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The ability of four different bacteria to synthesize new ATP upon exposure to different doses of pulsed-light (PL) irradiation was investigated. The bacterial cells were PL treated on a gel surface, resuspended in phosphate buffered saline (PBS) and subsequently incubated in Tryptic Soy Broth (TSB) at 37 °C. Cellular ATP levels were monitored during a 2 h incubation period and compared to the respective colony count data. Although PL affected ATP production in a dose dependent manner, the results showed that bacteria, which had rendered unculturable after PL exposure, are still capable of generating significant quantities of ATP. Escherichia coli and Listeria innocua proved to be more resistant to PL than Salmonella enterica and Staphylococcus aureus , which was supported by the colony count data and the ATP synthesis capacity. These findings underline that bacteria undetectable by culture-based methods may still show cellular activity and synthesize new ATP. [ABSTRACT FROM AUTHOR]

Nonthermal disinfection technologies are gaining increasing interest in the field of minimally processed food in order to improve the microbial safety or to extend the shelf life. Especially fresh-cut produce or meat and fish products are vulnerable to microbial spoilage, but, due to their sensitivity, they require gentle preservation measures. The application of intense light pulses of a broad spectral range comprising ultraviolet, visible and near infrared irradiation is currently investigated as a potentially suitable technology to reduce microbial loads on different food surfaces or in beverages. Considerable research has been performed within the last two decades, in which the impact of various process parameters or microbial responses as well as the suitability of pulsed light (PL) for food applications has been examined. This review summarizes the outcome of the latest studies dealing with the treatment of various foods including the impact of PL on food properties as well as recent findings about the microbicidal action and relevant process parameters. [ABSTRACT FROM AUTHOR]

Aims The objective of this study was a comprehensive characterization of physiological changes of Salmonella enterica induced by intense broad spectrum pulsed light (PL). After exposing the bacteria to this nonthermal decontamination technology on a gel surface, multiple viability parameters beyond culturability were assessed. Methods and Results By applying flow cytometry, a luciferin-luciferase bioluminescence assay and a microplate assay to measure the current redox activity, the impact of pulsed light on the membrane potential, membrane integrity, esterase activity, efflux pump activity, expression of the green fluorescent protein (GFP), respiration activity and ATP-content of Salm. enterica ATCC BAA-1045 was determined. These culture-independent methods for assessing the bacterial activity were compared to the ability to grow on tryptic soy agar. It is shown that this strain is rather sensitive to PL considering colony count reductions, while on the other hand unculturable bacteria still exhibit significant cellular energetic functions. However, this residual activity after PL exposure significantly decreases during sample storage in buffer for 24 h. This study also shows that the GFP expression of PL-treated cells which have rendered unculturable is severely reduced. Conclusions This study reveals that although not all cellular functions of Salm. enterica are immediately shut down after PL exposure, the synthesis of new GFP is strongly reduced and affected to a similar extent as the culturability. Significance and Impact of the Study It is shown for the first time, that even there is significant bacterial activity measurable after PL exposure, it is likely that nongrowing pathogenic bacteria like Salm. enterica are unable to express proteins, which is of great importance regarding their pathogenicity. [ABSTRACT FROM AUTHOR]

Aims Pulsed light ( PL) is an upcoming nonthermal decontamination technology mainly used for surface sterilization. The objective of this study was to investigate the extent of cellular damage caused by PL treatments of Listeria innocua and Escherichia coli on a polysaccharide surface in order to gain knowledge about the main inactivation pathways. Methods and Results The impact of PL on the cellular ATP level was investigated as well as the bacterial ability to take up fluorescently labelled glucose (2- NBDG). Furthermore, the extent of DNA damages was assessed by qPCR. The ability of L. innocua and E. coli to photorepair under artificial daylight exposure was quantified. Finally, the induction of reactive oxygen species ( ROS) and lipid peroxidation were studied by fluorometric detection of ROS and thiobarbituric acid reactive substances ( TBARS). It is shown that intracellular ATP levels and glucose uptake ability do not correlate with the immediate loss of bacterial reproducibility, which indicates that cellular activity and energy may remain on a relatively high level, although growth on tryptic soy agar is not observable. Sequence specific investigation of PL induced DNA damages by qPCR revealed distinct differences between L. innocua and E. coli although the observed inactivation efficacy of PL by the culture based method was similar. Photoreactivation has been observed for both bacteria, a higher recovery rate of up to 2 log was seen in case of E. coli. Intracellular ROS and lipid peroxides were both detectable at relatively high fluencies with E. coli so the contribution of oxidative damage to microbial inactivation of PL cannot be excluded. Conclusions Escherichia coli as well as L. innocua cells have proven to maintain residual cellular activity after having been exposed to PL even when they are not able to reproduce any more. High proportions of sublethal damages were also obvious with regard to occurring photoreactivation. The destruction of bacterial DNA seems to be the primary mechanism of inactivation of PL but the involvement of other factors like oxidative stress cannot be excluded. Significance and Impact of the Study The observed data underline that bacteria are not immediately inactivated after exposure to PL as different indicators of cellular energy are still detectable even when cells do not reproduce on solid media any more. DNA is the primary target of PL, but as the extent of damage among different bacteria may not reveal their actual sensitivity, other destructive effects should also be considered. [ABSTRACT FROM AUTHOR]

