| As the main undertaker and executor of life activities,protein is widely used in food system due to its high nutritional value and many functional properties.The functional properties of proteins depend not only on individual protein,but also on the synergistic interactions between proteins and various substances(e.g.proteins-proteins,proteins-polysaccharide,protein-phenol,etc.).These interactions affect the flavor,color,and nutritional function of a food.Zein andβ-lactoglobulin(β-LG)are two of the most common natural proteins,both of which have good biodegradability and biocompatibility.Both of them have been widely studied as drug carriers.Gum Arabic(GA)is an amphiphilic polysaccharide with complex molecular structure,and it is widely used in the food industry because of its good emulsification,thickening ability and low digestibility.Epigallocatechin gallate(EGCG)is the main component of tea polyphenols,which has many excellent effects such as anti-oxidation,anti-tumor and so on.However,EGCG is vulnerable to environmental factors and its properties are not stable enough,resulting in its low bioavailability,which greatly limits the application of EGCG in food,medicine and other fields.How to reveal the interaction mechanisms between proteins-proteins,proteins-small molecule and proteins-polysaccharide-small molecule at the molecular level still needs to be further explored,which can provide an important theoretical basis for the design of carriers for active molecules.In this thesis,the interaction mechanisms between zein andβ-lactoglobulin,two proteins and EGCG were studied by multi-spectroscopy and molecular dynamics simulation.Zein-Arabic Gum(GA)complex was constructed to encapsulate and protect EGCG in order to improve the encapsulation efficiency and bioavailability of EGCG.The main conclusions are as follows:(1)The interaction mechanism between Zein andβ-LG was studied by experiments combined with molecular dynamics simulation.When the mass ratio ofβ-LG to Zein changed from 9:1 to 2:8,the size ofβ-LG-Zein complex nanoparticles increased from 69.5 nm to 153.5nm.The complex nanoparticle showed good stability after 30 d of storage.Transmission electron microscope(TEM)showed thatβ-LG-Zein nanoparticles dispersed evenly whenβ-LG content was high.Fourier transform infrared spectroscopy(FTIR)and nanoparticle dissociation test showed that hydrogen bonding,hydrophobic and electrostatic interactions played important roles in the formation ofβ-LG-Zein complex nanoparticles.The structural model of Zein was constructed by homology modeling,and then the structural model ofβ-LG-Zein complex was obtained by molecular docking.Molecular dynamics simulation(MD)results showed thatβ-LG firmly grasped Zein like a clamp taking the P68 and G88 residues of Zein as the support point,and the binding Gibbs free energy reached-39.81 kcal/mol.In addition,the residues of V64,P65,P68,I69,G74,G75,G77 and G88 in Zein and the residues of P54,L103 and A104inβ-LG played critical roles in the binding of Zein andβ-LG.(2)Zein and Zein-EGCG models were constructed by homology modeling and molecular docking,and the detailed binding mechanism of Zein to EGCG was further investigated by multi-spectroscopy and molecular dynamics simulation.The results showed that the quenching of Zein by EGCG was mainly static quenching,and the secondary structure of Zein was slightly changed after the binding of EGCG to Zein.The formation of Zein-EGCG complexes was confirmed by ultraviolet-visible(UV-Vis),X-ray diffraction(XRD)and scanning electron microscopy(SEM).Molecular dynamics simulation clarified that the EGCG preferred to bind to the pocket of Zein formed by residues Y171,Q174,L176 and L205.Electrostatic and van der Waals interactions played a dominant role for the binding of EGCG to zein,which was consistent with the results of FTIR and thermodynamic analysis.(3)The pH values have an important influence on the aggregation state ofβ-LG,and the pH-dependent interaction mechanisms betweenβ-lactoglobulin and EGCG were explored by multi-spectroscopy and molecular dynamics simulation.The increase of absorbance and the blue shift of the maximum wavelength were observed in the UV-Vis spectroscopy,confirming the formation of theβ-LG-EGCG complexes.Fluorescence data showed that the quenching ofβ-LG by EGCG was mainly static quenching under three different pH(2.5,5.3 and 7)conditions,and the binding affinity from high to low was pH 7(K_a=1.83×10~4)>pH 5.3(K_a=1.6×10~4)>pH2.5(K_a=1.1×10~4),which was consistent with the results of molecular dynamics simulation.FTIR and circular dichroism(CD)studies showed that the interaction between EGCG andβ-LG resulted in slight changes in the secondary structure ofβ-LG.Molecular dynamics simulations indicated that EGCG preferred to bind to the binding pocket ofβ-LG at pH 7(dimer)and 5.3(tetramer),which consisted of two I lamellae and anα-helix.However,the binding site of EGCG at pH 2.5(monomer)was located on the outer surface ofβ-LG due to the closure ofβ-barrel structure ofβ-LG.(4)In order to encapsulate and protect EGCG,the Zein-GA complex nanoparticles were prepared using Zein and GA as wall materials.The effect of Zein:GA mass ratios on the physicochemical properties of Zein-GA-EGCG complex nanoparticles was investigated.The results showed that the complex nanoparticles were stable within the studied mass ratios(Zein:GA=5:1,3:1,2:1,1:1,2:3,1:2).The EGCG encapsulation efficiency reached the maximum of 75.23%when the mass ratio of Zein to GA was 1:1.The results of FTIR and XRD showed that the Zein-GA-EGCG complex nanoparticles were formed among Zein,GA and EGCG through electrostatic,hydrogen bonding and hydrophobic interactions.In vitro simulated gastrointestinal digestion experiments showed that Zein-GA complex nanoparticles had better sustained release effect on EGCG than that of only Zein nanoparticles.Therefore,using Zein-GA complexes to encapsulate EGCG can effectively improve the encapsulation efficiency of EGCG and achieve the purpose of sustained release in simulated gastrointestinal tract. |
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