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View of Literature Florida State University Libraries 2016 Lectin Analyses of Soybean and Soybean Products Qing Zhao Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES LECTIN ANALYSES OF SOYBEAN AND SOYBEAN PRODUCTS By QING ZHAO A Thesis submitted to the Department of Nutrition, Food & Exercise Sciences in partial fulfillment of the requirements for the degree of Master of Science 2016 Qing Zhao defended this thesis on June 21, 2016. The members of the supervisory committee were: Shridhar K. Sathe Professor Directing Thesis Qinchun Rao Committee Member John G. Dorsey Committee Member The Graduate School has verified and approved the above-named committee members, and certifies that the thesis has been approved in accordance with university requirements. ii ACKNOWLEDGMENTS This thesis becomes a reality with the kind support and help of many individuals. I would like to express my sincere appreciation to all of them. Foremost, I am very grateful to the mentor, philosopher, and the major professor, Dr. Shridhar K. Sathe, who gave me the opportunity to study and work in his lab in NFES at Florida State University. I am thankful to him for providing ideas and suggestions to make it possible for me to work on this topic that was of great interest to me, for providing the excellent and quality scientific instruments and materials in our lab, for sharing his experience and expertise with me, and making me aware the importance of thinking independently. I would like to thank Dr. Qinchun Rao for serving in my committee, providing me guidance and valuable thinking. I am also thankful for Dr. John G. Dorsey for willing to serve as my committee member and also providing suggestions during my research program at FSU. I would like to express my gratitude to the Department of Nutrition, Food, and Exercise Sciences for providing the opportunity to pursue my Masters degree. I also want to thank Ms. Tara Hartman, Ms. Ann Smith, Mr. David Parish for helping me during my study here at FSU. I am sincerely thankful to Changqi Liu (Ben) for taking the time to teach me the basic laboratory techniques and giving the advice for my experiments and thesis writing. My lab mates, Changqi, Valerie, Sahil, and Sepideh made my life in lab joyful and am thankful for their research support. Finally, I am extremely thankful to my parents and all family members for their unconditional love, encouragement and financial support. Without your support and love, I could not have come this far. iii TABLE OF CONTENTS List of Tables ...................................................................................................................................v List of Figures ................................................................................................................................ vi List of Abbreviations ..................................................................................................................... vii Abstract ........................................................................................................................................ viii 1. INTRODUCTION ......................................................................................................................1 Background ................................................................................................................................1 Statement of the Problem ...........................................................................................................3 Research Hypothesis ..................................................................................................................4 Specific Aims .............................................................................................................................4 2. REVIEW OF LITERATURE .....................................................................................................6 Food Allergies ............................................................................................................................6 Soybean ......................................................................................................................................7 Lectins ........................................................................................................................................9 Soy Lectin .................................................................................................................................10 Food Processing ........................................................................................................................11 3. MATERIALS AND METHODS ..............................................................................................14 Materials ....................................................................................................................................14 Methods .....................................................................................................................................15 4. RESULTS AND DISCUSSION ...............................................................................................22 Lectin Purification .....................................................................................................................22 Competitive ELISA ...................................................................................................................25 Hemagglutination Assay ...........................................................................................................40 5. CONCLUSIONS .......................................................................................................................49 APPENDICES ...............................................................................................................................50 A. ANIMAL CARE AND USE COMMITTEE APPROVAL MEMORANDUM .......................50 B. HUMAN SUBJECTS COMMITTEE APPRROVAL MEMORANDUM ...............................51 C. INFORMED CONSENT FORMS ............................................................................................52 References ......................................................................................................................................53 Biographical Sketch .......................................................................................................................65 iv LIST OF TABLES 1 Summary of lectin purification by column chromatography ...................................................24 2 Intra- and inter-assay variations for the pAb-based soybean lectin competitive ELISA .........28 3 Accuracy of constructed competitive ELISA ..........................................................................29 4 Soybean varieties used .............................................................................................................30 5 Raw soybean seed lectins analyzed by using the ELISA ........................................................31 6 Effects of thermal processing on SBA immunoreactivity as assessed by the competitive ELISA ............................................................................................................................................33 7 Germinated soybean sprouts ....................................................................................................36 8 Effects of germination on SBA immunoreactivity as assessed by the competitive ELISA ....38 9 The SBA immunoreactivity of commercial soybean products as assessed by the competitive ELISA ............................................................................................................................................39 10 Pure lectins measured by using the hemagglutination assay ...................................................41 11 Raw soybean seed lectins analyzed by using the hemagglutination assay ..............................41 12 Effects of thermal processing on SBA hemagglutination activity of GMO soybean AG5831 as assessed by the hemagglutination assay ....................................................................................43 13 Effects of thermal processing on SBA hemagglutination activity of Non-GMO soybean cobb as assessed by the hemagglutination assay ....................................................................................44 14 Effects of germination on SBA hemagglutination activity as assessed by the hemagglutination assay ..................................................................................................................46 15 The SBA hemagglutination activity of commercial soybean products as assessed by the hemagglutination assay ..................................................................................................................47 v LIST OF FIGURES 1 Soybean: acreage planted and value in United States, 1960-2013 (USDA oil crop yearbook, 2014) ................................................................................................................................................8 2 Elution profile for 40% - 80% ammonium sulfate precipitate soybean protein extract off a DEAE DE-53column (2.6 × 30.5 cm). ..........................................................................................22 3 Elution profile the soybean lectin (DEAE DE-53 pool) off a Sephacryl S200 column (2.6 × 91 cm) ............................................................................................................................................23
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