## 7 Discussion and Future Perspectives
Moreover, the technique presents limitations such as a complex sample pre-treatment, the need for enrichment steps, and advanced bioinformatics expertise, requiring improvements for its application to the study of VBNC cell physiology [55]. Limitations of VBNC molecular detection methods are the loss of some bacteria in the pretreatment steps and the interference of background noise. Therefore, IMS with immunomagnetic beads (IMB) conjugated with antibodies to capture target microorganisms was introduced but not applied to detect VBNC cells. The use of bentonite-coated activated carbon (BCAC) instead of IMB to remove DNA inhibitors also proved successful in sensitive detection of bacteria in milk. Other tools used to target foodborne pathogens were streptavidin magnetic beads-dual aptamers with high binding affinity, structural stability, ease of storage, and ease of synthesis in vitro and vancomycin-modified poly-l-lysine magnetic beads [30]. All these strategies should be evaluated in the ability to detect VBNC cells. Among the natural compounds that attract interest as substitutes for chemical preservatives in food, OVEO, ROEO, and LEO were tested and shown to favor VBNC _L. monocytogenes_ induction. Therefore, beyond attempting concentration and exposure time optimization of their usage in different foods to prevent this hazard, other natural compounds need to be tested for the capability to induce VBNC formation in _L. monocytogenes_. An example is luteolin, a flavonoid that showed efficacy in destroying the cell membrane integrity, with an increase in the number of non-viable cells, producing modifications in cell morphology, inhibitory effects on biofilm formation, and enhanced antibiotics diffusion within biofilms. This molecule was found to damage three tested strains of _L. monocytogenes_ treated with the MIC (32 to 64 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{\SIUnitSymbolMicro L}\)), with an increase in non-viable cells identified by confocal laser scanning microscope combined with the Live/Dead BacLight Kit. A 2 MIC concentration of luteolin induced remarkable cell membrane damage, as demonstrated by field emission scanning electron microscopy (FESEM), and an increase in dead cells based on fluorescence staining, thus proving suitable for preventing VBNC formation [56]. Green sanitizer formulations, such as one comprising the natural phenolic compound gallic acid, hydrogen peroxide, and DL-lactic acid, which induced the VBNC state in _L. innocytogenes_, should be evaluated for VBNC formation also in _L. monocytogenes_ strains, as well as erythromycinolarurate (EL), which showed promising results in the inactivation of _L. innocqua_ when coupled with mild heating [57,58]. Other antimicrobials to be tested in the capacity to inhibit VBNC induction are bacteriocins, particularly those inhibiting listerial growth such as nisin, enterocin CRL35, and lactocin [59]. To date, only nisin has been tested for death induction in two _L. monocytogenes_ strains in stationary phase by the BD Cell Viability Kit coupled with FC. It was observed that after 4 h of treatment with 75 \(\mathrm{\SIUnitSymbolMicro g}/\mathrm{mL}\) nisin, 25% of the cells were viable for the _L. monocytogenes_ A1 strain, a food isolate, but after 24 h, 91.7% of the cells were dead or VBNC/injured. For the collection strain _L. monocytogenes_ M5, 16.2% of cells were viable after 4 h and 95.2% of cells were dead after 24 h. Therefore, the capacity of nisin to leave a residual number of VBNC cells should be better investigated [54]. In addition, the effects on VBNC formation of alternatives to heat exposure to improve food microbiological safety should be defined. As an example, high-pressure technology (HPP) or pascalization, a low-temperature treatment of food that improves microbiological safety with minimal effects on the nutritional, functional, and/or sensory properties, and is largely applied to extend the shelf life of many liquid and solid foods at refrigeration temperature, should be tested for induction of the VBNC state in _L. monocytogenes_.