Mucin

Methanol, polyvinylpyrrolidone (K30; PVP), ketoprofen (KTP), flurbiprofen (FBP), ibuprofen (IBP), loxoprofen (LXP), mucin (from porcine stomach), phosphate-buffered saline (PBS), and trifluoroacetic acid were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Alginic acid sodium salt (Alg, from brown algae with low viscosity) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydroxypropyl methyl cellulose HPMC (TC-5^® M) was purchased from Shin-Etsu Chemical Co. Ltd. (Tokyo, Japan). All other chemicals used were of reagent grade.

Lactic acid bacteria cultures (2 mL) were added to 78 mL of simulated gastric acid containing 10 g of Proteose Peptone No. 3 (BD), 3 g of mucin (Wako, Osaka, Japan), 10 g of NaCl (Manac, Tokyo, Japan), 6 g of NaHCO₃ (Wako), 2 g of KH₂PO₄ (Wako), and a small amount of HCl (Wako) for pH adjustment per liter. Bacterial cultures were incubated in pH-4.0 simulated gastric acid with agitation at 75 rpm for 1 h, then 0.1 M NaHCO₃ was added to raise the pH to 4.4 and simulated bile containing 0.3% oxgall solution (BD) was added. This was followed by incubation for 5 min at 37°C. Then, 0.1 mL of the reaction fluid obtained after each incubation step was harvested in 0.9 mL of PBS and plate counting was performed as described above to determine the number of surviving LcS.