Aims The application of broad-spectrum intense light pulses is an innovative nonthermal technology for the decontamination of packaging materials, liquids or foodstuffs. The objective of this study was the fundamental investigation of the cellular impact of a pulsed light treatment on Listeria innocua and Escherichia coli. Methods and Results Flow cytometry in combination with different fluorescent stains, conventional plate count technique and a viability assay were applied to investigate the effects of a pulsed light treatment on the physiological properties of L. innocua and E. coli. The results showed that loss of cultivability occurred at considerably lower fluences than the shutdown of cellular functions such as the depolarization of cell membranes, the loss of metabolic, esterase and pump activities or the occurrence of membrane damage. Therefore, a considerable proportion of cells appeared to have entered the viable but nonculturable ( VBNC) state after the pulsed light treatment. A high percentage of L. innocua was able to maintain certain cellular vitality functions after storage overnight, whereas a further decrease in vitality was observed in case of E. coli. The loss of culturability was on the other hand directly accompanied by the formation of reactive oxygen species ( ROS) and DNA damages, which were assessed by the ROS-sensitive probe DCFH- DA and RAPD- PCR, respectively. Conclusions A significant discrepancy between conventional plate counts and different viability staining parameters was observed, which shows that a pulsed light treatment does not cause an immediate shutdown of vitality functions even when the number of colony-forming units already decreased for more than 6 log10 sample−1. Oxidative stress with concomitant damage to the DNA molecule showed to be directly responsible for the loss of cultivability due to pulsed light rather than a direct rupture of cell membranes or inactivation of intracellular enzymes. Significance and Impact of the Study The presented results suggest an UV light-induced photochemical rather than a photothermal or photophysical inactivation of bacterial cells by pulsed light under the applied experimental conditions. Flow cytometry in combination with different viability stains proved to be a suitable technique to gain deeper insight into the cellular response of bacteria to inactivation processes like a pulsed light treatment. [ABSTRACT FROM AUTHOR]

The impact of the fluence regulation on the inactivation efficiency of pulsed light (PL) surface disinfection treatments was investigated. E. coli and L. innocua were exposed to PL on a gel surface under variation of the applied voltage, the number of light flashes as well as the distance between the flash lamp and the sample surface. The results revealed deviations from the reciprocity law when the total fluence striking the sample surface was not applied at once, but subdivided into several successive light flashes. No differences were found when the fluence was delivered with only single light flashes, irrespective of the applied voltage. The pulse frequency did not have an impact on the microbial reduction within 1–5 Hz. Furthermore, the sensitivities of various bacterial strains, endospores and conidiospores were compared. Differences occurred for vegetative bacteria without a clear pattern, while bacterial endospores were more resistant. Dark pigmented mold spores were slightly more resistant than bacterial endospores. All dose-response curves exhibited a downward concavity, except for P. aeruginosa . Industrial relevance This study shows that the inactivation of bacteria on e.g. food surfaces by pulsed light systems depends on the way of fluence dosage. While it is irrelevant whether the fluence is regulated by the discharge voltage or the distance between the flash lamp and the treated surface, it is more effective to apply only single light flashes of high fluence instead of several consecutive light flashes. There is furthermore no distinct trend regarding the sensitivity of bacteria to PL, variations occur on species and strain level. Bacterial spores are in general more resistant while pigmented conidiospores show a slightly higher resistance than bacterial endospores. [ABSTRACT FROM AUTHOR]

This study evaluated the Pulsed Light technology (PL) for its efficiency to reduce the microbial loads on mung bean sprouts and endive salad during a simulated wash process for up to 60 s. The microbicidal action of PL proved to be more efficient than the sanitizers electrolyzed water (40 ppm free chlorine) and chlorine dioxide (15 ppm) within the same treatment time. Reductions of the microbial count up to 2.5 log were found in case of PL treated endive salad while both sanitizers only caused maximum reductions by about 1.5 log. A combination of PL and sanitizers did not have pronounced additional effect for both endive salad and mung bean sprouts. Overall, the decontamination treatments were more effective in case of endive salad compared to mung bean sprouts. It was also shown that the microbial loads in the washing water may be kept on a low level during PL treatments as count reductions of approx. 3 log were found in relation to simply washing in tap water. This study reveals that PL may be a suitable approach to reduce microbial hazards of industrially manufactured fresh produce. The application during the washing process not only reduces microbial loads on the product surface but also efficiently inactivates suspended microorganisms in the wash water and may therefore be an alternative to sanitizers for the prevention of cross contaminations. [ABSTRACT FROM AUTHOR]