EXTRACTION OPTIMIZATION AND IN-MATRIX STABILITY TESTING OF MEMBRANE-CONCENTRATED BETALAIN COLORANT FROM PRICKLY PEAR (OPUNTIA FICUS INDICA) A Thesis Presented to the Faculty of California State Polytechnic University, Pomona In Partial Fulfillment Of the Requirements for the Degree Master of Science In Human Nutrition & Food Science By Shannen N. Hilse 2018 SIGNATURE PAGE THESIS: EXTRACTION OPTIMIZATION AND IN-MATRIX STABILITY TESTING OF MEMBRANE-CONCENTRATED BETALAIN COLORANT FROM PRICKLY PEAR (OPUNTIA FICUS INDICA) AUTHOR: Shannen N. Hilse DATE SUBMITTED: Summer 2018 College of Agriculture Dr. Harmit Singh Thesis Committee Chair Human Nutrition & Food Science Dr. Jeremy T. Claisse Biological Sciences Stephen J. Lauro CEO; colorMaker, Inc. Anaheim, CA ii ACKNOWLEDGEMENTS First and foremost, I cannot express my gratitude and appreciation for my thesis advisor, Dr. Harmit Singh, Professor & Chair of the Human Nutrition & Food Science Department. Beyond his command of food chemistry and creativity in its applications, his support, compassion, and overall optimism during this project kept me going and I cannot thank him enough. To my committee members: Dr. Jeremy Claisse, who offered his statistical expertise in both the experimental design and data analysis of this project and Stephen Lauro, whose industrial input, support, and encouragement was pivotal to its completion. Thank you both for your willingness to be a member of my committee and for the time you have dedicated. Additionally, I would like to give thanks to Dr. Gabriel Davidov-Pardo, Associate Professor of the HNFS Department. Beyond his insight, enthusiasm, and genuine interest in this project, he served as a mentor for me during my time at Cal Poly Pomona and had a big hand in making the past two years such an invaluable experience. I would be remiss to not acknowledge those within the food science family that offered support in their own distinct ways. To my labmates Shirin Mal Ganji and Carol Pow Sang, and to my fellow graduate students Benjamin Steiner, Yuguang Zheng, Huiying Hu, and Franz Fernandez: your advice was always welcome, your experience was always helpful, and your friendship made the long days in lab just a little more entertaining. Also to my undergraduate researcher, Sarah Caballero, who offered a unique perspective, helped with data collection, and never once complained when asked to wash glassware. iii To my parents, Steve and Debra, who made me the person I am today and whose love and support never faltered. To my sister Taryn, who has offered wisdom and advice not just during this project, but throughout my entire adult life. To John, my beginning, and Ellie-Mae, my rock. To Christian, who has helped me in so many ways. And to my entire network of friends and family: your years of belief in me and my education got me here, and I am so grateful for all of you. And finally, to all the students, staff, and faculty at California State Polytechnic University, Pomona and those within the Human Nutrition and Food Science department – thank you for the most difficult, stressful, challenging, memorable, and rewarding years of my life. Research reported in this publication was supported by the MENTORES (Mentoring, Educating, Networking, and Thematic Opportunities for Research in Engineering and Science) project, funded by a Title V grant, Promoting Post-baccalaureate Opportunities for Hispanic Americans (PPOHA) | U.S. Department of Education, Washington, D.C. PR/Award Number: P031M140025. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Education. We would also like to thank Stephen Lauro at ColorMaker (Anaheim, CA) for funding and supporting this research. iv ABSTRACT Although they offer more stability and lower production costs, consumers perceive synthetic colorants as undesirable. Accordingly, companies are looking to natural color sources for their products. Prickly pears are cactus fruits that are rich in pigment molecules known as betalains. These water-soluble colorants are traditionally extracted from red table beets, but beet extract comes with complications – it imparts an undesirable taste and is high in nitrites, precursors to carcinogenic nitrosamines. In addition, sugar beets (as opposed to red table beets) are genetically modified to improve their yield of sugar. This further complicates the product development challenges facing companies looking to avoid artificial colors. Consequently, a commercial betalain source other than red table beets is desirable to industry. The purpose of this research was to increase the feasibility of a marketable betalain food colorant sourced from Opuntia ficus indica. This was achieved through three main objectives: (1) increased colorant yield with use of pectinase, cellulase, and hemicellulase enzymes to break down cell wall structures of the fruit solids, (2) the purification and concentration of the colorant with athermal crossflow membrane filtration, and (3) testing the resulting betalain colorant against comparable natural and artificial colorants on the market when used in a common food matrix. Interestingly, enzymatic treatments led to a decrease in betacyanin content the more crude enzyme was added. This could be explained by potential glucosidase activity of hydrolytic enzymes, leading to betalain degradation. Clarification with centrifugation and microfiltration (MF) caused transmittance to increase from the initial 4.1% to 14.33% post- centrifugation and an average 70.51% post-MF. Nanofiltration led to a 234.36% increase v in betacyanins. When used to color a common food matrix (gelatin dessert), both prickly pear and red beet saw rapid degradation at room temperature but showed significant stability under refrigerated conditions. This project resulted in a membrane-concentrated, natural betalain colorant sourced from prickly pear that can be used in refrigerated food items. vi TABLE OF CONTENTS Signature Page ................................................................................................................... ii Acknowledgements .......................................................................................................... iii Abstract .............................................................................................................................. v List of Tables .................................................................................................................... ix List of Figures ................................................................................................................... xi Chapter 1: Introduction ................................................................................................... 1 Relevance of the Topic ................................................................................................... 4 Specific Scope ................................................................................................................. 9 Chapter 2: Literature Review ........................................................................................ 11 Natural Colorants, Betalains ......................................................................................... 11 Phenolic Compounds .................................................................................................... 12 Red Beetroot & Alternatives ......................................................................................... 13 Enzyme Use for Pigment & Phenolic Extraction ......................................................... 15 Membrane Filtration ..................................................................................................... 18 Monitoring Physico-Chemical Properties of Prickly Pear Juice................................... 25 Stability & Model Food Matrices ................................................................................. 28 Chapter 3: Materials & Methods .................................................................................. 31 Objectives and Hypotheses ........................................................................................... 31 Flow Diagram ............................................................................................................... 32 Sample Preparation and Chemical Reagents ................................................................ 32 Methodology ................................................................................................................. 33 Physico-Chemical Parameters .................................................................................. 33 Enzymatic Pretreatments ........................................................................................... 36 Membrane Purification and Concentration via Micro- and Nano-filtration ............ 39 Stability of Prickly Pear Concentrate in a Model Food Matrix ................................ 42 Chapter 4: Results & Discussion ................................................................................... 45 Enzymatic Pretreatment ................................................................................................ 45 Varying Concentrations of Crude Enzymes............................................................... 45 vii Active Enzyme, Deactivated Enzyme, and a Blank Solution ..................................... 49 Membrane Purification and Concentration via Micro- and Nano-filtration ................. 55 Stability of Prickly Pear Concentrate