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: To study the mechanism of bacterial inactivation by carvacrol and the influence of genetic and environmental factors in its antimicrobial activity. Methods and Results: In general, bacterial inactivation by carvacrol was higher in the Gram-positive Listeria monocytogenes than in the Gram-negative Escherichia coli and at acidic pH. At pH 4Á0, 25 ll l À1 of carvacrol for 5 h inactivated 1 and more than 5 log 10 cycles of E. coli and L. monocytogenes populations, respectively. Genetic and environmental factors also influenced cell resistance to carvacrol: rpoS and sigB deletion decreased carvacrol resistance in E. coli and L. monocytogenes, respectively; a heat shock induced a phenomenon of cross-protection to carvacrol treatments. Repair of sublethal injuries in cell envelopes suggested that carvacrol targets lipid fractions and proteins of these structures. This result was corroborated by attenuated total reflectance infrared microspectroscopy analysis.Conclusions: This study shows critical genetic and environmental factors, such as rpoS or sigB and heat shocks, and reveals new microbial structures involved in the mechanism of bacterial inactivation by carvacrol. Significance and Impact of the Study: A better understanding of the mechanisms of microbial inactivation is of great relevance to design more appropriate carvacrol treatments with high antimicrobial effects.

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There is a pressing need to extend the knowledge on the properties of insect protein fractions to boost their use in the food industry. In this study several techno-functional properties of a black soldier fly (Hermetia illucens) protein concentrate (BSFPC) obtained by solubilization and precipitation at pH 4.0–4.3 were investigated and compared with whey protein isolate (WPI), a conventional dairy protein used to stabilize food emulsions. The extraction method applied resulted in a BSFPC with a protein content of 62.44% (Kp factor 5.36) that exhibited comparable or higher values of emulsifying activity and foamability than WPI for the same concentrations, hence, showing the potential for emulsion and foam stabilization. As for the emulsifying properties, the BSFPC (1% and 2%) showed the capacity to stabilize sunflower and lemon oil-in-water emulsions (20%, 30%, and 40% oil fraction) produced by dynamic membranes of tunable pore size (DMTS). It was proved that BSFPC stabilizes sunflower oil-in-water emulsions similarly to WPI, but with a slightly wider droplet size distribution. As for time stability of the sunflower oil emulsions at 25 °C, it was seen that droplet size distribution was maintained for 1% WPI and 2% BSFPC, while for 1% BSFPC there was a slight increase. For lemon oil emulsions, BSFPC showed better emulsifying performance than WPI, which required to be prepared with a pH 7 buffer for lemon oil fractions of 40%, to balance the decrease in the pH caused by the lemon oil water soluble components. The stability of the emulsions was improved when maintained under refrigeration (4 °C) for both BSFPC and WPI. The results of this work point out the feasibility of using BSFPC to stabilize O/W emulsions using a low energy system.