## 2 Materials and Methods
### In Vitro Determination of Antibiotic Resistance  
The antibiotic resistance of selected isolates was determined by the modified ISO 10932/IDF 223 standard [29] for the Determination of the Minimal Inhibitory Concentration (MIC) of antibiotics applicable to bifidobacterial and non-enterococcal LAB using the microdilution method. MICs were determined for nine antibiotics: kanamycin (Acros Organics Bvba, Geel, Belgium), gentamicin, streptomycin, tetracycline, clindamycin, chloramphenicol, ampicillin, erythromycinomycin (Merck KGaA, Darmstadt, Germany), and vancomycin (Merck KGaA). The MICs were assessed as the lowest concentration of the antibiotic that inhibited bacterial growth. Based on the MICs, strains were classified as either resistant or susceptible, adhering to the cut-off values and guidelines established by the FEEDAP [30] for LAB and by the SCAN [31] for the _Bacillus_ strain.  
The values were calculated from three independent experiments and were classified as susceptible (S) when the result was under the cut-off value or resistant (R) when the value was above the cut-off value phenotypes.  
Evaluation of the Cytotoxicity of the Cell-Free Supernatant of B. subtilis CNTA 517 on Epithelial Cells  
_In vitro_ assessments of the cytotoxicity of the CFS of _B. subtilis_ were conducted using Caco-2 and HT-29 cell lines; both were human colorectal cancer cells and were obtained from the Leibniz Institute DSMZ--German Collection of Micro-organisms and Cell Cultures GmbH (DSMZ) (Braunschweig, Germany) collection.  
The cytotoxicity was determined by the MTT method (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide) (Merck KGaA). The cell lines were seeded in a 96-well flat-bottom microtiter plate at varying concentrations: \(5\times 10^{3}\) cells/well for the Caco-2 cell line and \(1.7\times 10^{4}\) cells/well for the HT-29 cell line. Subsequently, they were incubated at \(37\)\({}^{\circ}\)C, \(5\%\) CO\({}_{2}\), and \(95\%\) relative humidity for 48 h in the CO\({}_{2}\) incubator SCA-165 DRS (ASTEC Co., Ltd., Fukuoka, Japan). In the case of the Caco-2 cell line, the medium was refreshed three times per week. After 15 days of growth to allow cell differentiation into enterocytes, _B. subtilis_ CFS was introduced at varying concentrations (5% and 20%). For the HT-29 cell line, the concentrations of 5%, 10%, and 20% of _B. subtilis_ CFS were added after 48 h of incubation. The entire volume of cell supernatants was transferred to 96-well U-bottom microtiter plates for subsequent analysis and stored at \(-80\)\({}^{\circ}\)C. Afterward, the culture medium was removed and 200 \(\mu\)L of phenol red-free medium (Thermo Fisher Scientific, Waltham, MA, USA) and 50 \(\mu\)L of MTT at a concentration of 5 mg/mL to each well were added. These plates were then placed at \(37\)\({}^{\circ}\)C, 5% CO\({}_{2}\), and 95% relative humidity in the incubator for 3 h until the observation of thread formation under the inverted microscope ECLIPSE TS100/TS100F (Nikon Microscopy, Melville, NY, USA). When the established time had elapsed, the culture medium was removed to eliminate unmetabolized MTT. Finally, 150 \(\mu\)L/well of dimethyl sulfoxide (DMSO) (Thermo Fisher Scientific) was added and agitated at 347.76 g for 5 min to dissolve the formazan deposits produced. Then, 100 \(\mu\)L of the supernatant was transferred to a new 96-well flat-bottom plate (DELTALAB, Barcelona, Spain), and the optical density (OD) was measured at a wavelength of 570 nm using the plate reader HEALES, MB-580 (Shenzhen Heales Technology Development Co., Ltd., Shenzhen, China). The cytotoxicity of the CFS of the strain _B. subtilis_ CNTA 517 was measured by the viability of the cell lines used following Equation (1):  
\[\%\;viability=\frac{OD570\;sample}{OD570\;control}\times 100 \tag{1}\]  
With this percentage as a basis, statistical significance studies were conducted to ascertain whether there was a decrease in cell cytotoxicity or not.