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LIN, Chîî-fa, 1940- THE CASEIN STABILIZING FUNCTION OF SULFATED HYDROCOLLOIDS. The Ohio State University, Ph.D., 1971 Food Technology 5 University Microfilms, A XEROX Company, Ann Arbor, Michigan | THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED THE CASEIN STABILIZING FUNCTION OF SULFATED HYDROCOLLOIDS A Dissertation Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of the Ohio State University By Chii-fa Lin, B. Sc., M. Sc., The Ohio State University 1971 Approved by Adviser Department of Foocf Science a Nutritio PLEASE NOTE; Some pages have small and indistinct print. Filmed as received. UNIVERSITY MICROFILMS. This investigation was supported in part by Public Health Service Research Grant No. FD-00117 from the Food and Drug Adminis tra tio n , Washington, D.C. and by a donation from Ross Laboratories, Columbus, Ohio. Dedicated to my wife, Kathy, and my daughter, Judy, for th e ir help, patience and encouragement. ACKNOWLEDGEMENTS My gratitude and sincere appreciation are expressed to Dr. P. M. T. Hansen, Associate Professor of the Department of Food Science and Nutrition, for his able guidance in this research and in assisting with the preparation of the manuscript and to Mrs. J ill Hansen for her help and encouragement offered to me and rny family during my graduate study. Without either of them, this work could not have been accomplished. Special thanks are due to Dr. I . Kristoffersen and Dr. W. J. Harper for their advice and keen personal interest in this study. November 25, 1940 ...................... Born - Hsinchu, Taiwan, China 1963 ............................................ B. Sc., National Taiwan University Taipei, Taiwan, China 1966-1968 ................................. Graduate Research Assistant, Department of Dairy Technology, The Ohio State University Columbus, Ohio 1968 ..................................... M. Sc., The Ohio State University, Columbus, Ohio Graduate Research Associate, Department of Dairy Technology, The Ohio State University Columbus, Ohio Graduate Research Associate, Department of Food Science & N utrition, The Ohio State University Columbus, Ohio TABLE OF CONTENTS Page ACKNOWLEDGMENTS........................................ iv VITA ..................... V LIST OF TABLES...................... ix LIST OF FIGURES................................................................................. x INTRODUCTION ..................................................................................... 1 LITERATURE REVIEW .... ............................................................... 3 A. Interaction between K-casein and calcium sensitive c a s e in s ........................................ 3 B. Interaction between sulfated polysaccharides and proteins ..................................................................................... 9 1. Chondroitin sulfate ....................................................... 11 2. H eparin ............................................................................. 12 3. Carrageenan .................................... 17 SCOPE OF INVESTIGATION............................................... 21 EXPERIMENTAL PROCEDURE......................... 22 A. Protein sa m p le s ..................................................................... 22 B. Fractionation of carrageenans from Chondrus crispus . 22 C. Polysaccharide samples .......................................................... 25 D. Stabilization t e s t .................................................................. 28 E. Determination of protein concentration ......... 29 F. Polysaccharide film s for infrared study ......................... 29 G. Butter solution ..................................................................... 29 H. Sedimentation velocity ........................................................... 30 v i TABLE OF CONTENTS co n tin u e d Page I. Viscosity measurement .................................................................. 30 J. Density measurement ....................................................................... 30 K. Sedimentation equilibrium ............................................................ 30 L. Mi H i pore electrophoresis ............................................................ 31 M. Polyacrylamide gel electrophoresis ............................................. 31 RESULTS.................................................................................................... 32 A. Isolation and properties of carrageenan fractions .................. 32 1. F ra c tio n a tio n ..................................................................... 32 2. Electrophoretic pattern ................................................... 34 3. Monomer/polymer formation ............................................... 