Browsing by Subject "Sterilisation <Hygiene>"

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Investigations on the mechanisms of sterilization by non-thermal low-pressure nitrogen-oxygen plasmas
(2011) Roth, Stefan; Hertel, Christian
Plastic based materials are increasingly used for packaging of pharmaceuticals (especially biologicals), food or beverages and production of medical devices. Their heat sensitivity requires safe and efficient non-thermal methods for decontamination. Plasma technology has the potential to provide a suitable means since it works at low temperatures and ? in contrast to conventional methods like application of ionizing radiation or ethylene oxide exposure ? is safe to operate, is free of residues and does not alter the bulk properties of the materials. Plasmas can generate various agents potentially active in decontamination like ultra-violet (UV) radiation, radicals and other reactive particles. To acquire an approval for plasma technology as a novel sterilization method, its process safety has to be proven. The research community has proposed hypotheses and models on its mechanisms of action, which are at least partially speculative. Still little is known about the details of the biologic effects of the combination of the various plasma agents on the components of microbial cells or spores. Especially, the question remains open which components of a cell or spore are the primary targets, and which of the agents are most effective in the inactivation process. The acquisition of such knowledge is necessary to identify parameters suitable to control, monitor, and assess the safety of plasma sterilization processes. The aims of the presented work are to elucidate which components of a cell or spore are the primary targets in low-pressure plasma sterilization, and which of the putative agents contained in the plasma are most effective in the inactivation process. To accomplish this, in the presented work suitable microbiological methods were established and the inactivation of bacterial spores and cells and fungal conidia by microwave induced low-pressure low-temperature nitrogen-oxygen plasmas was investigated. Moreover, two strategies were pursued that have hitherto not been applied in published plasma sterilization studies: (i) Using spores of Bacillus subtilis mutants to identify structural components serving as targets for sterilization with plasma and (ii) characterizing the response of Deinococcus radiodurans R1 cells to plasma treatment and identify repair processes during recovery from plasma induced damages in viable cells. Plasmas producing a maximum of UV emission were most effective in inactivating bacterial cells and spores. The inactivation followed a biphasic kinetics consisting of a log-linear phase with rapid inactivation followed by a slow inactivation phase. A continuous model fit was applied to the experimental data allowing reliable calculation of decimal reduction values for both phases. Cells of D. radiodurans were found to be more resistant than spores of B. subtilis. For B. subtilis spores, in the course of plasma treatment damage to DNA, proteins and spore membranes were observed by monitoring the occurrence of auxotrophic mutants, inactivation of catalase (KatX) activity and the leakage of dipicolinic acid, respectively. Spores of the wild-type strain showed highest resistance to plasma treatment. Spores of mutants defective in nucleotide excision repair (uvrA) and small acid-soluble proteins (ΔsspA ΔsspB) were more sensitive than those defective in the coat protein CotE or spore photoproduct repair (splB). Exclusion of reactive particles and spectral fractions of UV radiation from access to the spores revealed that UV-C radiation is the most effective inactivation agent in the plasma, whereby the splB and ΔcotE mutant spores were equally and slightly less sensitive, respectively, than the wild-type spores. The extent of damages in the spore DNA as determined by quantitative PCR correlated with the spore inactivation. Spore inactivation was effectively mediated by a combination of DNA damage and protein inactivation. DNA was identified to be the primary target for spore inactivation by UV radiation emitted by the plasma. Coat proteins were found to constitute a protective layer against the action of the plasma. For the investigation of the recovery from plasma-induced damages, cells of D. radiodurans R1 were subjected to short plasma treatments with various plasmas. A part of the survivors was sublethally injured as determined by their ability to form colonies on standard medium but not on stress medium and by the observation of a prolonged lag phase. Incubation of the cells in a recovery medium after plasma treatment allowed a part of the survivors to recover their ability to grow on stress medium. This recovery strongly depended on transcriptional and translational processes and cell wall synthesis, as revealed by addition of specific inhibitors to the recovery medium. Genes involved in DNA repair, oxidative stress response and cell wall synthesis were induced during recovery, as determined by quantitative RT-PCR. Damage to chromosomal DNA caused by plasma agents and in-vivo repair during recovery was directly shown by quantitative PCR. Plasmas with less UV radiation emission were also effective in killing D. radiodurans cells but resulted in less DNA damage and lower induction of the investigated genes. The response of D. radiodurans to plasma indicated that DNA, proteins and cell wall are primary targets of plasma, whose damage initially leads to the cells' death. Protein oxidation was more important for the killing of D. radiodurans cells than of B. subtilis spores. Thus, the plasma process parameters must regard the expected contaminating biological material in order to obtain a high-level sterilization. The results provide new insight into the interaction of non-thermal low-pressure plasmas with microorganisms. This knowledge supports the definition of suitable parameters for novel plasma sterilization equipment to control process safety. For example, monitoring the UV intensity below 280 nm and spectrometric online measurement of bands related to excited reactive gas particle species during the process is recommended.
