Study On The Preparation And Purification Of Whey Protein-derived Calcium-binding Peptide And Reaction Mechanism

In this thesis,whey protein was hydrolyzed by protease and whey protein hydrolysate-calcium chelate(WPH-Ca)was prepared with CaCl2.Then physicochemical properties and structural characteristics of WPH-Ca,as well as its biological activities in vitro,have been studied.The facilitating calcium absorption effect of WPH-Ca was invested by establishing the Caco-2 cell in vitro absorption model.The specific calcium-binding peptides were obtained and characterized through consecutive chromatography,mass spectrometry and other methods.Ultimately,the Calcium-binding mechanism was investigated through a combined method including spectroscopic methods,physical-chemical analysis and quantum chemical calculation.1.The response surface method was used to investigate the optimized hydrolysis conditions of whey protein with protamex and flavorzyme.The optimum hydrolysis process parameters were as follows:Whey protein concentration was 5.0%(w/v),the ratio of protease to whey protein was 4.0%(w/w),the mass ratios of protamex to flavorzyme(w/w)was 2:1,and the reaction temperature was 49?.The hydrolysate was obtained after the hydrolysis of 7 h,with a hydrolysis degree of 25.92%,possessed the highest Ca-binding capacity.2.To optimize the chelation technology of whey protein hydrolysate with calcium,the response surface method was used.The results showed that the optimum process parameters for the whey protein hydrolysate-calcium chelation were whey protein peptide and CaC12 ratio of 24:1(w/w),whey protein hydrolysate concentration 3.5%(w/v),pH 7.5,reaction time 20 min,and reaction temperature 30?,the maximum chelating rate of whey protein hydrolysate was 92.56±1.70%?3.The physicochemical properties and structural characteristics of whey protein hydrolysate-calcium chelation were researched.By qualitative detection,composition analysis and solubility test,the results proved that whey protein hydrolysate-calcium chelation was polypeptide chelate with high content of calcium,good solubility in aqueous solution with different pH.The structure properties of WPH-Ca were characterized by UV scanning spectrum,fluorescent spectrum,FT-IR measurements,laser diffraction particle size analyzer and atomic force microscope.The results indicated that the principal sites of calcium-binding corresponded to the carboxyl groups and carbonyl groups of whey protein hydrolysate.Moreover,particle size distribution and AFM images showed that the presence of calcium ions caused whey protein hydrolysate folding and aggregating,which led to the formation of WPH-Ca.4.Establishment of Caco-2 cell model.Effect of WPH-Ca on Caco-2 cell proliferation was studied using MTT assay.The result showed that WPH-Ca showed no effect on proliferation of Caco-2 cells in concentration range of 0-15mmol/L.The uptake content of calcium from WPH-Ca was significantly higher than that from CaCl2 or calcium gluconate5.Isolation of peptides from WPH was conducted through ion exchange chromatography(IEC),gel filtration chromatography and RP-HPLC.Four specific calcium-binding peptides were obtained,respectively designated as WPH-1?WPH-2a?WPH-2b and WPH-3.Their corresponding amino acid sequences were Glu-Gly(EG),Phe-Asp(FD),Gly-Tyr(GY)and Tyr-Asp-Thr(YDT).The corresponding calcium-binding activities of WPH-1?WPH-2 and WPH-3 were 67.81?g/mg,73.34 ?g/mg and 79.51 ?g/mg.6.The calcium-binding mechanism was investigated through a combined method including spectroscopic methods,physical-chemical analysis and quantum chemical calculation.The results demonstrated that,calcium ions could form dative bonds with carboxyl and carbonyl oxygen atoms as well as nitrogen-atom from amido bonds,and caused the charge redistribution and conformational change of the peptides,ultimately the calcium-binding peptides were formed.