Study On The Phenomenon And Mechanism Of Phosvitin Induced Calcium Mineralization On Chitosan Membrane

Phosvitin is an acidic glycoprotein found in egg yolk.Since phosvitin contains a large amount of serines and most of serines are phosphorylated,which brings strong emulsifiability,antioxidant resistance,and metal ions chelating ability.In this study,phosvitin was used to investigate its role in Ca²⁺mineralization in vitro.On this basis,phosvitin phosphopeptides were obtained by enzymolysis,and then the interaction between Ca²⁺and polypeptides was studied,and the further research was conducted at the cellular level.The main research contents and experimental results are as follows:（1）Chitosan and chitosan composite films with different graphene oxide contents were prepared.The microstructure,secondary structure,and swelling of the composite films were examined by scanning electron microscopy,total reflection infrared spectroscopy,and swelling rate test.Phosvitin induced precipitation of calcium ion on the composite films in the simulated body fluids,and the precipitate was observed by X-ray diffraction and scanning electron microscopy.The results showed that graphene oxide chemically reacted with chitosan,which made the surface of chitosan film roughened,and the cross-section appeared stratified.The swelling property of chitosan was changed,and the swelling rate was reduced by 27.90%when the concentration of graphene oxide was 0.5%.In vitro mineralization,it showed that phosvitin indeed promoted the conversion of minerals from amorphous calcium phosphate to hydroxyapatite during mineralization on graphene oxide/chitosan films.Conclusion:Graphene oxide could improve the properties of chitosan film.Both phosivtin and graphene oxide could promote the precipitation of simulated body fluid.（2）In this study,the graphene oxide/chitosan composite film was prepared and the effects of the phosvitin extracted from the yolk on the physiological activity of MC3T3-E1 cells on the matrix of the composite film were detected by CCK-8,Annexin V/PI double staining and PI staining.The results showed that the chitosan film had no toxic effect on the cells,and increased the activity of MC3T3-E1 cells by 31.92%by increasing the proportion of S cells,inhibiting apoptosis and reducing the apoptosis rate by 52.48%.The graphene oxide/chitosan films could increase the apoptosis rate of cells.The higher the content of oxidation stone,the more obvious the apoptosis could be promoted.The rate of apoptosis increased by 11.31 times when the content of graphene oxide was 2 wt%.On the matrix of chitosan film,100μg/mL phosvitin promoted the proliferation of MC3T3-E1 cells most significantly and the cell activity increased by81.59%,and the percentage of cell apoptosis decreased.The cell activity decreased and cell apoptosis promoted by 4.47%when the concentration of protein was 500μg/mL.Conclusion:chitosan and low concentration of phosphoprotein can promote the proliferation of MC3T3-E1 cells.（3）In order to find the active sites of phosvitin on calcium chelation,considing phosvitin is difficult to be hydrolyzed,different phosivtin phosphopeptides with different degrees of hydrolysis were obtained by enzymatic hydrolysis techniques,and the binding ability of phosphopeptides to Ca²⁺was studied.The results showed that the calcium chelation rate of phosvitin treated with pretreating agent followed high temperature and pressure was 68.57%,which was 62.9%higher than phosvitin.Compared with different time of enzymolysis,products of enzymatic hydrolysis after 10h had the highest binding capacity,the calcium chelation rate reached 48.69%.The secondary structure of both pretreatment samples and enzymolysis samples of phosvitin were changed.After binding to calcium,the microscopic appearance of proteins and peptides appeared granular.Conclusion:Non-enzymatic pretreatment could improve the calcium binding ability of phosvitin.（4）Fluorescence,SEM,CD and FT-IR were used to determine the microscopic appearance and secondary structure changes of PV digestion products and calcium chelate products of different treatment methods.The antioxidant activity of enzymolysis products were evaluated by DPPH scavenging free radical ability.The DPPH scavenging free radical ability of Pre-PV was the strongest with 52.98%.As shown in cell experiments,the treated PV could enhance the proliferation of MC3T3-E1 and inhibit its apoptosis.