Preparation,controlled Release And Antioxidant Of Chitosan/β-lactoglobulin Loaded EGCG Nanoparticles

Polysaccharide-protein nanocomposite delivery carriers have been widely used in food industry because of their advantages in protecting the stability of oral nutrients in gastrointestinal tract and promoting their intestinal absorption.The characteristics of nanoparticles,such as diameter and surface charge,had great influence on intestinal absorption.However,there were few reports about the effect of nanoparticle size of polysaccharide-protein composite nanoparticles on their intestinal absorption.Epigalocatechin gallate（EGCG）is the main esterified catechin in green tea,which has strong antioxidant and other biological activities.However,due to the instability of EGCG in weak alkali or neutral environment,and its easy decomposition in gastrointestinal tract after oral administration,it is necessary to improve the oral bioavailability of EGCG through appropriate food-grade delivery system to enhance its stability in harsh environment.In this study,two kinds of water-soluble chitosan（CS）derivatives chitosan hydrochloric（CHC）and carboxymethyl chitosan（CMC）and thermal denaturedβ-lactoglobulin（β-Lg）were used as wall materials and EGCG as core materials.Chitosan/β-lactoglobulin（EGCG-CS/β-Lg）composite nanoparticles loaded with EGCG were prepared by ion-gel method.The particle size effect of empty-loaded CS/β-Lg nanoparticles and the antioxidant activity of EGCG nanoparticles were evaluated by using Caco-2 cells as model.It provides a theoretical basis for the application of polysaccharide-protein nanocomposite delivery system and the improvement of oral bioavailability of polyphenols.The main contents of this paper are as follows:1.Preparation and characterization of empty-loaded CS/β-Lg composite nanoparticles with different particle sizes.Three kinds of empty-loaded CS/β-Lg nanoparticles with different particle sizes were prepared.The particles sizes were115.80,153.41 and 216.96 nm,respectively,and the nanoparticles size distribution of three kinds of empty-loaded CS/β-Lg nanoparticles with different particle sizes was concentrated and granular.2.The effect of different particle size on the uptake of Caco-2 cells.The Caco-2 cell uptake was analyzed at different concentration and incubation time with empty-loaded CS/β-Lg nanoparticles with different nanoparticle size.The results showed that the uptake rate of Caco-2 cells in small particle size empty-loaded CS/β-Lg nanoparticles（115.80 nm）was significant higher than that in control group（FITC-CHC）,medium particle size empty-loaded CS/β-Lg nanoparticles（153.41 nm）and large particle size empty-loaded CS/β-Lg nanoparticles（216.96 nm）,respectively（p<0.01）.In the range of 25～100μg/m L,the uptake rate of Caco-2 cells was significantly dependent on the concentration of empty-loaded CS/β-Lg nanoparticles（115.80 nm）.During the incubation time of 1-4 h,the uptake rate of Caco-2 cells reached saturation at～2 h.3.Preparation and characterization of the CS/β-Lg composite nano-carrier load EGCG.EGCG-CS nanoparticles（with a particle size of 190.63 nm,a Zeta potential of32.33 m V,an encapsulation efficiency of 59.79%）and an EGCG-CS/β-Lg nanoparticles（with a particle size of 157.20 nm,a Zeta potential of 30.51 m V,an encapsulation efficiency of 76.29%）were optimized,It was observed from TEM that EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles showed obvious core-shell structure and compact particles.4.Gastrointestinal stability and controlled release characteristics of CS/β-Lg composite nanocarrier loaded EGCG.The optimized EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles were used to simulate gastrointestinal digestion.EGCG quantitative analysis by HPLC and observed the microstructure of the nanoparticles by TEM.From the results of slow release,it could be seen that the EGCG release rates of EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles were 30%and10%,respectively,in the simulated gastric juice digestion.while the EGCG release rates of EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles were 66.97%and70.8%,respectively,in the simulated intestinal fluid digestion.In addition,under the observation of TEM,it can be seen that EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles change from core-shell compact structure to fluffy state during digestion.5.Research on antioxidant activity of EGCG loaded with CS/β-Lg composite nanocarrier in vitro.The antioxidant activity of free EGCG,EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles was detected by DPPH,FRAP and cellular antioxidant activity（CAA）.The results of DPPH and FRAP showed that the antioxidant activity of free EGCG,EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles in simulated intestinal environment（p H 7.2）120 min was significantly higher than that of free EGCG and EGCG-CS nanoparticles（p<0.01）;In addition,from the results of CAA,it could be seen that the CAA values of EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles were higher than those of free EGCG,and EGCG-CS/β-Lg nanoparticles are higher than those of EGCG-CS nanoparticles at the same concentration.The CAA value of EGCG-CS/β-Lg nanoparticles was significantly higher than that of free EGCG（p<0.01）.It was worth noting that the EC₅₀concentration of free EGCG nanoparticles was 15.53μg/m L,which was significantly higher than that of EGCG-CS nanoparticles and EGCG-CS/β-Lg nanoparticles,the concentration of EC₅₀decreased by 8.56%（14.2%μg/m L）and18.35%（12.68μg/m L）,respectively.The results showed that the antioxidant activity of EGCG was enhanced by nanoparticles,and the effect of CS/β-Lg nanoparticles was better than that of CS nanoparticles.