusion HPLC 33 Acidification 34 Study of Ca2+ and EGTA effects on proteinase 34 Activity staining 35 Results and Dicussion 36 pH and temperature profiles of proteinase in SWW 36 TABLE OF CONTENTS (Continued) Page Effects of proteinase inhibitors 36 Separation and characterizationof proteolytic activity 39 Effects of Ca2+ and EGTA on proteinase activity 46 Effect of acidification 47 Identification of proteinase by SDS-substrate gel electrophoresis 50 Acknowledgement 53 References 54 3. RECOVERY OF PROTEINASE FROM PACIFIC WHITING 58 SURIMI WASH WATER Abstract 59 Introduction 60 Materials and Methods 61 Preparation of surimi wash water 61 Ohmic heating of SWW 62 Ultrafiltration and freeze drying of OHSWW 62 Cathepsin activity assay 63 Protein determination 63 Results and Discussion 64 Optimization of ohmic heating 64 Effect of ohmic heating on SWW proteins 68 Effect of ohmic heating on cathepsin activities 70 Effect of ohmic heating time and temperature 70 Evaluation of proteinase recovery from SWW 76 Acknowledgment 77 References 77 TABLE OF CONTENTS (Continued) Page 4. CHARACTERIZATION OF CATHEPSIN L RECOVERED 80 FROM PACIFIC WHITING SURIMI WASH WATER Abstract 81 Introduction 82 Materials and Methods 83 Chemicals 83 Recovery of SWW proteinase 83 Cathepsin activity assays 84 Protein determination 84 Substrates specificity 84 Acidification of enzyme 85 Thermal stability of enzyme 85 pH stability of enzyme 86 Effect of chemicals on enzyme activity 86 Electrophoresis 86 Results and discussion 87 Efficiency of enzyme recovery 87 Protein patterns in SWW during enzyme recovery process 89 Substrate specificity of the recovered enzyme 92 Thermal stability of recovered enzyme 94 Effect of chemicals on recovered enzyme 94 pH stability of the recovered enzyme 97 Acknowledgement 100 References 100 5. PROTEOLYTIC ACTIVITIES IN SOLID WASTES AND 104 DIGESTIVE ORGANS OF PACIFIC WHITING Abstract 105 Introduction 106 TABLE OF CONTENTS (Continued) Page Materials and Methods 107 Reagents 107 Sample collection and preparation 108 Preparation of crude extracts 108 Enzyme assay 108 Chemical inhibitor study 109 Separation of enzyme by gel filtration 110 Effect of ATP and sodium molybdate on enzyme activity 110 SDS-substrate gel 111 Results 112 pH and temperature profile of proteases in solid wastes 112 and digestive organs Effect of chemical inhibitors 116 Substrate specificity of proteinases 119 Effect of ATP and molybdate on protease activity 119 Separation of proteolytic activity 121 SDS-substrate gel electrophoresis 125 Discussion 128 Conclusion 131 References 131 6. PROTEIN HYDROLYSATES FROM PACIFIC WHITING 136 SOLID WASTES Abstract 137 Introduction 138 Materials and Methods 139 Reagents 139 Materials 139 pH and temperature profile for Alcalase and Neutrase on PWSW 139 Enzymic hydrolysis of PWSW 140 TABLE OF CONTENTS (Continued) Page Electrophoresis 141 Determination of optimum condition for PWSW hydrolysate 141 production using Alcalase Determination of degree of hydrolysis (DH) 142 Determination of nitrogen recovery (NR) 143 Composition analyses 143 Color measurement 144 Statistical analyses 144 Results and Discussion 144 pH and temperature profile of Alcalase and Neutrase on PWSW 144 Enzymatic hydrolysis of PWSW 146 Hydrolysis pattern on SDS-PAGE 149 Effect of enzyme concentration on DH and NR 151 Effect of substrate/buffer ratio on hydrolysis and NR 154 Optimum condition for PWSW hydrolysis by Alcalase 160 Compositions of PWSW hydrolysate 162 References 165 7. PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF 167 PACIFIC WHITING SOLID WASTE HYDROLYSATE Abstract 168 Introduction 169 Materials and Methods 170 Reagents 170 Preparation of hydrolysate 170 Hydrophobicity 171 Total and surface sulfhydryl content (SH) 171 Solubility 172 Fat adsorption 172 Fat binding capacity 173 Emulsifying activity 174 Emulsion stability 174 Foaming capacity 175 TABLE OF CONTENTS (Continued) Page Foam stability 175 Antioxidant activity 176 Statistical analysis 176 Results and Discussion 176 Hydrophobicity 176 Total and surface sulfhydryl content 179 Solubility 181 Fat adsorption and fat binding capacity 181 Emulsifying activity 184 Emulsion stability 187 Foaming capacity 188 Foam stability 190 Antioxidant activity 190 References 195 8. SUMMARY 200 BIBLIOGRAPHY 202 LIST OF FIGURES Figure Page 2.1. pH activity profiles of Pacific whiting-SWW proteinase at various 37 temperatures. 2.2. Elution profile of SWW on SEC-HPLC. 40 2.3. Elution profile of HTSWW on SEC-HPLC. 41 2.4. Proteolytic activity of Biosep Sec-S3000 fractions of SWW analyzed 42 against casein at two different conditions. 2.5. Estimation of native molecular weight of SWW proteinase based on 43 elution volume on Biosep Sec-3000.