Yuting Fan

Fabrication of curcumin-loaded bovine serum albumin (BSA)-dextran nanoparticles and the cellular antioxidant activity

Bovine serum albumin (BSA)-dextran conjugate was prepared with glycation. Self-assembly nanoparticles were synthesized with a green, and facile approach. The effects of dry-heating time on the fabrication and characteristics of BSA-dextran conjugate nanoparticles were examined. Stable nanoparticles (<200nm) were formed after only 6h dry-heating because enough dextran was grafted onto the BSA to provide significant steric hindrance. Particle size decreased with the increase of dry-heating time and the lowest particle size (51.2nm) was obtained after 24h dry-heating. The nanoparticles were stable in a wide pH range (pH 2.0-7.0). The particle size of nanoparticles increased to 115nm after curcumin incorporation and was stable even after one-month storage. TEM results demonstrated that curcumin-loaded nanoparticles displayed a spherical structure and were homogeneously dispersed. Curcumin in BSA-dextran nanoparticle showed better stability, compared to free curcumin. In addition, BSA-dextran nanoparticles can improve the cellular antioxidant activity of curcumin in Caco-2 cells.

Thermal Degradation and Isomerization of β-Carotene in Oil-in-Water Nanoemulsions Supplemented with Natural Antioxidants

The goal of this study was to see the impact on the retention and isomerization of encapsulated β-carotene (BC) in nanoemulsions fortified with natural antioxidants (α-tocopherol (AT) and l-ascorbic acid (AA)). The physical stability of nanoemulsion, oxidative stability, and isomerization of all-trans-β-carotene (BC) in oil-in-water (O/W) nanoemulsions were determined in the presence or absence of natural antioxidants at 25 and 50 °C at certain intervals of time by high-performance liquid chromatography (HPLC). Sodium caseinate was used as the emulsifier, and corn oil (CO) was more protective than medium-chain triglycerides (MCT) and used for isomerization studies. Mean diameters of control (without antioxidants) and AA- and AT-fortified particles were similar. Mean particle diameter of nanoemulsions increased from 10 to 25 nm at 25 °C and from 40 to 50 nm at 50 °C during 30 days of storage. The isomerization from all-trans-BC to cis-BC isomers was inhibited by antioxidants. The isomerization rates were in the following order: 13-cis-BC > 15-cis-BC > 9-cis-BC. AT had better antioxidant activities than AA in inhibiting BC degradation in O/W nanoemulsions. The results indicated that BC encapsulated in nanoemulsions supplemented with antioxidants could significantly improve BCs chemical stability.

β-Lactoglobulin–chlorogenic acid conjugate-based nanoparticles for delivery of (−)-epigallocatechin-3-gallate

β-Lactoglobulin (BLG)–chlorogenic acid (CA) conjugates were generated by a free radical induced grafting method. BLG–CA conjugates showed better antioxidant activities than BLG. The antioxidant activity increased with the increase of CA substitution. The particle sizes of (−)-epigallocatechin-3-gallate (EGCG)-loaded nanoparticles prepared by the anti-solvent method were 110.3, 107.4, and 105.8 nm for BLG, BLG–CA (low), and BLG–CA (high), respectively. The encapsulation efficiencies of EGCG in BLG, BLG–CA conjugate (low), and BLG–CA conjugate (high) nanoparticles were 72.9%, 71.8%, and 73.5%, respectively. The chemical stabilities of EGCG in both BLG–CA nanoparticles were significantly higher than in BLG nanoparticles. BLG–CA conjugate (high) showed better EGCG retention than BLG–CA conjugate (low) in simulated gastrointestinal digestion fluid. Little EGCG was released in both BLG nanoparticles and BLG–CA nanoparticles under simulated gastric digestion. The release of EGCG in BLG–CA nanoparticles was less than that in BLG nanoparticles, indicating that CA conjugating protected BLG from the digestive enzymes.

