| In food industry,flavor agent,colorant,preservatives and nutrients are widely used.However,some bioactive agents are unstable or poor water-soluble,which makes it difficult to achieve expected effect in the application.As a good source of nutrient,protein has potential application in bioactive agents and drug delivery system.Furthermore,the interaction mechanism between protein and ligand determines the construction of the delivery system.Thus,this thesis systematically studied the interaction mechanism betweenβ-lactoglobulin and nobiletin.And the formation ofβ-lactoglobulin-nobiletin complex was uncovered under different conditions.The main finding are as follows:(1)The binding stoichiometry of complex was found to be 1:1 by analysis of intrinsic fluorescence experiment.And the results also confirmed that the binding behavior betweenβ-LG and Nob was a spontaneously endothermic process driving mainly by hydrophobic interaction.The analysis of fluorescence data was supported by isothermal titration calorimetry(ITC)measurement.Moreover,competitive binding and molecular docking method indicated the Nob was primary bound to internal calyx ofβ-LG at neutral pH.Meanwhile,the results obtained by UV spectrophotometry exhibited that the solubility of Nob was enhanced to 3 times by forming complex.Furthermore,Nob could alter secondary structure ofβ-LG by a transition fromα-helix toβ-sheet and lead to a slightly rose on surface hydrophobicity ofβ-LG.(2)Formation and interaction analysis ofβ-lactoglobulin-nobiletin complex under pH regulation.Molecular docking and molecular dynamics simulation were used to study the binding modes under different pH conditions.Fluorescence and CD were used to investigate the change ofβ-lactoglobulin conformation and effect on the urea stability ofβ-lactoglobulin under different binding modes.The results showed the nobiletin selectively bound to the surface hydrophobic pocket ofβ-lactoglobulin at acidic pH.And when complex formed under the neutral pH,the nobiletin still bound to calyx ofβ-lactoglobulin after pH was adjusted to acidic condition.In any case,the formation of complex led to changes ofβ-lactoglobulin conformation.Comparing with binding to surface hydrophobic pocket,the effect of binding into calyx onβ-lactoglobulin conformation was smaller(1%-2%)under the acidic pH.Finally,the addition of nobiletin made the urea stability ofβ-lactoglobulin increase.(3)Study on the interaction mechanism between heat-denaturedβ-lactoglobulin and nobiletin.Fluorescence and CD were used to research the change ofβ-lactoglobulin conformation and interaction betweenβ-lactoglobulin and nobiletin.The binding constant and binding site number were obtained by analysis of fluorescence date.UV-vis spectrophotometer was used to determine solubility of nobiletin bound to heat-denaturedβ-lactoglobulin.The results showed that heat-denaturedβ-lactoglobulin structure was more flexible,resulting in binding constant and binding site number was greater than that ofβ-lactoglobulin.However,when the temperature reached 80 ℃,they would no longer increase.In the aspect of solubility of nobiletin,the results exhibit the same trend.In addition,the surface hydrophobicity of heat-denaturedβ-lactoglobulin increased and reached maximum value at 75℃. |
