Ethylhexylglycerin Impairs Membrane Integrity and Enhances the Lethal Effect of Phenoxyethanol

Abstract

Preservatives are added to cosmetics to protect the consumers from infections and prevent product spoilage. The concentration of preservatives should be kept as low as possible and this can be achieved by adding potentiating agents. The aim of the study was to investigate the mechanisms behind potentiation of the bactericidal effect of a commonly used preservative, 2-phenoxyethanol (PE), by the potentiating agent ethylhexylglycerin (EHG). Sub-lethal concentrations of EHG (0.075%) and PE (0.675%) in combination led to rapid killing of E. coli (> 5 log reduction of cfu after 30 min), leakage of cellular constituents, disruption of the energy metabolism, morphological deformities of cells and condensation of DNA. Used alone, EHG disrupted the membrane integrity even at low concentrations. In conclusion, sub-lethal concentrations of EHG potentiate the effect of PE through damage of the cell membrane integrity. Thus, adding EHG to PE in a 1:9 ratio has a similar effect on membrane damage and bacterial viability as doubling the concentration of PE. This study provides insight about the mechanism of action of a strong potentiating agent, EHG, which is commonly used in cosmetics together with PE.

Fig 1. Log₁₀ for E. coli exposed to EUX, PE and EHG.

Results for three concentrations of EUX and corresponding concentrations of PE (90% of EUX-conc.) and EHG (10% of EUX-conc.) are shown. (A) 0.75% EUX, (B) 1.25% EUX, (C) 1.5% EUX. Mean values of three replicates and standard error of the mean are shown.

Fig 2. DNA-content of cells exposed to EUX, PE and EHG (% of total DNA pool measured for lysed cells).

Results for three concentrations of EUX and corresponding concentrations of PE (90% of EUX-conc.) and EHG (10% of EUC-conc.) are shown. (A) 0.75% EUX, (B) 1.25% EUX, (C) 1.5% EUX. Mean values of three replicates and standard error of the mean are shown.

Fig 3. ATP-content of E. coli exposed to EUX, PE and EHG (% of untreated control cells).

(A) and (C): Cells suspended in buffer. (B) and (D): Cells suspended in buffer with glucose. Results for two concentrations of EUX and corresponding concentrations of PE (90% of EUX-conc.) and EHG (10% of EUX-conc.) are shown. (A) and (B) 0.75% EUX, (C) and (D) 1.5% EUX. Mean values of three replicates and standard error of the mean are shown.

Fig 4. pH-drop of 10¹¹ cfu ml^-1 E. coli exposed to EUX (0.75%), PE (0.675%) and EHG (0.075%).

The cells were exposed for 5 min, 3 h and 24 h and added equal amounts of HCl (determined as the amount needed to reduce the pH of the control after 5 minutes with 4 units). Mean values of three replicates and standard error are shown.

Fig 5. Scanning electron microscopy of cells treated with biocides.

(A) Untreated control in Sodium phosphate buffer; (B) 0.75% EUX (0.675% PE + 0.075% EHG); (C) 1.125% PE; (D) 0.125% EHG.

Fig 6. Transmission electron microscopy of cells treated with biocides.

(A) Untreated control in Sodium phosphate buffer; (B) 0.75% EUX (0.675% PE + 0.075% EHG); (C) 1.125% PE; (D) 0.125% EHG. Inset in (B) shows leakage of cytoplasmic material out of the cell membrane (not to scale).