## 3 Results and Discussion
### Assessment of Sublethal Injury  
Figure 2 illustrates the SI of _L. monocytogenes_ for the different conditions mentioned in the previous section. In the case of growth (i.e., control, GSE-MIC, and GE-MIC), a peak in SI (i.e., approximately 20, 50, and 25%, in the control, GSE-MIC, and GE-MIC, respectively) always occurred when the cells were in their exponential growth phase. By the time the cells reached the stationary phase, they had recovered from almost all SI. This phenomenon was probably caused by the synthesis of stress proteins by the cells in their early stationary phase, causing them to be more stress-resistant than exponentially growing cells [2]. In pure GSE, SI increased radically until a treatment time of approximately 0.5 min. Afterwards, SI reached a constant level of approximately 100%. After 5 min, no viable counts were observed on the selective medium, meaning that all surviving cells were from that moment on sublethally injured. These cells were further inactivated, until a treatment time of 3 h, after which no more viable cells were detected on the nonselective medium, as seen in Figure 1C. In the case of inactivation in pure GE, SI increased during the loglinear part of the inactivation, with a peak of approximately 80%, and decreased again in the tailing phase. This phenomenon was possibly caused by a mechanism of injury accumulation that culminates in cell death [36]. A similar behavior was observed by [PERSON] et al. [2] for _Listeria innocua_ subjected to mild heating. Table 3 shows the Time-averaged Injured Cells Coefficient (TICC) for each condition, enabling a quantitative comparison of SI among the different conditions. It can be observed that TICC was always higher in GSE than in GE, both for the pure extracts and the extracts at their MIC, a trend which was confirmed by the height of the SI peaks in Figure 2. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Inactivation Parameter** & \multicolumn{2}{c}{**Pure GSE**} & \multicolumn{2}{c}{**Pure GE**} \\ \hline  & **TSAYE** & **Oxford** & **TSAYE** & **Oxford** \\ \hline \multirow{2}{*}{\(S_{L}\) [h]} & 0.0 \(\pm\) 0.1 & 0 \(\pm\) 0.0 & 11.4 \(\pm\) 0.7 & 5.2 \(\pm\) 1.7 \\  & A,a & A,a & B,b & B,a \\ \hline \multirow{2}{*}{\(k_{max}\) [1/h]} & 11.4 \(\pm\) 2.8 & 698 \(\pm\) 247 & 0.420 \(\pm\) 0.055 & 0.341 \(\pm\) 0.061 \\  & B,a & B,b & A,a & A,a \\ \hline \multirow{2}{*}{\(N_{res}\) [log(CFU/mL)]} & 2.7 \(\pm\) 0.2 & 2.2 \(\pm\) 0.3 & 5.7 \(\pm\) 0.1 & 5.2 \(\pm\) 0.2 \\  & A,a & A,a & B,b & B,a \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Inactivation parameters (i.e., the shoulder length \(S_{L}\), the maximum specific inactivation rate \(k_{max}\), and the residual cell population \(N_{res}\)) according to the model of [PERSON] et al. [26] for challenge tests of _L. monocytogenes_. For the different inactivation conditions (i.e., pure GSE and pure GE) on one specific plating medium (i.e., TSAYE or Oxford), values bearing different uppercase letters are significantly different (\(p\leq 0.05\)). For the different plating media at one specific inactivation condition, values bearing different lowercase letters are significantly different (\(p\leq 0.05\)). Figure 2: Sublethal injury (SI) of _Listeria monocytogenes_ for different bioactive extracts and their concentration as indicated for the control, GSE-MIC, pure GSE, GE-MIC, and pure GE. Since SI for Pure GSE could only be calculated up until a treatment time of 5 min, SI is represented over a period of 24 h (**A**) and 5 min (**B**). SI was calculated using Equation (9) with the modeled values from Figure 1 as input. TICC was quite similar in GE-MIC and in the control, with TICC in GE-MIC even being slightly lower. Consequently, the risk of underestimating food contamination due to SI would be rather limited when GE is used to inhibit _Listeria_ growth at a low extract concentration (e.g., as part of a hurdle approach). The use of free or encapsulated GE in milk to inhibit _L. monocytogenes_ growth, as studied by [PERSON] and [PERSON] [PERSON] et al.