STRUCTURAL AND FUNCTIONAL PROPERTIES OF HEMP SEED STORAGE PROTEINS By Comfort F. Ajibola A Thesis Submitted to the Faculty of Graduate Studies of the University of Manitoba In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Food and Human Nutritional Sciences Faculty of Agricultural and Food Sciences University of Manitoba Winnipeg, Manitoba Canada Copyright @ 2020 by Comfort Funmilayo Ajibola I ABSTRACT This study aimed to determine the structural and functional properties of hemp seed storage protein fractions (2S, 7S, and 11S) in comparison to hemp seed protein isolate (HPI). The protein content of 11S, 7S, 2S, and HPI were 91%, 90%, 83%, and 90% respectively. The 11S fraction was the predominant protein in hemp seed and accounted for 72% of the total seed protein. In vitro protein digestibility of the hemp seed proteins ranged from 72.54 to 88.28%. Hemp seed proteins were rich in glutamic acid and sulfur-containing amino acids while limiting in tryptophan and lysine. The SDS-PAGE profiles revealed that the 7S fraction consists of basic subunits (18 kDa to 20 kDa) and high MW polypeptides (47 kDa and 85 kDa). The 11S and HPI fractions had similar polypeptides bands that consist of the basic subunit (18 to 20 kDa), acidic subunit (30 to 40 kDa) and other high MW polypeptides. The 2S profile has seven polypeptides (15 kDa, 19, 80 and 125 kDa) with the 15 kDa in the highest proportion. The intrinsic fluorescence and near-UV data showed that the aromatic amino acids of the protein samples were more exposed to the polar environment. The far-UV data showed that the secondary structures of all the proteins were mostly dominated by b-sheet conformation at all the pH values. The 2S fraction was relatively soluble at all the pH values. The 11S fraction and HPI were more soluble at the acidic pH while 7S fraction was soluble at alkaline pH. The 2S and HPI exhibited good oil absorption capacity. The 7S fraction showed better gelling properties when compared to HPI and other fractions. The 2S fraction exhibited higher foaming capacity at all the pH values when compared to HPI and other fractions. The 2S, 7S, and 11S fractions formed better emulsions when compared to HPI emulsions. Overall, the hemp seed protein fractions differ from one another in terms of amino acid composition, polypeptide composition, and protein conformation while their functional properties are superior to those of HPI. II ACKNOWLEDGMENTS Glory, honor, and majesty be to Almighty God that gave me the grace, strength, and resources to start and finish this program. May His name be praised forever and ever. I am indeed grateful to my supervisor, Dr. R.E. Aluko, who gave me thorough supervision, necessary guidance, corrections and suggestions throughout this study. God will continue to bless and strengthen you. Thank you, sir. I will also like to acknowledge the contributions of my thesis committee members, Mr. Alphonsus Utioh and Dr. Rob Duncan, for taking the time to read and evaluate this thesis. My gratitude goes to Dr. Monisola Alashi and all the members of Dr. Aluko’s research group for their contributions and supports. You are all wonderful. My thanks go to my loving, caring, wonderful and handsome husband, Mr. Olayiwola Ajibola for always being there for me. I appreciate your contributions in terms of financial assistance, prayers, and sacrifices. God will continue to bless you, my dear. To my wonderful and loving children, Oluwanifemi, Oluwadarasimi, and Oluwafayokemi thank you for your support and sacrifices. You always put a smile on my face. I thank God for having you in my life. Thank you all and God bless you richly. III DEDICATION This work is dedicated to the God Almighty, the master of the universe. IV TABLE OF CONTENTS ABSTRACT....................................................................................................................................II ACKNOWLEDGMENTS ............................................................................................................III DEDICATION ..............................................................................................................................IV TABLE OF CONTENTS..............................................................................................................V LIST OF TABLES ......................................................................................................................VII LIST OF FIGURES ......................................................................................................................IX LIST OF ABBREVIATIONS.......................................................................................................XI CHAPTER ONE 1. INTRODUCTION ......................................................................................................................1 