The Studies On The Interaction Of Kiwifruit Glycoprotein And Polyphenol

The interactions of kiwifruit glycoprotein and polyphenol （quercetin, quercetin-3-rhamnoside,thymol, catechol and pyrogallic acid） were studied in this thesis. First, the kiwifruitglycoprotein was extracted by the ethanol leaching extraction, and the response surfaceanalysis was used to optimize the extraction technology of kiwifruit glycoprotein. Secondly,the crude glycoprotein was separated and purificated by Sepharose CL-6B columnchromatography technique with the appropriate elution requirement. Finally, The interactionsof kiwifruit glycoprotein with the plyphenols were separately investigated by fluorescencequenching spectrum、 synchronous fluorescence spectrum and UV-vis absorbancespectrometry under different temperatory, the quenching mechanism was determined, andcalculated the apparent binding constants, binding sites values, thermodynamic parameters,combinding force, binding distance and other relevant parameters, and discussed the thecomformatinal influence of kiwifruit glycoprotein by synchronous fluorescence spectrum andUV-vis absorbance spectrometry. Through the experiment, the following results wereobtained.The optimum conditions of protein extraction was obtained when material/solvent ratio,ethanol concentration, extraction temperature and extraction time were1:6.5（v/v）,24%,24℃and45min through the response surface analysis. Under such conditions, theexperimental maximum protein extracted was63.052%. The extraction times was once. Theoptimum precipitation conditions were ammoniun sulfate addition of40g/100mL, pH of4.2and pricipitated9h, the glycoprotein precipitation rate was94.6%. The crude glycoproteinwas separated and purificated by Sepharose CL-6B column chromatography, twoglycoprotein components were obtained after the purification, kiwifruit glycoprotein Ⅰandkiwifruit glycoprotein Ⅱ.Through the experiments of fluorescence quenching spectrum、synchronous fluorescencespectrum and UV-vis absorbance spectrometry, the following results were obtained. Thefluorescence experiment concentrations of kiwifruit glycoprotein Ⅰand kiwefruitglycoprotein Ⅱ were1×10^-5mol/L and1×10^-4mol/L, the experiment temperatures was27℃and37℃, and water bath time was45min. The results indicated that the interaction ofkiwifruit glycoprotein with polyphenols formed new complex that lead to the fluorescence quenching of kiwifruit glycoprotein Ⅰa nd Ⅱ, the fluorescence quenching mechanismsmostly were static-based quenching of dymamic-static joint quenching, and there wereindividual static quenching process too. The binding constants and the binding sites valueswere determined respectively, the binding constands showen a little strong combin and thebinding sites values were all about1. the interacting forces mainly were Van der Waals forceand hydrogen bonding force, and the Hydrophobic force and electrostatic force were existed.