关键词: 抗菌肽BCp12；大肠杆菌；生长繁殖；壁膜损伤机制；DNA

Abstract: The milk-derived antimicrobial peptide BCp12, separated from buffalo casein hydrolysate by affinity adsorption to living bacterial cells, has a broad-spectrum antibacterial effect, but its antibacterial mechanism is yet unclear. This study investigated the destructive effect of BCp12 on the cell wall and membrane of Escherichia coli using ultraviolet absorption spectroscopy, flow cytometry, Fourier transform infrared spectrometer (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and the binding capacity and mode between BCp12 and bacterial DNA were determined by gel retardation assay and fluorescence spectroscopy. The results showed that 2 mg/mL BCp12 caused an increase in the hydrophilicity of the cell wall and membrane, and decreased the adsorption rate of bacterial cells to 64.73%. The 2 mg/mL antimicrobial peptide had a significant impact on the fatty acid, protein, peptide amide, polysaccharide and fingerprint information areas of the bacterial cell wall, damaged the cell membrane (the ratio of?cells with membrane damage to total cells was 25.1%), and resulted in leakage of intracellular ultraviolet-absorbing substances. Moreover, BCp12 could make the cell surface rough and wrinkled, and severely damage the internal structure of the cytoplasm and result in cavitation. BCp12 could compete with bromine ethidium bromide (EB) for binding sites and bind to DNA in an intercalated manner, thus resulting in gel retardation, affecting DNA replication and ultimately inhibiting the growth and reproduction of bacteria. The findings of this study partially explained the antibacterial mechanism of BCp12.

Key words: antimicrobial peptide BCp12; Escherichia coli; growth and reproduction; cell wall and membrane damage mechanism; DNA