Nh4 2co3

All commercially available reagents were used as received unless otherwise stated. Such as methanol, ethanol, diethyl ether, ethyl acetate. Zn(NO₃)₂·6H₂O, ZrO(NO₃)₂·xH₂O, K₂PdCl₄, and (NH₄)₂CO₃ were purchased from Sigma-Aldrich, Alfa Aesar, and Fisher Scientific Co. Ltd., respectively, and were used without further treatment.

Analytical grade (NH₄)₂CO₃, CaCl₂·2H₂O and L-aspartic acid sodium salt monohydrate were purchased from Sigma-Aldrich and used as received. Na₂SiO₃ solution (1.35 g cm⁻³) was from Merck Chemicals, and aqueous stock solutions were prepared using Milli-Q water, 18.2 MΩcm. Stock solutions of L-α PC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, >99%, Sigma) were prepared in HPLC-grade chloroform. Glassware used to prepare solutions was soaked overnight in 10% w/v NaOH, followed by rinsing with dilute HCl and finally washing with Milli-Q water. Glass slides and crystallizing dishes were placed overnight in Piranha solution (70:30 wt% H₂SO₄: H₂O₂) and then washed copiously with Milli-Q water before drying with acetone.

All materials were standard analytical grade. α-amylase was obtained from Bacillus licheniformis (E-BLAAM EC.3.2.1.1; Megazyme International Ireland Ltd., Wicklow, Ireland) and was supplied at a concentration of 10000 U/ml on soluble starch at pH 6.5 and 40°C. Glucoamylase was obtained from Aspergillus niger (AMG Conc. BG; Novozymes, Bagsværd, Denmark) and had an activity of 1300 AGU/g. Pepsin (P6887; Sigma-Aldrich S.r.l. Milan, Italy) was obtained from porcine gastric mucosa and had an activity between 3200 and 4500 U/mg protein. Creon® 10000 U.Ph.Eur. (Abbott Products S.p.A; Sesto San Giovanni (Mi), Italy), which is normally used in the treatment of pancreatic exocrine insufficiency, was available as brown/clear capsules. Each capsule contained lipase (10000 Ph.Eur. units), amylase (8000 Ph.Eur. units), and protease (600 Ph.Eur. units). CaCl₂, KCl, KH₂PO₄, NaHCO₃, NaCl, MgCl₂(H₂O)₆, (NH₄)₂CO₃ and HCl were purchased from Sigma-Aldrich (Sigma-Aldrich, Sigma-Aldrich S.r.l. Milan, Italy).

The survival kinetics of the strains in simulated gastric juice (SGJ) were measured during 2 h of incubation, as previously described by [29 (link)]. The SGF was prepared with KCL 6.9 mM, HCl 15.6 mM, KH₂PO₄ 0.9 mM, NaHCO₃ 25 mM, NaCl 47.2 mM, MgCl₂ 0.1 mM, (NH₄)₂CO₃ 0.5 mM (all from Sigma-Aldrich, Saint Quentin Fallavier, France) and sterilized through filtration. The pH was then adjusted to 3 using HCl 1 M, and CaCl₂ and porcine pepsin (Sigma-Aldrich, Saint Quentin Fallavier, France) were added to achieve a final concentration of 0.075 mM and 2.000 U/mL, respectively, in the final digestion mixture. The bacterial suspension was centrifugated (2000 rpm, 10 min), washed twice with PBS, and standardized at 10⁹ CFU/mL. Then, 950 µL of SGF (with pepsin and at pH 3) was inoculated with 50 µL of the bacterial suspension to reach a final concentration of 5 × 10⁷ CFU/mL and incubated at 37 °C for 2 h with sampling at 0 and 120 min. Bacterial viability was measured by the numeration of a serial dilution plated on BBA plates after 48 h incubation in anaerobiosis. The death rate was calculated by dividing the number of CFU/mL at 120 min by the CFU/mL measured at time zero, as the 14 studied strains have been previously shown to survive under aerobic conditions without any indication of mortality for 6 h [26 (link)].

An Fe₂O₃-Al₂O₃ mixed oxide was synthesized by co-precipitation. Fe(NO₃)₃·9H₂O (8 g, 0.02 mol) and Al(NO₃)₃·9H₂O (7.5 g, 0.02 mol, 98%, Sigma-Aldrich) were first dissolved in 80 mL deionized water. Separately, (NH₄)₂CO₃ (7.7 g, 0.08 mol, 99%, Sigma-Aldrich) was dissolved in 80 mL deionized water. The two aqueous solutions were combined in 30 mL water at 50 °C under stirring for 3 h, and then aged static for a further 2 h. The resulting solid was recovered by filtration, washed with deionized water, dried at 60 °C overnight, and finally calcined at 800 °C (ramp rate 3 °C min⁻¹) for 5 h under flowing air (100 mL min⁻¹).