Research On The Separation And Purification Of Phosphopeptide And Its Functional Properties

Phosvitin phosphopeptides (PPPs) that contain phosphoserines are kinds of bioactive peptides with many biological functions such as promoting the absorption and utilization of calcium, they have very important research value. Immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as Fe₃O₄ (PEG+CS) @ Fe³⁺) were successfully obtained and applied for the adsorption and enrichment of phosvitin phosphopeptides. And the PPPs'ability to prevent the formation of calcium phosphate deposits was studied in this paper.Firstly, monodispersed and functional immobilized metal affinity magnetic chondroitin sodium sulfate nanoparticles (short as Fe₃O₄ (PEG+CS) @ Fe³⁺) were obtained by employing Fe³⁺ ion solutions for the processing of chondroitin sodium sulfate (CS) nanoparticles (Fe₃O₄ (PEG+CS)) prepared by chemical co-precipitating. The morphology of magnetic Fe₃O₄ (PEG+CS) nanoparticles was observed by transmission electron microscope (TEM). It was found that the nanoparticles have the good nanometer size and superparamagnetic properties. From the FTIR spectra analysis, it can be concluded that CS was decorated on the surface of Fe₃O₄ successfully, and Fe³⁺ was decorated on the surface of the Fe₃O₄ (PEG+CS) successfully.Secondly, solvent extraction was carried on using 95% ethanol. The results showed that the yield of egg yolk lipid was 94.97%. The dried defatted egg yolk powder was suspended in 0.1 M NaOH solution for dephosphorization, the dephosphorization rate of crude egg yolk and phosvitin were 36.7% and 38.6%, respectively. After chymotrptic hydrolysis, the protein hydrolysis degree of crude egg yolk and phosvitin were 13.02% and 14.97%, and the nitrogen/phosphorus molar ratios (N/P) of crude egg yolk peptides and phosvitin peptides were 38.36 and 33.76, respectively.Thirdly, in the PPPs enrichment study, the results obtained emphasize the role of pH, time, the initial concentration of the peptides solution, the temperature and the concentration of Fe₃O₄ (PEG+CS) @ Fe³⁺ magnetic nanoparticles in governing the enrichment efficiency, The nitrogen/phosphorus molar ratio (N/P) which at the maximum enrichment capacity for PPPs was 5.78 when the time was 150min, the concentration of peptides was 5mg/mL, the pH was 4.5, the temperature was 37℃, the concentration of Fe₃O₄ (PEG+CS) @ Fe³⁺ magnetic nanoparticles was 1.5 mg/mL.Traditional organic solvent precipitation method and metal affinity adsorption method was compared. The final nitrogen/phosphorus molar ratios (N/P) of PPPs1 from metal affinity adsorption method was low to 5.78 and the final nitrogen/phosphorus molar ratios (N/P) of PPPs2 from organic solvent precipitation method was low to 14.98. The PPPs'ability to prevent the formation of calcium phosphate deposits was studied in this paper. The subsequent research suggested that PPPs1'ability to prevent the formation of calcium phosphate deposits was obviously better than PPPs2'ability to prevent the formation of calcium phosphate deposits.Finally, Imidazole was used as an eluent to evaluate the repeated use of the Fe₃O₄ (PEG+CS) @ Fe (III) nanoparticles in this study. It indicated that the stability of the Fe₃O₄ (PEG+CS) @ Fe (III) nanoparticles was not so good.