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Application of flow cytometry to study mechanism of microbial inactivation/stabilisation

This work deals with the application of flow cytometric analysis to evaluate the mechanisms of leading to viability loss of Lactobacillus rhamnosus GG (LGG) following exposure to physical treatments. A multiple staining strategy, which is composed of physiological dyes carboxyfluoresceindiacetate (cFDA) and propidium iodide (PI) was applied to examine specific cellular metabolic activities and their relative changes following various treatments, which may lead to loss of viability of probiotic organisms. It was expected that additional insights on process-induced changes in cellular integrity or metabolic activities, which were not explicitly assessable by culture techniques, could be achieved using this measurement technique.

From the results of flow cytometric measurement following exposure to heat and pressure one could elucidate the mode of action of these physical stressors on cellular activities or integrity. Ultimately, the use of this technique might open a possibility to an improved design of bacterial inactivation processes, i.e. one could then choose a certain type of cellular damage preferred and then select the type of treatments required to achieve this goal. Particularly in the field of probiotic research, the importance of the ingestion of viable bacteria in eliciting health effect is sometimes questioned, since non-viable bacteria were reported to be effective as well. Non-viable probiotic cells are of interest due to easy-handling and longer shelf life. However, systematic studies on the efficacy of inactivation methods to produce non-viable cells are still lacking. In this context, high pressure killed cells might be one of the promising candidate to be investigated, since the fluorescence pattern of pressure inactivated cells - which is indicative for a lower extent of damage on metabolic activity and on membrane - is quite similar to the one of viable cells.

Furthermore, flow cytometric analysis in combination with liposomes loaded with fluorescence marker was used to evaluate the protective effect of different types of sugars on model membranes. With this technique the sugars present in the spray drying media was evaluated on their stabilizing effect on liposomes during dehydration. Principally, the size distribution and the retention of incorporated flurochrome in lipid vesicles in the presence of lactose, polydextrose, Raftilose®P95 or in absence of sugar before and after dehydration can be monitored. In the case of drying without sugar, an increase in liposome size was found, as evidenced by a broadening of the histogram in the direction of higher liposome sizes. This behaviour was thought to be highly related to fusion and/or aggregation of liposomes. On the other hand the size distributions of liposomes dried in the presence of all evaluated sugars were nearly identical to the one before dehydration. This indicates that the addition of sugars could effectively prevent fusion and/or aggregation.

On the other hand, not all sugars tested had the good capacity in preventing leakage of cF out of liposomes. In the absence of sugar there was only a small fraction of liposomes which still had fluorescence intensity values equivalent to that before dehydration. It is obvious that drying increased the permeability of the phospholipid bilayer, thus allowing cF to leak from the liposomes.

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Berlin University of Technology (TUBER LMT)
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