34 4. Infrared sp e ctra ................................................... ... 39 5. Stabilizing ability ........................................................... 42 B. Factors affecting the s ta b ility of carrageenan/ag-casein s y s te m ......................... 49 1. pH e f f e c t .......................................................... 52 2. Heating time and temperature effects ............................. 55 3. Polymer size e f f e c t ........................................................... 60 (a) V is c o s it y ...................................................................... 61 (b) Molecular weight ........................................................... 64 4. Concentration effects ....................................................... 66 (a) Hydrocolloid concentration ......................................... 69 (b) Protein concentration ................................................ 69 5. Sedimentation velocity . ............................................ 74 C. Interaction between synthetically sulfated polysaccharides and ag-casein ................................................................................. 74 1. Comparison of chemically derived polysaccharide s u lfa te s ................................................................................. 74 2. pH e f f e c t ..................................................................• • • • ^0 3. Heat e f f e c t .......................................................................... 83 4. Concentration e f f e c t ............................. 83 DISCUSSION................................................................................ 96 TABLE OF CONTENTS c o n tin u e d Page REFERENCES............................................................................................... HI APPENDIX................................................................................................... H 8 LIST OF TABLES Table Page 1. RECOVERY AND PROPERTIES OF FRACTIONATED CARRAGEENANS FROM CHONDRUS CRISPUS .............................................................. 33 2. VISCOSITY AND MOLECULAR WEIGHT OF CARRAGEENAN FRACTIONS OF VARYING STABILIZING A B IL IT Y.................................................... 65 3. SOLUBILITY OF FREEZE-DRIED STABILIZED COMPLEX FROM ALBG/as-CASEIN SYSTEM .............................................................. 93 4. COMPARISON OF THE STABILIZING PROPERTIES OF KNOWN STABILIZERS OF as-CASEIN ....................................................... 104 5. AN EXAMPLE OF THE COMPUTER OUT-PUT .................................... 121 6. AN EXAMPLE OF THE IN-PUT DATA CARDS.................................... 125 LIST OF FIGURES Fi gure Page 1. FAG-ELECTROPHORETIC PATTERNS OF as"CASEIN AND K-CASEIN............................................................................................ 23 2. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF CARRAGEENAN EXTRACT FROM CHONDRUS CRISPUS .................... 26 3. FLOW DIAGRAM OF THE PROCEDURE FOR THE FRACTIONATION OF k-CARRAGEENAN................................................... 27 4. ELECTROPHORETIC PATTERNS OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS (CELLULOSE ACETATE) ................................ 35 5. ELECTROPHORETIC PATTERNS OF ks“ AND Ki-CARRAGEENAN AT DIFFERENT CONCENTRATION (CELLULOSE ACETATE) .................... 3 7 6 . ELECTROPHORETIC PATTERNS OF k- AND x-CARRAGEENANS AT DIFFERENT CONCENTRATIONS (CELLULOSE ACETATE) ................. 4 0 7. INFRARED SPECTRA OF CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS............................................................................................ 43 8 . STABILIZATION OF as-CASEIN BY CARRAGEENAN FRACTIONS FROM CHONDRUS CRISPUS (0.15% p ro te in , pH 7.2 and 0.01 M CaCl2) . ............................... 50 9. EFFECT OF pH ON THE STABILIZING ABILITY OF K-CASEIN, k-carrageenan and X-CARRAGEENAN ............................................ 5 3 10. EFFECT OF HEATING TIME AT 85 C ON THE STABILIZING ABILITY OF K-CASEIN, K-CARRAGEENAN AND X-CARRAGEENAN . 55 11. EFFECT OF TEMPERATURE OF THE STABILIZING ABILITY OF k- casein, k- carrageenan and X-CARRAGEENAN ........................ 58 12. RELATIONSHIP BETWEEN VISCOSITY AND STABILIZING ABILITY . 62 13. RELATIONSHIP BETWEEN MOLECULAR WEIGHT OR CARRAGEENANS AND ITS STABILIZING A B IL IT Y.................................................... 57 14. STABILIZATION OF Og-CASEIN BY K-CARRAGEENAN AT THREE DIFFERENT PROTEIN CONCENTRATIONS ............................................ 70 LIST OF FIGURES continued Figure Page 15. EFFECT OF ag-CASEIN CONCENTRATION ON THE STABILIZING ABILITY OF K-CARRAGEENAN AND K-CASEIN ................................ 72 16. SEDIMENTATION VELOCITY PAHERNS