1.1 Hypothesis……………………………………………………………………………..........7 1.2 Objective……………………………………………………………. …………………….7 CHAPTER TWO………………………………………………………………………………….9 2. LITERATURE REVIEW............................................................................................................9 2.1 Industrial Hemp…………………………………………………….....................................9 2.2 Industrial Hemp seed: Uses and Composition…………………………………………….10 2.3 Nutritional and chemical properties of Hempseed protein………………………………..11 2.4 Structural conformation of protein………………………………………………………...14 2.4.1 Intrinsic Fluorescence……………………………………………………………….15 2.4.2 Extrinsic Fluorescence: Surface Hydrophobicity…………………………………...18 2.4.3 Circular dichroism…………………………………………………………………..19 2.4.4 Electrophoresis………………………………………………………………………21 2.5 Food proteins functionality………………………………………………………………..23 2.5.1 Protein solubility…………………………………………………………………....24 2.5.2 Water absorption capacity (WAC)………………………………………………….25 2.5.3 Oil absorption capacity (OAC)……………………………………………………..26 2.5.4 Gelation properties (least gelation concentration)………………………………….26 2.5.5 Foaming capacity…………………………………………………………………...27 2.5.6 Emulsifying activity and stability…………………………………………………..29 V CHAPTER THREE 3. MATERIALS AND METHODS .............................................................................................32 3.1 Materials..............................................................................................................................32 3.2 Methods .................................................................................................………………….32 3.2.1 Preparation of defatted hemp seed flour (DHF)…………………………………….32 3.2.2 Preparation of Hemp Seed Protein Isolates (HPI)…………………………………..32 3.2.3 Preparation of 11S, 7S, and 2S hemp protein fractions……………………………..33 3.2.4 Determination of Protein Content of HPI, 11S, 7S, and 2S…………………………33 3.2.5 Proximate composition analysis…………………………………………………….34 3.2.6 In vitro protein digestibility method………………………………………………..34 3.2.7 Amino acid composition analysis…………………………………………………..35 3.2.8 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)………..35 3.2.9 Total and exposed sulfhydryl contents……………………………………………...36 3.2.10 Total carbohydrate content………………………………………………………...37 3.2.11 Determination of Surface hydrophobicity (So)…………………………………….37 3.2.12 Intrinsic fluorescence emission…………………………………………………….38 3.2.13 Measurements of circular dichroism (CD) spectra………………………………..38 3.2.14 Determination of Functional Properties…………………………………………...39 3.2.14.1 Protein solubility (PS) of HPI, 2S, 7S, and 11S…………………………39 3.2.14.2 Water and oil holding capacities (WHC and OHC)……………………..39 3.2.14.3 Least gelation concentration…………………………………………….40 3.2.14.4 Foam capacity (FC)……………………………………………………..40 3.2.14.5 Emulsion formation and oil droplet size measurement………………....40 3.2.15 Statistical Analysis...................................................................................................42 VI CHAPTER FOUR 4. RESULTS AND DISCUSSION ..............................................................................................43 4.1 Proximate compositions of defatted flour (DHF), spent flour (SF), HPI, 7S, 11S and 2S…………………………………………………………………………………………….43 4.2 Percentage protein content as determined by Kjehdahl, Lowry and amino acid methods…………………………………………………………………………………..45 4.3 Percentage gross yield, protein yield and protein digestibility of HPI, 11S, 7S, and 2S………………………………………………………………………………………..45 4.4 Total and exposed Sulfhydryl (SH)……………………………………………………...49 4.5 Amino acids composition………………………………………………………………..49 4.6 Electrophoresis (SDS-PAGE)……………………………………………………………52 4.7 Surface hydrophobicity (So)……………………………………………………………..56 4.8 Intrinsic Fluorescence emission………………………………………………………….58 4.9 Secondary structure conformation……………………………………………………….61 4.10 The effect of pH on tertiary structure conformation……………………………………64 4.11 Protein solubility profiles of hemp seed proteins………………………………………67 4.12 Water and oil absorption capacity………………………………………………………69 4.13 Least Gelation Concentration…………………………………………………………..72 4.14 Foaming properties……………………………………………………………………..72 4.15 Emulsion formation and stability………………………………………………………76 CHAPTER FIVE CONCLUSION..............................................................................................................................84 SIGNIFICANCE OF RESEARCH ...............................................................................................87 FUTURE RESEARCH .................................................................................................................88 REFERENCES .............................................................................................................................89