Mrs broth agar

Colonies from Lacticaseibacillus paracasei (CECT 9610) and Lactiplantibacillus plantarum (CECT 9608) were grown in MRS broth/agar (Oxoid, Dublin, Ireland) at 37 °C in aerobiosis (Lactiplantibacillus plantarum) or anaerobiosis (Lacticaseibacillus paracasei) for 48 h. Cultures were heat‐inactivated at 80 °C for 2 h and microbial cells were collected after centrifugation at 1036 g for 10 min. Two washes with sterile water were carried out to remove the remains of the culture medium. Cell pellets were adjusted to a O.D.600 nm (Optical Density) of 1 in sterile water.

Colonies from the selected lactobacilli isolates were grown in MRS broth/agar (Oxoid) at 37°C without any aeration for 24–48 h. Two Mycobacterium bovis Bacillus Calmette-Guérin (BCG) strains were used to determine the antagonistic effect of the lactobacilli isolates against M. bovis: BCG Pasteur and BCG ΔleuD pAS^OriMXF (Brosch et al., 2000 (link); Stedman, 2017 ). Both BCG strains were cultured in Middlebrook 7H9 (Difco) broth supplemented with 10% Oleic acid-Albumin-Dextrose-Catalase enrichment (OADC, Sigma-Aldrich), 0.05% Tween 80 (Sigma-Aldrich), 0.2% glycerol and 40 μg/ml kanamycin at 37°C in an orbital shaker at 225 rpm for 5–7 days. BCG ΔleuD pAS^OriMXF was generated by transforming the BCG Pasteur ΔleuD strain with pAS^OriMXF as previously described (Stedman, 2017 ). pAS^OriMXF is a mycobacterial episomal vector that complements the leucine mutation to correct auxotrophy, and enable stable expression of GFP under the control of the constitutively expressed promoter pL5X (Borsuk et al., 2007 (link)). All bacterial strains were maintained as −80°C frozen stocks in their appropriate media with the addition of 15% glycerol.