THE UNIVERSITY OF NEW SOUTH WALES FACULTY OF APPLIED SCIENCE DEPARTMENT OF FOOD SCIENCE AND TECHNOLOGY THE UTILISATION OF COWTAIL RAY VISCERA A thesis submitted for the degree of Doctor of Philosophy by ACHMAD POERNOMO BSc (Agricultural Technology, Gadjah Mada University) Ir (Agricultural Technology, Gadjah Mada University) MAppSc (Food Engineering, UNSW) Submitted Sydney, January 1997 U N S W 1 7 JUL 1337 LIBRARY ACKNOWLEDGEMENT I am greatly indebted to Professor Ken A. Buckle for his supervision throughout the study. Sincere gratitude is due to the Government of the Republic of Indonesia for granting me study leave and to the Australian Agency for International Development (AusAID) for the award of a scholarship for my study. My thanks go to the Head and staff of The Research Station for Marine Fisheries, Slipi, Jakarta and to the staff of The Department of Food Science and Technology, The University of New South Wales for their assistance during my fieldwork in Indonesia and finishing of the work in Australia. I am also grateful to the dried salted ray processors in Muara Angke, Jakarta and Labuhan Maringgai, Lampung, Indonesia, for supplying me the cowtail ray viscera used in this study. Finally, I wish to express my indebtedness to my wife, Farida Ariyani, who has been constantly supporting me while she herself was suffering from her study creating the agony that she has patiently and strongly borne. This thesis is dedicated to her and also to my parents, Mr. and Mrs. Djarnawi Hadikusuma, who had been sending us their encouragement from a distance, but will never know that their son has finally finished the work. All praises be to Allah, the One and Only God. TABLE OF CONTENTS Page ACKNOWLEDGEMENT i TABLE OF CONTENT ii ABSTRACT ix 1 INTRODUCTION 1 2 LITERATURE REVIEW 5 2.1 Aquatic food with special reference to fish 5 2.1.1 Classification of fish 5 2.1.2 Basic fish anatomy 11 2.1.2.1 Shape and skin 11 2.1.2.2 Skeleton and muscle 12 2.1.2.3 Digestive and internal organs 14 2.1.3 Chemical composition of fish 17 2.2 Aquatic food utilisation 21 2.2.1 Utilisation as food 21 2.2.2 Utilisation as feed 27 2.2.3 Others 28 2.3 Aquatic food processing waste 29 2.3.1 Production and characteristics 29 2.3.2 Waste treatment 30 2.3.3 Waste utilisation 33 2.3.3.1 Fin-fish waste 33 2.3.3.1.1 Fish meal 33 2.3.3.1.2 Minced fish and surimi 34 2.3.3.1.3 Fish protein preparations 35 2.3.3.1.4 Pharmaceutical products 37 2.3.3.1.5 Fertiliser and compost 37 2.3.3.1.6 Others 38 2.3.3.2 Crustacean and molluscan wastes 39 2.3.3.2.1 Animal feeds 39 2.3.3.2.2 Chitin and chitosan 40 2.3.3.2.3 Others 41 2.3.3.3 Liquid Wastes 42 2.4 Viscera and its utilisation 43 2.5 The stingrays (Dasycitididcie spp) 47 2.5.1 General description 47 2.5.2 Utilisation 49 2.5.3 Waste products 54 3 ENSILATION OF COWTAIL RAY (T. sephen) VISCERA 58 3.1 Introduction 58 3.2 Materials 63 3.2.1 Viscera 63 3.2.2 Chemicals and solvents 65 3.2.3 Equipment 65 3.3 Methods 65 3.3.1 Preparatory 65 3.3.1.1 Experiment 1 65 3.3.1.2 Experiment 2 66 3.3.2 Analytical 67 3.3.2.1 Proximate analysis 67 3.3.2.1.1 Moisture content 67 3.3.2.1.2 Protein content 67 3.3.2.1.3 Fat content 68 3.3.2.1.4 Ash content 68 3.3.2.2 pH 69 3.3.2.3 Viscosity 69 3.3.2.4 Liquefaction 69 3.3.2.5 Soluble nitrogen 69 3.3.2.6 Enzyme activity 70 3.3.3 Statistical analysis 70 3.4 Results 70 3.4.1 Experiment 1 70 3.4.2 Experiment 2 77 3.5 Discussion 81 3.5.1 pH and spoilage of silage 81 3.5.2 Proteolysis 85 3.5.3 Fish protein hydrolysis 90 4 USE OF CRUDE PEPTONES FROM COWTAIL RAY (T. sephen) VISCERA SILAGE AS MICROBIAL GROWTH MEDIA 93 4.1 Introduction 93 4.2 Materials 98 4.2.1 Viscera 98 4.2.2 Equipment 98 4.2.3 Chemicals and microbiological media 98 IV 4.2.4 Sources of microorganisms 99 4.3 Methods 100 4.3.1 Preparatory 100 4.3.1.1 Ensilation of cowtail ray viscera and preparation of ray viscera peptones 100 4.3.1.2 Culture media 101 4.3.1.3 Microbial growth test 101 4.3.1.3.1 Preparation of cultures, inoculation and incubation 101 4.3.1.3.1.1 Bacteria and mixed population of microorganisms 101 4.3.1.3.1.2 Yeast 102 4.3.1.3.1.3 Fungi 102 4.3.1.3.2 Growth