## 2 Results
### Effects of CAR1 and THY5 on Caco-2 Cell Permeability  
In order to evaluate if EOs exposure could perturb intestinal epithelial permeability, transepithelial electrical resistance (TEER) and phenol red apparent permeability (Papp) were measured in differentiated Caco-2 cells after treatment with several concentrations (ranging from 0.01 to 0.1%) of the two representative EOs CAR1 or THY5, dissolved in a complete culture medium for up to 24 h. The results show that only the 0.01 and 0.02% CAR1 treatments did not affect cell permeability up to 24 h, while the higher concentrations (0.025-0.03-0.05-0.1%) already induced a TEER drop after 1 h (less than 20% of the control), which was maintained until the end of the experiment (Figure 4A). This TEER decrease was associated with a biologically relevant phenol red Papp increase, as the corresponding values were in the order of magnitude of 6 \(\times\) 10\({}^{-6}\) cm s\({}^{-1}\), indicating that the tight junctions were open (Figure 4B). Indeed, permeability coefficients higher than 5 \(\times\) 10\({}^{-7}\) cm s\({}^{-1}\) are considered to be indicative of compromised cell monolayers, while values above 1 \(\times\) 10\({}^{-6}\) cm s\({}^{-1}\) indicate destroyed cell monolayer integrity [27]. Concerning THY5, a drop in TEER values was already observed after 1 h treatment with 0.03, 0.05 and 0.1% concentrations (Figure 4C). Differently from CAR1, the 0.025% concentration of THY5 induced a TEER decrease less rapidly, but regardless, was indicative of important and irreversible damage to the monolayer, since from 4 h treatment, the TEER values were lower than 50% of the control and they continued to decrease. At the lower concentrations tested (0.02 and 0.01%), the TEER values remained above 100% of the control up to 24 h, indicating the integrity maintenance of the monolayer (Figure 4C). The TEER data were confirmed by the results of the paracellular passage of phenol red, shown in Figure 4D. The Papp values were indeed higher than the threshold value (1 \(\times\) 10\({}^{-6}\) cm s\({}^{-1}\)) for THY5  
Figure 2: Direct contact test of CAR1 and THY5 antimicrobial activity **(A)** against _L. monocytogenes_ OH and **(B)** S. Typhimurium LT2. Each EO was tested at 0.12, 0.25 and 0.5% concentrations. C: 0% EO; CTw: 0% EO + 0.125% Tween 80; CTw + Amp: 50 ug/mL ampicillin + 0.125% Tween 80. Bacterial cell viability is expressed as the geometric mean of CFU/mL \(\pm\) SD of one experiment carried out in triplicate. Means without a common letter significantly differ, \(p<\) 0.05.  
concentrations between 0.1 and 0.025%, while those related to the treatment with 0.02 and 0.01% were in the order of 1 \(\times\) 10\({}^{-7}\) cm s\({}^{-1}\), indicating that the tight junctions between cells were functionally sealed. From these data, it appears that among the concentrations tested, the highest concentration not damaging the Caco-2 cell monolayer was 0.02% for both EOs. This concentration was therefore used for the experiments of reduction in pathogen adhesion and for the evaluation of anti-inflammatory activity. The presence of 2% ethanol, corresponding to the concentration contained in the higher EO dilution tested, did not affect cell permeability for up to 24 h.  
Figure 3: Challenge test of CAR1 against **(A)**_L. monocytogenes_ OH and **(B)**_S. Typhimurium LT2 and of THY5 against **(C)**_L. monocytogenes_ OH and **(D)**_S. Typhimurium LT2. The assay was performed in minced cow meat artificially contaminated with _L. monocytogenes_ OH or _S._ Typhimurium LT2, with or without the addition of 0.5% CAR1 or THY5. The meat was stored at 4 \({}^{\circ}\)C for up to 7 days, homogenized at the indicated timepoints and appropriate dilutions were plated. Bacterial cell viability is expressed as the geometric mean of CFU/g \(\pm\) SD of one experiment carried out in triplicate. Means without a common letter significantly differ, \(p\) < 0.05.