Critical Concentration of Lecithin Phospholipids Synergistically Improves the Antimicrobial Activity of Eugenol against Escherichia coli

Open Access
Author:
Zhang, Haoshu
Graduate Program:
Food Science
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
December 15, 2017
Committee Members:
  • Federico Miguel Harte, Dissertation Advisor
  • Federico Miguel Harte, Committee Chair
  • Edward G Dudley, Committee Member
  • John Neil Coupland, Committee Member
  • Yong Wang, Outside Member
Keywords:
  • lecithin
  • essential oil
  • e. coli
  • synergistic
Abstract:
Essential oils (EOs), such as eugenol, are natural antimicrobials from plants. They are receiving increasing attention, due to the challenges to create “all natural” and “clean labels” foods. However, the application of EOs in foods is still limited by their hydrophobicity and strong aroma. Therefore, researchers are developing methods to enhance the antimicrobial properties of EOs, resulting in lower amount of needed EOs. Lecithin is a mixture of different amphiphilic phospholipids. It has been used in a wide range of foods as an emulsifier (>100 mg/100 mL) to improve the food physical stability without known bioactive effects. However, two preliminary studies from our group demonstrated that lecithin phospholipids at concentrations below 10 mg/100 mL increased the antimicrobial property of eugenol against bacteria, without affecting the physical stability of EOs. The present study focused on demonstrating the ability of lecithin and different individual lecithin phospholipid at low concentrations (<10 mg/100 mL) to enhance the antimicrobial activities of eugenol against three E. coli strains and to elucidate the underlying mechanisms. In the first study, the effect of lecithin on the antimicrobial performance of a constant eugenol concentration was tested against three E. coli strains (C600, 0.1229, and O157:H7 strain ATCC 700728). For all three cultures, significant synergistic antimicrobial effects were observed when E. coli cultures were exposed to a constant eugenol concentration (ranging from 0.043 to 0.050% w/w), together with lecithin at critical synergistic concentrations (CSCs) ranging from 0.5 to 1 mg/100 mL. Increasing the concentration of lecithin above 1 mg/100 mL (up to 10 mg/100ml lecithin) diminished the antibacterial effect to values similar to eugenol-only treatments. The formation of aggregates (<100 nm) at the critical lecithin concentration was observed using Cryo-TEM together with an increase of light absorbance at 284 nm. At CSC, the iv formation of nano-scale aggregates was suggested to be responsible for improving eugenol antimicrobial activities. In the second study, the effect of individual lecithin phospholipids on the antimicrobial properties of eugenol against E. coli C600 was investigated. Five major phospholipids common in soy or egg lecithin (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC; 1,2-distearoyl-snglycero-3-phosphocholine, DSPC; 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, DPPE; 1,2-dipalmitoyl-sn-glycero-3-phosphate sodium salt, DPPA and 1,2-dipalmitoyl-sn-glycero-3- phospho-L-serine sodium salt, DPPS) and one synthetic cationic phospholipid (1,2-distearoyl-snglycero-3-ethylphosphocholine chloride salt, EPC 18:0) were tested. Among six phospholipids, DPPC, DSPC, DPPE, DPPA, and the cationic 18:0 EPC showed CSCs that significantly improved the inactivation effect of eugenol against E. coli after 30 min of exposure. At CSC, an additional ca. 0.4-1.9 log reduction (ca. 0.66-2.17 log CFU/mL reduction) in microbial population was observed when compared to eugenol-only (control) treatments (ca. 0.25 log reduction). In all cases, increasing the phospholipid amount above the CSC resulted in antimicrobial properties similar to eugenol-only (control) treatments. DPPS did not affect the antimicrobial properties of eugenol at the tested concentrations. The CSC of phospholipids was correlated to their critical micelle concentrations (CMC). In the third study, the mechanisms of the synergistic antimicrobial effect between eugenol and DSPC against E. coli were investigated. After exposing E. coli C600 to increasing levels of DSPC-only samples for 30 min, E. coli C600 showed more than 20% additional uptake of hydrophobic fluorescence dye (1-N-phenylnaphthylamine, NPN) than that in the control sample (without DSPC). However, the subsequent eugenol-only (0.043% v/v) treatment did not show any significantly different inactivation in control and DSPC-only pre-treated samples. The solubility of eugenol in phosphate buffered saline (PBS) was higher than 0.086% v/v, which v indicated that DSPC did not solubilize eugenol (applied at 0.043% v/v) to enhance its antimicrobial property. 1 H-NMR signal of several eugenol protons revealed interaction between eugenol and DSPC at CSCs, indicating the eugenol-DSPC complex formation. 0.043% v/v eugenol together with 0.63 µM DSPC demonstrated similar synergistic antimicrobial effects (ca. 1 log additional inactivation) against two E. coli strains with distinctly different charge on cell membranes. Therefore, electrostatic attraction between eugenol-DSPC complex and E. coli was not supported to be the reason for the synergistic effect. The present work supports the bioactivity of lecithin phospholipids at low concentrations (<10 mg/100 mL) to improve the antimicrobial effects of eugenol, which will contribute to a rational design of EO-phospholipid delivery systems.