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The Optimization and Scale-Up of Pea Protein Extractions and Impact on Structural and Functional Properties

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The Optimization and Scale-Up of Pea Protein Extractions and Impact on Structural and Functional Properties The Optimization and Scale-Up of Pea Protein Extractions and Impact on Structural and Functional Properties A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Lucy Hansen IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Baraem (Pam) Ismail, PhD August 2020 © Lucy Hansen 2020 Acknowledgements First, I would like to thank my advisor, Dr. B. Pam Ismail, for her consistent support and encouragement throughout my education. Though I did not realize it at the time, the course of my life was changed when you accepted me to work in your lab as an undergraduate researcher. Since the very beginning, you have provided extensive guidance and opened numerous opportunities for me, enabling me to become the food scientist I am today, and I am truly grateful. I would also like to thank Dr. Gary Reineccius and Dr. Dan Gallaher for making time to serve on my committee and for the valuable insight I have gained from taking your courses during my time at the University of Minnesota. Thank you to Mitch Maher and Ray Miller in the pilot plant, along with Yara Benavides, for helping me with the scaled-up protein extractions. I would also like to recognize my funding sources, the USDA Pulse Crop Health Initiative and the Schwan’s Company, for making this research possible. Additionally, I want to thank my lab mates for making the lab feel like a second home. Thank you to Claire Boyle for being my point-person from the start and training me on many of the analytical methods used for this thesis. Your patience and positivity are deeply appreciated and something I strive to emulate. I also want to thank Chelsey Hinnenkamp and Rachel Mitacek for helping me navigate graduate school and brainstorming with me when I got stuck in my research, despite your own busy schedules. And thank you to everyone else in the Ismail Army for your friendship. I am fortunate to have coworkers that I can talk to about science during the day and watch The Bachelor with at night. I would also like to thank my loved ones, whom I would not be here without. To my mom, thank you for believing in me fully, sometimes more than I believed in myself. To my dad, thank you for keeping me smiling with your jokes and daily Cash photos. To my siblings, Leo, Louie, Josie, and Callie, thank you for energizing me with your zeal for life. To my best friend, Monica, thank you for all the stories. And to my boyfriend, Chris, thank you for being everything I needed you to be. I love you all. i Dedication This thesis is dedicated to my parents for supporting me every day of my life, even when they did not know what “food science” was. ii Abstract As the demand for plant proteins continues to grow, there is a need to develop alternative sources of protein other than soy protein, which is limited by being sourced from a GMO crop and a “Big Eight” allergen. Yellow field peas (Pisum sativum L. subsp. arvense) are similar to soybeans in their agricultural benefits and protein profiles but are non-GMO and of low allergenicity. While soy protein has undergone decades of research to optimize extraction conditions and evaluate structural and functional properties, pea protein is less researched. Currently, pea protein is only mass produced by alkaline solubilization with isoelectric precipitation, which may cause damage to the protein by altering its native structure, thus reducing protein functionality in food applications. Therefore, in order to make pea protein competitive with soy protein, there is a need to optimize both the conditions used for pea protein extractions, as well as the methods of extraction, to produce pea protein isolates (PPI) with high protein purity and preserved structural properties. Additionally, the scalability of optimized extraction methods must be evaluated to determine industrial feasibility. Therefore, the objectives of this study were: (1) optimize pea protein extraction conditions to maximize protein purity and yield following an alkaline solubilization with isoelectric precipitation and a salt solubilization coupled with membrane filtration; (2) characterize the impact of the two extraction methods on the protein structure and relate structure to functionality; (3) produce pea protein isolates on a pilot scale following the optimized benchtop extraction methods and evaluate the impact of a larger-scale production on protein structure and functionality. Extraction conditions including solubilization pH, isoelectric precipitation pH, solubilization duration, number of solubilizations, and use of dialysis were optimized for PPI production by alkaline solubilization coupled with isoelectric precipitation (pH-extraction), based on protein purity