The Saponin Composition of Common Canadian Pulses By Beiyi Shen A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science In Food Science and Technology Department of Agricultural, Food and Nutritional Science University of Alberta © Beiyi Shen, 2020 Abstract Pulses are high in nutritional value but are underutilized as foods due to their undesirable flavor attributes. These include bitterness, which may in part due to their saponin content. In this research, a simple and rapid method using high-performance liquid chromatography-mass spectrometry (HPLC-MS) and requiring the minimum sample preparation was developed for the identification and quantification of saponins. This was applied to 8 common Canadian pulses including 3 pea varieties, 4 faba bean varieties, pinto beans, black bean, kidney beans, chickpeas, and green and red lentils. The method was validated according to linearity, accuracy, detection limit, quantification limit, inter- and intra-day precision. The calibration curve for a soyasaponin Bb standard with added internal standard (ginsenoside Rb1) showed correlation coefficients of 0.994 and a linear range of 0.2-5 g/mL. Saponin recoveries from the extraction method applied to faba bean samples ranged from 95 -105%, with n= 6 and RSD <12%. Saponin composition and content varies depending on the pulse type and variety. Four types of group B saponins including DDMP-conjugated soyasaponin g and g, and non- DDMP- conjugated soyasaponin Bb and Ba were identified in several pulse samples by HPLC-MS according to their relative retention times, and their molecular and fragment ions, as compared to standards and literature. All of the pulses tested contained soyasaponin Bb and g with different amounts and percentile distribution. Amongst all of the 8 types of pulses, the total saponin content ranged from 30 to 8566 g/g, where the lowest saponin content was found in faba bean variety Fabelle and the highest saponin content was found in black beans. Within the 3 pea varieties, the total saponin content varied from 550 to 2144 g/g, and for the 4 faba bean varieties tested it varied between 30 and 388 g/g. In all pulse samples, either soyasaponin Bb or g was the predominant ii saponin type. Soyasaponin Ba and g was only present in small amounts, except for black beans and pinto beans where 3145 and 1306 g/g of g was found, respectively. The effects of pulse processing methods (sprouting, drying, baking and pressure cooking) on the saponin profile was investigated. Four cultivars of faba beans grown in Alberta, Canada were germinated and subjected to a range of sprouting times (0, 48, 54, 60, and 72 h and drying times (0, 24, 36, 48, and 60 h). The saponin profiles of raw and sprouted faba bean seeds were measured, along with those found in baked faba bean flour-based crackers. Soyasaponin Bb and soyasaponin βg were the only two types of saponin found in the faba bean varieties studied. Soyasaponin Bb reaches the highest abundance after 54 h of sprouting in most cultivars, whereas the highest for βg was observed at 60 h in most cases, except for FB9-4 (54 h). A significant reduction of soyasaponin Bb was observed after 24 h of drying the sprouted seeds at 60C. The total saponin content after 72 h of sprouting significantly increased in Snowdrop and Fabelle, decreased in FB9-4 and no change in Snowbird compared to unsprouted seeds. Prolonged drying times of up to 60 h significantly reduced the soyasaponin Bb content of 48 hours sprouted faba beans, whereas a slight increase was observed in the soyasaponin βg content in Snowdrop and Fabelle. Regardless of the sprouting condition, both baking and cooking of faba bean flour led to significant reductions in both total saponin and individual saponin content. In addition, the combination of germination and pressure cooking is more effective in reducing saponin content in faba bean comparing to pressure cooking alone. The present research into the saponin profile of pulses and how this is changed by common food processing methods, contributes fundamental knowledge which may be beneficial in the utilization of pulses and pulse flour in food. iii Preface This thesis is an original work by Beiyi Shen under the supervision of Dr. Jonathan Curtis. There are a total of five chapters in this thesis, where Chapter 1 is literature review provides an overall introduction on topics related to this research and the hypothesis and objective of this study; Chapter 2 developed and validated an analytical method for quantification of saponin from faba bean samples by using HPLC-MS; Chapter 3 investigated the effects of sprouting, drying and baking on the saponin composition and content of faba bean samples; Chapter 4 conducted a survey on the saponin profile of 8 common Canadian pulses and the effects of pressure-cooking on saponin content in those pulse samples; and Chapter 5 is the overall summary of the key findings and recommendations for future study. In Chapter 2 of this thesis, the optimization of HPLC-MS was achieved with the assistance of Dr. Yuan Yuan Zhao. In Chapter 3 of this thesis, the preparation of sprouted faba bean seeds and baked cracker were prepared at Food Processing Development Centre (Leduc, AB, CA). Chapter 3 of this thesis was part of Sprouted Faba Bean Project-820039 (2017F108R), sponsored by Agriculture Funding Consortium- Alberta Pulse Growers Commission (AFC-APGC). In Chapter 4 of this thesis, the sensory test was conducted and studied at Food Processing Development Centre (Leduc, AB, CA). The performance of all the other experiments, data analysis and literature review were Beiyi Shen’s original work. Manuscripts based on Chapter 2, 3 and 4 are in the preparation for submission to Food Research International and Journal of Food Composition and Analysis for consideration of publication. iv Dedication To my parents and grandfather For your support, love and guidance v Acknowledgements I would especially like to thank my supervisor, Dr. Jonathan M. Curtis for the great opportunity and learning experience working as a graduate student in his Lipid Chemistry Group team at the University of Alberta. Thank you greatly for his unstinting guidance, support, encouragement and patience throughout the time it has taken me to complete this project. I would also like to thank my mentor Dr. Rami Akkad and lab technician Dr. Yuan Yuan Zhao for their knowledge, support, and valuable advice on my research projects for the past two years. I would also like to thank Dr. Wendy Wismer who is my committee member for her generous support, knowledge, and great suggestions to my research at committee meetings. Besides, she also supervised my review paper titled: The Application of Gas Chromatography-Olfactometry in Legume Aroma Analysis. During the preparation of that review paper, she shared her experience in sensory science and gave me great advice and feedback. In addition, I appreciate Dr. Thava Vasanthan being my external examining committee member and chair for my final oral exam. I greatly appreciate all of the team members of the Lipid Chemistry Group. I would like to thank Mr. Ereddad Kharraz for the great help in the lab. All the happy and fun times we had together will always be precious memories for me. Thank my lab mates and friends Magdalena Hubmann, Nuanyi Liang, Vinay Patel and Savanna Won for standing by my side and cheering me up when I was under stress. I am also grateful for the funding sponsored by Agriculture Funding Consortium- Alberta Pulse Growers Commission (AFC-APGC). Lastly, I would like to thank my family and friends for their endless emotional support and love, which helped me throughout some difficult times. vi Table of Contents Chapter 1 Literature review .....................................................................................................1 1.1 Canadian grown pulses..................................................................................................... 1 1.1.1 Beans ............................................................................................................................. 4 1.1.1.1 Faba bean (Vicia faba var. minor) ............................................................................... 4 1.1.1.1.1 Production, consumption and significance .............................................................. 4 1.1.1.1.2 Plant structure and characteristics ........................................................................... 5 1.1.1.1.3 Major genotypes grown in Canada ......................................................................... 6 1.1.1.1.4 Nutritional composition and properties ................................................................... 7 1.1.1.2 Pinto beans ............................................................................................................... 11 1.1.1.3 Black beans .............................................................................................................. 12 1.1.1.4 Kidney beans ........................................................................................................... 12 1.1.2 Peas.............................................................................................................................. 13 1.1.2.1 Chickpeas ................................................................................................................ 13 1.1.2.2 Green and yellow peas ............................................................................................. 16 1.1.3 Lentils .........................................................................................................................