Study Of The Competitive Binding Interactions Between Polyphenols And Amaranth With β-lactoglobulin By Multi-spectroscopic And Molecular Docking Methods

Functional dairy products are rich in bioactive compounds like polyphenols,which are beneficial to human body,but at the same time,in order to improve the appearance of food,the addition of synthetic azo pigment amaranth increases the health risk of food to some extent.The interaction between small molecular bioactive compounds and milk protein in functional dairy products was studied,in order to clarify the micro changes of bioactive components in related products;the scientific research on the competitive effect between small molecules also provides a theoretical basis for the practical application of controlling the absorption of some food additives by processing methods.In this paper,curcumin was selected from the five kinds of polyphenols as the strongest competition effect with amaranth by molecular docking and fluorescence spectrometry;while the effects of interaction with curcumin and amaranth on the secondary structure ofβ-lactoglobulin were studied by UV-Visible absorption spectroscopy,synchronous fluorescence spectroscopy,excitation-emission matrix spectroscopy and Fourier transform infrared spectroscopy;and combined with heat treatment process in the production of functional dairy products,to explore the thermal sensitivity ofβ-lactoglobulin binding with ligand.The results showed that curcumin,resveratrol,quercetin,caffeic acid phenylethanol ester,caffeic acid and amaranth could all form complexes withβ-lactoglobulin.The binding constants of several ligands withβ-lactoglobulin were all in the order of 10⁵～10⁷M^-1,which showed strong binding ability.Among them,amaranth and curcumin bind toβ-lactoglobulin through hydrogen bond and hydrophobic interaction,respectively.In neutral environment,both curcumin and amaranth bind to the site at the entrance ofβ-lactoglobulin hydrophobic cavity and compete with Asn90 with strong competitive effect.The interaction between amaranth and curcumin changed the secondary structure ofβ-lactoglobulin,which showed that curcumin reduced the hydrophilicity of the microenvironment of aromatic amino acid residues inβ-lactoglobulin,and the content of C=O bond inβ-lactoglobulin decreased.The formation ofβ-lactoglobulin-ligand complex improved the stability of the protein,and the thermal denaturation temperature ofβ-lactoglobulin was increased by 2.27℃on account of binding with amaranth.At the same time,binding withβ-lactoglobulin also protected the ligand,and the UV retention rate of curcumin increased by more than 26.25%.Binding to amaranth and curcumin is conducive to the extension ofβ-lactoglobulin’s structure under certain heat treatment conditions,and the ligand to adapt to the change of ambient temperature will change the binding site ofβ-lactoglobulin,in which the heat treatment at 80℃for 20 min directly eliminated the competition between amaranth and curcumin.At the same time,the different heat treatment conditions are accompanied by the change of the thermal force type.However,prolonged high temperature treatment will destroy the stable structure of the complex formed byβ-lactoglobulin and ligand.Therefore,the protection and transportation of bioactive compounds by milk protein in functional dairy products can be controlled by optimizing the processing conditions.