Co-assembly Modification Mechanism Of Rice Proteins And Application Of Apigenin Loading And Release

Rice proteins（Rice Proteins,RPs）are recognized high-quality plant proteins with excellent hypoallergenicity and physiological activity.The low content of hydrophilic albumin and the high content of hydrophobic gluten result in poor water solubility of RPs,which in turn limits their commercial application in the food field.Chemical,enzymatic and physical methods are the current mainstream modification methods to improve the solubility of RPs,all of which have their own disadvantages,such as low efficiency,structural damage,hidden safety hazards and high cost.In this study,non-covalent interactions were used to prepare food natural glue-rice proteins composite materials（PmRs and SmRs）,and the construction mechanism and solubilization mechanism were analyzed;hydrophobic functional compounds（Apigenin）were loaded with the co-assemblies to explore the effect of the self-assembly system（AP@PmRs and AP@SmRs）;the calcium modified carrier microstructure was used to evaluate the release effect and biological activity of apigenin through in vitro experiments.This study aimed to design an environmentally friendly,safe and convenient protein modification technology,develop a binary or ternary complex and provide a theoretical basis for its application in the fields of food,biology and medicine.The specific research contents are as follows:First,the pH cycle preparation method of modified rice proteins co-assemblies was developed.RPs,Sodium carboxymethyl cellulose and pectin were neutralized after deprotonation at pH 12.0 to obtain stable ternary complex PmRs.The solubility of RPs increased to 24 times.Microscopic morphology showed that the co-assembled effect inhibited the aggregation of RPs,and the hydrophilic groups that were easily hydrogen-bonded to water were fully exposed.Polyacrylamide gel electrophoresis showed that the primary structure of PmRs remained unchanged,and its structure was maintained by non-covalent action.Circular dichroism spectroscopy,fluorescence spectroscopy,ANS fluorescence spectroscopy,and ultraviolet spectroscopy showed that the disaccharide was combined with RPs through hydrophobic interaction at pH 9.0,and the hydrogen bonding effect made the RPs resistant to folding during the neutralization process,and part of the structure was unfolded.Zeta potential and surface hydrophobicity confirmed that PmRs exposed charged groups and had colloidal stability.RPs and shellac were co-solvent through pH cycle to obtain binary complex SmRs.The solubility of RPs increased from 1.74%to 90.26%.Polyacrylamide gel electrophoresis showed that the primary structure of the protein remained unchanged,and non-covalent interactions involved in co-framed binding.Fourier transform infrared spectroscopy,X-ray diffraction,autofluorescence spectroscopy,and ANS hydrophobic probe fluorescence spectroscopy showed that shellac binded to RPs between pH 12.0 and 10.0through hydrophobic interaction,and the electrostatic interaction between hydrophobic groups resisted co-folding.The resistance to folding increased with the increase of shellac and there was a concentration dependence of shellac.The decrease in surface potential and the increase in surface hydrophobicity during the pH cycle confirmed that controlling the folding process of RPs was beneficial to improve the structure.Secondly,the self-assembly behavior of apigenin-loaded co-assemblies and the modification of the carrier structure were studied.Through a single factor experiment,SmR1.0 was determined to be the best carrier for loading apigenin.When apigenin was initially put into 0.2%（w/v）,the maximum loading was 91.2 mg/g,and the maximum loading rate was about 90%.X-ray diffraction verified that the crystallinity disappeared after apigenin was loaded.0.1 mmol/L or 0.2 mmol/L Ca²⁺induced SmRs to shield the surface charge,and the adjacent particles were connected to each other,so that the spherical structure turned into a ribbon or a network.The carrier structure had little effect on the loading performance.Finally,the in vitro digestibility and biological activity of apigenin-loaded co-assemblies was characterized.Simulated gastric digestion experiments showed that the release rate of apigenin reached 75%～95%within 0～10 min due to the influence of shellac content.In vitro cell uptake experiments showed that the affinity between SmR1.0 and HepG-2 cells was very strong.Cell activity experiments showed that apigenin had a concentration-dependent anti-proliferative effect,and AP@SmRs had higher cytotoxicity.The microstructure of the carrier affected cell activity,in the order of sphere<ribbon<reticulate.