| Epigallocatechin gallate(EGCG)was one of the abundant catechins in green tea.It had lots of biological activities such as antioxidation,antibacterial activity,and antispasm.Therefore,it could be used as food additives,anticancer drugs and so on.However,EGCG was easily degraded in the air under the light,alkaline environment or high temperature condition.?-lactoglobulin(?-LG)was a major protein in mammalian whey.As a member of the lipocalin family,it could bind and transport a variety of ligands in vitro.Protein-ligand complexes could improve properties of ligand and prevent degradation of ligand.However,the functional properties of ?-LG were easily altered by heat,high pressure,enzymatic hydrolysis and so on.Binding to the ligand also depended on the structure and polymerization state of the protein associated with environmental factors such as pH,temperature and so on.Although there were many studies on the interaction between protein and ligands,the study on the interaction between unfolded-?-lactoglobulin(U-?-LG)and EGCG was unclear.Furthermore,the changes of U-?-LG-EGCG complexes in the digestion process were rarely reported.Therefore,in this study,U-?-LG was obtained after ?-LG treated by microwave.The interaction mechanism of U-?-LG combined with EGCG and structural characteristics was investigated by using fluorescence,ultraviolet and circular dichroism(CD)spectroscopy.Bioavailability of EGCG was investigated by vitro digestion.Structural characteristics of digesta was investigated by using fluorescence and circular dichroism(CD)spectroscopy.The main results were the following three aspects:1.The fluorescence quenching ability of EGCG on U-?-LG is stronger than that of ?-LG.EGCG quenched U-?-LG fluorescence strongly in static mode.EGCG could interact with ?-LG or U-?-LG and then form ?-LG-EGCG complex or U-?-LG-EGCG complex.The polarity of tryptophan and tyrosine residues increased and the hydrophobicity decreased.The binding constant and number of binding sites for the interaction of U-?-LG with EGCG were greater than that of ?-LG.The main interaction force of EGCG binding with ?-LG or U-?-LG was hydrophobic interaction.The donor-acceptor proximity in ?-LG-EGCG complex and U-?-LG-EGCG complex were 3.188 nm and 2.875 nm at 25 ?,respectively,based on the F?rster's theory of non-radiative energy transfer.2.The results of UV-Visible,Synchronous fluorescence and Circular dichroism spectroscopy showed that the polarity of tryptophan and tyrosine residues of ?-LG and U-?-LG increased the and hydrophobicity of ?-LG and U-?-LG decreased after binding with EGCG.The binding site was closer to the tryptophan residue than the tyrosine residue.The binding affinity between U-?-LG and EGCG was stronger than ?-LG and EGCG.The secondary structure of U-?-LG changed more than ?-LG.After forming U-?-LG-EGCG complexes,the ?-helix content of U-?-LG increased,the ?-sheet content and sum content of ?-turn and random coil decreased.However,change of secondary structure of ?-LG binding with EGCG was not significant.It showed that ligand-induced structural changes only occurred near the binding sites.The surface hydrophobicity of ?-LG and U-?-LG decreased after binding with EGCG.However,the surface hydrophobicity of U-?-LG-EGCG complexes was higher than that of ?-LGEGCG complexes at different EGCG concentrations.3.After simulating gastric digestion,the intrinsic fluorescence intensity value of ?-LG-EGCG complex and U-?-LG-EGCG complex slightly increased;the maximum emission wavelength red shifted,and the surface hydrophobicity decreased.The ?-helix content of the ?-LG-EGCG complex increased,and the content of ?-turn and random coils decreased.The ?-sheet content of U-?-LG-EGCG complex increased,and ?-turn angle and random coil content decreased.The EGCG recovery of the ?-LGEGCG complex and the U-?-LG-EGCG complex increased.After simulating intestinal digestion,the intrinsic fluorescence intensity value and surface hydrophobicity of the ?-LG-EGCG complex and U-?-LG-EGCG complex decreased,compared to gastric digesta.The ?-sheet content of ?-LG-EGCG complex decreased and content of ?-turn increased.The ?-helix content of the U-?-LG-EGCG complex increased,and the content of ?-turn decreased.The EGCG recovery of the ?-LG-EGCG and U-?-LGEGCG complexes decreased.However,after stomach digestion and further intestinal digestion,U-?-LG had better protective effect on EGCG than ?-LG and had higher bioavailability.In vitro digestion,U-?-LG could protect EGCG.And protective effect of U-?-LG was better than ?-LG.Bioavailability of U-?-LG-EGCG complex was higher than ?-LG-EGCG complex. |
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