and yield as well as industrial feasibility. Similarly, purification conditions including use of ultrafiltration (UF) and dialysis, individually or combined, were optimized for PPI production by salt iii solubilization coupled with membrane filtration (salt-extraction). Optimized benchtop methods were then scaled-up to pilot plant production. The scaled-up (SU) protein extractions had some notable differences compared to the benchtop counterparts. Differences included overnight solubilization, use of diafiltration instead of dialysis, pasteurization, homogenization, and spray drying. The structural characteristics of the benchtop and SU pH- and salt- extracted PPIs were compared by determining the protein profile using SDS-PAGE, protein denaturation by DSC, surface charge by measuring zeta potential, and surface hydrophobicity as measured by a spectrophotometric method. Additionally, the functional properties of the PPIs were compared by measuring protein solubility, gelation, emulsification, and foaming properties. The optimized pH extraction conditions were double solubilization for one hour at pH 7.5, followed by isoelectric precipitation at pH 4.5, and dialysis of the neutralized extract. The optimized purification conditions for salt extraction were ultrafiltration followed by dialysis. The benchtop pH-PPI and salt-PPI had protein purity of 87.6% and 92.8%, and protein yield of 64.7% and 72.0%, respectively. The SU-pH and SU-salt PPIs had comparable protein purity, at 88.7% and 92.4%, while protein yields were not determined due to sampling throughout the protein extractions, as well as the recovery of only the high solids retentate from UF. Protein profiles were generally similar for all PPIs, except that the salt- extracted PPIs contained albumin proteins, while the pH-PPIs did not. Salt-PPIs, therefore, had a slightly higher isoelectric point than pH-PPIs, leading to lower surface charge at pH 7. Because of the presence of albumins, salt-PPI had slightly lower surface hydrophobicity than pH-PPI. Compared to benchtop PPIs, the protein in SU PPIs were partially denatured, had higher surface hydrophobicity, and were more aggregated. Differences in structural characteristics led to observed differences in functionality. Salt-PPIs had slightly lower protein solubility at pH 7, and comparable or higher solubility at pH 3.4. Though the benchtop salt-PPI had higher gel strength than the pH-PPI, the gel strength of the SU-salt PPI was comparable to that of the iv SU-pH PPI due to similar surface charges and levels of denaturation and aggregation. Emulsification capacity (EC) of the benchtop and SU PPIs was similar. Due to differences in relative amounts of globulins and albumins, the pH- PPIs had higher emulsion stability (ES), yet lower EAI, than the salt-PPIs. Similarly, higher albumin content in salt-PPIs potentially contributed to higher foaming capacity (FC) and foaming stability (FS) than pH-PPIs. In comparison to SU PPIs, commercially available SPI and PPI (cSPI and cPPI, respectively) were completely denatured and extensively aggregated. Compared to cSPI, the SU PPIs had superior solubility at pH 3.4. However, cSPI had superior gelation and emulsification properties. On the other hand, both SU- pH and SU-salt PPIs had superior functional properties, in general, compared to cPPI. Overall, this study demonstrated successful optimization and scalability of two pea protein extraction methods: alkaline solubilization with isoelectric precipitation and salt solubilization coupled with membrane filtration. Both optimized benchtop methods achieved high protein purity and yield, while using relatively nondenaturing conditions. Scaled-up extractions achieved similar protein purity to the benchtop counterparts. Further work investigating complete recovery of all fractions, while monitoring levels of protein denaturation, is needed to determine scaled-up extraction yields. The slight differences in structural and functional properties between benchtop and SU PPIs were mostly due to the thermal treatment the SU PPIs received that caused partial denaturation and aggregation. However, compared to cPPI, SU PPIs were less denatured, resulting in generally superior functionality that should be considered advantageous to industry. This study is significant in demonstrating that PPI, with superior functionality to commercially available PPI, can be produced on a large scale through both the traditional pH extraction and the novel salt extraction coupled with membrane filtration, under optimized conditions. v Table of Contents Acknowledgements .............................................................................................. i Dedication ............................................................................................................ ii Abstract ..............................................................................................................
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