| Protein-based Pickering emulsions have high loading capacity and biocompatibility,but a single particle is highly sensitive to adverse environments,and the emulsion is prone to flocculation and demulsification and loss of stability.Through reasonable modification,the development of protein-polyphenol composite nanoparticles with excellent performance is of great significance for the application of Pickering emulsion,but the effectiveness as an active substance delivery carrier remains to be further explored.In vitro gastrointestinal simulation system and cell model can be used to evaluate the physiological efficacy of active substances,but it is difficult to clarify the utilization effect after absorption and metabolism in vivo.As an ideal model organism for anti-oxidation and anti-aging,the C.elegans model can avoid the high cost and ethical problems of using large animals.This paper aims to expand the preparation and application value of Pickering emulsions based on protein-polyphenol matrix,and provide new strategies for biological delivery and metabolic efficacy evaluation of active substances.Firstly,casein-caffeic acid was used as the model compound to prepare nanoparticles.The size,distribution,stability and emulsification performance were used as evaluation indexes.At 25℃,the concentration of casein and caffeic acid were 10.0 g/L and 8.0 g/L,and the p H was 7.0,the non-covalent composite nanoparticles could be obtained with size of 174.08±5.81 nm,Zeta potential of-35.33 m V,emulsifying activity index of 56.36 m~2/g and emulsification stability index of 72.56%.The covalent nanoparticles with size of 171.11±6.83 nm,Zeta potential of-37.73 m V,emulsifying activity and stability index of 64.29 m~2/g and 79.47%were obtained at p H 9.0.The results of fourier transform infrared spectroscopy,circular dichroism and fluorescence spectroscopy showed that the phenolic hydroxyl group of caffeic acid bound to casein,reduced theα-helix content and enhanced fluorescence quenching.The non-covalent interaction between casein and caffeic acid was dominated by hydrogen bonds and hydrophobic interactions.The covalent interaction was based on the C-N bond formed by casein amino group and caffeic acid phenolic hydroxyl group.Caffeic acid was found to bind to the amino acid residue site on the surface of casein by fluorescence probe method,and the surface hydrophobicity was reduced.The three-phase contact angles of non-covalent and covalent nanoparticles were increased to 83.4°and 89.8°,close to 90°,measured by the sessile drop method.The wettability of the particles tended to be hydrophilic and lipophilic balance,which showed the potential as an excellent Pickering emulsifier.Two kinds of Pickering emulsions were stabilized by composite particles prepared by non-covalent and covalent reactions.When the particle concentration increased from 10 g/L to 30 g/L,the size of the emulsion decreased to about 2.23μm,and the microscopic microstructure also verified this concentration effect.The composite emulsion could withstand high temperature(>60℃)and high ionic strength(>600 mmol/L).After storing 28 d,the absolute value of the Zeta potential was still higher than 30 m V,the droplet state remained stable.The above non-covalent and covalent Pickering composite emulsions were used to encapsulate curcumin,and it was found that the initial loading percentage of curcumin by the two emulsions was more than 93.50%.Compared with the single protein emulsion,the retention percentage increased by 1.42 and 1.15 times,the DPPH·scavenging ability increased by 1.27 and 1.39 times,the ABTS~+·scavenging ability added by 1.20 and 1.24 times,and the iron ion reduction ability enhanced by about 1.87 and 1.96 times after 28 days of storage,showing strong loading effect and oxidative stability.In vitro digestion characteristics showed that the release percentage of curcumin in non-covalent and covalent composite emulsions in the stomach were reduced by 24.68%and46.91%,respectively,and could achieve more than 71.34%slow release in the intestine for 4 h,and the bioavailability was significantly increased by 1.39 and 1.25 times.The in vivo model of Caenorhabditis elegans(C.elegans)was constructed.Laser confocal microscopy showed that the autofluorescence intensity of curcumin in C.elegans in the composite emulsion was enhanced,which helped to the intake and accumulation of curcumin.Compared with the single protein emulsion,the non-covalent and covalent composite emulsions reduced the reactive oxygen species level by 27.28%and 32.89%,respectively,the superoxide dismutase activity increased by 30.85%and 33.64%,the catalase activity added by 19.74%and 27.57%,and the malondialdehyde content decreased by 5.66%and 14.61%.The composite emulsion could significantly inhibit the oxidation markers accumulation and reduce oxidative damage by up-regulating the antioxidant enzymes activity.The composite emulsion could weaken the fluorescence expression of lipofuscin and delay the senescence in nematodes within 2 weeks of growth by fluorescence microscope.By inducing oxidation and heat stress,it was found that the composite emulsion helped to enhance the stress resistance of C.elegans and significantly improved their survival percentage.The composite emulsion could effectively protect curcumin,improve its bioavailability in vivo,and help to exert the physiological activity.In summary,through the non-covalent and covalent reactions of casein-caffeic acid,composite nanoparticles with superior emulsifying properties could be obtained,and as an excellent emulsifier matrix,Pickering emulsion with high stability could be obtained.The composite emulsion exhibited high loading percentage and high retention percentage for curcumin,and slow release in the gastrointestinal tract by in vitro simulation experiments.With the help of C.elegans,a convenient and efficient in vivo model,it showed that Pickering composite emulsion contributed to the in vivo delivery of curcumin,effectively exerting physiological activity,improving bioavailability,and demonstrating the application potential as a new delivery system. |
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