Study On Interaction Mechanism Of β-lactoglobulin And β-galactosidase With Phenolic Compounds

Milk tea and all kinds of functional dairy products contained fruit are becoming more and more popular.The tea with milk or adding fruit to milk not only gives milk unique flavor,but also is coincided the requirements of public in nutrition.This work investigated the interaction between β-lactoglobulin(β-LG)and three polyphenols based on multi-spectroscopy and modeling methods.These three polyphenols were chlorogenic acid(CGA),ferulic acid(FA)and epigallocatechin-3-gallate(EGCG).The influence of these three polyphenols on β-LG was also discussed including its surface hydrophobicity,particle size and the ability calcium binding.In addition,the inhibition kinetics and mechanism of EGCG on β-galactosidase(β-Gal)was explored.This study is help to understand the interaction between polyphenols and milk protein or lactose,which has an important significance for the functional dairy products processing and drinking.The main contents of this paper are as follows:In the chapter one,it summarized the definition and physiological function of polyphenols,the structure and functions of protein;described the methods about studying the interaction between small molecules and protein;introduced the research on the interaction between polyphenols and protein in recent years and the significance and innovation of this research.In the chapter two,this work investigated the interaction between CGA,FA and EGCG and β-LG based on multi-spectroscopy and modeling methods.Analysis of the fluorescence quenching data found that all of them quenched the intrinsic fluorescence of β-LG strongly in static mode,and EGCG had stronger binding affinity toward β-LG than CGA and FA.The hydrogen bonding and van der Waals interactions played major roles in the β-LG binding process with CGA and FA,while hydrophobic interaction was the main force with EGCG.The binding distences of β-LG with CGA,FA and EGCG were 4.91 nm,4.92 nm and 4.02 nm.Synchronous fluorescence and EEM spectra showed that these three polyphenols changed the conformation of β-LG;the binding all changed the secondary structure of the β-LG,inducing a-helix to β-structures transition,based on the FTIR and CD results.In the chapter three,this work investigated the influence of CGA,FA and EGCG on β-LG surface hydrophobicity,particle size and ability of binding calcium by fluorescence spectroscopy and particle size measure methods.The experiments on surface hydrophobicity showed that CGA,FA and EGCG might lead to different changes on β-LG surface hydrophobicity: the FA binding with β-LG might increase surface hydrophobicity,while the CGA/EGCG binding with β-LG might destroy the originally hydrophobic structure of the protein.Adding these three polyphenols increased the particle size of β-LG,which further suggested that they could combine with β-LG.In addition,FA promoted β-LG binding with calcium,while CGA and EGCG weakened the binding ability of β-LG with calcium,presumably being related with their influence on β-LG surface hydrophobicity.In the chapter four,we investigated the inhibitory kinetics and mechanism of EGCG on β-Gal by multi-spectroscopy and modeling methods.The inhibitory kinetics results indicated that the β-Gal activity was strongly inhibited by EGCG and exhibited a reversible and mixed type of inhibition on β-Gal.Fluorescence spectroscopy revealed that the interaction belonged to a static quenching mechanism and the main force was hydrophobic interaction.In addition,EGCG could change the polarity,secondary structure and conformation of β-Gal.The resonance light scattering and nanoparticle size data showed that EGCG could increase the particle size and scattering effect of β-Gal by binding with it.Finally,molecular modeling showed that EGCG could prevent substrate interacting with it by entered the hydrophobic active region of β-Gal.