Phosphorylation Modification Of Collagen Peptide And Effect Of Its Calcium Chelate On Proliferation And Differentiation Of Human Osteoblasts

Hypophthalmichthys molitrix bone is the main solid by-product produced during the processing of aquatic products such as surimi.It is often discarded directly as waste or processed into fertilizer and feed,which causes great waste of resources and environmental pollution.Fish bone is rich in collagen,which is a safe source.In this paper,collagen peptides were prepared by enzymatic hydrolysis of white mullet bone,phosphorylated by pyrophosphate to obtain phosphorylated collagen peptides,and the preparation process,structural characteristics of phosphorylated collagen peptides,and the structure after binding with calcium was explored.Finally,the effect of collagen peptide chelated calcium and phosphorylated collagen polypeptide chelated calcium on the proliferation and differentiation of human osteoblasts was compared.The results are as follows:1.The optimal process conditions for preparing phosphorylated collagen peptide were determined by single factor experiment and response surface experiment:mass ratio of sodium pyrophosphate to collagen polypeptide was 1:2,reaction temperature was 83°C,reaction pH was 5.9,reaction time 11.35 h,the degree of phosphorylation under this condition is 0.4592 g/100g.The order of influence of various factors on the phosphorylation degree of collagen polypeptide:reaction temperature>reaction pH>reaction time>mass ratio of sodium pyrophosphate to collagen polypeptide.2.The structure of phosphorylated collagen polypeptides is characterized by modern instruments.High performance liquid chromatography showed that the molecular weight of the phosphorylated modified collagen peptide increased significantly.UV and endogenous fluorescence spectra indicated that the collagen peptide structure was loose at high temperature.The introduction of phosphate ions causes the esterification reaction to occur,and the interwoven network structure inhibits the unfolding of the structure to some extent.Infrared spectroscopy and ³¹P nuclear magnetic resonance spectrum confirmed that the phosphate ion was successfully grafted into the collagen polypeptide molecule and bound to the collagen polypeptide in the form of polyphosphate.3.Determination of calcium mass fraction and electron microscopy showed that the ability of phosphorylated modified collagen peptide to bind calcium ions was significantly enhanced.Infrared spectrum analysis showed that collagen peptide was linked to calcium ion through-NH₂ bond and-COOH bond.In addition to this connection,phosphorylated collagen peptides participate in the binding of calcium ions through phosphate groups.The calcium content of collagen polypeptide chelated calcium and phosphorylated collagen polypeptide chelated calcium after gastrointestinal simulated digestion in vitro was above 70%,indicating that they all have certain digestion stability.4.By detecting the relative proliferation rate,alkaline phosphatase staining rate,alkaline phosphatase activity,calcium nodule count and osteocalcin content of human osteoblasts after injection,it is known that both the collagen polypeptide chelated calcium and the phosphorylated collagen polypeptide chelated calcium have a promoting effect on the proliferation and differentiation of human osteoblasts.The order of influence is as follows:phosphorylated collagen polypeptide chelated calcium>collagen polypeptide chelated calcium>phosphorylated collagen peptide>collagen polypeptide,indicating that collagen peptide,calcium ion and phosphate ion play an important role in the proliferation and differentiation of human osteoblasts.