Analyses On The Interaction Between Rice Glutelin And Small Molecule Bioactive Components With Or Without Dynamic High Pressure Microfluidization Using Spectroscopy And Molecular Simulation

The interactions of native and unfolded rice glutelin(RG)with small molecule bioactive components,epigallocatechin gallate(EGCG)and conjugated linoleic acid(CLA),were studied in this paper,respectively.The objective of this research was to focus on the binding mechanism of the interactions between protein macromolecules and small bioactive molecules,the differences of thermodynamic parameters and the conformational changes of RG.The interaction between RG and EGCG was investigated.Ultraviolet spectroscopy,fluorescence spectroscopy,circular dichroism and molecular docking simulation were used to investigate the binding mechanism and protein structure between RG and EGCG.The main results are as follows: EGCG could inhibit the fluorescence of RG,which preliminarily proved the combination of RG and EGCG.Thermodynamic calculations showed that the hydrogen bonding was the main driving force in the interaction between EGCG and RG and the process was spontaneous.Fluorescence emission spectroscopy indicated that the surface hydrophobicity of RG decreased with the increase of EGCG concentration in RG-EGCG mixed system.Circular dichroism and simultaneous fluorescence spectroscopy provided more information on the conformation of RG and the microenvironmental changes of RG-EGCG system.The ratio of α-helical structure reduced and the ratio of random coil structure increased during the interaction.The binding site was simulated between the amino acid residue of RG and EGCG by molecular docking technology.The interaction between RG and CLA was investigated.The intrinsic fluorescence intensity of RG was quenched by increasing CLA concentrations,which preliminarily indicated the interaction between RG-CLA mixed system.Thermodynamic analysis showed that the RG-CLA binding process also occurred spontaneously,and hydrogen bonding and van der Waals force were the main driving forces.It was calculated by fluorescence spectroscopy that there is only one binding site between RG and CLA.The surface hydrophobicity of RG was decreased with increasing CLA.Circular dichroism and simultaneous fluorescence spectroscopy indicated that microenvironmental changes existed in the RG-CLA solution.After the interaction of RG and CLA,the α-helix rose while the β-sheet was decreased.Molecular simulation programs displayed a target site where CLA and amino acid residues of RG might bind on.The correlation between the conformation,microstructure and functional characteristics of RG treated by dynamic high pressure microfluidization techniques(DHPM)under different pressures was further investigated.The study found that DHPM treatment did not disintegrate the subunits of RG.The particle size of the DHPM-treated RG solution was reduced.After treatment with 40 MPa and 80 MPa,the solubility of RG was improved,the emulsifying activity index(EAI)was increased,and the emulsifying stability index(ESI)was decreased with increasing pressure.In terms of structure,the β-sheet content of RG decreased at first and increased subsequently.Combined with the observation of atomic force microscopy,it was speculated that RG partially unfolded under the DHPM treatment of 80 MPa,and when the pressure rose to 160 MPa,the protein molecules was aggregated.The unfolded RG was prepared by DHPM at 80 MPa,and the potential interaction between unfolded RG and EGCG was investigated by fluorescence spectroscopy.The result showed that EGCG could still act as a quencher for unfolded RG,indicating that EGCG has combined with unfolded RG.Thermodynamic analysis showed that the hydrogen bonds dominated the interaction spontaneously,and the binding constant of unfolded RG with EGCG was 14.25 times that of natural RG at 298 K.Fluorescence analysis showed that the increase of the surface hydrophobicity was caused by the DHPM treatment under 80 MPa,and the surface hydrophobicity of the unfolded RG-EGCG solution was reduced with the increase of EGCG concentration.The interaction of unfolded RG and CLA was investigated using a fluorescence spectroscopy system.The results showed that CLA could still quench the fluorescence intensity of unfolded RG.Thermodynamic analysis showed that the spontaneous binding of unfolded RG-CLA dominated by hydrogen bonding,and the number of binding site of native RG and unfolded RG with CLA kept constant at 298 K.The binding constant of unfolded RG and CLA was 2.98 times that of native RG.Fluorescence analysis showed that the surface hydrophobicity of unfolded RG was decreased with the increase of CLA,which was caused by the change of the microenvironment of amino acid residues.