L cysteine

The populations of the main groups of microorganisms were determined by plating the brines and their decimal dilutions (in 0.9% NaCl) on the appropriate solid media with a spiral plater (Easy Spiral Dilute, Interscience, Saint Nom la Bretèche, France). The culture media used were de Man–Rogosa–Sharpe agar (MRS, Oxoid, Basingstoke, UK) supplemented with 0.02% (w/v) sodium azide (Sigma-Aldrich, St. Louis, MO, USA) and 0.05% (w/v) L-cysteine (AppliChem GmbH, Darmstadt, Germany) for LAB; glucose-yeast extract agar containing oxytetracycline (0.01% w/v; AppliChem; OGYE) for yeast; VRBG agar (Laboratorios Conda S.A., Torrejón de Ardoz, Spain) for Enterobacteriaceae; and plate count agar (PCA, Labkem, Premià de Dalt, Spain) for mesophilic aerobic bacteria (MAB). MRS plates were incubated under anaerobic conditions using a DG250 Anaerobic Workstation (Don Whitley Scientific Ltd., Shipley, West Yorkshire, UK), with a gas mixture consisting of 10% H₂-10% CO₂-80% N₂ at 30 °C for 72 h. OGYE plates were incubated aerobically at 25 °C for 72 h. VRBG plates were incubated aerobically at 37 °C for 24 h. PCA plates were incubated aerobically at 22 °C for 72 h. The resulting numbers of colony-forming units were counted with a Scan 500 (Interscience, Saint-Nom-la-Bretèche, France) colony counter. The detection limit was established in 10 CFU/mL.

Bifidobacteria were routinely propagated on de Man Rogosa Sharpe media (MRS; BD Difco) with 0.05% (w/v) of L-cysteine (Sigma Aldrich, St. Louis, MO, USA). Cultures were incubated at 37 °C in an anaerobic chamber maintained with a gas mix of 7% H₂, 10% CO₂, and N₂ to balance (Coy Laboratory Products, Grass Lake, MI, USA). Genomic DNA was extracted from liquid cultures using the MasterPure Gram Positive DNA Purification Kit according to the manufacturer’s protocol (Epicentre, Madison, WI, USA). In order to isolate bifidobacteria, fresh feces were mixed into 5 mL of peptone water. Serial dilutions of 10x and 100x were performed on bifidobacterial-specific media agar plates which consisted of MRS, 0.05% (w/v) L-cysteine, and 0.05% (w/v) mupirocin (AppliChem Panreac, Chicago, IL, USA). Isolated colonies were identified via the bifidobacteria-specific colorimetric fructose-6-phosphoketolase assay and PCR of the 16s and ITS rRNA gene regions (PCR primers: F-GGTGTGAAAGTCCATCGCT, R-GTCTGCCAAGGCATCCACCA; Sanger sequencing primers: F-GGTGTGAAAGTCCATCGCT, R-CATGCCCCTACGTCCAG) according to Turroni et al. and Milani et al. [21 (link),22 (link),23 (link)].