Preparation And Functional Evaluation Of Lactoferrin-cichoric Acid Nano-complexes

As the main active ingredient in echinacea,cichoric acid belongs to coffee acid derivative,and has the functions of antioxidant,scavenging free radicals,anti-inflammatory,and immunomodulatory.The instability of cichoric acid leads to its vulnerability to the surrounding environment in the process of food production,and cichoric acid in the stomach will be degraded by pepsin,resulting in low bioavailability.Lactoferrin is widely used in the construction of nano-complexes due to its good biocompatibility,high biodegradability,low cytotoxicity,and high dielectric points.In addition,lactoferrin has a certain regulatory effect on the growth of small intestine cells because of its specific receptor in the small intestine.In this study,lactoferrin-cichoric acid nano-complexes were constructed by heat treatment.And the stability of the gastrointestinal tract of cichoric acid was improved after the interaction between cichoric acid and lactoferrin.It was proved from the cellular level that lactoferrin-cichoric acid nano-complexes promoted the ability of intestinal epithelial cells to uptake cichoric acid.The main research contents and results of this thesis are as follows:(1)The interaction between lactoferrin and cichoric acid:the lactoferrin-cichoric acid nano-complexes were constructed by heat treatment method.And the concentration of cichoric acid,the heat treatment temperature,and the p H value of lactoferrin solution were used as variables to optimize the experimental conditions.The decrease of fluorescence intensity in fluorescence spectroscopy and the increase of absorbance at the maximum absorption wavelength in UV-Vis absorption spectra proved that cichoric acid interacted with lactoferrin to form lactoferrin-cichoric acid nano-complexes.Further analysis of fluorescence spectra showed that lactoferrin-cichoric acid nano-complexes were mainly formed by hydrophobic force and electrostatic force to form.(2)Characterization of lactoferrin-cichoric acid nano-complexes:the binding rate of lactoferrin and cichoric acid(97.87%～99.87%)was determined by ultrafiltration combined with high performance liquid chromatography(HPLC),and the results showed that the nano-complexes prepared under p H 7 and 95℃had the minimum particle size(67.20±1.86 nm)and the maximumζ-potential(28.3±0.93 m V);FTIR and CD showed that the combination of lactoferrin and cichoric acid affected the secondary structural compositions of lactoferrin,and the results of DSC showed that the addition of cichoric acid improved the thermal denaturation temperature of lactoferrin(4～10℃).The SEM results showed that both lactoferrin and lactoferrin-cichoric acid nano-complex were spherical.(3)Study on the stability and antioxidant resistance of lactoferrin-cichoric acid nano-complexes:the results of DPPH·scavenging experiment and ABTS~+·scavenging experiment were proved that the antioxidant ability of lactoferrin-cichoric acid nano-complexes was better than the sum of cichoric acid and lactoferrin,they may played a synergistic antioxidant effect.The results of storage stability showed that lactoferrin-cichoric acid nano-complexes had smaller changes in particle size andζ-potential.The results of simulated digestion experiments in vitro showed that lactoferrin-cichoric acid nano-complexes significantly improved the gastrointestinal stability of cichoric acid,improving the retention rate of cichoric acid in the stomach from 67.39%to 33.82%,and the degradation rate in intestine from 28.80%to 76.18%.(4)Study on the ingestion rate of intestinal epithelial cells of lactoferrin-cichoric acid nano-complexes:Ht-29 cells and Caco-2 cells were selected for lactoferrin-cichoric acid nano-complexes MTT cell-proliferation assay measures,the results showed that the complexes had a significant toxic effect on Ht-29 cells while the complexes had no cytotoxic effect on Caco-2 cells.The lactoferrin-cichoric acid nano-complexes had a more significant inhibitory effect on LPS induced cell inflammatory response,possibly because higher content of cichoric acid reached the inside of Caco-2 cells.So that the cells played a more significant role in scavenging free radicals.After adding lactoferrin-cichoric acid nano-complexes and equivalent cichoric acid culture Caco-2 cells,the HPLC was used to analysis cells intake content of cichoric acid.The results showed that lactoferrin-cichoric acid nano-complexes promote the absorption of cichoric acid,and the absorption rate increased from 18.2±2.09%to 38.92±6.77%.In summary,the lactoferrin-cichoric acid nano-complexes were successfully constructed by heat treatment,which improved the antioxidant properties,gastrointestinal stability of cichoric acid,and absorption in intestinal cells.This research could lay a foundation for improving the bioactivity of cichoric acid and provide new ideas for the targeted absorption of cichoric acid in vivo.