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Development of Simple and Rapid Procedures for Purification of Egg Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 Development of simple and rapid procedures for purification of egg yolk immuno-globulin and ovotransferrin from chicken egg and antimicrobial activity of ovo-transferrin against Escherichia coli O157:H7 and Listeria monocytogenes Kyung Yuk Ko Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Food Science Commons Recommended Citation Ko, Kyung Yuk, "Development of simple and rapid procedures for purification of egg yolk immuno-globulin and ovotransferrin from chicken egg and antimicrobial activity of ovo-transferrin against Escherichia coli O157:H7 and Listeria monocytogenes" (2007). Retrospective Theses and Dissertations. 15913. https://lib.dr.iastate.edu/rtd/15913 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Development of simple and rapid procedures for purification of egg yolk immuno- globulin and ovotransferrin from chicken egg and antimicrobial activity of ovo- transferrin against Escherichia coli O157:H7 and Listeria monocytogenes. by Kyung Yuk Ko A dissertation submitted to the graduate faculty in paritial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Meat Science Program of Study Committee: Dong U. Ahn, Major Professor James S. Dickson Joseph G. Sebranek Joan E. Cunnick Elisabeth J. Huff-Lonergan Iowa State University Ames, Iowa 2007 Copyright © Kyung Yuk Ko, 2007. All rights reserved. UMI Number: 3274855 UMI Microform 3274855 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii DEDICATION Firstly, I would like to dedicate this dissertation to my loving God. During my Ph. D. study, He was my perfect supporter, refuge and fortress. Whenever I was frustrated for my weakness or a variety of works unexpected during the study in Ames, He lifted me from the mire and taught me about his deep thoughts and love. Through such difficulties and sufferings faced during ph. D study, I realized that true my pleasure and satisfaction come from only God not the circumstances or any other people. Also, I knew that there is no any shortness in my life if I have to be beside Him because He is a perfect father who knows precisely my need and joyfulness. Also He wants readily to fill them plentifully in my life. Furthermore, I am thankful to God for providing the privilege to study for me and I would like to remember forever the lessons and his love showed during ph. D study. Due to memories of his deep love rendered to me, I would like to accomplish a beautiful life to share my knowledge and privileges gladly for other people. Secondly, I want to dedicate this dissertation to my respected father, Tae-jun Ko and my mother, Aeok Hong in heaven. Even though they passed away before starting ph. D study, I believe that they have been looking at my life in heaven and want to help me readily anywhere. It was the largest suffering and sorrow in my entire life that I could not the chance to reward their love and devotion. However, I thought that only the way to reward their love is to do my best during given life including my Ph. D. study. Thirdly, I would like to dedicate this dissertation in loving memory of my niece, Min-ji Kim. Her death which happened in last year has been one of the hardest things that I have dealt with during my Ph.D. study. Although Minji’s life was cut short, she left behind a lifetime of memories. She was a special loving niece. Even though she was 21 year-old, she was my friend. During her short life, she gave many joys and pleasures to my family. The sorrow losing Min-ji made me perform more diligently and sincerely in my research. I believe that her death will not be in vain. iii TABLE OF CONTENTS ABSTRACT v CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 3 CHAPTER 2. LITERATURE REVIEW 4 I. Escherichia coli O157:H7 and Listeria monocytogenes 4 I-1. Escherichia coli O157:H7 4 I-2. Listeria monocytogenes 6 II. Natural antimicrobial proteins in chicken eggs 8 II-1. Egg yolk immunoglobulin (IgY) 9 II-1.1. Characteristics of IgY 9 II-1.2. Passive immunizations of animals using IgY 10 II-1.3. Separation and purification methods of IgY 12 II-2. Lysozyme 15 II-3. Ovotransferrin 17 II-3.1. Chemical and physical characteristics of ovotransferrin 18 II-3.2. Iron uptake and release of ovotransferrin 19 II-3.2.1. Iron uptake of ovotransferrin 19 II-3.2.2. Iron release of ovotransferrin 21 II-3.2.3. Effect of sodium bicarbonate on iron uptake and release 23 II-3.3. Purification methods of ovotransferrin 23 II-3.4. Activated lactoferrin 26 II-3.5. Antimicrobial activity of ovotransferrin 27 II-3.5.1. Mechanism of antibacterial action of ovotransferrin 28 II.3.5.2. Factors affecting antibacterial activity of ovotransferrin 