Date of Award

8-2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Food Science

Committee Chair

Jozef L. Kokini

Committee Member 1

Lia Stanciu

Committee Member 2

Yuan Yao

Committee Member 3

Kee-Hong Kim

Committee Member 4

Owen G. Jones

Committee Member 5

Arun K. Bhunia

Abstract

Nanotechnology techniques are enabling food scientists to create functional ingredients with micro- and nanostructures, displaying unique properties that are finding applications in encapsulation of bioactive compounds/phytochemicals, food flavors, essential oils, and other important high value ingredients. The ability of edible proteins and polysaccharides to fabricate different nanostructures was investigated using three different methods: 1) desolvation using water as solvent, and acetone, ethanol and methanol as non-solvent agents, 2) complex coacervation using the combination of two biopolymer polyelectrolytes, and 3) layer-by-layer deposition technique to fabricate nanotubes using the combination of two biopolymer polyelectrolytes. These nanoparticles were evaluated for their capacity to encapsulate and deliver curcumin as a model for hydrophobic bioactive compounds.

First, desolvation was used to fabricate spherical nanoparticles from ovalbumin (OVA) and α-lactalbumin (LAC) using different solvent/non-solvent volume ratios (1:3, 1:4, 1:5, 1:10, and 1:20). In this study, the effects of protein solution temperature (25 ℃, 50 ℃, and 80 ℃) and desolvating agent types (acetone, methanol or ethanol) on the size of nanoparticles and their stability for 30 days were studied. The optimization of these parameters permitted the fabrication of sub-100 nm OVA particles using simple desolvation method for the first time, while high-quality α-lactalbumin particles were achieved without using many separation steps as determined by DLS and SEM. This study provided important information regarding the manufacture parameters for production of nanoparticles with desired characteristics using the desolvation method.

Second, the interaction between two pairs of biopolymer polyelectrolytes was evaluated and used to fabricate nanoparticles using complex coacervation. BSA and poly-D-lysine (PDL) were used to obtain soluble coacervate nanoparticles at pH 7. It was found that the particle size was affected by the molecular weight of PDL, the mass ratio of polyelectrolytes (PEs), and salt concentration. Nanoparticles were fabricated by mixing BSA and PDL with low (LMW-PDL) and high molecular weights (HMW-PDL). The smallest nanoparticles with relatively spherical shapes had a diameter of ~200 nm, as confirmed by DLS and SEM. The encapsulation efficiency (EE) of curcumin of these particles was dependent on the curcumin to BSA molar ratio. The highest EE of 60% was achieved with a curcumin to BSA molar ratio of 10 with a loading capacity of 22 μg of curcumin per mg of coacervate nanoparticles. These curcumin-loaded BSA:LMW-PDL nanoparticles were found to be fairly stable over a period of 21 days. To gain more insights on the mechanism of interaction and the effects of molar charge ratio and order of addition on the formation of polyelectrolyte complexes (PECs), sodium alginate and chitosan were used as biopolymer polyelectrolytes at pH 4. Through ITC and DLS, it was demonstrated that the stoichiometry and enthalpy of reactions were strongly affected by the order of addition and influenced the average particle size and zeta potential of PECs. The addition of alginate (-) into chitosan (+) gave positively charged particles and resulted in stronger interactions characterized by larger enthalpy and entropy of complexation, which led to smaller particles. Additionally, the regularity in shape and particle size was also affected by the selected charge ratio as observed in the SEM images. The smallest PECs were obtained when alginate was added into chitosan at a charge ratio of 0.1. Overall, these studies offer new and clearer mechanistic insights with new explanations related to how the polyelectrolyte complexes are formed and how the thermodynamic forces define the particle size and particle size distribution as a function of molar charge ratio and order of addition.

The last part of this research studied the transition from PECs to polyelectrolyte nanolayers. The constitutive interactions between two polyelectrolytes and their ability to form nanotubes using the layer-by-layer deposition technique were evaluated and then divided into two parts. First, BSA and sodium alginate were used to study and optimize the manufacture parameters necessary to form nanotubes at diameters of 200, 400, 600 and 800 nm. It was found that the formation of nanotubes was strongly influenced by the zeta potential difference, pore size of the template, flow rates through the template and the concentration and ratio of each biopolymer. DLS and specially ITC results showed that most of the driving force of the interaction between BSA and sodium alginate at pH 3 and 4 was electrostatically driven, while strong electrostatic repulsion occurred at pH 6 and 7, where nanotubes were not formed. This information was then used to fabricate nanotubes from BSA/κ-carrageenan and α-lactalbumin/chitosan at pH 4 and 7, respectively. The topography and nano-mechanical properties of these nanotubes were characterized using AFM. The mechanical properties of the nanotubes were affected by their diameter as well as the type of polyelectrolytes used during their fabrication. BSA/CAR nanotubes were more robust than nanotubes fabricated with LAC/CHI. The entrapment/encapsulation ability of these nanotubes was also evaluated using curcumin. These nanotubes achieved entrapment efficiencies around 40-45% with subsequent release in physiological conditions. The curcumin-loaded nanotubes exhibited a concentration-dependent toxicity on HeLa cells with no apparent differences between the two types of nanotubes or their diameter. The cell viability values obtained were near 60-65% when the concentration of encapsulated curcumin was 60 μg/ml.

In general, this research proved that nanotechnology techniques using edible biopolymers can be used to fabricate nanostructures with different geometries that offer a repertoire of flexible carrier systems to encapsulate and deliver bioactive compounds. The findings of this research, therefore, can be used to control the manufacturing parameters of different nanoparticles using edible biopolymers with different chemical properties.

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