Study On The Affinity Absorption Separation Of Bovine Bone Collagen Peptides And It Promotion Of Osteogenic Activity

Bovine bone collagen peptides are highly valued in the food,health,and medical industries due to their biocompatibility,bioavailability,and functional activity.To enhance the product’s calcium holding capacity,promote bone activity,and increase its value,bovine bone collagen peptides were isolated and purified using affinity adsorption.The components were screened based on their calcium chelating ability and their structure was analyzed.The purified components were then evaluated for their ability to promote bone activity,and the results were as follows:1.Affinity adsorption separation of bovine bone collagen peptides.Bovine bone collagen peptides were isolated and purified using a hydroxyapatite column.The optimal elution conditions were determined by optimizing the elution conditions as follows:5 mmol/L K3PO4 equilibrium;Eluent a,1 mol/L KCl(pH=7.0);Eluent B,100 mmol/L K3PO4(pH=8.0).Finally,the components F1 and F2 were isolated and purified.The calcium chelating ability of F1 and F2 was measured,and the results showed that F2 had significantly higher calcium chelating ability than F1.To investigate the cause of this phenomenon,molecular weight distribution measurement and amino acid composition measurement were conducted.The molecular weight distribution of the purified component was significantly increased compared to that of the unpurified component at 1-3 kDa,as revealed by the measurement results.Furthermore,the amino acid composition of F2 had changed,with a significant increase in the content of acidic amino acids.These results suggest that changes in amino acid composition and molecular weight distribution may account for the observed increase in calcium chelating ability.2.This study aimed to prepare and characterize bovine bone collagen peptide-calcium chelate.Based on previous research,the chelation reaction was conducted at a reaction temperature of 60℃,a peptidecalcium ratio of 1:1(M/M),a reaction time of 60 min,and a reaction solution pH of 7.The structure of the purified sample and the prepared calcium peptide chelate were analyzed to determine changes in amino acid composition,molecular weight distribution,and other indicators of each sample and to analyze the calcium binding mode.Fourier transform infrared spectroscopy and fluorescence spectroscopy were used to investigate the formation of the peptide-calcium chelate,which was confirmed by significant changes in characteristic peaks.Scanning electron microscopy was used to observe the microstructure of bone collagen peptide before and after chelation,revealing significant changes in surface characteristics after calcium chelation.The amino acid composition results showed a significant decrease in the contents of Gly,Pro,Ile,Leu,and Phe after calcium chelation,whereas the content of amino acids such as Asp and Lys increased significantly,suggesting that the hydroxyl groups of Asp and Glu may be the main chelating sites of the peptide-calcium chelates.Mass spectrometry was used to support these findings,and Autodock was employed to analyze the chelating mode of bone collagen peptide with calcium,indicating that bone collagen peptide may bind with calcium ion through the amino or carboxyl groups on the amino acid residues of Asp,Lys,and others.3.The study aimed to investigate the effects of purified(F2)and unpurified collagen peptides(CPs)extracted from bovine bone on the proliferation,differentiation,and mineralization of MC3T3-E1 cells,which are a type of osteoblast.Results showed that both CPs and the purified component F2 significantly promoted osteoblast activity compared to the control group.Furthermore,F2 exhibited stronger osteogenic activity than CPs.To elucidate the underlying mechanism,the amino acid sequence of F2 was analyzed using Nano-HPLC MS/MS,and a peptide structure file was generated through structural simulation.Molecular docking was then used to identify the binding of F2 to relevant receptors,which revealed that the identified peptide can bind to transforming growth factor beta receptor I(TGFBR1A)and bone morphogenetic protein receptor(BMPR1A),suggesting that the peptide may promote bone activity through BMP and TGF-β signaling pathways.