Author ORCID Identifier
Year of Publication
Doctor of Philosophy (PhD)
Agriculture, Food and Environment
Animal and Food Sciences
Dr. Youling L. Xiong
Oat (Avena sativa L.), a specialty cereal grain belonging to the Poaceae family, is relatively abundant in nutritious protein (12–20%) and dietary fiber (β-glucan). The balanced hydrophilic/hydrophobic amino acid profile in oat protein relative to other plant sources provides numerous possibilities for formulating oat protein isolate (OPI) into a wide range of food products. Yet, there has been limited research on OPI and the application of this valuable protein source is hampered by its low solubility at neutral and mildly acidic conditions. This dissertation research aimed to elucidate the efficacy of high-intensity ultrasound (HIU) for structural modification of OPI and investigate the complementary roles of high pressure, disulfide-breaking agents (mainly cysteine), and flavonoids for the ultimate improvement of OPI functionalities, including solubility, emulsification, and gelation.
OPI (mainly 12S globulins) was isolated from oat groats by alkaline extraction–isoelectric precipitation. Because protein functionality is influenced by environmental factors, such as pH, ionic strength, and temperature, it is essential to understand the surface properties of native protein (charge, polarity, and amphiphilicity). Therefore, Experiment 1 was conducted to examine the solubilization behavior of OPI in relation to surface properties when subjected to varying pH and ionic strength conditions. A characteristic U-shaped solubility curve was observed within pH 2.0–8.0 at low ionic strengths (I < 0.01) and a minimum solubility was found at pH 4.0–5.0 and I of 0.03–0.2. Particle size, ξ-potential, and SDS–PAGE patterns supported the solubility profile.
In seeking effective modification strategies, Experiment 2 was carried out to determine the effect of internal disulfide cleavage on OPI solubility and emulsifying activity. Differential scanning calorimetry revealed that disulfide bonds contributed to the remarkable high stability (Tm of 111.1 °C) of OPI but impeded its molecular flexibility and functionality. Hence, cysteine was applied to disrupt inter-subunit S–S bonds of OPI. Cysteine at concentrations higher than 1.0 mM/mg protein induced disulfide cleavages, and the 12S globulin subunit dissociation reached a maximum (80%) at 3.3 to 6.7 mM/mg protein. Correspondingly, emulsions prepared with cysteine-treated OPI showed superior interfacial protein coverage (0.170 m2/g compared to 0.092 m2/g protein for control) and reduced emulsion particle size from 4722 to 2238 nm.
While cysteine was able to modulate OPI emulsifying activity, more robust techniques were necessary to modify protein structure for functionality improvement. Hence, Experiment 3 sought to apply high-intensity ultrasound (HIU) to dissociate 12S globulins and disrupt tertiary structure to overcome the low-solubility constraints. Through cavitation-induced microscopic shearing and pressure, HIU treatment (5 minutes at 70% amplitude) significantly reduced OPI particle size (up to 37%) and increased solubility (up to 48%) at pH 5.5–8. Fluorescence spectrometry and hydrophobicity measurements revealed protein particle dissociation and major conformational changes, with the exposure of previously occluded hydrophilic/hydrophobic groups predisposing OPI to increased interfacial activity.
To enhance the effectiveness of HIU, high pressure (HP) at 30 MPa was applied to complement the effect on modulating OPI functionality (Experiment 4). With further increased emulsifying activity of OPI by the HUI+HP combination treatment at 20 and 60 °C, the emulsions prepared exhibited an additional particle size reduction (from 1653 to 955 nm). Confocal microscopy revealed increased distribution uniformity of oil droplets and emulsion stability. Although in theory both HP and the moderate temperature (60 °C) could aid HIU in dissociating protein aggregates, the improvement on emulsifying activity remained predominated by the HIU process.
Based on the above findings, cysteine was reintroduced into Experiment 5 to assist in the formation of oat protein-based gel networks with the pretreatment of HIU. Native OPI samples treated with cysteine alone up to 400 mg/g protein had a significantly increased gel strength (from 0.0049 to 0.23 N) and decreased cooking loss (up to 44%). For HIU treated OPI, the efficacy of cysteine increased 3-fold (gel strength improved to 0.68 N). The combination of cysteine and HIU also enhanced the rheological properties (storage modulus), structural, and water-immobilizing capacity of the heat-induced gel.
Polyphenols are naturally present in oat groats and are increasingly utilized to modulate plant protein functionality, Experiment 6 was conducted to investigate the potential collaborative effect of three di-phenol flavonoids with varying hydroxyl groups (1-kaempferol, 2-quercetin, and 3-myricetin) with HIU for enhancing OPI emulsifying activity. Binding with flavonoids (fluorescence quenching) subjected HIU treated OPI to further increased hydrophobicity with 0.5 mmol/L myricetin producing the maximum effect. The HIU+flavonoid treatment improved the emulsion stability. Additionally, all flavonoid-treated samples exhibited a higher oxidative stability against free radicals.
Overall, this dissertation research demonstrated that natural chemical compounds (cysteine and flavonoids) and physical treatment (high pressure) provided synergistic effects for HIU on increasing structural flexibility and improving amphiphilicity of oat globulins. The physicochemical changes led to enhanced emulsifying and gelling properties accentuating the potential of these structure-modifying techniques for oat protein.
Digital Object Identifier (DOI)
This work was supported by the USDA National Institute of Food and Agriculture, Hatch project (no: 1020736) from 2017 to 2023.
Li, Runnan, "ULTRASOUND-BASED STRUCTURAL AND FUNCTIONAL MODIFICATIONS OF OAT PROTEIN: COMPLEMENTARY EFFECTS OF HIGH PRESSURE, CYSTEINE, AND FLAVONOIDS" (2023). Theses and Dissertations--Animal and Food Sciences. 143.
Available for download on Wednesday, July 17, 2024