Effect Of Tuna Bone Collagen Peptides And Its Calcium Chelates On Osteoblasts Activity

Tuna,as a kind of deep-sea fish,has the title of “ocean gold”.With the advantages of high nutritional value,pure nature and no pollution,tuna is well-known in the international market and welcomed by consumers.However,during processing,a large number of leftovers including fish bones are discarded at will.This not only wastes resources but also pollutes the environment.In this study,we used tuna bone as raw material to prepare collagen peptides by enzymatic hydrolysis and further obtained tuna collagen peptides calcium chelates through chelation process.Meanwhile,the effect and mechanism of bone collagen peptides(BCP)and peptides calcium chelates(Ca-BCP)on osteoblasts activity were investigated,which provided theoretical basis for the development of functional food with BCP or Ca-BCP as efficacy factor.The main research conclusions are as follows:1.Through single factor test and response surface test,the optimal process conditions for preparing BCP by animal protease hydrolysis are as follows: enzyme addition amount2.5 %,enzymolysis time 8 h,material-to-liquid ratio 0.09 g/m L,temperature 53 ?,p H7.0.Under these conditions,the degree of hydrolysis of the enzymatic hydrolysate is 65.43%.2.BCP with different molecular weight could promote the proliferation of MC3T3-E1Subclone14 cells.And through the detection of ALP activity,OCN,COL I content and alizarin red staining experiment,we can know that BCP can promote the differentiation and mineralization of osteoblasts.The low molecular weight of bone collagen peptides(BCP1)have better effect.Furthermore,QPCR assay showed that BCP1 could promote osteoblasts differentiation by up regulating the expression of ALP,OCN and COL I genes.3.Chelate BCP1 with anhydrous calcium chloride,and determine the optimal process conditions for Ca-BCP1 through single factor test and response surface test: peptides calcium mass ratio 3.3:1,p H 6.1,chelating time 2 h,temperature 69 ? and the chelation rate is 94.27 % under these conditions.In addition,the chelates was analyzed by UV and IR spectra.It was found that the formation of Ca-BCP1 was the result of the chelation of carboxyl group,amino group with calcium ion.4.Through the detection of MC3T3-E1 Subclone14 cells proliferation activity,ALP activity,OCN,COL I content and alizarin red staining experiment,it can be known that CaBCP1 can promote the proliferation,differentiation and mineralization of osteoblasts.Also,it can up-regulate the expression of ALP,OCN and COL I genes.5.Both BCP1 and Ca-BCP1 can promote the secretion of OPG and inhibit the secretion of RNAKL in the supernatant of MC3T3-E1 Subclone14 cells.QPCR and Western blot were used to study the effects of the BCP1 and Ca-BCP1 on the expression of OPG,RANKL and RUNX2.It was found that BCP1 and Ca-BCP1 can up-regulate the expression of gene and protein in OPG and RUNX2,inhibit the expression of gene and protein in RANKL.This may be through increasing the ratio of OPG/RANKL to activate the signal pathway,then promote the differentiation of osteoblasts and bone formation.What's more,The effect of Ca-BCP1 is better than that of BCP1.