Structure, assembly, and applications of peanut oil body and oil body protein extracts
Creators: name: Pan, Yijun
Publisher: name: No Publisher Supplied
Year: 2022

description: Emulsion formulations containing two immiscible liquids, one of which is dispersed in the other, have been widely used in food, cosmetic and pharmaceutical industries. Surface-active compounds are needed to form a stable emulsion. Natural surface-active molecules are more preferred than synthetic surfactants due to the clean labeling requirements and safety concerns. Different from traditional emulsions stabilized by small molecular emulsifiers or amphiphilic polymers, Pickering emulsions stabilized by colloidal particles have attracted more and more attention in recent years. These colloidal particles are insoluble in either the continuous phase or the dispersed phase but have proper partial wettability in both phases. There is an increasing research trend of protein-based Pickering stabilizers due to their good capabilities to form emulsions of high physical stability and high loading capacity. Oil bodies (OBs) are subcellular organelles which widely exist in oleaginous plants, algae, fungi, and bacteria. They are spherical droplets composed of a triacylglycerol core covered by a phospholipid-protein layer, which makes OBs natural emulsified oils. OB proteins (mainly oleosins) contribute to the unique physical and chemical stability of OBs and regulate their sizes. The overall goal of my Ph.D. research was to extract, characterize, assemble and apply peanut oil body and oil body proteins for delivery of nutraceuticals. In this work, OBs were extracted from peanut seeds and used as emulsifiers. The extracted peanut OB cream was dispersed in deionized water, and the same volume of peanut oil was added as oil phase to prepare OB emulsions, which may undergo creaming during storage. Different polysaccharides were added to improve the creaming stability of OB emulsions. Adding low content of ɩ-carrageenan (0.5% w/v) into the aqueous phase not only greatly improved the freeze-thaw stability of OB emulsions, but also improved their physical stability over a wide range of pH (4-8) and ionic strength (0-300 mM). OB emulsions were used as a delivery system for lipophilic nutraceuticals by adding them to the oil phase before emulsion preparation. In vitro lipolysis results indicated that nobiletin encapsulated in ɩ-carrageenan stabilized OB emulsions had the highest bioaccessibility compared to that in either the OB emulsions or the oil suspension. OB proteins (oleosins, caleosins, steroleosins, and oil body associated proteins) and phospholipids are necessary to maintain the structural integrity of OB. Oleosins are mandatory to avoid the coalescence of OBs. Emulsions stabilized solely by OB proteins have been rarely studied. Because of its unique structure and insolubility in water, oleosin has the potential to be used as a Pickering stabilizer to construct oil-water interfaces. In this study, OBPEs were successfully extracted from peanut seeds and their profiles were characterized by LC-MS/MS (64.7% oleosin, 23.4% steroleosin and 9.1% lipoxygenase). OBPEs can be dispersed in buffer solution to form colloidal particles, which can reduce the interfacial tension at the oil-water interface. OBPEs were dispersed into 10 mM pH 7.4 phosphate buffer solution to form the aqueous phase. The wettability of OBPEs in phosphate buffer indicated that they were more inclined to form oil in water type of emulsions. Pickering emulsions with different oil fractions were prepared by mixing two phases with a high-speed homogenizer. The morphology, size, and stability of emulsion droplets at different oil fractions were characterized. The formulated oil-in-water emulsions with high internal oil phase fraction exhibited stability against coalescence. Emulsion with high nobiletin loading (2%) was successfully developed and the bioaccessibility of nobiletin was improved as a result of encapsulation. The application of OBPEs in aqueous environment is greatly limited by their highly hydrophobic structures. OBPEs were insoluble in water and most of the common organic solvents including acetic acid, ethanol, butanol, chloroform and dimethyl sulfoxide. After detailed research, formic acid is the only solvent we found so far that can completely dissolve OBPEs. OBPEs nanoparticles were successfully assembled in aqueous environment by the antisolvent precipitation method for the first time. The mean diameter of OBPEs nanoparticles was 215.6±1.8 nm and the polydispersity index was 0.238±0.005. Transmission electron microscopy (TEM) confirmed that the colloidal particles were spherical in shape. As far as we know, this is the first work to report the formic acid as the good solvent for OBPEs and to use the fabricated OBPEs nanoparticles as Pickering-type stabilizers. Oil-in-water (O/W) Pickering emulsions with good stability against coalescence could be formed at protein concentration as low as 0.1 mg/mL. Cryo-scanning electron microscopy (cryo-SEM) confirmed that spherical nanoparticles were packed at the oil-water interface. Formic acid can be removed through dialysis. After dialysis, the oil-water interface can still be stabilized by the dispersion of nanoparticles. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted to prove that 1 mg/mL OBPEs nanoparticles had no cytotoxicity against Caco-2 cells (a colon carcinoma cell line). Pickering emulsion loaded with 2% nobiletin was prepared and characterized. Compared with the simple dispersion of OBPEs in phosphate buffer, 1/10 of protein concentration was used in the dispersion of OBPEs nanoparticles to stabilize the same amount of nobiletin. In vitro evaluation by TIM-1 model showed that nobiletin encapsulated in Pickering emulsion could significantly improve its bioaccessibility.Oleosins are usually the most abundant proteins on the surface of plant oil bodies (OBs). The ability of oleosins to stabilize OBs and prevent their coalescence is well known. OBs can be irreversibly adsorbed at the lipid/water interface. Peanut seeds contain a variety of storage proteins, different oleosins and oil body associated proteins. The purity of oleosin obtained by extracting proteins from OBs cannot be too high. Peanut oleosin 1 (Q647G5), which has the highest similarity to the other seven peanut oleosins, was chosen as the representative oleosin. Recombinant oleosin 1 with 6 histidine gene added to the C-terminal of the protein was successfully expressed in E. coli BL21 (DE3). After separation by immobilized metal affinity chromatography (IMAC), it was proved that high purity recombinant oleosin 1 was obtained. Purified oleosin 1 was dispersed in 10 mM tris buffer solution with different pH values, and the effect of pH values on charge, morphology, and surface activity of oleosin 1 was characterized. Oleosin 1 could be dispersed in pH 2 tris buffer solution, forming round to oval-shaped colloidal particles. They could be rapidly adsorbed to the oil-water interface, forming an elastic interfacial film. Seven commonly used solubility-enhancing fusion tags were screened. From the expression of protein and solubility evaluation by SDS-PAGE gel, SUMO (Small Ubiquitin-like Modifier) and AFV (hypothetical protein from Acidianus filamentous virus 1) could potentially be used as fusion tags to improve the solubility of peanut oleosin 1.In conclusion, peanut oleosin 1 is a promising candidate to help understand how the oil body proteins may affect the interfacial structure and rheological properties at the oil/water interface. Successful construction of oleosin 1 fusion protein may solve the solubility problems of oleosins and provide a new approach to biosynthesize novel protein-based emulsion stabilizers. This dissertation greatly expands the applications of peanut oil bodies and oil body proteins in structuring the oil-water interfaces and developing novel formulations in the food, cosmetic and pharmaceutical fields.