Research On Preparation And Osteogenic Activity Of Hen Egg Yolk Protein Peptide

Egg yolk contain various kinds of proteins.Egg yolk proteins hydrolysate,known as EYPH,is hydrolyzed from protease under certain conditions.EYPH contain a great deal of active substances,which as reported,have functions like antioxidation,relieving hypertension and combination with some mineral materials.This paper focuses on the preparation of EYPH as the research object.First through the screening of protease hydrolysis and optimization of preparation conditions of EYPH,and study effects of EYPH on osteoblast differentiation and mineralization of MC3T3-E1 reaction and its effect on bone mineralization.This research could be theoretical basis for functional food for promoting bone growth.Results and conclusions are as followed:We cultured the mouse anterior parietal osteoblasts 14(MC3T3-E1).We got the hydrolysate by adding into MC3T3-E1 cultured cells with 4 kinds of protease hydrolysis of egg yolk protein under different concentration to detect the cell proliferation activity and found no relationship between the dose of hydrolysis and their effects on the proliferation of MC3T3-E1 cells.What’s more,high concentration will decrease the osteoblastic cell proliferation promoting effect.After comparing 4 kinds of hydrolysis products on the proliferation of MC3T3-E1 cells,we conclude that the trypsin preparation EYPH on osteoblast proliferation promoting effect is the best,the proliferation of the cells was 130.27%,followed by flavourzyme prepared EYPH,whose cell proliferation activity was 123.69%.We optimized the process of preparing EYPH from trypsin and flavor protein.The results showed that hydrolysate hydrolysis degree(DH)increased as time goes on.High DH not necessarily results in high activity.Hydrolysate afte 6h of hydrolysis have the highest promotion rate for proliferation rate of MC3T3-E1.These hydrolysate ’ s hydrolysis degree is 18.6 %.We optimize the hydrolysis condition for higher proliferation rate of MC3T3-E1.Single factor experiments were carried out for two kinds of protease hydrolysis conditions,as well as the orthogonal experiments of two kinds of enzymes.We find that the best activity was obtained by a single trypsin reaction and the best condition of hydrolysis is 6hs at 37℃,PH 8.0 and rate of enzyme and substrate is 1:50.EYPH was prepared under the optimal hydrolysis condition of trypsin.Then the effect of EYPH on the proliferation,differentiation and mineralization of MC3T3-E1 cells was investigated.EYPH can effectively promote the proliferation MC3T3-E1,the best effect shows up at concentration of 1 mg/m L,the highest proliferation rate was 129.87%.Under the stimulation of EYPH,S phase in cell cycle increased significantly.Determination of ALP activity in osteoblasts showed that EYPH can promote the differentiation of osteoblast,and the activity of ALP increased by 9.1% during 7 days stimulation.Testing of RT-PCR toward Osteogenesis genes showed the positive effect of EYPH on osteoblast differentiation.Besides,alizarin red staining results showed that EYPH can also help the mineralization process of osteoblast.In this test,concentration of 1 mg/mL has better effects than 2 mg/m L.From this series of results,EYPH can be shown to possess osteogenic growth activity.Use Superdex peptides gel column and ultrafiltration to separate the EYPH and compare the effects of different components on MC3T3-E1 proliferation and differentiation.The molecular weight of the most active osteoblasts in the hydrolysate was found to be between 10-30 kDa.Analysis results from automatic amino acid analyzer implied that the active peptide which has the best promotion effect for MC3T3-E1 contains abundant of aspartic acid,glutamic acid,alanine,valine,leucine,tyrosine,lysine and arginine.Through MALDI-TOF-MS analysis,the results showed that there were a large number of common peptide segments in the two constituents,the high abundance peptides include VSGTPVKLSFVR、AVPATEFVTTAVLPEER、AGQFLLDVSQTTVVSGIR 、 LVIESMGINIPSQEYR 、 EKEEGLLPDTLPK.NLRGVDDGADIPR is a difference in the high abundance peptide segment in the 30 kDa component.