UV-C-Behandlung von Traubenmost zur Inaktivierung von Mikroorganismen
(2018) Diesler, Kathrin; Scharfenberger-Schmeer, Maren
The development of new preservation process techniques to protect ingredients and maintain a high quality standard is always a main goal in the food industry. In course of this, microbial safety has top priority. UV-C technology is a modern, non-thermal process with high efficiency. It has been used for sterilization and treatment of drinking water for many years. Also, ultraviolet radiation for disinfection purposes is already being used in other areas of food production. To what extent this method can be successfully applied in the field of grape must production, will be investigated in this dissertation. For this purpose, several yeasts and bacteria, relevant in this area, were examined for their inactivation potential by UV-C treatment. To ensure the best possible microbial inactivation in must, it is essential to determine an ideal treatment dose for both yeasts and bacteria. The results have confirmed that bacteria are far more sensitive to UV C treatment, than yeasts. It was also shown that there are major differences in UV-C stability within the seven yeast species and six bacteria species used in this study. The analyses have identified Metschnikowia pulcherrima and Acetobacter aceti as the most UV-C stable and Brettanomyces custerianus and Pediococcus sp. as the most sensitive organisms. Furthermore, three morphologically different Brettanomyces strains were used to show that there are also strain-specific variances in the response to UV-C treatment. Using Saccharomyces cerevisiae as an example, a potential formation of UV-C resistance was also ruled out. For this purpose, yeast cells were exposed to a dose, that did not result in complete inactivation. The surviving cells were cultured and retreated. Even after repeating this process eight times, no change in the UV-C response of the yeast cells could be detected. For the application of UV-C technology in the juice and wine industry it has to be ensured, that microorganisms are killed directly and their enzymatic activities are directly inhibited. Yeasts and bacteria could further convert sugar to alcohol or form unwanted metabolic byproducts. Therefore, the enzymatic activity after the initial treatment and during the inactivation process of Saccharomyces cerevisiae was analyzed in more detail. HPLC was used to determine the content of glucose, fructose and ethanol. No enzymatic activity could be detected in the UV-C treated samples from the moment after the initial UV-C treatment, up to the day of complete destruction. However, the effectiveness of UV-C treatment of must and wine cannot be attributed solely to the responsiveness of the various microorganisms. Other product parameters such as grape variety, turbidity and optical density also play a decisive role. In this context, four different musts with different optical density and turbidity were treated and the inactivation kinetics of Saccharomyces cerevisiae were compared. In this work it could be proved, that with an increasing optical density and a higher turbidity, the efficiency of the UV-C treatment in must decreases strongly. The success of a treatment is also directly dependent on the initial contamination rate of the product. Tests with different starting cell numbers have shown, that the required inactivation dose also has to be increased, as the number of cells increases. In the winemaking process, however, not only yeasts and bacteria can be a potential source of danger. The fungal infection of grapes by Botrytis cinerea also carries a high risk. The polyphenol oxidase laccase, produced by the fungus, damages ingredients and leads to a colour change in must and wine. In the investigations it could be proven, that it is possible to strongly reduce or completely inactivate the enzymatic activity in Botrytis infected must, depending on the starting concentration. In summary, UV-C technology represents an effective alternative and extension for current oenological practice. It offers the possibility to inactivate a large number of wine relevant microorganisms without causing resistance. In addition, this work has created a new framework for the application of must specific parameters. The results for the inactivation of the enzyme laccase are also proved to be extremely promising.