Purification of novel peptide antibiotics from human milk

A strategy was established for the identification of novel antimicrobial pe ptides from human milk. For the generation of bioactive peptides human milk was acidified and proteolyzed with pepsin simulating the digest in infants stomachs. Separation of proteins and resulting fragments was performed by means of reversed-phase chromatography detecting the antimicrobial activity of each fraction using a sensitive radial diffusion assay. In order to avo id the purification of the known abundant antimicrobial milk protein lysozy me, it was identified in HPLC fractions by its enzymatic activity and by ma trix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). On condition that lysozyme was not detectable and antibacterial activity of HP LC fractions was caused by a peptide, which was confirmed by proteolytic cl eavage leading to a loss of activity, further purification was performed by consecutive chromatographic steps guided by the antibacterial assay. Using this strategy, an as yet unknown casein fragment exhibiting antimicrobial activity was purified in addition to antimicrobial lactoferrin fragments. T he new antimicrobial peptide resembles a proteolytic fragment of human case in-le (residues 63-117) and inhibits the growth of Gram-positive, Gram-nega tive bacteria, and yeasts. Our results confirm that antimicrobially-active peptides are liberated from human milk proteins during proteolytic hydrolys is and may play an important role in the host defense system of the newborn . (C) 2001 Elsevier Science B.V. All rights reserved.