Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2013 Effects of Maillard Reaction on the Immunoreactivity of Almond Major Protein in the Food Matrices Guneet Singh Chhabra Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF HUMAN SCIENCES EFFECTS OF MAILLARD REACTION ON THE IMMUNOREACTIVITY OF ALMOND MAJOR PROTEIN IN THE FOOD MATRICES By GUNEET SINGH CHHABRA A Thesis submitted to the Department of Nutrition, Food and Exercise Science in partial fulfilment of the requirements for the degree of Master of Science Degree Awarded Fall Semester, 2013 Guneet Singh Chhabra defended this thesis on October 18, 2013. The members of the supervisory committee were: Shridhar K. Sathe Professor Directing Thesis Yun-Hwa Peggy Hsieh 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 master’s thesis is dedicated to my niece, Noor who has come in my life as a blessing of the almighty. Your smile is my remedy. I wish to see you grow all your life and live all your dreams, for that will make my one dream come true, to see you happy. God bless you always. I love you! The journey that has lead to the completion of this Thesis has been long and challenging and could not have been accomplished without several people. Thank you first to Dr. Sathe, who gave me a chance to come back and supported me to finish my master’s degree. Sir, you literally gave me a new beginning in life and when I needed it the most. I owe my life to my family and friends, whose moral support kept me going when I got discouraged or frustrated. The encouragement I received outside of the academic world overwhelms me. To perhaps my biggest supporter throughout my life, the almighty, I call him Waheguru, whose blessings carried me through many tough times and kept me going when the end was not insight, thank you. Thank you to my Mom, Harpreet Kaur and Dad, Jasbir Singh, who got me where I am today and always have stood with me in my decisions. To my sister, Gursimran Kaur and brother-in-law, Harpreet Singh for being the best advisors all the time and bringing the angel of my life, Kulpreet Kaur aka Noor, who is most precious to me. To my brother, Mandeep Singh, my best friend for ever, thank you for always being there with all your love. Thank you so much to my committee members, Dr. Hsieh and Dr. Dorsey, who provided valuable suggestions and guidance in this research and otherwise. I could not have completed this Thesis without the academic and moral support of my lab mates. Coming to lab everyday was so easy when you visited an environment of learning, thinking, and encouragement. I was so lucky to be a part of a lab with so many caring and gifted people that have become dear friends. iii They have taught me not only about protein research, but have taught me the value of peer support. Thank you to Changqi Liu “Ben”, who always helped me selflessly and gave me valuable suggestions during this research. There is so much to learn from you and you are a good man my friend. Thank you to Sahil Gupta for being a friend and a colleague and always being there to celebrate good and bad days. I would take this opportunity to thank Ying Zhang for being an inspiration to work hard. Thank you to my colleagues Jasamrit Bakshi and Sepideh Alasvand. I will cherish all of you. Finally, thank you again to the person who has had the biggest impact on me through the process of completing my Master’s Thesis, my major professor, Dr. Shridhar Sathe. I cannot thank you enough for everything that you have done for me. Dr. Sathe loves research, and he loves his graduate students. Thank you for giving me the finest education that one could receive in this area of research and for always finding a way to support me financially and giving me the opportunity to teach. Thank you for serving as my major professor for the past three years, for being a mentor and a friend. Most of all, thank you for believing in me, even when I did not. You have instilled in me a confidence that is worth more than I can begin to explain. iv TABLE OF CONTENTS List of Tables................................................................................................................................vii List of Figures.............................................................................................................................viii Abstract..........................................................................................................................................ix INTRODUCTION..........................................................................................................................1 Statement of the Problem................................................................................................................5 Research Hypothesis.......................................................................................................................5 Limitations of the Study…..............................................................................................................6 Significance of the Study................................................................................................................6 Definitions.......................................................................................................................................7 REVIEW OF LITERATURE.......................................................................................................10 Maillard Reaction Mechanism......................................................................................................11 MATERIALS AND METHODS..................................................................................................16 Isolation of Almond Major Protein (AMP)...................................................................................21 Protein Extraction..........................................................................................................................22 HunterLab LabScan XE Spectrophotometer.................................................................................22 Water Activity (Aw)….................................................................................................................22 Soluble Protein Content................................................................................................................23 Immunoassay.................................................................................................................................24 Statistical Analysis........................................................................................................................28 RESULTS AND DISCUSSION...................................................................................................29 Water Activity (Aw) of Food Matrices.........................................................................................29 Color of Food Matrices.................................................................................................................29 Effect of Maillard Reaction and Food Matrix on the Immunoreactivity of Amandin.................33 Sandwich ELISAs.........................................................................................................................35 v Effect of Maillard Reaction on Immunoreactivity of Food Matrices............................................49 Dot Blots........................................................................................................................................50 Western Blots.................................................................................................................................51 Conclusion.....................................................................................................................................59 APPENDIX: The Animal Care and Use Committee Approval Memorandum.............................61 REFERENCES..............................................................................................................................64 BIOGRAPHICAL SKETCH.........................................................................................................74 vi LIST OF TABLES 2.1 Peptides recognized by murine mAbs......................................................................................11 3.1 List of food matrices for analysis.............................................................................................17 3.2 Gradient gel recipe.................................................................................................................. 26 4.1 Water activity (Aw at 25 °C) and browning of food matrices containing almonds................30 4.2 Sandwich ELISA using 4C10..................................................................................................35 4.3 Sandwich ELISA using 4F10...................................................................................................40 4.4 Sandwich ELISA using 2A3....................................................................................................44 vii LIST OF FIGURES 4.1 Relationship between the L* and the Aw of the processed food matrices.............................34 4.2 Dot blot for the food matrix samples