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Title:Whey and soy amyloid fibrils as a novel iron delivery nanosystem
Author(s):Lopez Barrera, Emely Cristina
Director of Research:Helferich, William G
Doctoral Committee Chair(s):Engeseth, Nicki J
Doctoral Committee Member(s):Andrade, Juan E; Lee, Youngsoo
Department / Program:Food Science & Human Nutrition
Discipline:Food Science & Human Nutrition
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Amyloid
Fibrils
Iron
Fortification
Chelation
Reduction
Abstract:Iron deficiency anemia (IDA) is the most common micronutrient deficiency disorder in the World; it afflicts half of the 2.2. billion total anemic population. The most common cause is insufficient iron consumption through the diet. In a context where common iron fortificants have had limited success, the exploration of functional and novel ingredients to deliver iron in an inexpensive, safe, effective, and culturally accepted manner is a critical need. Recently, food-based biomimetic nanomaterials, such as amyloid fibrils (AFs), have acquired much attention due to their rheological and metal-binding functionality. From food proteins, these structures have been mainly produced from ultra-specific protein fractions and subunits such as β-lactoglobulin, glycinin, lysozyme, and β-conglycinin (β, α, and α’), which makes their synthesis a time-consuming and cost-prohibitive enterprise. Limited information exists, however, on the production and functional properties of AFs originated from non-fractionated proteins such as whey and soy protein isolates. Furthermore, the most utilized protocol to manufacture AFs (i.e., random self-assembly by magnetic stirring) is limited in that it does not provide controlled shear, replicability, and scalability. Therefore, the objective of this research was to identify the optimal shear rate and time combination to create AFs from common protein sources such as whey and soy protein isolates, and to further determine their influence on iron speciation, chelation, and dialyzability. First, to determine the optimal shear-time combination and, a study was conducted utilizing whey protein solutions (2%, pH 2), which were heated at 90˚C and stirred at different shear rates (0, 325, and 650 s-1) from 0 to 20 h. Aggregation kinetics were analyzed with Thioflavin T (ThT). A two-phase in vitro digestion was performed to evaluate the stability of β-structure, which is characteristic of AFs, upon exposure to digestive enzymes. It was found that a controlled shear rate significantly increases the aggregation of protein monomers into amyloid aggregates. AFs were completely digested after exposure to gastric + intestinal digestive enzymes. Optimal shear and time combination was found at 325 s-1 for 10 h, a point where fibrils were long and mature strands. To determine the functionality of AFs, the aforementioned optimal conditions were used to self-assemble whey and soy protein AFs (W-AF and S-AF). Morphological, chemical, and functional characterization was conducted using the Thioflavin T, TEM, SDS-PAGE, EDX-ESEM, FerrozineTM chromogenic test, and in vitro digestion coupled to an iron dialyzability test. ThT assay revealed that both proteins form AFs. Original protein monomers (70-100 nm thickness) converted into nanofibrils (<10 nm thickness and micrometer-sized lengths). W-AF and S-AF reduce iron in an acidic environment (pH 2) over time. The highest recorded ferrous iron yield was 1.32 and 0.93 µg Fe2+/mg protein for S-AF and W-AF, respectively after 20 h of iron-reduction reaction. Native protein solutions exhibited a much lower iron-reducing capacity of <0.1 µg Fe2+/mg protein than AFs. Iron chelating ability of W-AF and S-AFs was 1.31 and 5.02 µM EDTA equivalents, respectively, and was significantly higher compared to non-treated protein (p<0.05). Adding W-AF or S-AF to FeCl3 increased iron dialyzability compared to a control with no AFs. Iron dialyzability (6-8 kDa cutoff) was significantly higher for whey (25.98%) and soy (21.82%) AFs compared to ferric iron alone (5.70%). Furthermore, a proportion of the iron crossing the dialysis membrane does so in a Fe2+ state. This article represents a novel set of data to elucidate the extensive interest in the iron-functionality of AFs. Additionally, we prove that these nanomaterials can be synthesized from inexpensive food sources such as whey and soy protein isolates, which could be the first step in scaling up the production of the next generation of functional nanomaterials for biomedical, food, and nutritional applications.
Issue Date:2020-12-01
Type:Thesis
URI:http://hdl.handle.net/2142/109606
Rights Information:All the material included in this dissertation is the intellectual property of Emely Lopez Barrera and is Copyright of the University of Illinois at Urbana Champaign © 2020. Download and use of this material are restricted to individuals for educational and non-commercial purposes only. Any commercial use of the materials in this dissertation, in part or in whole, requires written permission from the author and/or the copyright holder. Please report any unauthorized use to helferic@illinois.edu or eclb@illinois.edu
Date Available in IDEALS:2021-03-05
Date Deposited:2020-12


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