## 3 Results and Discussion
### Assessment of Sublethal Injury

Table 3: Time-average Injured Cells Coefficient (TICC) of _L. monocytogenes_, according to Equation (10), for challenge tests in the control (TSBYE), GSE-MIC, GE-MIC, pure GSE, and pure GE. incorporated with nisin, GSE, malic acid, and EDTA was applied to turkey frankfurter systems. Finally, log reductions of up to 2 log were reported by [PERSON] et al. [17] for Turkey frankfurters coated with soy protein films containing GSE and nisin. Similar to what was mentioned before for the studies involving growth reductions, the inactivation of _L. monocytogenes_ in the five aforementioned studies was solely studied by means of viable plate counts on a selective medium. Since sublethal injury was hence not taken into account, the antilisterial effect of those novel applications was possibly overestimated. For pure GE, the amount of SI induced to _L. monocytogenes_ was also significant, albeit less pronounced than in pure GSE. While some laboratory-scale studies have reported the inactivation of _L. monocytogenes_ at relatively high concentrations of GE [35; 38], to the best knowledge of the authors, no studies have reported inactivation of the bacterium using novel applications involving GE concentrations suitable for food products. At those GE concentrations, a bacteriostatic effect (i.e., no growth of _L. monocytogenes_, but also no inactivation) is reported at best [39; 40; 41; 42]. However, inactivation of _L. monocytogenes_ has been reported for combinations of GE and nisin (e.g., in milk, soymilk, and hummus) [39; 40; 42]. Log reductions ranging from 1 to 4 log have been accomplished using this GE-nisin combination, which is similar to or higher than the log reductions observed for pure GE in the current study. Taking into account the observed trend that more severe inactivation treatments entail more SI, SI caused by GE-nisin combinations could be even more pronounced than SI caused solely by pure GE. Since the occurrence of sublethally injured cells was also not taken into account in the mentioned studies, reported antilisterial effects (i.e., both bacteriostatic and bactericidal) were possibly also overestimated. Even though the current study only focused on SI inflicted to one specific strain of _L. monocytogenes_ (i.e., LMG23775) and results should also be validated for other strains, the large amount of SI observed after treatments with GSE-MIC, pure GSE, and pure GE confirms the risk of over-estimating the antibacterial effect of natural plant extracts in novel food preservation applications. However, real food products contain background microflora, making it impossible to use a combination of nonselective and selective media to quantify SI, since the growth of nontarget microorganisms is also supported on nonselective media. In order to take SI into account when assessing the antilisterial effect of natural plant extracts, future research in the field should therefore include a resuscitation step to allow the quantification of both injured and healthy cells. Examples of such resuscitation techniques include liquid-repair (e.g., two-fold dilution) and solid-repair (e.g., thin agar overlay) methods [43]. In addition, the large amount of SI related to the use of natural plant extracts at relatively high concentrations confirms the advantages of using extra antilisterial measures (i.e., as part of a hurdle approach) in order to enable the use of lower extract concentrations which entail a limited amount of SI.