Beta-Glucanase Activity and its Impact on Beta-Glucan Molecular Weight Degradation in Cereal Products Fortified with Beta-Glucan by Azadeh Vatandoust A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Food Science Guelph, Ontario, Canada © Azadeh Vatandoust, January, 2012 ABSTRACT β-GLUCANASE ACTIVITY AND ITS IMPACT ON β-GLUCAN MOLECULAR WEIGHT DEGRADATION IN CEREAL PRODUCTS FORTIFIED WITH β-GLUCAN Azadeh Vatandoust Advisor: University of Guelph, 2012 Dr. Koushik Seetharaman Health benefits of high molecular weight (MW) β-glucans are well documented. Therefore, understanding and controlling depolymerization of β-glucan in baked products, would increase the effectiveness of β-glucan to confer health benefits. In this study we demonstrated that endogenous β-glucanase in wheat kernels are responsible for the depolymerization of β-glucans. A protocol was developed based on the Megzayme procedure to detect low levels of β-glucanase activity in wheat flour. This was confirmed by using HPLC-Calcofluor detection to monitor molecular weight changes. The distribution of β-glucanase in wheat kernels was also investigated. The effect of genotype, location, planting season and environmental factors on the level of endogenous β-glucanase in selected wheat cultivars was investigated using different wheat varieties planted under different condition and different seasons. Furthermore, kinetics of β-glucan depolymerization by endogenous glucanase in two dough systems with different moisture content was investigated. The results demonstrated that enzymes with β-glucanase activity are concentrated primarily in the outer layer of wheat kernels. Also genotype, environmental conditions and agronomic practice all had significant effects on the β- glucanase activity in wheat flours and poor harvesting conditions can significantly increase β-glucanase activity level in wheat. The kinetics results demonstrated that moisture content, incubation time and levels of endogenous β-glucanase activity of the system had significant impact on the final MW of β-glucan in the dough. Among all factors investigated, moisture content had the greatest impact. This study presents opportunities for industry to fortify baked products with high molecular weight β-glucan. ACKNOWLEDGEMENTS Venturing on an unknown journey of studies abroad initially seemed frightening. There were numerous moments that I didn’t know where the journey would end, but having faith that throughout the journey God is with me, was a constant source of strength and inspiration. First and foremost I would like to express my gratitude to my supervisor Dr. Koushik Seetharaman. The generous opportunity that he provided by accepting me as his student made my dreams of graduate studies become a reality. His advice, insightful criticisms, and patient encouragement aided me throughout my studies in innumerable ways. His truly scientific intuitions inspired me and enriched my life both as a student, and an aspiring scholar. One simply could not wish for a better or friendlier supervisor. I would like to express my sincere appreciation to Dr. Sanaa Ragaee for her support, supervision and encouragement which made her the true backbone of this study. Her long hours of discussion and her constant urge to learn, made me understand my research problem in greater depth. She believed in me and taught me to believe in myself. Her understanding and help towards me to reach beyond academic boundaries is greatly appreciated. I learned from her not just how to research and finish my thesis but how to inhale the downside of life while living abroad. I was continually amazed by her willingness to help and teach me all the small details in life, and I am grateful to her in every possible way. I would also like to thank Dr. Susan Tosh, my committee member, whose support for this project is greatly appreciated. She made her lab available for my experiments, which was the reason I was able to conduct all the experiments related to β-glucan molecular weight; also for her helpful inspiration in β-glucan which led me to read and educate myself further. Special thanks also go to Yolanda Brummer who thought me how to use the HPSEC. Her useful suggestions and comments during my research, her help in editing and correcting my literature review and finally her friendship and sense of humor made working in Agriculture and Agri Food Canada lab more than mere fun. iv It has been an honor to be a student of the late Dr. Peter J. Wood. Despite his ill health, he generously shared his time and ideas with me. He was my primary source for getting β-glucan questions answered. His unflinching courage and conviction will always inspire me, and I hope to continue to work with his noble thoughts. The joy and enthusiasm he had for β-glucan was contagious and served as a source of motivation during difficult times. I am most grateful for having known him during my studies and I will never forget him as a remarkable individual. I need to extend my gratitude and acknowledgement for the financial support provided by the Ontario Ministry of Agricultural, Food and Rural Affairs and the Department of Food Science. I would also like to thank all my lab mates at the University of Guelph and Agriculture-Agri Food Canada that became a part of my life. I am grateful to them for the sleepless nights we worked together before deadlines and for all the fun we had during our studies. My thanks go to my dear roommate and friend Mina Kaviani who put up with my difficult times and my crazy schedule. I can’t even imagine how I could have completed my work without her. I am indebted to Renuka Waduge for her friendship, support and valuable advices during my research; to George Annor who helped me understand every concept of my thesis, to Farzaneh Farah-Bakhs for her generous care and ultimate support, to Sepideh Khalili who has been a great friend and constant support since we were high school classmates, and to Neda Barjesteh, Mehdi Emam and Davood Rezaie for their genuine friendship which brightened my life at Guelph. Lastly, this thesis is dedicated to my father, Gholamreza, who taught me that nothing can be more joyful than learning for the sake of learning. I would like to thank him for inspiring in me the love to learn. It is also dedicated to my mother, Shayesteh, who taught me that even the most difficult task can become easy if I have faith and advance one step at a time. The thesis is also dedicated to my brother, Ardeshir, whose advice lightened my journey in life and his sense of humor made life all the more colorful. My family supported me in all my endeavors and crazy, self-centered decisions and pushed me time after time. Knowing that they will always be there to pick up the v pieces has given me the strength and confidence to repeatedly risk getting shattered. I am grateful and fortunate to have their constant support. vi TABLE OF CONTENTS Acknowledgements ............................................................................................................ iv Table of Contents .............................................................................................................. vii List of Tables ..................................................................................................................... xi List of Figures ................................................................................................................... xii 1. Chapter One Introduction .............................................................................................. 1 2. Chapter Two Litreature Review...................................................................................... 3 2.1 Dietary Fiber ..................................................................................................... 3 2.1.1 Fibers Health Benefits ............................................................................... 3 2.1.1.1 Role of Dietary Fiber in Obesity ........................................................ 4 2.1.1.2 Role of Fibers in Diabetes .................................................................. 5 2.1.1.3 Role of Fibers in Coronary Heart Diseases ........................................ 5 2.1.2 Structure of Dietary Fiber .......................................................................... 5 2.1.3 Examples of Dietary Fiber ......................................................................... 6 2.1.3.1 Cellulose ............................................................................................. 6 2.1.3.2 Arabinoxylan ...................................................................................... 7 2.1.3.3 β-Glucan ............................................................................................. 7 2.2 β-Glucan as a Dietary Fiber .............................................................................. 8 vii 2.2.1 β-Glucan Sources....................................................................................... 8 2.2.2 Extraction of β-Glucan .............................................................................. 9 2.2.2.1 Determination of β-Glucan Concentration ....................................... 10 2.2.2.2 β-Glucan Molecular Weight ............................................................. 11 2.2.3 β-Glucan Health Benefits ........................................................................ 11 2.3 β-Glucanase Activity .....................................................................................