Author ORCID Identifier

https://orcid.org/0000-0003-3426-2669

Date Available

2-8-2021

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture; Engineering

Department/School/Program

Biosystems and Agricultural Engineering

First Advisor

Dr. Akinbode Adedeji

Abstract

More than one-third of Americans today incorporate plant-based protein into their diet and about 40% believed that plant-based protein is healthier than animal protein, especially Millennials. The increasing global demand for plant-based proteins driven by the high cost of animal proteins, consumers’ desire for lean protein, vegetarianism, and the need for more sustainable green protein products have necessitated research into alternate emerging and underutilized sources of protein to complement or supplement the major plant protein in the market- soy, pea, and gluten. Therefore, this dissertation is focused on the valorization of the proteins in proso millet. Specifically, this work focused on the identification and structure-function characterization of the protein fractions in proso millet to include the determination of the three-dimensional structure of its glutelin fraction isoform (glutelin-type B 5-like protein) and finally, on the application of molecular dynamic modeling simulation to elucidate the effects of simulated processing stresses on the behavior of glutelin-type B 5-like protein at the molecular level. This dissertation is made up of eight chapters with four major objectives from chapter three through to chapter seven.

In objective one, four major proteins fractions from two cultivars (Dawn and Plateau) of proso millet flour (albumin, globulin, prolamin, and glutelin) were identified and characterized for their physicochemical properties and functionalities. Prolamin and glutelin were identified as the major protein in proso millet with respective percent composition of 47.2, 39.1 for Dawn cultivar and 50.8, and 34.5 for Plateau cultivar. The average denaturation temperature of 82.1±3.5°C requiring an average enthalpy of 0.1±0.06 J/g was reported for all fractions. Most of the protein fractions showed the highest solubility at pH 9 ranging from 5.7 to 100%; however, these protein fractions showed poor solubility at pH 7 and below (less than 40%). Emulsifying activity index of less than 25 m2/g was recorded for most fractions, while the highest emulsion stability index recorded was about 60 min.

In objective two, the effects of three levels ultrasound treatments (50%, 75%, and 100% amplitude and constant 20kHz for 5- and 10-min treatment times) were applied to the two major protein fraction from proso millet flour of Dawn and Plateau cultivars and selected functionality (solubility, emulsion, foam, thermal properties, and invitro-digestibility) of the two major fractions (prolamin and glutelin) were evaluated. It was observed that the ultrasound treatment (US) increased the solubility of the prolamin and the glutelin protein significantly (p < 0.05) for both cultivars. For instance, Dawn prolamin showed a protein solubility from 8.21±0.13% to 22.1±0.78%, 23.9±0.41%, 27.5±6.57%, 24.3±5.03%, 31.1±5.03% and 49.2±1.80% for 50, 75, 100% amplitude for 5 and 10 min, respectively. Additionally, the pepsin digestibility of both prolamin and glutelin also improved compared to their native protein. Dawn prolamin increased by about 30% for ultrasound treatment 100% amplitude for 10 min and Plateau glutelin pepsin digestibility increased by 48.6% for the same level of treatment. Foaming capacity was improved by the US at a higher treatment time of 10 min but there was inconsistency in the emulsion activity index.

Objective three presents the results on work carried out to determine the three-dimensional structures of glutelin-type B 5-like protein, a type of glutelin protein using comparative homology, and the attempt made at an empirical approach. The result showed that the structure of glutelin-type B 5-like is a trimer in its natural state with amino acid residue length of 256 and conserved regions (one jelly-like β-barrel and two extended helix domains) with the globulin proteins (11s or pro-11s globulin proteins) of pea, soybean, pumpkin, amaranth, and rapeseed and between 35 - 45% structural similarity with the globulins of these proteins. The attempt made at purifying the glutelin-type B 5-like for X-ray crystallography revealed that the protein was aggregating in solution. The obtained three-dimensional structure contains 4.6% alpha-helix, 2.5% 3/10 helix, 23.8% beta-sheet and 69.1% coils/turns/bends/bridges. The structure was deposited in the protein model database (PMDB) (http://srv00.recas.ba.infn.it/PMDB/main.php) with PMDB identifier PM0083241.

Finally, objective four and five reveal the effects of temperature levels (300, 350, and 400 K) combined with electric field levels (0, 0.1, 1 and 3 v/nm) as well as the effects of temperature levels (300, 350, and 400 K) combined with pressure levels (1b, 3kbar, and 6kbar) on the three-dimensional structure conformations of glutelin-type B 5-like in silico. The results showed that the root mean square deviations (RMSD) increased as the intensity of the stresses increased, except for increasing pressure where the RMSD decreased when the temperature was held constant. Moreover, we showed that the amino acid at the terminals of the protein fluctuates more with stresses and the alpha-helix fluctuates more than beta-sheet. While some losses of the amino residue that make up the secondary structure of the glutelin type-B 5-like protein were observed obvious disruption to this secondary structure were not noticed which may suggest that higher processing stress intensity and/or higher simulation time may be needed to cause a major and irreversible disruption of the protein secondary structure.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.354

Additional Files
Akharume_Felix_Dissertation_ Appendices.pdf (1265 kB)
Structural data of glutelin type-B 5-like protein
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