Zirconia glass beads

Intracellular Mtb cAMP levels were measured using the Direct cAMP Enzyme Immunoassay Kit according to the acetylated version of the manufacturer's protocol (Sigma‐Aldrich). Sample culture aliquots were recovered and resuspended to ~1x10⁸ CFU/ml in TBST, pH 6.50, centrifuged at 4500 × g for 10 min at 4°C, resuspended in 0.10 M HCl, and boiled for 10 min at 100°C (Kahramanoglou et al., 2014 (link)). Whole‐cell lysates were transferred to 1.50 ml screw‐cap microcentrifuge tubes (USA Scientific) filled with 200 μl 0.10 mm diameter zirconia glass beads (BioSpec Products) and exposed to three rounds of bead beating (2400 oscillations in 30 s), using the Mini‐BeadBeater‐1 (BioSpec Products), with cooling on ice for at least 2 min in between each round. Bacterial cell debris was removed via centrifugation at 12,500 × g for 15 min at 4°C, and clarified lysates were stored at −20°C until further use. Intracellular cAMP levels were measured by reading the optical density at 405 nm (OD₄₀₅) of 100 μl of immunoassay whole cell lysates using an EnSpire Multimode microplate reader (PerkinElmer). Intracellular cAMP levels were estimated from standard curves generated from reading the OD₄₀₅ of 0–20 pmol/ml of cAMP in 0.10 M HCl, and cAMP per 10⁸ CFU was calculated by dividing pmol cAMP/ml by CFU/ml, similarly to prior reporting (VanderVen et al., 2015 (link)).

RNA was extracted from UHT full fat milk fermented for 5 days at 37 °C with the strains L. casei 2138 and L. paracasei 2333, which showed opposite trends for the production of the volatile compound acetoin after growth in UHT full fat milk. RNA was extracted using the TRIzol^® Reagent (Invitrogen) protocol. In brief, 250 µL of fermented milk were placed in a 2 mL screw cap tube containing a 500 µL volume of 0.1 mm zirconia/glass beads (BioSpec Products, Bartlesville, USA) and immediately added with 1.5 mL of TRIzol (Invitrogen, Milan, Italy).
Sample processing was performed with a Mini-BeadBeater 8 (BioSpec Products, Bartlesville, OK, USA), with three repeated intervals of 60 s mixing at maximum speed followed by 60 s pauses on ice. After homogenization, TRIzol manufacturer’s instructions were followed to extract total RNA. Total RNA samples were treated with TURBO DNA-free™ Kit (Ambion, Life Technologies, Milan, Italy). Digested RNA samples were then quantified spectrophotometrically, and 1 µg was used for reverse transcription using SuperScript™ IV Reverse Transcriptase (ThermoFisher Scientific, Milan, Italy) with random hexamers priming strategy, according to the manufacturer’s protocol.