Antimicrobial activity of suspensions and nanoemulsions of citral in combination with heat or pulsed electric fields.

The application of essential oils in form of nanoemulsions has been proposed as a method to improve their solubility in aqueous solutions, and hence their antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of citral, applied directly or in combined treatments with heat or pulsed electric fields (PEF), as a function of the inoculation procedure assayed: (i) a simple, vigorous shaking method by vortex agitation (suspension of citral; s-citral) or (ii) the previous preparation of nanoemulsions by the emulsion phase inversion (EPI) method (nanoemulsion of citral; n-citral). n-Citral was more effective in either inhibiting or inactivating Escherichia coli O157:H7 Sakai than s-citral. However, when combined with heat, a greater synergistic effect was observed with s-citral rather than with n-citral, either in lab media (pH 7·0 and 4·0) or apple juice. For instance, while almost 5 log10 cell cycles were inactivated in apple juice after 15 min at 53°C in the presence of 0·1 µl ml-1 of s-citral, the use of n-citral required 30 min. The use of nanoemulsions did not modify the slight synergism observed when citral and mild PEF were combined (150 µs, 30 kV cm-1 ). SIGNIFICANCE AND IMPACT OF THE STUDY: The exploration of different delivery systems of antimicrobial compounds such as citral in aqueous food products aids in the establishment of successful combined treatments for food preservation. While at room temperature, citral in form of a nanoemulsion shows a higher antimicrobial activity; its combination with heat would imply a partial loss of the outstanding synergistic lethal effect achieved when added in suspension form. Therefore, the most suitable procedure to magnify the synergism between heat and citral when processing juices would merely require an intense homogenization step prior to the combined treatment.

Resistance of Staphylococcus aureus to UV-C light and combined UV-heat treatments at mild temperatures.

In this investigation, the resistance of enterotoxigenic Staphylococcus aureus to short-wave ultraviolet light (UV-C) and to combined UV C-heat (UV-H) treatments in buffers and in liquid foods with different physicochemical characteristics was studied. Microbial resistance to UV-C varied slightly among the S. aureus strains tested. The UV-C resistance of S. aureus increased in the entry of stationary growth phase, which in part was due to the expression of the alternative sigma factor σ(B). The UV-C resistance of S. aureus was independent of the treatment medium's pH and water activity, but it decreased exponentially as the absorption coefficient increased. UV-C bactericidal efficacy in liquids of high absorption coefficients was improved synergistically when combined with a mild heat treatment at temperatures ranging from 50.0 to 57.5 °C. pH of the treatment medium modified the lethality of UV-H treatments and therefore the temperature of maximum synergy. The advantage of combined UV-H treatments was demonstrated in fruit juices and vegetable and chicken broths, inactivating 5 Log10 cycles of S. aureus by applying UV-C treatments of 27.1 mJ/L for 3.6 min at 52.5 °C or 13.6 mJ/L for 1.8 min at 55.0 °C.

New insights in mechanisms of bacterial inactivation by carvacrol.

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 4.0, 25 µl l(-1) 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.

Role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in bacterial heat resistance as a function of treatment medium pH.

This investigation aimed to determine the role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in heat resistance and the occurrence of sublethal injuries in cell envelopes of stationary-phase Escherichia coli BJ4 and Listeria monocytogenes EGD-e cells, respectively, as a function of treatment medium pH. Given that microbial death followed first-order inactivation kinetics (R(2)>0.95) the traditional D(T) and z values were used to describe the heat inactivation kinetics. Influence of rpoS deletion was constant at every treatment temperature and pH, making a ΔrpoS deletion mutant strain approximately 5.5 times more heat sensitive than its parental strain for every studied condition. Furthermore, the influence of the pH of the treatment medium on the reduction of the heat resistance of E. coli was also constant and independent of the treatment temperature (average z value=4.9°C) in both parental and mutant strains. L. monocytogenes EGD-e z values obtained at pH 7.0 and 5.5 were not significantly different (p>0.05) in either parental or the ∆sigB deletion mutant strains (average z value=4.8°C). Nevertheless, at pH 4.0 the z value was higher (z=8.4°C), indicating that heat resistance of both L. monocytogenes strains was less dependent on temperature at pH 4.0. At both pH 5.5 and 7.0 the influence of sigB deletion was constant and independent of the treatment temperature, decreasing L. monocytogenes heat resistance approximately 2.5 times. In contrast, the absence of sigB did not decrease the heat resistance of L. monocytogenes at pH 4.0. The role of RpoS in protecting cell envelopes was more important in E. coli (4 times) than SigB in L. monocytogenes (1.5 times). Moreover, the role of σ(S) in increasing heat resistance seems more relevant in enhancing the intrinsic resilience of the cytoplasmic membrane, and to a lesser extent, outer membrane resilience. Knowledge of environmental conditions related to the activation of alternative sigma factors σ(S) and σ(B) and their effects on heat resistance would help us to avoid and/or identify situations that increase bacterial stress resistance. Therefore, more efficient food preservation processes might be designed.

Effect of citral on the thermal inactivation of Escherichia coli O157:H7 in citrate phosphate buffer and apple juice.

Inactivation and sublethal injury of Escherichia coli O157:H7 cells induced by heat in citrate phosphate buffer and apple juice (both at pH 3.8) were studied, and the effect of a combined preservation treatment using citral and heat treatments was determined. Heat resistance of E. coli O157:H7 was similar in both treatment media; after 27 min at 54°C, 3 log units of the initial cell population was inactivated in both treatment media. However, under less harsh conditions a protective effect of apple juice was found. Whereas inactivation followed linear kinetics in the citrate phosphate buffer, when cells were treated in apple juice the survival curves were concave downward. Heat treatment caused a great degree of sublethal injury; 4 min at 54°C inactivated less than 0.5 log CFU/ml but sublethally injured more than 3 log CFU/ml. The addition of 18 and 200 ppm of citral to the treatment medium acted synergistically with heat at 54°C to inactivate 3 × 10(4) and 3 × 10(7) CFU/ml, respectively. Addition of citral thus reduced the time needed to inactivate 1 log unit of the initial E. coli O157:H7 population from 8.9 to 1.7 min. These results indicate that a combined process of heat and citral can inactivate E. coli O157:H7 cells and reduce their potential negative effects.