## 3 Results
### In Vitro Assessment of Probiotic Characteristics of L. plantarum CNTA 628
plantarum_ CNTA 628. Figure 3: Morphology of _Lactobacillus plantarum_ CNTA 628 colonies in agar plates containing MRS (**A**) or MRS supplemented with 0.5% sodium salt of taurocholic (**B**), taurodeoxycholic (**C**), glycolocholic (**D**) or glycodeoxycholic acids (**E**). The potential biofilm-forming capability of _L. plantarum_ CNTA 628 was evaluated in vitro. This strain displayed a very strong biofilm-forming capacity on an abiotic surface (Figure 4), with absorbance values higher than 0.5, ranging from 2.32 \(\pm\) 0.55 (24 h) to 4.54 \(\pm\) 0.50 (48 h). To sum up, the probiotic strain _L. plantarum_ CNTA 628 displays tolerance to simulated gastric and intestinal environments in vitro, exhibits susceptibility to antibiotics, demonstrates \(\beta\)-galactosidase and bile salt hydrolysate enzymatic activities, forms biofilms, and is able to produce acetic acid as a metabolic SCFA. L. plantarum CNTA 628 Competes with Pathogenic Bacteria and Exhibits Immunomodulatory Capacity In Vitro  
In our study, a possible effect of _L. plantarum_ on modifying the adhesion of _E. coli_ O157:H7 onto Caco-2 cells was addressed. Thus, the individual adhesion of the bacteria (_L. plantarum_ CNTA 628 and _E. coli_ O157:H7) to Caco-2 cells was determined, resulting in about 2.4 \(\pm\)1.6; 2.5 \(\pm\)1.5 and 3.7 \(\pm\) 0.9, respectively. The percentage of adhesion of _E. coli_ to Caco-2 cells (3.7 \(\pm\) 0.9) was used to normalize the results of _E. coli_ adhesion in competence, displacement and exclusion assays (Figure 5). For the cell/bacteria 1:10 ratio, the ability of _L. plantarum_ to reduce _E. coli_ adhesion on Caco-2 cells was also investigated. When _L. plantarum_ is added prior to the pathogenic bacteria (exclusion), _E. coli_ adhesion is not affected. However, when _L. plantarum_ is added at the same time as _E. coli_ (competence), its adhesion is reduced by 53%. Furthermore, _L. plantarum_ was capable of significantly reducing the adhesion of _E. coli_ by 77% in displacement assays. These results indicate that our _L. plantarum_ strain is capable of competing with and displacing _E. coli_ on the cellular model Caco-2. On the other hand, we also investigated the in vitro potential immunomodulatory activity of _L. plantarum_ CNTA 628 directly on the immune system using RAW 264.7 cells and under an intestinal scenario using HT-29 cells. For a correct assessment of cytokine production, we first determined the potential cytotoxic effect of the probiotic strains on both RAW 264.7 and HT-29 cell lines, maintaining the same conditions as for the cytokine assay quantification. Macrophage cell line RAW 264.7 did not show a decrease in cell viability under the anti-inflammatory control hydrocortisone or _L. plantarum_ CNTA 628 (Figure 6A). In a similar manner, HT-29 cell viability was unaffected by the presence of the lactic bacteria (Figure 6B). Once we demonstrated the absence of the cytotoxicity  
Figure 4: _L. plantarum_ CNTA 628 exhibited the property of biofilm formation. The data are presented as mean \(\pm\) SD. Three independent experiments were performed, with three replicates per assay. of the cells when cultured in the presence of _L. plantarum_, we determined the immunomodulatory activities of the probiotic. In RAW 264.7 cells, _L. plantarum_ did not significantly alter the secretion of IL-6 (Figure 6C) or TNFa (Figure 6D) under basal conditions. However, when cells were stimulated with LPS, _L. plantarum_ significantly increased TNF-\(\alpha\) secretion. Additionally, we analyzed the levels of proinflammatory cytokine IL-10. Under basal conditions, _L. plantarum_ drastically increased IL-10 production (Figure 6E). Interestingly, under LPS stimulation, _L. plantarum_ did not significantly modify IL-10 secretion compared to only LPS control cells (Figure 6E). Figure 5: Results of adhesion of _E. coli_ to Caco-2 cells individually, in competition with _L. plantarum_, exclusion displacement (_E. coli_/_L. plantarum_). Data represented as mean \(\pm\) standard deviation.