Study On The Interaction Mechanism Of ?-lactoglobulin B With Three Inhibitory AGEs Active Components And Their Complex Properties

Some foods rich in protein and reducing sugars are prone to non-enzymatic glycosylation and Maillard reactions during processing and storage,ultimately producing advanced glycation end products(AGEs).Not only does it reduce the quality of the food,but it also exacerbates the formation of AGEs in the body after entering the body,which in turn induces chronic diseases such as diabetes,kidney disease,and Alzheimer's disease.Therefore,it has become a research hotspot to add plant polyphenols with antioxidant activity in foods to inhibit the production of AGEs.However,most of the polyphenols have poor solubility and stability,if the polyphenol monomer is added directly to the food,although the formation of AGEs can be inhibited at the initial stage of storage,the inhibition effect is weakened as the storage time is prolonged.Moreover,the color of the food is deepened and the hardness of the food is lowered.In this paper,P-lactoglobulin B(?-LG B)with multiple ligand binding sites is used as a carrier,ferulic acid(FA),quercetin(QT)and vanillic acid(VA),which are plant-derived and have AGEs inhibitory activity were used as ligands.The protein-tri-ligand complex model was established and analyzed,revealing the binding mechanism of ligands and proteins with different properties.At the same time,the effect of protein-ligand complexes on the control of AGEs in different models was studied.It provides a reference for the preparation of protein-polyphenol complexes and the problems that the system produces in controlling foods in AGEs.The main research contents and results are as follows:(1)Molecular fluorescence,ultraviolet absorption spectroscopy,isothermal titration calorimetry(ITC)were used to study the binding types and thermodynamic parameters of?-lactoglobulin B and three polyphenols.The results showed that ferulic acid(FA),quercetin(QT)and vanillic acid(VA)and P-lactoglobulin B were statically bound by hydrogen bonding and van der Waals force,and the binding reaction between them was spontaneous.And all of them are exothermic reactions,?H are-32.27 KJ/mol(FA),-31.09 KJ/mol(QT),-32.27 KJ/mol(VA),respectively.In addition,ferulic acid,quercetin and vanillic acid have one and only one binding site on P-lactoglobulin B,and the binding constants are 5.24×104 L/mol,2.85×104 L/mol,3.74×104 L/mol,respectively.(2)Detailed information on the binding sites of ferulic acid,quercetin and vanillic acid in ?-lactoglobulin B was analyzed using AutoDock software in combination with experimental data and references.The results showed that the binding site of ferulic acid was near the gap of(3-barrel,quercetin was combined with the hydrophobic pocket formed by a-helix and ?-barrel,and vanillic acid was combined with the ligand binding site in p-barrel.There are 9(FA),8(QT)and 7(VA)amino acid residues involved in the binding.(3)The effects of binding ligands on protein secondary structure were investigated by three-dimensional fluorescence spectroscopy.circular dichroism and Fourier transform infrared spectroscopy.The results showed that the combination with ferulic acid and quercetin resulted in the conversion of the a-helix of the(3-lactoglobulin B to the ?-sheet.Binding to vanillic acid resultsed in the conversion of the ?-turn and random coiling of the portion of ?-lactoglobulin B to ?-sheet.(4)The changes in solubility,photostability and thermal stability of ferulic acid,quercetin and vanillic acid after binding to ?-lactoglobulin B were studied by ultra-high performance liquid chromatography.The results showed that the solubility of quercetin and vanillic acid increased by 1844 times and 75 times,respectively,after binding to ?-lactoglobulin B.Their photostability and thermal stability are also greatly improved after binding to ?-lactoglobulin B.(5)The preparation process of ?-lactoglobulin B-tri-ligand complex was studied by fluorescence spectroscopy,isothermal titration calorimetry(ITC)and ultra-high performance liquid phase.According to the results,the binding sequence of the ligand will affect the stability of the protein-tri-ligand complexes,and the optimal binding order is FA>QT>VA or QT>FA>VA.The above two processes are completely spontaneous,and the?H is-215.2 KJ/mol and-226.1 KJ/mol,respectively.The loading rates of the three ligands in the protein-tri-ligand complexes were 94.72%(FA),89.81%(QT)and 94.91%(VA),respectively.(6)The stability,antioxidant activity and inhibitory activity of ?-lactoglobulin B-tri-ligand complexes in different models were investigated by various methods.The results showed that the average particle size of the ?-lactoglobulin B-tri-ligand complexes was smaller than that of ?-lactoglobulin B,and the particle size did not change significantly after 10 days of storage,and the loading efficiency does not change much.Antioxidant experiments showed that the ?-lactoglobulin B-tri-ligand complexes has high antioxidant activity,and its DPPH free radical and ATBS free radical scavenging ability are 154.21 and 163.19 ?M Trolox equivalent(TE)/g samples,respectively.The inhibitory rates of AGEs in serum albumin(BSA)-fructose,BSA-methylglyoxal and arginine-methylglyoxal models were 100.00%,68.30%,and 92.86%,respectively,which indicates that the complexes has a good inhibitory effect on the initial reaction and the terminal reaction of AGEs formation.Based on the above results,?-lactoglobulin B can combine with ferulic acid,quercetin and vanillic acid to form a stable complexes,which improves the solubility and stability of the ligand,and also has significant antioxidant activity and AGEs inhibition activity.The results of this study have important reference value for the development of protein-tri-ligand complexes and the problems of the system in controlling AGEs in foods.