29 II-3.6. Bactericidal peptides of ovotransferrin 31 III. Summary 32 References 33 CHAPTER 3. PREPARATION OF EGG YOLK IMMUNOGLOBULIN (IGY) FROM CHICKEN EGGS USING AMMONIUM SULFATE PRECIPITATION AND IRON EXCHANGE CHROMATOGRAPHY 60 Abstract 60 Introduction 61 Materials and Methods 63 Results and Discussion 65 Conclusion 70 References 70 CHAPTER 4. AN ECONOMIC AND SIMPLE PURIFICATION PROCEDURE FOR THE LARGE-SCALE PRODUCTION OF OVOTRANSFERRIN FROM EGG WHITE 88 Abstract 88 iv Introduction 89 Materials and Methods 91 Results and Discussion 95 Conclusion 99 References 99 CHAPTER 5. THE EFFECTS OF ZN2+, SODIUM BICARBONATE, CITRIC ACID ON THE ANTIBACTERIAL ACTIVITY OF OVOTRANSFERRIN AGAINST ESCHERICHIA COLI O157:H7 AND LISTERIA MONOCYTOGENES IN VITRO AND HAMS 114 Abstract 114 Introduction 115 Materials and Methods 117 Results and Discussion 121 Conclusion 129 References 130 CHAPTER 6. EDTA AND LYSOZYME IMPROVES ANTIMICROBIAL ACTIVITIES OF OVOTRANSFERRIN AGAINST ESCHERICHIA COLI O157:H7 143 Abstract 143 Introduction 144 Materials and Methods 146 Results and Discussion 151 Conclusion 157 References 158 CHAPTER 7. THE EFFECT OF EDTA AND LYSOZYME ON THE ANTIMICROBIAL ACTIVITY OF OVOTRANSFERRIN AGAINST LISTERIA MONOCYTOGENES 170 Abstract 170 Introduction 171 Materials and Methods 173 Results and Discussion 177 Conclusion 183 References 184 CHAPTER 8.GENERAL CONCLUSIONS 196 ACKNOWLEDGEMENTS 199 v ABSTRACT The overall goal of this study was to develop practical, natural, and efficient antimicrobials from egg. To accomplish the goal, novel protocols for large-scale production of egg yolk immunoglobulin and ovotransferrin were developed. Also antibacterial activity of ovotransferrin against Escherichia coli O157:H7 and Listeria monocytogenes was investigated and the antimicrobial activity of ovotransferrin was improved by combinations with other antimicrobials. The developed protocol for large-scale production of egg yolk immunoglobulin (IgY) consisted of several steps such as using 0.01 % charcoal to remove lipoproteins from IgY extract, ultrafiltration to concentrate IgY solution, and 40% ammonium sulfate to precipitate IgY following dialysis. Morevoer, apo-ovotransferrin was isolated from 2 time-diluted egg white by following steps: as iron saturation of egg white, extraction of iron-bound (holo) ovotransferrin using 43% ethanol, precipitation of holo- bound ovotransferrin by 59% ethanol, dissolving the precipitate with distilled water, and ® removal iron from holo-ovotransferrin using AG 1-X2 ion exchange resin. After separating apo-ovotransferrin, the effects of citric acid, Zn- and Fe- binding, and sodium bicarbonate (NaHCO3) on the antibacterial activity of ovotransferrin against E. coli O157:H7 and L. monocytogenes in model systems using BHI broth were investigated. Synergistic effects of EDTA and lysozyme on the antimicrobial activities of ovotransferrin were investigated toward E. coli O157 and L. monocytogenes on model systems using BHI broth and pork chops and hams. According to this study, IgY obtained by developed protocol was approximately 70 ~ 80% recovery and approximately 80% purity. The new procedure seems to be economical and practical for a large-scale purification of IgY from egg yolk since ammonium sulfate precipitation could accommodate of large volume of egg yolk. Also, the recovery rate and purity of ovotransferrin produced by the newly developed protocol was around 94% and 80% respectively. As the procedure is consisted of only a few steps, it is suitable to a large-scale ® production of ovotransferrin. Also becasuse ethanol and AG 1-X2 ion exchange resins can be regenerated, it seems to be economical and efficient. In study related to antibacterial activity of ovotransferrin, 100 mM-NaHCO3 significantly increased the antibacterial activity of ovotransferrin against both E. coliO157:H7 and L. monocytogenes. 0.5% citric acid vi enhanced the antibacterial activities of ovotransferrin against E. coli O157:H7, Zn-bound ovotransferrin appeared to inhibit the growth of L. monocytogenes. So, this study suggests that the iron binding capacity of ovotransferrin was not the major mechanism of antimicrobial actions of ovotransferrin because acidic pH or Zn2+ promotes antibacterial activity of ovotransferrin toward E. coliO157:H7 or L. monocytogenes respectively. Also, 2 mg/ml EDTA improved antibacterial activity of ovotransferrin containing 100 mM-NaHCO3 against E. coli O157:H7. Also when lysozyme was combined with OS, the antibacterial activity of OS against L. monocytogenes increased slightly in vitro test. However, synergistic effect of lysozyme on antimicrobial activity of OS against L. monocytogenes did not show significantly in this study.
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