Development of β-Carotene-Loaded Organogel-Based Nanoemulsion with Improved In Vitro and In Vivo Bioaccessibility

β-Carotene (BC), a naturally occurring lipophilic carotenoid, is beneficial for human health. However, its water solubility and bioavailability are low. In this study, organogel-based nanoemulsion was successfully prepared to improve the loading amount, solubility, and bioavailability of BC. Corn oil was selected as the oil phase for the organogel as a result of the greatest release amount of BC. Tween 20 was optimized as the emulsifier based on the highest extent of lipolysis and BC bioaccessibility. The nanoemulsion was a better alternative than the organogel according to both the extent of lipolysis and BC bioaccessibility. Cellular uptake of BC was significantly improved through organogel-based nanoemulsion compared to BC suspension. Caveolae-/lipid-raft-mediated route was the main endocytosis pathway. Pharmacokinetic results confirmed that the in vivo bioavailability of BC in nanoemulsion was 11.5-fold higher than that of BC oil. The information obtained suggested that organogel-based nanoemulsion may be an effective encapsulation system for delivery of insoluble and indigestible bioactive compounds.

Purification and Characterization of a Black Walnut (Juglans nigra) Allergen, Jug n 4

Tree nuts as a group cause a significant number of fatal anaphylactic reactions to foods. Walnuts (Juglans spp.) are one of the leading causes of allergic reactions to tree nuts in the U.S. and Japan. The purpose of this study was to purify and characterize potential food allergens from black walnut. Here, we report the isolation of the black walnuts allergen Jug n 4 (an 11S globulin) by ammonium sulfate precipitation, hydrophobic interaction, and size exclusion chromatography. Reducing SDS-PAGE analysis indicated that purified Jug n 4 consists of three major bands. N-Terminal sequencing data of these bands indicated that they were the results of a post-transcriptional protease cleavage of the mature protein at a site that consists of a known conserved protease recognition motif, NGXEET. Western blot experiments revealed that 32% of the sera from 25 patients with double-blind, placebo-controlled clinical walnut allergy contained IgE antibodies that recognized Jug n 4, indicating that it is a walnut allergen. Identifying this and additional allergens may facilitate the understanding of the allergenicity of seed storage proteins in tree nuts and their cross-reactivity.

Identification, characterization, and initial epitope mapping of pine nut allergen Pin k 2

The aims of this study were to predict, identify and characterize pine nut allergens. Korean pine (Pinus koraiensis) vicilin was predicted to be a pine nut allergen. Recombinant Korean pine vicilin was expressed in E. coli and purified. Natural Korean pine vicilin isolated from pine nuts (which displayed multiple bands in SDS-gels due to posttranslational digestion) and its full length recombinant counterpart were used to test whether it is a food allergen. The recognition of the protein (and its fragments) by patient serum IgE was analyzed by Western blot. The study included fourteen patients diagnosed with clinical pine nut allergy. Twenty nine percent of the patient sera recognized both the natural and recombinant pine nut vicilin, indicating that Korean pine vicilin is a bona fide food allergen. The serum recognition patterns of the naturally occurring protein fragments suggested that some of linear IgE epitopes may be mapped to the fragment boundaries. The chemical and thermal stability of the recombinant protein was investigated. It underwent a thermal transition with a Tm=76.6°C. The transition was accompanied by an increase in the amplitude of the circular dichroism signal at 220nm. Urea induced unfolding of the recombinant protein had a Cm of 4.6M.

Improved chemical stability and cellular antioxidant activity of resveratrol in zein nanoparticle with bovine serum albumin-caffeic acid conjugate

In this study, bovine serum albumin (BSA)-caffeic acid (CA) conjugate was prepared with free radical-induced grafting method. The CA to BSA ratio of the conjugate was 115.7 mg/g. In vitro antioxidant activity assays suggested that BSA-CA conjugates had stronger antioxidant activity than BSA. Resveratrol-loaded zein encapsulated with BSA and BSA-CA conjugate core-shell nanoparticles were prepared with antisolvent method. Particle sizes were 206.3 nm, and 217.2 nm for BSA and BSA-CA, respectively. The encapsulation efficiencies (EEs) were 85.3% and 86.5% for zein-BSA and zein-BSA-CA nanoparticles, respectively. SEM results indicated that both nanoparticles were spherical with mean diameter approximately 200 nm and smooth surfaces. Both thermal and UV light stability of resveratrol was significantly improved after nanoencapsulation. BSA-CA conjugate showed remarkably greater protection than BSA against resveratrol degradation. Cellular antioxidant activity (CAA) study confirmed that resveratrol in both zein-BSA and zein-BSA-CA nanoparticles had significant higher antioxidant activities than resveratrol alone.

