Study On The Covalents Of Four Catechins And Milk ?-lactoglobulin

Since ?-lactoglobulin(?-LG)has multiple binding sites,interactions between ?-LG and other ligands is one of popular studies in recent years.The coexistence of ?-LG and catechins in food systems is very common.At present,there were two forms of binding about proteins and polyphenols,which were non-covalent binding and covalent bonding.A large number of scholars have studied the non-covalent interaction of proteins and polyphenols.Besides,the non-covalent interaction between protein and polyphenols has been studied thoroughly.Covalent bonding has become one of the research hotspots in recent years.The covalent effect was to make irreversible covalent bonds between proteins and polyphenols,and the covalent substance formed were more stable than non-covalent complexes.The covalent substance of ?-LG and polyphenol prepared by alkali method,free radical method,enzyme catalytic method,and so on.The alkali method was widely used in the covalent bonding of protein and polyphenol because it was easy to operate and did not need to add additional catalyst.In addition,the difference structure of catechins will inevitably affect the structure and function of the complex formed by its combination with ?-LG.However,it was still unclear about the influence of different structures of catechin on the structure and functional properties of the covalent substance formed with ?-LG and its mechanism.Therefore,four catechins,including epicatechin(EC),epicatechin gallate(ECG),epigallocatechin(EGC)and Epigallocatechin gallate(EGCG)were prepared to conjugate with ?-LG by alkaline method(pH 9)in this study.Mass spectrometry and electrophoresis were used to study its mechanism.Fluorescence,circular dichroism and other techniques were used to compare its structural changes.The antioxidant assays and indirect ELISA were used to study their functional properties.The main conclusions were as follows:1.The experimental results of binding mechanism: ?-LG can conjugate with four catechins(EC,ECG,EGC,EGCG)under alkaline conditions(pH 9).Different color were formed mainly due to the self-oxidation of catechins.Covalently bound to catechin led to an increase in the molecular weight of ?-LG.One ?-LG molecule could covalently crosslink one EC or EGC molecule and two ECG or EGCG molecules.Covalent binding reaction resulted in a decrease in the sulfhydryl and free amino groups of ?-LG.The decreased degree of sulfhydryl and free amino groups in the complexes of ?-LG-ECG and ?-LG-EGCG was higher than than in the ?-LG conjugated with EC and EGC.The catechin binding equivalent experiment showed that catechin combined with ?-LG of the highest equivalent was ECG,followed by EGCG,EGC and EC.The mechanism of the catechins conjugate with ?-LG under alkaline conditions was presumed to be: catechins were actively oxidized into hydrazine in pH 9 solution.Because of the different catechin structure,the time of oxidation to hydrazine was different.EC and EGC were slowly oxidized to hydrazine.When oxidized to hydrazine,they were covalently bound to ?-LG.However,when EC and EGC continued to oxidize to dimer,?-LG binding site was saturated and no more sites can be bound to the dimers of EC and EGC.But ECG and EGCG were automatically oxidized to hydrazine for a short period of time,which immediately continue to oxidize to a dimer and then covalently bound to ?-LG.2.The experimental results of structural characterization: The covalent substance led to fluorescence quenching which accompanied by the maximum fluorescence intensity red shift.Besides,the quenching intensity of ?-LG-ECG and ?-LG-EGCG were much greater than that of ?-LG-EC and ?-LG-EGC.Fourier transform infrared spectroscopy indicated that the covalent binding of catechin to ?-LG resulted in the shift of characteristic absorption peak of the ?-LG amide A band and the amide I band and the broadening of peak shape.The circular experiment showed that the secondary structure of the four covalent substance changed in the varying degrees,which mainly reflected in the decrease of ?-sheet and the increase of ?-turn and unordered.Furthermore,the tertiary structure also changed in the varying degrees.The covalent attachment of ECG and EGCG to ?-LG results in a bigger change of protein tertiary structural than that in the ?-LG-EC and ?-LG-EGC.Covalent binding of different catechins to ?-LG also led to a change in the hydrophobic environment of the protein,which was manifested by a decrease in hydrophobicity and an increase in hydrophilicity.The surface hydrophobicity of ?-LG-ECG and ?-LG-EGCG was lower than ?-LG-EC and ?-LG-EGC.3.The experimental results of functional characterization: when ?-LG and four catechins covalently combined under alkaline conditions(pH 9),the antioxidant performance was significantly improved.DPPH,ABTS,FRAP experiments showed that the antioxidant properties of ?-LG-ECG and ?-LG-EGCG were superior to those of ?-LG-EC and ?-LG-EGC.At the same time,when ?-LG was covalently bound to four catechins,the antigenic reactivity was increased,which led to an increased risk of sensitization.Additionally,the antigenic responses of the covalent complexes ?-LGECG and ?-LG-EGCG were higher than ?-LG-EC and ?-LG-EGC.These changes were related to the binding form and conformational changes in their covalent binding processes.