## 3 Results and Discussion
### Determination of Propidium Iodide Uptake  
In order to investigate possible membrane damage induced by the plant extracts, cells were stained with propidium iodide (PI), a component which binds to single and double stranded nucleic acids. Since PI only penetrates cells with damaged membranes, it can be used as an indicator of cytoplasmic membrane damage [44]. Figure 3 illustrates the evolution of the amount of membrane damage of _L. monocytogenes_ in the control, pure GSE, and pure GE, employing the ratio of fluorescence (indicating cells with a damaged cell membrane) over optical density (OD) (indicating the total amount of living and dead cells). In the control, membrane damage was detected at time 0 min. The damage decreased over time, disappearing almost completely at sampling times of 12, 18, and 24 h. Since this membrane damage was observed at time 0 min, it originated from membrane damage present in the preculture, probably caused mostly by cell death after the cells had reached the stationary phase. In this regard, precultures of 24 h were used for this experiment, while a Listeria preculture already reaches the stationary phase after approximately 9 h at 30 \({}^{\circ}\)C [45], resulting in a considerable amount of dead cells. The membranes of these dead cells are damaged [46], causing an increase of the total membrane damage, which is detected by the PI staining, similarly to what is the case in live/dead staining. The apparent decrease in membrane damage over time in the control was due to the growth of the cells. Since the total amount of cells increased, OD increased over time. In comparison, the amount of dead cells did not increase significantly and fluorescence remained constant. Therefore, fluorescence/OD decreased. As a consequence, no conclusions can be drawn about the nature of the small SI in the control during the exponential growth phase, observed in Figure 2. If this SI would be caused by membrane damage, an increase in membrane damage between sampling times of 0 min and 2 h would be expected in Figure 3. The absence of this increase would mean that the increase of SI in the control was caused by metabolic damage. However, due to the membrane damage in the dead cells, this increase might not have been detectable by this experimental method. Similar to the cells in the control, the cell membranes in pure GSE were also damaged at the start of the experiment due to the dead cells originating from the preculture. However, the statistical analysis indicates that a clear increase in membrane damage occurred after 15 min, remaining approximately constant until a treatment time of 2 h. No samples were taken of cells in GSE at 12, 18, and 24 h, but the challenge tests (as seen in Figure 1C) demonstrated that the cells did not recover from their damage in pure GSE, since no viable cells were observed after 3 h in GSE. Therefore, it can be concluded that GSE causes a significant amount of membrane damage to _L. monocytogenes_. The membrane damage caused to microorganisms by GSE is described extensively in literature, since the phenolic compounds in GSE are reported to be detrimental for the membrane stability of bacteria as they interact with bacterial cell walls in three different ways: (i) physical disruption of the membrane, (ii) dissipation of the proton motive force, and (iii) inhibition of membrane-associated enzyme activity [9]. In Figure 2, it is demonstrated that SI in pure GSE already reached a level of approximately 100% after 1 min, while Figure 3 illustrates that the maximum amount of membrane damage only occurred after 15 min. Since the amount of membrane damage of cells in GSE remained constant during the first 10 min, the rapid increase of SI in the first minute was most likely caused by damage of a metabolic nature.