Novel Antibacterial Peptides Isolated from the Maillard Reaction Products of Half-Fin Anchovy (Setipinna taty) Hydrolysates/Glucose and Their Mode of Action in Escherichia coli

Abstract

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.

Figure 1

Isolation of HAHp(9.0)-G MRPs and activity of separated fractions. (A) The size exclusion chromatography (SEC) of MRPs isolated by high performance liquid chromatography (HPLC) method, detected at 220 nm. (B) The percentage inhibition of isolated fractions against E. coli cells. (C) H₂O₂ self-produced concentration of isolated fractions. The actual peptide concentration of isolated fractions was 0.18 mg/mL in the percentage inhibition and H₂O₂ production assays. Spots in (B,C) represent the raw data. The results are expressed as the mean ± standard deviation (n = 3). Different letters (a–c) in (B,C) represent significant differences among isolated fractions (p < 0.05).

Figure 2

Purification of active fraction F2 using reverse phase high performance liquid chromatography (RP-HPLC) and the activities of sub-fractions assay: (A) percentage inhibition of hydrophilic and hydrophobic extracts of F2; (B) chromatogram of active fraction F2 by RP-HPLC, measured at 280 nm; (C) percentage inhibition of F2-1 and F2-2; and (D) H₂O₂ production capacity of F2-1 and F2-2. Spots in (A,C,D) represent the raw data. The results are expressed as the mean ± standard deviation (n = 3). The symbol of “**” and “*” in (A,C,D) represent significant differences of p < 0.01 and p < 0.05, respectively.

Figure 3

ESI-MS and helical wheel projection of synthetic peptides: (A) HGM-Hp1, FEDQLR; (B) HGM-Hp2, ALERTF; and (C) HGM-Hp3, RHPEYAVSVLLR. The helical wheel projection of HGM-Hp1, HGM-Hp2, and HGM-Hp3 (insert diagrams in Figures (A–C)) were performed using the online website tool (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). In helical wheel projection, circles and diamonds represent hydrophilic and hydrophobic residues, respectively. The green color, whose intensity decreases proportionally to the hydrophobicity, represents the most hydrophobic residue. Non-hydrophobic portions are encoded in yellow. The red color is used to encode hydrophilic residues, whose intensity represents the extent of hydrophilicity. The charged residues are encoded in light blue. Negatively charged and positively charged residues are displayed as triangles and pentagons, respectively. The hydrophobic moment is denoted in the center.

Figure 4

CD spectra of synthetic peptides in membrane-mimicking solution (1.6 mmol/L sodium dodecyl sulfate (SDS), dissolved in 10 mmol/L PBS, pH 7.4): (A) HGM-Hp1; (B) HGM-Hp2; and (C) HGM-Hp3. The peptide concentration was 0.5 mg/mL.

Figure 5

Antibacterial activity and H₂O₂ self-production of synthetic peptides: (A) percentage inhibition against E. coli cells; (B) H₂O₂ self-production of synthetic peptides at the actual peptide concentration of 0.25 mg/mL; and (C) percentage inhibition of synthetic peptides, nisin A and ε-poly-lysine against E. coli at different concentrations. Raw data in (A,B) are displayed as spots. The results are expressed as the mean ± standard deviation (n = 3). Different letters (a–c) in (A,B) indicate significant differences among samples (p < 0.05).

Figure 6

Antibacterial activity of synthetic peptides on the logarithmic growth phase of E. coli cells: (A) percentage inhibition; (B) extracellular H₂O₂ concentration; (C) intracellular H₂O₂ concentration; and (D) extracellular potassium ion (K⁺) content. Raw data are displayed as spots. E. coli cells and peptides treated under the same conditions were used as the bare bacteria and peptide control, respectively. The results are represented as the mean ± standard deviation (n = 3). Different letters (a–d) indicate significant differences among samples (p < 0.05).