2.5. Simulated GI Conditions

Simulated gastric conditions were performed using phosphate buffer saline (PBS: 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.76 mM KH₂PO₄) at pH 2.5.

Simulated intestinal conditions were performed using MRS Broth supplemented with 0.3% bile (Sigma, product n. B8381) and indicated as MRS/bile.

In detail, 1 mL of free or microencapsulated cells (sample before exposure) was suspended in 9 mL of PBS at pH 2.5 or MRS/bile and incubated at optimal growth temperature for the tested strain. Incubation duration was 3 and 4 h for simulated gastric and intestinal conditions, respectively. Samples were analysed at the start (T0) and end (T3 or T4) of the incubation period. Surviving bacteria were enumerated by serially diluting 1 mL aliquots of the test materials, pour plating on MRS Agar and incubating at optimal growth temperature for the tested strain, for 48 h in anaerobic conditions. Three independent experiments were replicated. Residual population at T0 and T3 (gastric condition) or T4 (intestinal condition) was compared with population before the exposure, and t-test was performed on the mean values to ascertain significative difference (see Section 2.9).