Objective To analyze the biological activity of bioactive peptides in human breast milk and to find the polypeptides so as to investigate the preventive and therapeutic effects of breast milk-derived bioactive peptides on neonatal necrotizing enterocolitis(NEC).Methods Six mothers who gave birth to preterm neonates were enrolled in this study and 5 mL of their breast milk secreted within 2-5 postnatal days were collected for 6 times and blended subsequently.Bioactive peptides from maternal milk of the preterm infants were separated by ultrafiltration and analyzed by using tandem mass spectrometry.Polypeptides possibly with biological function were screened out by using bioinformatics software and the protein function cluster online analysis software was used to predict the polypeptides associated with infection according to the biological function of their precursor proteins.The ATCC25922,an Escherichia coli strain commonly associated with infection in NEC and drug solution (sulbactarr/cefoperazone) were used to conduct the drug susceptibility testing and bactericidal kinetics testing,so as to verify the antibacterial effects of bioactive peptides in the breast milk.Results Four thousand three hundred and eleven peptides contained in breast milk were identified successfully,of which 1 370 were non-differential peptides,and 188 peptides possibly with biological activity and 11 peptides were associated with infection.The peptide compound in the breast milk had antimicrobial activity and bactericidal power against Escherichia coli.Conclusions The active peptide compounds in the breast milk have antimicrobial activity,which play an important role in the prevention of NEC.Finding the true antimicrobial peptides with in vivo and in vitro biological activity by using antimicrobial spectrum test is expected.