M. Somolinos

Inactivation of Escherichia coli by citral

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.

Effect of environmental factors and cell physiological state on Pulsed Electric Fields resistance and repair capacity of various strains of Escherichia coli.

The aim was to determine the resistance variation of four strains of Escherichia coli to Pulsed Electric Fields (PEF), the role of the sigma factor RpoS in PEF resistance, as well as the influence of several environmental factors and the cell physiological state on the PEF resistance and repair capacity. The rpoS null mutant, E. coli BJ4L1, exhibited decreased PEF resistance as compared with its wild-type parent, BJ4. W3110 and O157:H7 were the most PEF-resistant strains: whereas 2 and more than 3 Log10 cycles of BJ4 and BJ4L1 cells, respectively, were inactivated after 50 pulses at 35 kV/cm, only 0.5 Log10 cycle of inactivation of W3110 and O157:H7 was attained. A different pattern was observed and the resistance variation among strains was largely reduced, when selective recovery media were used. At exponential growth phase, the resistance of the four strains was lower, and more than 4 Log10 cycles of inactivation of all strains tested were attained at 30 kV/cm. Previous heat and cold shock treatments scarcely influenced cell PEF resistance. PEF survival increased with the reduction in water activity of the treatment medium to 0.94: the occurrence of sublethally injured cells was negligible, and less than 1 Log10 cycle of inactivation was attained at 35 kV/cm. PEF-treated cells were sensitive to a subsequent storage at pH 4.0 or in the presence of sorbic acid, attaining a final inactivation of 4-5 Log10 cycles after 24 hour-incubation. In conclusion, the work confirms the role of rpoS in PEF resistance. E. coli strains exhibit large differences in PEF resistance. These differences were less important when cells were recovered under selective conditions. Both resistance variation among strains and occurrence of sublethal damage were noticeably influenced by the environmental factors tested.

Relationship between Sublethal Injury and Microbial Inactivation by the Combination of High Hydrostatic Pressure and Citral or tert-Butyl Hydroquinone

ABSTRACT The aim was to investigate (i) the occurrence of sublethal injury in Listeria monocytogenes, Escherichia coli, and Saccharomyces cerevisiae after high hydrostatic pressure (HHP) treatment as a function of the treatment medium pH and composition and (ii) the relationship between the occurrence of sublethal injury and the inactivating effect of a combination of HHP and two antimicrobial compounds, tert-butyl hydroquinone (TBHQ) and citral. The three microorganisms showed a high proportion of sublethally injured cells (up to 99.99% of the surviving population) after HHP. In E. coli and L. monocytogenes, the extent of inactivation and sublethal injury depended on the pH and the composition of the treatment medium, whereas in S. cerevisiae, inactivation and sublethal injury were independent of medium pH or composition under the conditions tested. TBHQ alone was not lethal to E. coli or L. monocytogenes but acted synergistically with HHP and 24-h refrigeration, resulting in a viability decrease of >5 log10 cycles of both organisms. The antimicrobial effect of citral depended on the microorganism and the treatment medium pH. Acting alone for 24 h under refrigeration, 1,000 ppm of citral caused a reduction of 5 log10 cycles of E. coli at pH 7.0 and almost 3 log10 cycles of L. monocytogenes at pH 4.0. The combination of citral and HHP also showed a synergistic effect. Our results have confirmed that the detection of sublethal injury after HHP may contribute to the identification of those treatment conditions under which HHP may act synergistically with other preserving processes.

Inactivation of Escherichia coli O157:H7 in fruit juices by combined treatments of citrus fruit essential oils and heat

This work approaches the possibility of combining mild heat treatments with citrus fruit essential oils (EOs) to improve the effectiveness of heat treatments and thus to reduce treatment intensity. Concentrations between 10 and 200 μL/L of lemon, mandarin, or orange EO were tested at 54 °C for 10 min in laboratory media, determining that 200 μL/L of each EO was necessary to achieve a 5 log(10) reduction of the initial Escherichia coli O157:H7 concentration. A relationship could be established between sublethally injured cells after the heat treatment and inactivated cells after the combined process. In apple juice, the synergism in the inactivation of E. coli O157:H7 when adding 200 μL/L of lemon EO might suppose a reduction in the treatment temperature (of 4.5 °C) or in the treatment time (by 5.7 times) within the range of temperature assayed (54-60 °C). Addition of 75 μL/L of lemon EO was determined to achieve the same synergistic effect of the combined treatment when the initial inoculum was reduced from 3×10(7) to 3×10(4) CFU/mL. Since the addition of lemon EO did not decrease the hedonic acceptability of apple juice, the proposed combined treatment could be further studied and optimized for the production of new minimally processed juices.

Pulsed electric fields cause sublethal injuries in the outer membrane of Enterobacter sakazakii facilitating the antimicrobial activity of citral

Aims:  The objective was to evaluate the relation of sublethal injury in the outer membrane of Enterobacter sakazakii to the inactivating effect of the combination of pulsed electric fields (PEF) treatments and citral.

