Characterizations of Zein: Evaluating Functionality and Potential uses in Food Structuring by Kristin Danielle Mattice A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Food Science Guelph, Ontario, Canada © Kristin Danielle Mattice, January, 2020 i ABSTRACT CHARACTERIZATIONS OF ZEIN: EVALUATING FUNCTIONALITY AND POTENTIAL USES IN FOOD STRUCTURING Kristin Danielle Mattice Advisor: University of Guelph, 2020 Professor Alejandro G. Marangoni This thesis encompasses a thorough investigation in the potential use of zein networks as a structuring ingredient in plant-based meat and dairy alternatives. The popularity of plant-based products is continually increasing, however the majority of meat analogues and plant-based cheeses lack properties that are characteristic to the conventional products. Attempts to improve upon these products focused on the use of zein, a protein that had not been studied in this application previously. For the creation of whole muscle meat analogues, three techniques were developed to produce fibrous material from zein, either as fibrous networks or individual protein fibres. These techniques are: electrospinning, antisolvent precipitation of zein from ethanol using water and mechanical elongation of self-assembled zein networks. It was determined that the zein fibres produced were able to contribute a chicken-like texture to soy protein isolate gels when fibres were of uniform size and orientation. Despite this promising result, in depth investigation into the rheology of zein networks revealed inherently brittle behaviour, particularly when compared to the ductility of chicken muscle tissue. Efforts to reduce the brittleness of zein networks through enzymatic crosslinking with microbial transglutaminase proved unsuccessful and instead caused the brittleness of the network to increase even further. As an alternate technique, an increase in ductility was observed when zein networks were formed ii through antisolvent precipitation from acetic acid. Even further increases in ductility were achieved through antisolvent precipitation from ethanol. Throughout analysis, observations of softening and melting with increasing temperature were made. This led to the additional application of plant-based cheeses made with zein. Plant-based cheese samples containing a sufficient content of zein were shown to soften and stretch with increasing temperature in a statistically similar manner as conventional cheddar cheese. This result was emphasized further when currently available plant-based cheddar-style products were unable to stretch or perform with any similarities to conventional cheese. This research has therefore provided an increased understanding of the potential structural and rheological contribution of zein in food products. In addition, it provides the basis for the development and production of highly functional zein based meat analogues and plant-based cheeses. iii ACKNOWLEDGEMENTS As my time spent as a graduate student at the University of Guelph comes to an end, I need to thank all of the people who have helped me get to this point in my academic career. To Dr. Marangoni, thank you for all of your support over the past few years. I am extremely thankful to have been given the opportunity to study in an area that I truly find fascinating. It is because of your guidance that I was able to accomplish what I previously considered to be impossible goals. I will forever value my time spent as a researcher in your lab, learning, improving and succeeding as a team. To my advisory committee members, Dr. Mario Martinez, Dr. Loong-Tak Lim, and Dr. Paul Spagnuolo, thank you for providing such thoughtful insight and support for the duration of my PhD studies. I am very appreciative of all your constructive feedback and encouragement, all of which greatly enriched my research and thesis. To all of the members of the Marangoni lab, thank you for creating such a positive work environment. I am lucky to have worked with such genuinely welcoming and collaborative people. I would also like to thank Andrew and Saeed for always keeping the lab so organized, and Preethi for all of the help in running my many electrophoresis gels. To my parents, thank you for your endless love and support. Your unwavering encouragement during my degree fuelled me through even the toughest times. To Greg, Kyulim and the many voices of Finnigan, thank you for always making me laugh during the long days of writing. Finally, to Ryan, thank you for loving me unconditionally, for believing in me and for supporting all that I do. Having you by my side makes me feel like I can accomplish anything. And this just may be the proof. iv TABLE OF CONTENTS ABSTRACT ............................................................................................................................ i ACKNOWLEDGEMENTS ................................................................................................... iv TABLE OF CONTENTS .........................................................................................................v LIST OF TABLES ..................................................................................................................x LIST OF FIGURES .............................................................................................................. xii LIST OF ABBREVIATIONS AND SYMBOLS.................................................................... xv Chapter 1: Introduction ............................................................................................................1 1.1 Objectives .................................................................................................................................. 3 1.2 Hypotheses ................................................................................................................................ 4 1.3 Organization of this Dissertation ................................................................................................ 4 1.3 References ................................................................................................................................. 5 Chapter 2: Comparing Methods to Produce Fibrous Material from Zein ..................................7 2.1 Abstract ..................................................................................................................................... 7 2.2 Introduction ............................................................................................................................... 8 2.3 Materials and Methods ............................................................................................................. 10 2.3.1 Materials .......................................................................................................................... 10 2.3.2 Zein Fibre Preparation ..................................................................................................... 10 2.3.2.1 Electrospinning ............................................................................................................. 10 2.3.2.2 Antisolvent Precipitation ............................................................................................... 11 2.3.2.3 Mechanical Elongation .................................................................................................. 11 2.3.3 Polarized Light Microscopy .............................................................................................. 11 2.3.4 Scanning Electron Microscopy ......................................................................................... 12 2.3.5 Soy-Zein Composite Gel Preparation ............................................................................... 12 2.3.6 Texture Profile Analysis ................................................................................................... 13 v 2.3.7 Moisture Content .............................................................................................................. 13 2.3.8 Water Holding Capacity ................................................................................................... 14 2.3.9 Statistical Analysis ........................................................................................................... 14 2.4 Results and Discussion ............................................................................................................. 14 2.4.1 Structure .......................................................................................................................... 14 2.4.2 Texture Profile Analysis ................................................................................................... 18 2.4.3 Moisture Content and Water Holding Capacity ................................................................ 21 2.5 Conclusions ............................................................................................................................. 23 2.6 Acknowledgements .................................................................................................................. 23 2.6 References ............................................................................................................................... 24 Chapter 3: Evaluating the Use of Zein in Structuring Plant-Based Products ........................... 28 3.1 Abstract ..................................................................................................................................