A1043

During the experiment, a medium [24 (link),25 (link)] of the following composition (g L⁻¹) was used: 6.43 sodium chloride (S9888; Sigma-Aldrich, Munich, Germany); 0.50 potassium chloride (191494; PanReac Applichem, Barcelona, Spain); 0.21 calcium chloride (131232; PanReac Applichem, Barcelona, Spain); 0.05 potassium dihydrogen phosphate (A1043; PanReac Applichem, Barcelona, Spain); 0.05 magnesium chloride (131396; PanReac Applichem, Barcelona, Spain); 5.67 sodium hydrogen carbonate (131638; PanReac Applichem, Barcelona, Spain); 0.15 urea (U5378; Sigma-Aldrich, Munich, Germany); albumin (CAS Number 9048-46-8; “Sonac Loenen BV”, GD Loenen, Netherlands); 10.00 pork bile (48305; Sigma, St. Louis, MO, USA); 1.00 lipase (CAS Number 9001-62-1; Caglificio Clerici S.p.A., Cadorago, Italy); and 6.00 pancreatin (A0585; Panreac, Barcelona Spain), pH 6.0.

During the experiment, simulated gastric juices of the following composition (g L⁻¹) were used: 3.50 dextrose (361105; Roqquette, Lestrem, France); 2.05 sodium chloride (S9888; Sigma-Aldrich, Munich, Germany); 0.60 potassium dihydrogen phosphate (A1043; PanReac Applichem, Barcelona, Spain); 0.11 calcium chloride (131232; PanReac Applichem, Barcelona, Spain); and 0.37 potassium chloride (191494; PanReac Applichem, Barcelona, Spain), pH 2.0. The solution was autoclaved at 115 °C for 20 min. The following components were sterilized by filtration (0.2 μm, Millipore, St. Louis, MO, USA) and added to the solution after cooling: 0.05 g L⁻¹ pork bile (48305; Sigma, St. Louis, MO, USA); 0.10 g L⁻¹ lysozyme (CAS Number 9066-59-5; Caglificio Clerici S.p.A., Cadorago, Italy), and 13.30 mg L⁻¹ pepsin (12762; JSC “Plant of Endocrine Enzymes”, Moscow, Russia) [23 (link)].

The tolerance of microorganisms to pepsin and pancreatin was evaluated separately. The bacterial cell precipitate was resuspended in the buffered enzyme (pancreatine or pepsin) solution in a 1:1.33 ratio. The enzyme solutions were previously sterilized by filtration (0.22 μm, Pall Corporation, Port Washington, NY, USA) [22 ,27 ].
To obtain the pancreatin (A0585; Panreac Applichem, Barcelona, Spain) solution (6 g L⁻¹), a phosphate buffer of the following composition (g L⁻¹) was used: 8.00 sodium chloride (S9888; Sigma-Aldrich, Munich, Germany); 0.24 sodium hydrogen phosphate (131679; PanReac Applichem, Barcelona, Spain); 0.20 potassium chloride (191494; PanReac Applichem, Barcelona, Spain); 0.24 potassium dihydrogen phosphate (A1043; PanReac Applichem, Barcelona, Spain) [28 ], pH 7.0. Pepsin (12762; JSC “Plant of Endocrine enzymes”, Moscow, Russia) was dissolved in the citrate–HCl buffer (pH 3.0) at 13.5 g L⁻¹ [23 (link),25 (link)]. To prepare the citrate–HCl buffer at pH 3.0, 21.0 g of citric acid (C2404; Sigma-Aldrich, Munich, Germany) was dissolved in 200 mL of 1 M NaOH, brought up to 1 L with distilled water, and 40.3 mL of this solution was diluted with 0.1 M HCl to 100.0 mL [29 ].