Isolation, identification, and mode of action of antibacterial peptides derived from egg yolk hydrolysate.

Egg yolk is a coproduct of egg white processing. The protein hydrolysis of egg yolks to exhibit antimicrobial activity is a strategy for its valorization. The objective of this study is to fractionate antibacterial peptides from pepsin-hydrolyzed egg yolks using flash chromatography. In addition, the mode of actions of the fractionated peptides were elucidated and plausible antibacterial peptides were reported. The fraction 6 (F6) obtained from a C18-flash column exhibited antibacterial activity against Staphylococcus aureus ATCC 29213 and Salmonella typhimurium TISTR 292 at minimal inhibitory concentration (MIC) values of 0.5 to 1 mmol/L (Leucine equivalent). The fractionated peptides induced DNA leakage as monitored by 260 nm. Propidium iodide and SYTO9 staining observed under a confocal microscope suggested the disintegration of cell membranes. Synchrotron-based Fourier-transform infrared spectroscopy analysis revealed that the egg yolk peptides at 1 × MIC induced an alteration of phospholipids at cell membranes and modified conformation of intracellular proteins and nucleic acids. Scanning electron microscopy revealed obvious cell ruptures when S. aureus was treated at 1 × MIC for 4 h, whereas damage of cell membranes and leakage of intracellular components were also observed for the transmission electron microscopy. Egg yolk peptides showed no hemolytic activity in human erythrocytes at concentrations up to 4 mmol/L. Peptide identification by LC-MS/MS revealed 3 cationic and 10 anionic peptides with 100% sequence similarity to apolipoprotein-B of Gallus gallus with hydrophobicity ranging from 27 to 75%. The identified peptide KGGDLGLFEPTL exhibited the highest antibacterial activity toward S. aureus at MIC of 2 mmol/L. Peptides derived from egg yolk hydrolysate present significant potential as antistaphylococcal agents for food and/or pharmaceutical application.

Anti-Salmonella Activity of a Novel Peptide, KGGDLGLFEPTL, Derived from Egg Yolk Hydrolysate.

The present study aimed to characterize the mode of action of a novel antimicrobial peptide isolated from egg yolk hydrolysate. The EYHp6, KGGDLGLFEPTL, exhibited inhibition against Salmonella enterica serovar Typhimurium TISTR 292 and S. enterica serovar Enteritidis DMST 15679 with a MIC value of 2 mM. In contrast, S. enterica serovar Newport ATCC 6962 and other strains of Typhimurium and Enteritidis were inhibited at 4 mM. EYHp6 increased the cell membrane permeability of S. Typhimurium TISTR 292, leading to DNA leakage. Membrane integrity determined by propidium iodide and SYTO9 staining visualized by confocal microscopy demonstrated that EYHp6 at 1 × MIC induced disruption of cell membranes. Electron microscopy revealed that treatment of S. Typhimurium with EYHp6 led to damage to the cell membrane, causing the leakage of intracellular contents. Synchrotron-based Fourier-transform infrared spectroscopy indicated that EYHp6 killed S. Typhimurium by targeting fatty acids and nucleic acids in the cell membrane. The peptide did not show hemolytic activity up to 4 mM. These findings suggest that EYHp6 could be a promising antibacterial agent for controlling the growth of S. enterica.

Boesenbergia rotunda (L.) Mansf. extract potentiates the antibacterial activity of some ß-lactams against ß-lactam-resistant staphylococci.

OBJECTIVES: The purpose of this study was to investigate the effect of Boesenbergia rotunda (L.) Mansf. extract (BRE) and peptidoglycan inhibitor antibiotics, alone and in combination, against ß-lactam-resistant staphylococci. METHODS: Antibacterial and synergistic activities of BRE alone and in combination with ampicillin (AMP), cloxacillin (CLX), cefazolin (CZO) or vancomycin (VAN) were evaluated against two ß-lactam-resistant Staphylococcus aureus (BRSA) isolates and one ß-lactam-resistant Staphylococcus epidermidis (BRSE) isolate. The activities were confirmed by killing curve assays. The preliminary antimicrobial action was elucidated by transmission electron microscopy (TEM) and cytoplasmic membrane (CM) permeability assay. RESULTS: All tested staphylococci were inhibited by BRE at a minimum inhibitory concentration (MIC) of 16µg/mL. Two BRSA strains showed high resistance to CLX, AMP and CZO, whilst BRSE was resistant to CLX and AMP. All tested isolates remained susceptible to VAN. Chequerboard assay demonstrated a fractional inhibitory concentration index (FICI) of 0.50 for the BRE+CLX combination against the BRSA strains. Killing curve determinations confirmed the antibacterial and synergistic activities. TEM revealed collapse of the CM in BRE-treated cells and damage both of the CM and peptidoglycan (PG) in BRE+CLX-treated cells. The CM permeability assay showed that either BRE or nisin alone as well as BRE+CLX significantly induced leakage of OD260nm-absorbing materials. CONCLUSIONS: BRE potentiated the activity of ß-lactams, particularly CLX, against ß-lactam-resistant staphylococci by damaging the CM and PG layer, leading to leakage of intracellular material. Combination of BRE and ß-lactams provides a potential way forward in developing novel antistaphylococcal agents.