This work explores the bactericidal effect of (+)-limonene, the major constituent of citrus fruits' essential oils, against E. coli. The degree of E. coli BJ4 inactivation achieved by (+)-limonene was influenced by the pH of the treatment medium, being more bactericidal at pH 4.0 than at pH 7.0. Deletion of rpoS and exposure to a sub-lethal heat or an acid shock did not modify E. coli BJ4 resistance to (+)-limonene. However, exposure to a sub-lethal cold shock decreased its resistance to (+)-limonene. Although no sub-lethal injury was detected in the cell envelopes after exposure to (+)-limonene by the selective-plating technique, the uptake of propidium iodide by inactivated E. coli BJ4 cells pointed out these structures as important targets in the mechanism of action. Attenuated Total Reflectance Infrared Microspectroscopy (ATR-IRMS) allowed identification of altered E. coli BJ4 structures after (+)-limonene treatments as a function of the treatment pH: β-sheet proteins at pH 4.0 and phosphodiester bonds at pH 7.0. The increased sensitivity to (+)-limonene observed at pH 4.0 in an E. coli MC4100 lptD4213 mutant with an increased outer membrane permeability along with the identification of altered β-sheet proteins by ATR-IRMS indicated the importance of this structure in the mechanism of action of (+)-limonene. The study of mechanism of inactivation by (+)-limonene led to the design of a synergistic combined process with heat for the inactivation of the pathogen E. coli O157:H7 in fruit juices. These results show the potential of (+)-limonene in food preservation, either acting alone or in combination with lethal heat treatments.

Aims: The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. Methods and Results: The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 μl l⁻¹ of citral at pH 4·0 for 24 h at 20°C caused the inactivation of more than 5 log₁₀ cycles of cells starting at an inoculum size of 10⁶ or 10⁷ CFU ml⁻¹, whereas increasing the cell concentration to 10⁹ CFU ml⁻¹ caused <1 log₁₀ cycle of inactivation. Escherichia coli showed higher resistance to citral at pH 4·0 than pH 7·0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild‐type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. Conclusions: This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Significance and Impact of the Study: Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

Aims: The objective was to investigate the occurrence of sublethal injury after pulsed electric field (PEF) depending on the treatment time, the electric field strength and the pH of the treatment media in two Gram-positive (Bacillus subtilis ssp. niger, Listeria monocytogenes) and six Gram-negative (Escherichia coli, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella serotype Senftenberg 775W, Salmonella serotype Typhimurium, Yersinia enterocolitica) bacterial strains. Methods and Results: A characteristic behaviour was observed for the Gram-positive and Gram-negative bacteria studied. Whereas Gram-positive bacteria showed a higher PEF resistance at pH 7·0, the Gram-negative were more resistant at pH 4·0. In these conditions, in which bacteria showed their maximum resistance, a large proportion of sublethally injured cells were detected. In most cases, the longer the treatment time and the higher the electric field applied, the greater the proportion of sublethally injured cells that were detected. No sublethal injury was detected when Gram-positive bacteria were treated at pH 4·0 and Gram-negative at pH 7·0. Conclusions: Sublethal injury was detected after PEF so, bacterial inactivation by PEF is not an ‘all or nothing’ event. Significance and Impact of the Study: This work could be useful for improving food preservation by PEF.