curves, growth rate and total growth 103 4.3.1.3.3 Biomass production 103 4.3.2 Analytical 104 4.3.2.1 Proximate composition 104 4.3.2.2 Amino acid composition 104 4.3.2.3 Minerals 104 4.3.3 Statistical analysis 105 4.4 Results 105 4.4.1 Protein concentration and proximate composition 105 4.4.2 Growth curves 105 4.4.3 Biomass production 114 4.5 Discussion 115 4.5.1 Proximate composition 115 4.5.2 Growth of mixed population of microorganisms 119 4.5.3 Growth of single microorganisms 121 4.5.4 General discussion 123 5 ISOLATION AND CHARACTERISATION OF COWTAIL RAY (T. Sepheri) VISCERA PROTEASES 126 5.1 Introduction 126 5.2 Materials 130 5.2.1 Viscera 130 5.2.2 Chemicals 130 5.2.3 Equipment 131 5.3 Methods 132 5.3.1 Preparatory 132 5.3.1.1 Enzyme extraction 132 5.3.1.2 Fractionation of alkaline and acidic proteases 132 5.3.1.3 Acetone precipitation 133 5.3.1.4 Ion exchange chromatography 133 5.3.1.5 Electrophoresis 134 5.4 Analytical 135 5.4.1 General acidic and alkaline proteases assay 135 5.4.2 Trypsin activity 135 5.4.3 Chymotrypsin activity 135 5.4.4 Carboxypeptidase activity 136 5.4.5 Protein analysis 136 5.4.6 Amino acids 136 5.4.7 pH optima 136 5.4.8 pH stability 137 5.4.9 Temperature optima 137 5.4.10 Temperature stability 137 5.4.11 Effect of inhibitors 137 5.4.12 Effect ofNaCl on enzyme activity 138 5.4.13 Effect ofNaCl on enzyme stability 138 5.4.14 Milk clotting activity 138 5.4.15 Effect of substrate concentration 138 5.5 Results 139 5.5.1 Isolation and partial purification 139 5.5.1.1 Fractionation of alkaline and acidic proteases 139 5.5.1.2 Acetone precipitation and dialysis 141 5.5.1.3 Ion exchange chromatography 141 5.5.1.4 Polyacrylamide gel electrophoresis and molecular weight estimation 144 5.5.2 Characterisation of alkaline and acidic proteases 144 5.5.2.1 Optimum pH 144 5.5.2.2 Stability of alkaline and acidic proteases at different pH 144 5.5.2.3 Temperature optima of alkaline and acidic proteases 149 5.5.2.4 Stability of alkaline and acidic proteases at different temperature 149 5.5.2.5 Effect ofNaCl of enzyme activity 149 5.5.2.6 Stability of enzyme in NaCl solution 150 5.5.2.7 Effect of inhibitors 150 5.5.2.8 Substrate specificity 150 5.5.2.9 Milk clotting activity 150 5.5.2.10 Effect of substrate concentration 152 5.5.2.11 Amino acid composition 154 5.6 Discussion 154 vii 5.6.1 Isolation and partial purification of alkaline and acidic proteases 154 5.6.2 Characterisation of acidic and alkaline proteases from cowtail ray viscera 160 5.6.2.1 Acidic proteases 160 5.6.22 Alkaline protease 168 6 CONCLUSIONS AND RECOMMENDATIONS 173 6.1 Conclusions 173 6.2 Recommendations 176 7 REFERENCES 178 8 APPENDICES 227 8.1 Summary of results of statistical analysis for silages from Muara Angke and Labuhan Maringgai 227 8.2 Raw data of cowtail ray viscera silage from Muara Angke 228 8.3 Raw data of cowtail ray viscera silage from Labuhan Maringgai 232 8.4 Raw data of fish protein hydrolysates 234 8.5 Summary of results of statistical analysis for effects of peptones on growth rate, total growth and biomass production of test microorganisms 235 8.6 Raw data of use of cowtail ray viscera peptones as microbial growth media 236 viii ABSTRACT The classification and utilisation of aquatic food are reviewed, including its waste treatment and utilisation. The general description of stingrays and their utilisation in Indonesia are described. Cowtail ray viscera was preserved by acid ensilation. Parameters investigated included origin of cowtail ray viscera, levels and types of acids, temperature and time of incubation. Changes in pH, soluble nitrogen, solubilisation and viscosity were monitored. Hydrochloric acid liquefied but failed to preserve the viscera. Organic acids liquefied and preserved the viscera for at least 120 days. The pattern of nitrogen solubilisation was comparable to those of cold and temperate water fish. Propionic and formic acids (1:1 v/v; 3% v/w) are recommended. Peptones were produced from cowtail ray viscera silages prepared with 3% organic acids and 4% HC1, and their ability to support the growth of selected microorganisms investigated.