Interaction Between Phenolic Acid And Ovalbumin And Application Of Modified Ovalbumin To Construct Transport Vector

Phenolic acid is a kind of plant active ingredient,which widely exists in vegetables and fruits.Because of its many beneficial biological activities to human body,it has attracted extensive attention.However,phenolic acids are poor in water solubility and stability,and their biological activities are greatly affected by adverse environmental factors.Therefore,it is instant to seek for methods to improve the stability and bioavailability of phenolic acids.Nano delivery system is an effective method to protect drug or food functional factors,and many studies have been carried out on it.Ovalbumin accounts for 54%of the protein in egg white.Owing to its high frequency of use in daily life and rich nutrition,ovalbumin has become a model protein in physical and chemical research and is often used as a raw material for building transport systems.In addition,the functional properties of proteins also affect the stability of the nanotransport system,and glycosylation modification is a proven method to improve the functional properties of proteins.At present,there are few reports on the use of glycosylated modified proteins to construct delivery systems.In this study,caffeic acid and ferulic acid,two typical phenolic acid compounds of plants,were selected as the research objects,combined with multiple spectral methods and computer simulation technology to study their interaction with ovalbumin and explore the binding mechanism.Besides,ovalbumin was modified by ultrasonic-assisted glycosylation to improve the functional properties of ovalbumin.In addition,the nano-delivery system of ferulic acid was constructed by using the modified ovalbumin to explore the effects of modified ovalbumin on ferulic acid encapsulation and in vitro digestion cumulative release rate.The main results are as follows:1.The interaction of two phenolic acids（ferulic acid and caffeic acid）with ovalbumin was investigated.Both phenolic acids could quench the endogenous fluorescence of ovalbumin.The binding constants of ferulic acid and caffeic acid to ovalbumin at 25℃were 1.69×10⁴L mol^-1and 5.25×10⁴L mol^-1,respectively,indicating that ferulic acid and caffeic acid had a moderate affinity with ovalbumin.Both the phenolic acids have a binding site on ovalbumin and the main driving forces of the interaction are hydrogen bonding and van der Waals forces.There were no significant differences in hydrophobicity and polarity in the microenvironments of tyrosine and tryptophan residues of ovalbumin in ferulic acid or caffeic acid.The synchronous fluorescence quenching ratio showed that the tyrosine residues were closer to the binding sites of the two phenolic acids to ovalbumin,and caffeic acid had a greater effect on the conformation of ovalbumin than ferulic acid at the same concentration.In addition,the binding of ferulic acid and caffeic acid resulted in a decrease inα-helix content and an increase inβ-sheet content of ovalbumin,resulting in a loose structure of ovalbumin.Molecular simulations showed that both ferulic acid and caffeic acid were attached to the hydrophobic cavity of ovalbumin.Ferulic acid formed hydrogen bonds with Arg84,Asn88,Leu101 and Ser103 residues of ovalbumin,while caffeic acid formed hydrogen bonds with Arg84,Leu101 and Ser103 residues of ovalbumin.Molecular dynamics simulation results revealed that ferulic acid enhanced the stability of protein carbon skeleton of ovalbumin more than caffeic acid,and the solvent accessible surface area did not change significantly before and after the combination of caffeic acid and ferulic acid,and the hydrogen bond played a vital role in stabilizing the complex during the interaction.2.The effects of ultrasonic pretreatment on the glycosylation of ovalbumin were compared,and the effects of ultrasonic assisted glycosylation on the structure and functional properties of ovalbumin were studied.The outcomes revealed that ultrasound could improve the degree of grafting and accelerate the glycosylation process of ovalbumin.The endogenous fluorescence intensity of ovalbumin modified by glycosylation or ultrasonic glycosylation decreased.Circular dichroism spectroscopic analysis showed that glycosylation or ultrasonic glycosylation increased theα-helix content of ovalbumin and made the structure of ovalbumin denser.In addition,the surface hydrophobicity of ovalbumin treated by glycosylation and ultrasound-assisted glycosylation decreased,while the content of free sulfhydryl groups increased.Compared with natural ovalbumin,the denaturation temperature of modified ovalbumin was increased by 9-10℃,indicating that both glycosylation and ultrasound-assisted glycosylation could enhance the thermal stability of ovalbumin.After modification,emulsification and oxidation resistance of ovalbumin were improved greatly.The emulsifying activity and stability of ultrasonic glycosylation were increased by 47.63%and 5.17%,respectively.Ultrasonic cavitation and the formation of advanced glycosylated products are the main reasons for the improvement of these physicochemical and functional properties.3.The preparation and delivery system of modified ovalbumin for ferulic acid and its digestion in vitro were studied.The particle size and polydispersity index were used as measurement indexes to optimize the p H（3,4,5,6,7,8,9）,temperature（70,80,90,100℃）and protein concentration(1,2,3,4,5,6 mg m L^-1)of the preparation system of modified ovalbumin nanoparticles.The optimal preparation conditions were p H 6,protein concentration 4 mg m L^-1and temperature 90℃.The nanoparticles loaded with ferulic acid were prepared under the optimal preparation conditions.When the mass ratio of ovalbumin to ferulic acid was 10:1,the encapsulation efficiency and loading capacity of the nanoparticles prepared by ultrasonic assisted glycosylation of ovalbumin were 69.87%and 22.40%,respectively.Scanning electron microscopy（SEM）observed that the nanoparticles prepared by ultrasonic assisted glycosylation had stable microstructure.In vitro digestion experiments manifested that nanoparticle prepared by modified ovalbumin could significantly increase the stability of ferulic acid and effectively control the release of ferulic acid in intestinal tract.