Endocytosis of Corn Oil-Caseinate Emulsions In Vitro: Impacts of Droplet Sizes

The relative uptake and mechanisms of lipid-based emulsions of three different particle diameters by Caco-2 cells were studied. The corn oil-sodium caseinate emulsions showed little or no cytotoxicity even at 2 mg/mL protein concentration for any of the three droplet size emulsions. Confocal laser scanning microscopy (CLSM) of Nile red containing emulsions showed that the lipid-based emulsions were absorbed by Caco-2 cells. A negative correlation between the mean droplet size and cellular uptake was observed. There was a time-dependent and energy-dependent uptake as shown by incubation at different times and treatment with sodium azide a general inhibitor of active transport. The endocytosis of lipid-based emulsions was size-dependent. The internalization of nanoemulsion droplets into Caco-2 cells mainly occurred through clathrin- and caveolae/lipid raft-related pathways, while macropinocytosis route played the most important role for 556 nm emulsion endocytosis as shown by the use of specific pathway inhibitors. Permeability of the emulsion through the apical or basal routes also suggested that active transport may be the main route for lipid-based nanoemulsions. The results may assist in the design and application of lipid-based nanoemulsions in nutraceuticals and pharmaceuticals delivery.

Structure analysis of a glycosides hydrolase family 42 cold-adapted β-galactosidase from Rahnella sp. R3

The β-galactosidase isolated from a psychrotrophic bacterium, Rahnella sp. R3 (R-β-Gal), exhibits high activity at low temperature and has potential in the dairy industry. R-β-Gal is a member of the glycoside hydrolases family 42 (GH42), and it forms a 225 kDa trimeric structure in solution. The crystals of R-β-Gal were acquired via hanging-drop vapor-diffusion method, and the X-ray crystal structure of the native R-β-Gal was determined at a 2.5 A resolution. It is the first structure of a cold-adapted GH42 enzyme. In the crystallographic asymmetric unit, there were two homotrimers of the enzyme. Each monomer consists of three domains, an N-terminal catalytic domain which is a (β/α)8 barrel, a mixed β-sheet and α-helices domain, and a C-terminal β-sandwich domain. Two putative residues might be involved in catalysis, a proton donor E157 and a nucleophile E314, were superimposed well with the catalytic residues of other β-galactosidases. Site-directed mutagenesis targeting these residues abolished the activity of the enzyme. Structure and sequence comparison of R-β-Gal with two mesophilic β-gals and a thermophilic β-gal indicated that intramolecular force and higher structural flexibility might result in the cold-adaptation of R-β-Gal.

Oxidative stability and in vitro digestion of menhaden oil emulsions with whey protein: Effects of EGCG conjugation and interfacial cross-linking

The goal of this study was to improve the chemical stability of menhaden oil and control the lipolysis in emulsions with whey protein during in vitro digestion through EGCG conjugation and genipin-mediated interfacial cross-linking (CL). WPI-EGCG conjugate was successfully synthesized, confirmed by SDS-PAGE, ESI-MS, and phenolic group quantifications (125.3 mg/g), and characterized with far UV CD and ATR-FTIR. Emulsion particle diameter with WPI-EGCG is lower than with WPI. Compared to the native emulsion, WPI CL increased particle diameter and physical stability. Higher oxidative stability was observed for emulsions stabilized with WPI-EGCG conjugate than that with interfacial cross-linking due to the great antioxidant activity. Whereas, WPI CL is more effective than WPI-EGCG conjugate in hindering the rate and extent of lipolysis. The combination of EGCG conjugation and interfacial CL showed both the highest protection of menhaden oil against degradation and highest inhibition on the rate and extent of lipolysis of menhaden oil.