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Functional Properties of Triticale Bran Bioactives Using in Vitro and in Vivo

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Functional Properties of Triticale Bran Bioactives Using in Vitro and in Vivo Functional properties of triticale bran bioactives using in vitro and in vivo model systems by Rania Agil A thesis submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry (Food Science and Nutrition) Carleton University Ottawa, Ontario © 2016, Rania Agil Abstract Part of the solution to reducing the prevalence of chronic diseases is the employment of food as medicine. Whole grain consumption has been associated with a variety of health benefits owing to their bioactive constituents, namely fibre and phytochemicals. The overall aim of this study was to discover the underlying mechanisms behind the positive physiological effects of whole grains and further elucidate their nutritional value. In particular, the bioactive properties of alkylresorcinols (ARs), phenolic lipids present almost exclusively in cereal grains, and soluble dietary fibre, potential prebiotics, were investigated using triticale bran (TB), an underestimated cereal crop. Specific objectives were met through the following projects. Conditions for large-scale extraction of ARs from cereal grains were optimized using response surface methodology (RSM). Isolation of ARs for 16-24 h at a solid-to-solvent ratio of 1:40 (weight per volume) and temperature of 24 °C produced the highest yield with the least amount of co-extractives/unknowns. The antioxidant and anti-inflammatory activities of ARs were evidenced by in vitro model systems, ORAC, DPPH, and COX assay whereby results were in agreement with ex vivo model systems, RAW 264.7 macrophage cell cultures induced by AAPH or LPS. Subsequently, the in vivo effects of ARs were evaluated and oxidative stress markers in liver and heart tissues of mice supplemented 0.5% ARs and mice fed 10% TB demonstrated antioxidant protection. Additionally, water extractable polysaccharides (WEP) from cereal samples were isolated by varying treatments; the highest yield with the least amount of impurities was achieved by boiling water extraction followed by successive enzyme treatments, dialysis and successive ethanol fractionations. Using yogurt as fermentation model, probiotic yogurt in the presence of TB produced significantly higher TTA values, lower pH levels, and improved microbial viability compared to controls. In addition to the prebiotic potential of TB, likely owing to its high fibre content, isolates of WEP also demonstrated antioxidant activity as measured by ORAC assay. ii Findings of this study will contribute to a better understanding of the underlying mechanisms behind the health benefitting properties of whole grains. iii Acknowledgements First and foremost, I would like to thank my academic supervisor Dr. Farah Hosseinian for this incredible and life changing opportunity that has helped me learn and grow as a researcher, student, teacher, team player, and as an individual overall. I am very fortunate to have a supervisor like Dr. Hosseinian who encouraged me to work hard and push forth during trying times. She gave me confidence in my abilities, guidance, and shared all of her knowledge and wisdom while still challenging me to push myself beyond my perceived limits. Additionally, I am very grateful to Dr. Dave B. Oomah from Agriculture and Agri-Food Canada (AAFC) in Summerland, BC, for taking a chance on me as an intern in his lab and for introducing me to Dr. Farah Hosseinian. The valuable research experience I acquired in Dr. Oomah’s lab opened a series of windows of opportunity that helped me strengthen my CV and lead me to where I am today. I feel very fortunate to have had the opportunity to collaborate with such exceptionally talented, kind, and knowledgeable faculty and scientists, Dr. Alfonso Abizaid from the Department of Neuroscience and Dr. William B. Willmore from the Department of Biology at Carleton University (CU). Other scientists I have been fortunate enough to collaborate with include Dr. Judith Fregeau-Reid from AAFC Ottawa, Dr. Tyler Avis from the Department of Chemistry at CU and Dr. Koushik Seetharaman at the University of Guelph. I have also learned a great deal in my graduate level courses from Dr. Veronic Bezaire and Dr. Apollinaire Tsopmo who have taught me a great deal as teachers and mentors, providing guidance and wisdom that has helped prepare me for the next steps in my career. For initiating the Food Science and Nutrition program at Carleton University and maintaining its success, thank you Drs. Robert Burke and Robert Crutchley, I inspire to be a successful educator and leader like you. To the amazing and hard-working lab co-ordinators, Mastaneh Azad, Shari Levac, and Elena Munteanu, thank you for training me to be an excellent teaching assistant by being kind and patient iv with students. To the wonderful administrative and technical staff, Peter Mosher, Marilyn Stock, Chantelle Gravelle, Ann Anderson, and Jim Logan, thank you for never failing to respond to and resolve promptly my urgent inquiries. Thank you to the following students for their assistance and contribution to my research: Harry Mackay, Zackary R. Patterson, Mengle Wang, Yiran Zhang, Julia Gliwa and my dearest friend/ colleague, Aynur Gunenc. To my family, thank you for providing me with the foundation of support I needed to be able to progress in this journey and build towards my career in science. I will always be grateful to my Mom who may as well earn a doctoral degree for all of the trying times and sleepless nights she has endured with me along the way. For supporting my decisions, encouraging me to further my education, for always knowing the right words to say during trying times, for being so understanding, and for never leaving my side, I thank you Mom. I would like to conclude my acknowledgements by dedicating this thesis to my mother; I could not have done this without her. v List of Publications This thesis is based on the following original publications: 1. Agil R, Oomah DB, Mazza G, and Hosseinian F, Optimization of alkylresorcinols extraction from triticale bran using response surface methodology. Food and Bioprocessing Technology. 5:2655-2664 (2012). 2. Agil R and Hosseinian F, Dual functionality of triticale as a novel dietary source of prebiotics with antioxidant activity in fermented dairy products. Plant Foods for Human Nutrition. 67: 88-93 (2012). 3. Agil R, Hosseinian F, Determination of water-extractable polysaccharides in triticale bran. Journal of Food Composition and Analysis. 34: 12-17 (2014). 4. Agil R, Patterson ZR, Mackay H, Abizaid A, and Hosseinian F, Triticale bran alkylresorcinols enhance resistance to oxidative stress in mice fed a high-fat diet. Foods. 5(1): 5 (2016). 5. Agil R, Gliwa, J, Willmore WG, and Hosseinian F, Protective effects of triticale bran alkylresorcinols against inflammation and oxidative stress measured by in vitro and ex vivo model systems. Manuscript submitted to Journal of Functional Foods vi Table of Contents Abstract ................................................................................................................................... ii Acknowledgements ............................................................................................................... iv List of Publications ............................................................................................................... vi Table of Contents ................................................................................................................. vii List of Tables ....................................................................................................................... xiv List of Abbreviations ........................................................................................................ xxiii Chapter 1 Literature Review ........................................................................................ 26 1.1 Background ...................................................................................................................... 26 1.2 Wholegrain Structure and Nutrition ............................................................................. 26 1.3 Triticale ............................................................................................................................ 28 1.3.1 History.................................................................................................... 28 1.3.2 Varieties and Production ........................................................................ 29 1.3.3 Applications ........................................................................................... 30 1.4 Alkylresorcinols ............................................................................................................... 32 1.4.1 Sources and Structure ............................................................................ 32 1.4.2 Biosynthesis ........................................................................................... 35 1.4.3 Alkylresorcinols as biomarkers of wholegrain consumption ................. 38 1.4.4 Pharmacokinetics ................................................................................... 38 1.4.4.1 Digestion and Absorption .......................................................................... 38 1.4.4.2 Metabolism and Elimination ...................................................................... 41 1.4.5 Health Benefits....................................................................................... 43 vii 1.4.5.1 Antioxidant Activity
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