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Download .Pdf University of Nigeria Research Publications OKEZIE, Stanley M. PG/M.Sc./0027858 Author Production of Cocoa Butter Substitutes/Equivalents from Palm Oil, Shea Butter and Dika Fat using Title Aspergillus niger Lipase Biological Sciences Faculty Faculty ent Biochemistry Departm May, 2007 Date Date Signature PRODUCTION OF COCOA BUTTER SUBSTITUTESIEQUIVALENTS FROM PALM OIL, SHEA BUTTER AND DIKA FAT USING Aspcigoilltrs niger LIPASE. OKEZIE, STANLEY MADUAKOLAM (PGIMSc/00/27858) DEPARTMENT OF BIOCHEMISTRY UNIVERSITY OF NIGERIA NSUKKA MAY, 2007 TITLE PAGE PRODUCTION OF COCOA BUTTER SUBSTITUTES/EQUIVALENTS FROM PALM OIL, SHEA BUTTER AND DIKA FAT USING Aspergillus tziger LIPASE. CERTIFICATION PAGE OKEZIE, MADUAKOLAM STANLEY, a post-graduate student of the Department of Biochemistry and with the Registration Number PG/M.Sc/00/27858, has satisfactorily completed his requirement for research work for the Degree of Master of Science (M.Sc) in Industrial BiochemistryA3iotechnology. The work embodied in this thesis is original and has not been submitted in part or fill1 for any other diploma or degree of this or any other University. J Prof. I. C. Ononogbu Prof. 0. U. Njoku (Supervisor) (HOD) Externd Examiner DEDICATION This work is dedicated to the Almighty God, who in his infinite mercies and powers, have made it possible for me to complete this project. ACKNOWLEDGEMENT I am particularly grateful to my Supervisor Prof. I.C. Ononogbu who not only made this work possible but also provided the guide. I also thank him for all his generosity and contribution which saw this work to completion. My most profound gratitude also goes to Prof. 0.11. Mioki~for his efforts and support in the entire project. Despite his tight schedules, he still found time to read, criticize and correct the entire project work. I must express my indebtedness to Mr. O.E. lkwuagwu ~vhoby his cosupervision, commitment and guide played a major role in the sirccess of this work. He was also helpful in many other ways too numerous to mention. For various kinds of' contributions and suggestions, I am equally gratefill to Prof. 0. Obidoa, Dr. O.F.C. Nwodo. Dr. F.C Chilaka, Dr. P.N. Uzoegbu, Dr. L.U.S. Ezeanyika, Dr. B.C. Nwanguma, Dr. 1.N.E Onwurah, Dr. E.O. Aiumanah, Dr. V. Ogugua, Dr. S. Eze, Dr. Onwubiko, Mr. P.A.C., Egbuna and Mr. C.U. Anyanwu. I am also grateful to the entire academic and technical staff members of the Depart~nent for all their efforts My colleagues are also warmly acknonfledged-particularly Chinedu Nwuke, Chibuzor Nwaka, Parker Joshua, Dr. Ikgene. Mr. Sabastine, Chukwu Jude. I,. Onyenweaku (Prof.) Mr. I. Iroha and a host of others. Special appreciation goes to Miss Perpetua Offor for all her efforts in the typing of this Project. Special thanks go to my parents, Mr. and Mrs. Okezie, my brothers and sisters, and also my friends. Lastly but by no mems the least, my special ack~iowIedgemntgoes to God Almighty whose plans and purposes must surely prevail. Okezie S. M. B.Sc (Hons) Nig May 2007 ABSTRACT Cocoa butter Substitutes/I~quivalents were prepared by enzymatic interesterification of fractions from partially refined palm oil, shea butter and dika fat. The partially retined oil samples were fractionated by crystallization in a solvent medium of 11-hexane. Aspergillus niger lipase immobilized by adsorption on keseilguhr was used as the interesterification catalyst. The lipase was found to have pH and temperature range stability of 6-9 and 25-55" C respectively. Optimal activity of the enzyme was found to be at 30mM oil concentration, pH of 7.2 and temperature of 40' C. The V,,, and K,, of the enzyme was found to be 462u/l and 47mM respectively. Temperature and pH Stability suggest an enzyme suitable for industrial application. Incubation of the oil fractions with the i~nrnobilizedenzyme For 3 days, yielded interesteritied products. Analysis of the products obtained was done using melting point and TLC analysis of their fatty acid concentration. Interesterified products of Palm mid fraction + shea butter crude and palm mid fraction + shea olein were found to have stearic and oleic acid content respectively that compared with that of cocoa butter. The melting point range of palm mid fraction+ shea stearin. palm mid fraction + shea olein, palm olein + shea butter crude, palm mid fraction + shea butter crude, and palm olein + dika olein were found to compare u~ll with the melting point range of cocoa butter. TABLE OF CONTENT CONTENTS PAGE Title page.. ....................................................................................i . Certification Page.. ..........................................................................II ... Dedication.. .................................................................................,111 