Research On The Preparation And Characteristics Of β-carotene Nanoparticles Stabilized By Proteins

β-carotene is widely used in food systems due to the highest vitamin A activity and lipophilic antioxidant activity which can scavenge free radicals among carotenoids. Almost no aqueous solubility, weak stability under heat, light, and oxygen, and low bio availability strictly restricted the application in food industry and reduced the absorption by human beings. In this study, β-carotene nano-delivery system was prepared based on biomacromolecules, proteins, to study the factors that affect the physicochemical stability, simulated in vitro release and absorption mechanism by Caco-2 cells. The potential cell toxicity of β-carotene nanoparticles was also evaluated. The chemical and cellular antioxidant activities of β-carotene nanoparticles were also analyzed. On these basises, glycation products was made with β-lactoglobulin and dextran to prepare β-carotene nanoparticles and show the possible improved stability under stree environments. The controlled release properties of encapsulated β-carotene and Caco-2 monolayer cells transport mechanism were evaluated.Six different mean particle size oil-in-water β-carotene emulsions were prepared with microfluidizer to evaluate the impacts of particle size on the physicochemical stability and in vitro bioaccessibility of β-carotene in oil-in-water emulsions. Homogenization at different pressures produced droplet diameters(Dz = 368-124 nm) that were linear and inversely related to homogenization pressures in the pressure range 10-100 MPa. The slow rate of volume increase was found to be related to the square of the initial droplet radius following Stokes velocity of setting equation. β-carotene stability towards oxidation was lower as droplet diameter decrease. The extent of lipolysis in an in vitro system was higher and linearly related to the inverse of droplet diameter. β-carotene bioaccessibility, as defined by the amount of β-carotene recovered in the aqueous phase after ultracentrifugation, was linearly related to smaller emulsion droplet diameter, providing experi mental foundation for nano-delivery systems.In order to decrease particle size further to improve β-carotene bioavailability, β-carotene loaded nanoparticles were prepared with different contents of food-grade sodium caseinate, whey protein isolate, or soy protein isolate with a homogenization-evaporation method and evaluated for their physiochemical stability, in vitro cytotoxicity, and cellular uptake by Caco-2 cells. The particle diameters of the β-carotene of β-carotene loaded nanoparticles with sodium caseinate or whey protein isolate emulsifiers were both below 100 nm, w hile the particle diameter was 350 nm based on soy protein isolate. This was attributed to the different emulsifying activities and emulsifying stability. During 30 days of storage, al l nanoparticles were resistant to flocculation or sediment and mean particle diameters of three β-carotene nanoparticles increased less than 10% at 4°C. The oxidative stability of β-carotene loaded nanoparticles encapsulated by proteins decreased in the following order: SC>WPI>SPI. The β-carotene’s chemical stability was improved by increasing the concentration of protein. Almost no cytotoxicity due to β-carotene loaded nanoparticles cellular uptake was observed. The uptake of β-carotene was significantly improved through nanoparticle by 2.6-, 3.4- and 1.7-fold increase, respectively, for SC, WPI, and SPI, as compared to the free β-carotene. A significant negative correlation was observed between β-carotene uptake and the nanoparticles’ negative surface charge.The FTIR-ATR showed the presence of β-carotene in the nanoparticles and hydrogen bonding was found between proteins and β-carotene. XRD and DSC analyses suggested that β-carotene is amorphous in the nanoparticles.Freeze-drying did not adversely affect the size and the re-dissolving activity of β-carotene nanoparticles since particles resuspended in water after freeze-drying had similar mean particle diameters. The β-carotene antioxidant activities in proteins nanoparticles were evaluated through chemical and cellular methods and the relationship between β-carotene antioxidative activities and emulsifiers. The reducing power, DPPH, and hydroxyl radical scavenging activity were significantly improved by nanoencapsulation. And the cellular antioxidant activities of β-carotene were also improved by proteins-based nano-delivery system. The cellular antioxidant activities of three proteins β-carotene nanoparticles were in the following order: WPI>SC> SPI.The simulated gastrointestinal in vitro digestion systems were built to explore the characteristics and release rate of β-carotene in nanoparticles. In both gastric and intestinal digestion stage, the rates and extents of β-carotene in SC nanoparticles were greater than in SPI nanoparticles. However, the rate and extent of β-carotene in WPI nanoparticles was close to 0, and the release extent was 69.95% for WPI nanoparticles during intestinal digestion tract. Release was low with pepsin but high with trypsin suggesting that WPI might be the best protein delivery vehicle to deliver β-carotene to the intestine.New emulsifiers based on β-lactoglobulin-dextran conjugates were obtained through “dry heating method”. The effects were evaluated in improving the physicochemical stability of β-carotene in proteins-based nanoparticles and the potential mechanism was analyzed. Theβ-lactoglobulin-dextran conjugates were verified with SDS-PAGE. The TNBS assay showed that the extent of glycation increased with increasing “dry heating” treatment time. The highly ordered structure content(α-helix and β-sheet) was increased slightly by glycation with far-UV Circular Dichroism. There were no significant differences in particle diameter. However, the β-carotene nanoparicles’ ζ-potential values were different. Glycation with dextran appreciably decreased the apparent ζ-potential, showing the surface charge was blocked by conjugated dextran. The encapsulation efficiency of β-carotene was 98.6 and 98.4% for native β-lactoglobulin and β-lactoglobulin-dextran conjugates, respectively, and the loading capacity was 1.07 and 1.06 for native β-lactoglobulin and β-lactoglobulin-dextran conjugates, respectively. The stability at p H close to isoelectric point was significantly improved by dextran glycation by steric hindrance with large carbohydrate-dextran from aggregation and flocculation. Almost no β-carotene was released during gastric digestion in both nanoparticles. The releases of β-carotene were 60.9 and 51.8% for β-lactoglobulin and β-lactoglobulin-dextran conjugates, respectively. The β-carotene release was inhibited by dextran glycation through steric hindrance. Both β-lactoglobulin β-carotene nanoparticles and β-lactoglobulin-dextran conjugates nanoparticles were non-cytotoxic to Caco-2 cells, even at 10mg/m L. Compared to free β-carotene suspension, the apparent permeability coefficient(Papp) of Caco-2 cells to β-carotene was appreciably improved by nanoencapsulation. There were no appreciably differences in the permeability rate(Papp) between β-lactoglobulin and β-lactoglobulin-dextran conjugates, indicating that polysaccharide molecules on the surface of nanoparticles had no adverse effects on β-carotene bioavailability.