Relationship between sublethal injury and inactivation of yeast cells by the combination of sorbic acid and pulsed electric fields.

ABSTRACT The objective of this study was to investigate the occurrence of sublethal injury after the pulsed-electric-field (PEF) treatment of two yeasts, Dekkera bruxellensis and Saccharomyces cerevisiae, as well as the relation of sublethal injury to the inactivating effect of the combination of PEF and sorbic acid. PEF caused sublethal injury in both yeasts: more than 90% of surviving D. bruxellensis cells and 99% of surviving S. cerevisiae cells were sublethally injured after 50 pulses at 12 kV/cm in buffer at pHs of both 7.0 and 4.0. The proportion of sublethally injured cells reached a maximum after 50 pulses at 12.0 kV/cm (S. cerevisiae) or 16.5 kV/cm (D. bruxellensis), and it kept constant or progressively decreased at greater electric field strengths and with longer PEF treatments. Sublethally PEF-injured cells showed sensitivity to the presence of sorbic acid at a concentration of 2,000 ppm. A synergistic inactivating effect of the combination of PEF and sorbic acid was observed. Survivors of the PEF treatment were progressively inactivated in the presence of 2,000 ppm of sorbic acid at pH 3.8, with the combined treatments achieving more than log10 5 cycles of dead cells under the conditions investigated. This study has demonstrated the occurrence of sublethal injury after exposure to PEF, so yeast inactivation by PEF is not an all-or-nothing event. The combination of PEF and sorbic acid has proven to be an effective method to achieve a higher level of yeast inactivation. This work contributes to the knowledge of the mechanism of microbial inactivation by PEF, and it may be useful for improving food preservation by PEF technology.

Recovery of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields

The objective was to investigate the influence of the recovery liquid medium on the repair of Saccharomyces cerevisiae sublethally injured cells after Pulsed Electric Fields (PEF) in media of different pH. Sublethal injury was detected in the yeast S. cerevisiae after 50 pulses at 12.0 kV cm(-1) in both pH 4.0 and 7.0, by using a selective medium plating technique. PEF treatments cause a repairable sublethal injury in S. cerevisiae. Injured cells showed their maximum repair capacity when suspended in Sabouraud Broth compared to Peptone Water or citrate-phosphate buffer of pH 4.0. The extent to which cells repair their injuries depended on the treatment medium pH, and on the nature of the storage medium. No repair was detected when the recovery liquid medium was citrate-phosphate buffer of pH 7.0. Acid conditions favour repair and survival of PEF-treated S. cerevisiae cells. This work contributes to the description of the mechanisms of PEF injury and inactivation, which would be useful in defining adequate PEF treatments, alone or in combination with additional hurdles, to assure food stability.

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.

sigB absence decreased Listeria monocytogenes EGD-e heat resistance but not its Pulsed Electric Fields resistance.

This study shows the behaviour of Listeria monocytogenes under heat and Pulsed Electric Fields (PEF) treatments, as well as the influence of sigB in bacterial resistance and recovery. Absence of the sigB gene resulted in a decreased heat tolerance, showing that sigma(B) influences L. monocytogenes heat survival. Heat treatments at 60 degrees C (40s) caused sublethal membrane injuries in 99.99% of survivors. The repair of heat damage required energy production and lipid, protein, and RNA synthesis, and it lasted 6h. Furthermore, deletion of sigB did not affect the heat injuries repair. PEF resistance at pH 4.0 and 7.0 was not influenced by sigB. Sublethal damage after PEF treatments was only detected when PEF-treated cells had previously been heat-shocked (45 degrees C/1h). The membrane repair only required energy production, and it was independent of sigB. Although both heat and PEF treatments have an effect on cellular membrane, the repair of the sublethal damages suggests different membrane targets, and thus we propose a different mechanism of inactivation by these food preservation technologies.

Organic acids make Escherichia coli more resistant to pulsed electric fields at acid pH.

Stationary growth phase cells of Escherichiacoli were more pulsed electric fields (PEF) resistant in citrate-phosphate McIlvaine buffer at pH 4.0 than at pH 7.0. The greater PEF resistance was also confirmed in fruit juices of similar acid pH. In this work we studied whether the higher PEF resistance of E. coli at acid pH was due to the low pH itself or to the interaction of the components of the treatment medium with the cells. The protective effect on E. coli cells was due to the presence of organic acids such as citric, acetic, lactic or malic at pH 4.0. The protective effect of citric acid at pH 4.0 depended on its concentration. A linear relationship was observed between the Log(10) of the citric acid concentration and the degree of inactivation. Organic acids contained in laboratory treatment media (citrate-phosphate buffer) or in fruit juices did not sensitize E. coli cells to PEF but, on the contrary, they induced a protective effect that made E. coli cells more resistant at pH 4.0 than at neutral pH. This work could be useful for improving food preservation by PEF technology and it contributes to the knowledge of the mechanism of microbial inactivation by PEF.