The Maillard reaction products (MRPs) of half-fin anchovy hydrolysates and glucose, named as HAHp(9.0)-G MRPs, were fractionated by size exclusion chromatography into three major fractions (F1⁻F3). F2, which demonstrated the strongest antibacterial activity against Escherichia coli (E. coli) and showed self-production of hydrogen peroxide (H₂O₂), was extracted by solid phase extraction. The hydrophobic extract of F2 was further isolated by reverse phase-high performance liquid chromatography into sub-fractions HE-F2-1 and HE-F2-2. Nine peptides were identified from HE-F2-1, and two peptides from HE-F2-2 using liquid chromatography-electrospray ionization/multi-stage mass spectrometry. Three peptides, FEDQLR (HGM-Hp1), ALERTF (HGM-Hp2), and RHPEYAVSVLLR (HGM-Hp3), with net charges of -1, 0, and +1, respectively, were synthesized. The minimal inhibitory concentration of these synthetic peptides was 2 mg/mL against E. coli. Once incubated with logarithmic growth phase of E. coli, HGM-Hp1 and HGM-Hp2 induced significant increases of both extracellular and intracellular H₂O₂ formation. However, HGM-Hp3 only dramatically enhanced intracellular H₂O₂ production in E. coli. The increased potassium ions in E. coli suspension after addition of HGM-Hp1 or HGM-Hp2 indicated the destruction of cell integrity via irreversible membrane damage. It is the first report of hydrolysates MRPs-derived peptides that might perform the antibacterial activity via inducing intracellular H₂O2 production.
Keywords: Maillard reaction products; antibacterial peptide; half-fin anchovy hydrolysates; identification; membrane damage; self-production of hydrogen peroxide.