Evaluation of Breadfruit (Artocarpus altilis and A. altilis X A. mariannensis) as a Dietary Protein Source by Ying Liu B.Sc. (Hons.), Dalhousie University, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The College of Graduate Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan Campus) April 2016 © Ying Liu, 2016 The undersigned certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis entitled: Evaluation of Breadfruit (Artocarpus altilis and A. altilis x A. mariannensis) as a Dietary Protein Source Submitted by Ying Liu in partial fulfillment of the requirements of The degree of Doctor of Philosophy . Dr. Susan Murch, Chemistry Supervisor, Professor (please print name and faculty/school above the line) Dr. Deanna Gibson, Biology Supervisory Committee Member, Professor (please print name and faculty/school in the line above) Dr. Paula Brown, Biology Supervisory Committee Member, Professor (please print name and faculty/school in the line above) Dr. Ian Walker, Biology University Examiner, Professor (please print name and faculty/school in the line above) Dr. Nyree Zerega, Northwestern University External Examiner, Professor (please print name and university in the line above) April 20, 2016 (Date submitted to Grad Studies) Additional Committee Members include: Dr. Fred Menard, Chemistry Please print name and faculty/school in the line above Dr. Miranda Hart, Biology Please print name and faculty/school in the line above Abstract Protein malnutrition is a leading cause of child mortality and chronic development disorders in the tropics. This study evaluated the protein quality of breadfruit using three standards measures: (1) yield of fruit and production of protein, (2) the protein content and amino acid profile, and (3) the protein digestibility/amino acid availability. I analyzed field data collected from 2006 to 2012 from 24 breadfruit cultivars in National Tropical Botanical Garden in Kauai, Hawaii. Based on a plant density of 100 trees/ha, breadfruit can yield 10-14 tons of fruit/ha. The commercial cultivar Ma’afala can produce up to 0.52 t/ha of protein. This is higher than wheat, rice, cassava, and potato. To determine the quality of breadfruit protein, I conducted an amino acid analysis of 49 breadfruit cultivars using high performance liquid chromatography with fluorescence detection and determined that the full spectrum of the essential amino acids were present in all cultivars. Breadfruit is especially rich in phenylalanine, leucine, isoleucine, and valine. Ma’afala contained a higher total essential amino acid content than many other staples including wheat, corn, rice, soybean, and yellow pea. To determine breadfruit protein digestibility, I developed a human digestion model, and found that Ma’afala protein was 10-25% more digestible than wheat protein. Ma’afala and wheat protein were compared using a human intestinal epithelial cell model. The results showed that Ma’afala induced similar impact on cell morphology and cytokine production in the model. To determine the overall effect on health, I compared breadfruit and wheat diet in a standard rodent model. The results show that the breadfruit diet did not induce any adverse effects on the overall health or growth of the mice. Breadfruit-fed mice gained more weight iii overall than wheat-fed mice but the crumbly nature of the diet made accurate food intake measures impossible. Chemical and histological analysis did not reveal any significant difference between the two diet groups suggesting that breadfruit is not toxic. Together, these data demonstrate that breadfruit has potential as a staple protein source for reducing malnutrition in tropical countries and as an ingredient in nutritional products, in particular gluten-free food products, for world markets. iv Preface A version of the thesis has been prepared for a GRAS application to the U.S. Food and Drug Administration for the regulation of breadfruit as a safe food ingredient. Ying Liu and Susan Murch (2015). Breadfruit (Artocarpus altilis and Artocarpus altilis x Artocarpus mariannensis) Generally Recognized As Safe (GRAS) notice. USDA. Some of the information in Chapter 1 has been published in the review article: Christina Turi, Ying Liu, Diane Ragone, and Susan Murch. (2014). Breadfruit (Artocarpus spp.): A traditional crop with the potential to prevent hunger and mitigate diabetes in the Tropics. Trends in Food Science and Technology, 45, 267-272. I critically reviewed the literature, and shared this information with the author groups. I also summarized and wrote some significant sections for the paper. A version of Chapter 2 has been published. Ying Liu, Maxwell P. Jones, Diane Ragone, and Susan Murch. (2014). Crop productivity, yield and seasonality of breadfruit (Artocarpus spp., Moraceae). Fruits 69,345-361. I conducted the data analysis, and wrote most of the original draft of the paper. A version of Chapter 3 has been published. Ying Liu, Diane Ragone, and Susan Murch. (2015). Breadfruit (Artocarpus altilis): A source of high quality protein for food security and novel food products. Amino Acids, Online, DOI 10.1007/s00726-015-1914-4. v I modified and developed the methods, conducted the majority of the experiment and analyzed all of the data. I also critically reviewed the literature, summarized and graphed all of the data and wrote the original draft of the paper. A version of Chapter 4 is being prepared for publication. A version of Chapter 5 is being prepared for publication. Presentations: Data from Chapter 2, 3, and 4 were presented at: Liu Y., Ragone D., Gibson D., Murch S. Breadfruit: a source of high quality protein. Annual Biology Graduate Symposium. University of British Columbia, Kelowna, BC. September 11th 2015. Liu Y. and Murch S. Breadfruit: Breadfruit (Artocarpus altilis, Parkinson (Fos)) for Food Security and Novel Food Products. Botany 2015, Edmonton, AB, Canada, July 25th – 29th 2015. Liu Y. and Murch S. Love it or hate it, you gotta eat it. Three Minutes Thesis, University of British Columbia, Kelowna, February 26th 2014 vi Table of Contents Abstract ................................................................................................................... iii Preface ...................................................................................................................... v Table of Contents ................................................................................................... vii List of Tables .......................................................................................................... xii List of Figures ........................................................................................................ xv List of Symbols and Abbreviations ..................................................................... xix Acknowledgements .............................................................................................. xxi Dedication ........................................................................................................... xxiii Chapter 1. Introduction ......................................................................................... 1 1.1. History of breadfruit consumption ......................................................... 1 1.2. Current uses and significance of breadfruit in the world ....................... 4 1.3. Breadfruit botanical description ............................................................. 5 1.4. Current breadfruit nutritional analysis ................................................... 6 1.4.1. Carbohydrates .................................................................................... 14 1.4.2. Carotenoids ........................................................................................ 16 1.4.3. Minerals .............................................................................................. 18 1.4.4. Vitamins .............................................................................................. 21 1.4.5. Anti-nutrients ...................................................................................... 21 1.5. Study rationale and objectives ............................................................ 24 Chapter 2. Seasonality, Productivity, and Yield of Breadfruit ......................... 25 2.1. Synopsis ............................................................................................. 25 2.1.1. Objectives ........................................................................................... 26 2.2. Materials and methods ....................................................................... 26 vii 2.2.1. Germplasm repository ......................................................................... 26 2.2.2. Data collection ..................................................................................... 27 2.3. Results ................................................................................................ 29 2.3.1. Time from planting to fruit production .................................................. 29 2.3.2. Breadfruit seasonality .......................................................................... 33 2.3.3. Breadfruit productivity/fruit number ...................................................... 41 2.3.4. Breadfruit fruit weight........................................................................... 44 2.3.5. Breadfruit yield ...................................................................................