Preparation And Properties Of Collagen-based Antimicrobial Peptides

Bacteria are a serious threat to health.In recent years,researchers have discovered a new class of antimicrobial agents-antimicrobial peptides.Antimicrobial peptides can not only resist bacteria,but also have low cytotoxicity and good cytocompatibility,so they have potential development.In this study,the antimicrobial peptides with protease-resistant ability were obtained by selective enzymatic hydrolysis of tuna collagen sequences.In addition,we preliminarily investigated the antimicrobial mechanism of the antimicrobial peptides.In order to increase the application range of antimicrobial peptides,this study provides an exploration on their stability,safety and therapeutic effect.Firstly,in order to obtain peptides resistance to enzymatic hydrolysis,tuna collagen sequence was selectively digested by stepwise enzymatic hydrolysis.More specifically,Tuna bone collagen was enzymolyzed by alkaline protease and pepsin,respectively.Tuna bone hydrolysate peptides(TBP)were finally obtained by preparation optimization.The MIC and MBC values of TBP were 4 mg/mL and 8 mg/mL.This study also investigated the safety and stability of TBP.The result of the cytotoxicity and hemolytic toxicity to mouse embryonic fibroblasts(NIH)showed that TBP basically did not have cytotoxicity and hemolytic toxicity at the antibacterial concentration.In addition,it is found that TBP has a certain environmental stability by testing the stability of protease hydrolysis,serum environmental and salt ion existence.In order to explore the antimicrobial mechanism of the antimicrobial peptides,TBP was isolated and purified by using ion-exchange chromatography and gel permeation chromatography.The sequence of TBP was determined by liquid chromatography-tandem mass spectrometry.It is speculated that the antibacterial activity of TBP is related to the special sequence structure of peptide.We also found that TBP has certain cationic characteristics and can exist stably in water by using Heliquest and ProtParam tool to carry out helical projection and related parameter calculation on the special sequence structure.Membrane integrity test and Escherichia coli morphology test,it was found that the killing of Escherichia coli by TBP was closer to carpet damage model.Finally,its antioxidant capacity and the ability to promote osteoblasts proliferation were tested.The IC50 value of DPPH free radical scavenging and ABTS free radical scavenging were 6.50±0.29 mg/mL and 3.97±0.13 mg/mL,respectively.The results of NIH cell injury model induced by H2O2 showed that H2O2 could protect NIH cells.When the concentration of peptides was 1mg/mL,the cell viability increased from 44%to 76%in the control group.Moreover,TBP is demonstrated the ability to promote the proliferation of osteoblasts.Based on the above results,the antimicrobial peptides from tuna bone collagen prepared by the two-time enzymatic hydrolysis have the potential to be used as an antimicrobial agent and to treat bone loss.