2.2. Preparation of Cell-Free Culture Supernatant (CFCS)

The assay for obtaining the CFCS was adapted from Pessoa et al. [21]. After overnight cultures of Lactobacillus strains were centrifuged (3,000 × g, 15 min), the supernatants were discarded; cell pellets (lactobacilli) were washed twice with sterile saline (0.9% NaCl) and resuspended to a final concentration (10⁸ CFU.mL⁻¹). After this, suspensions (1.5 mL) of each Lactobacillus sp. were inoculated (10%, v/v) in sterile MRS broth (15 mL), and after incubation (24 h, 37°C, 5% CO₂ atmosphere), the cultures were centrifuged (3000 × g, 15 min); supernatants were aspirated using sterile syringes and sterilized by filtration (0.22 μm nitrocellulose filter; Merck, Darmstadt, Germany) for obtaining CFCS.