Acknowledgement.. .........................................................................iv Abstract.. ................................................................................... .v Table of Contents .................................................................................vi List of Tables.. .................................................................................... vii List of Figure.. .............................................................................. .ix CHAPTER ONE-INTRODUCTION AND LITERATURE REVIEW Introduction .........................................................................I Lipids an overview.. ................................................................3 Fats and oil ...........................................................................3 Nature composition and uses of selected fats and oils.. ......................6 Industrial Processing of Fats and Oils.. ...........................................I I Characterization of fats and oils ................................................I5 Lipases.. ............................................................................I9 CHAPTER TWO-MATERIALS AND METHODS Materials. ......................... ................................................26 Oil seeds .............................................................................26 C helnicals.. ........................................................................ .26 Methods. ...........................................................................26 Preparation of reagents.. .........................................................26 Extraction of oil from samples ..................................................28 Partial refining of oil samples.. .................................................. 28 Determination of % oil yield ....;... ..............................................36 Solvent fractionation (crystallization) .......................................... 29 Characterization of ttie partially refined oil samples.. .......................29 Production and extraction of Fungal Iipase.. ..................................33 2.2.8 Characterization of lipase ......................................................34 2.2.9 Immobilization of the Iipase ...................................................35 2.2.10 Interesterification reaction system ............................................36 2.2.1 1 Fatty acid analysis of hydrolysates ...........................................36 2.2.12 Statistical analysis ............................................................... 38 3.0 CHAPTER THREE-RESULTS 3.1 Physical properties of crude and partially refined oil samples ............39 3.2 Chemical properties of partially refined oil estracts and their Fraction ...........................................................................46 3.3 Enzyme properties of A.spergillw nigw lipase ............................SO 3.4 Physical and chemical properties of intercsterified products ...............55 4.0 CHAPTER FOUR-DISCUSSION Discussion ................................................................................58 Conclusion ................................................................................65 Suggestion ................................................................................66 References ...............................................................................67 LIST OF TABLES Table 1.1 a Foods uses ol'fats and oils ....................................................... 4 Table I . 1 b Industrial uses of fats and oils ..................................................5 Table 1.2 Fatty acid composition of dika fat .............................................7 Table 1.3 Fatty acid composition of shea butter ..........................................9 Table 1.4 Some specific lipases for the production of specific structured triacylgycerols ....................................................... 24 Table 3.1 Yield of oil fiom different oil fruitstseeds .................................... 39 Table 3.2 Acylglycerol fractions of palm oil at varying temperati~re and solvent omcentrations ......................................................40 Table 3.3 Acylglycerol fractio~isof shea butter at varying temperature and solvent concentrations ...........................................41 Table 3.4 AcylglyceroI fractions of dika fat at varying temperature and solvent clmcentrations ......................................................42 Table 3.5 physical properties of partially refined (PR) palm oil and its fracticns ..................................................................43 Table 3.6 Physical properties of PR shea butter and its fractions .....................44 'Table 3.7 Physical properties of PR dika fat and its fractions
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