Aims: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. Methods and Results: In laboratory media, MICs of 15, 75 and 35 mmol l )1 vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l )1 vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l )1 vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l )1 vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l )1 vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l )1 vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. Conclusions: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. Significance and Impact of the Study: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.

Membrane permeabilization due to pulsed electric field (PEF) treatment of gram-positive Lactobacillus cells was investigated by using propidium iodide uptake and single-cell analysis with flow cytometry. Electric field strength, energy input, treatment time, and growth phase affected membrane permeabilization of Lactobacillus plantarum during PEF treatment. A correlation between PEF inactivation and membrane permeabilization of L. plantarum cells was demonstrated, whereas no relationship was observed between membrane permeabilization and heat inactivation. The same results were obtained with a Lactobacillus fermentum strain, but the latter organism was more PEF resistant and exhibited less membrane permeabilization, indicating that various bacteria have different responses to PEF treatment. While membrane permeabilization was the main factor involved in the mechanism of inactivation, the growth phase and the acidity of the environment also influenced inactivation. By using flow cytometry it was possible to sort cells in the L. plantarum population based on different cell sizes and shapes, and the results were confirmed by image analysis. An apparent effect of morphology on membrane permeabilization was observed, and larger cells were more easily permeabilized than smaller cells. In conclusion, our results indicate that the ability of PEF treatment to cause membrane permeabilization is an important factor in determining inactivation. This finding should have an effect on the final choice of the processing parameters used so that all microorganisms can be inactivated and, consequently, on the use of PEF treatment as an alternative method for preserving food products.A high-voltage pulsed electric field (PEF) can inactivate microorganisms under reduced-temperature conditions. Consequently, food products have a fresher appearance and lose less flavor and other functional food components, factors that are currently in high demand by consumers (1,16,27). PEF treatment is the application of pulses with very high field strength for a short time (microseconds) to foods placed between two electrodes. Due to technical and technological developments during the last few years, it is now possible to perform PEF treatment in a continuous-treatment chamber. This has increased the efficiency of the treatment process and offers more possibilities for scaling up the technology (4, 26, 40), which has enhanced interest by the food industry.Recently, microbial inactivation kinetics were systematically studied under a range of conditions in continuous-PEF systems (8,29,39). Furthermore, inactivation kinetics were determined under close-to-isothermal conditions to study the effect of field strength and energy input independent of heat (12). It was concluded that electric field strength and the amount of energy input, (i.e., the number of pulses) were important in determining the inactivation level. Other important process factors were pulse length and inlet temperature (39). Product factors (pH and conductivity) and the physiological s...

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. 1997. Flow cytometry in combination with fluorescent molecular markers 5-(and 6-) carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied to determine lag times, numbers of cell divisions and injury after mild heat (50°C, 5 min) and nisin treatments (0·1 and 1·0 mg ml −1 ) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 mg ml −1 ) allowed determination of lag times and cell proliferation for up to eight generations. Double-labelling with CFSE and PI (5 mg ml −1 ) provided additional information about damage levels and distributions within populations. Subpopulations surviving treatment could be identified easily and selectively sorted.