Gastric mucin

Simulated gastric and intestinal fluids were prepared according to published reports [24 (link),38 (link)]. In total, 1 g pepsin (pepsin, from porcine gastric mucosa, Sigma-Aldrich, St. Louis, MO, USA), 1.5 g gastric mucin (mucin from porcine stomach Type II, Sigma-Aldrich), 8.7 g NaCl, and 5 g bile salts (bile extract porcine, Sigma-Aldrich) were dissolved in 1 L water. After 3 h, 1 mL of the suspension was inoculated into 9 mL of simulated intestinal fluid (pH 8.0 and incubated at 37 °C). The viability (log cfu/mL) of Lacticaseibacillus rhamnosus GG ATCC 53103, Lactiplantibacillus plantarum ATCC14917, Lactobacillus gasseri ATCC 14917, and Levilactobacillus brevis ATCC 8287 was tested after 3, 6, 9, and 12 h of incubation at pH 8 (HCl 2M) in the presence of 0.5% (w/v) bile salts derived from dried pig bile. The samples were incubated anaerobically at 37 °C and retrieved for enumeration at their respective end points.

Artificial rat saliva and gastric juice were prepared following the method of Chen et al. [10 (link)] and Wu et al. [14 (link)]. Artificial pancreatic juice and bile juice were prepared according to Wu et al. [15 (link)]. Glucose concentration was determined with a D-glucose assay kit (GOPOD-Format) (Megazyme International Ireland Ltd., Wicklow, Ireland). Gastric mucin and α-amylase were obtained from Sigma (Sigma-Aldrich, Saint Louis, MO, USA). Amyloglucosidase, pancreatin, and pepsin were obtained from Solarbio (Shanghai Solarbio Bioscience & Technology Co. Ltd., Shanghai, China). All other chemical reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Beijing, China).
The DIVRSD-II model was applied to digestion of HPH-treated starch [15 (link)]. Starch samples (200 mg, dry basis) were dispersed in 2.0 mL of water and cooked for 15 min until gelatinized. Then 2.0 mL of artificial saliva (37 °C) was added to mimic oral digestion [16 (link)]. The food samples were digested in the DIVRSD-II model in batches as reported by Wu et al. [15 (link)] for 0, 10, 20, 30, 40, 50, 60, 90, 120 and 180 min.

For the systemic infection model, ICR male mice (four-week-old male weighing 18 to 20 g) (Daehan Bio Link Co., Ltd., Eum sung Gun, Korea) were used. Mice were maintained in animal chambers kept at 23 ± 2 °C with 55% ± 20% relative humidity. A systemic infection mouse model was established as described previously [20 (link)]. K. pneumoniae strains were cultured in CAMHB for 18 h at 37 °C. For inoculation, the cultured bacteria were suspended in 0.9% NaCl containing 5% gastric mucin (Sigma-Aldrich). Groups of five male ICR mice were intraperitoneally (i.p.) injected with a single 0.5 mL dose of the bacterial suspension. The challenge inoculum was sufficient to kill 100% untreated control mice within 24 h after infection. LCB10-0200 and ceftazidime were subcutaneously administered at 1 and 4 h after the bacterial infection. Mortality was recorded for 7 days, and the median effective dose required to protect 50% of mice (ED₅₀) was calculated using the probit method with BioStat version 6 (Walnut, CA, USA).