Limosilactobacillus Balticus Alleviates DSS-Induced Colitis by Promoting indole-3-propionic Acid Production and Regulating the IL-6/JAK2/STAT3/SOCS3 Pathway
Description
We diluted fresh fecal samples using phosphate-buffered saline (PBS). Coarse particulate matter was eliminated by filtration through a 0.4 mm mesh. De Man–Rogosa–Sharpe (MRS) broth was transferred into anaerobic culture bottles specifically designed for oxygen-free cultivation. After flushing with nitrogen gas, the bottles were sealed and sterilized at 121 °C by autoclaving. Sterile MRS broth received the fecal suspension, followed by anaerobic incubation at 37 °C. Single colonies obtained after 48 h of anaerobic growth were chosen and then underwent two successive rounds of purification on MRS agar plates, followed by 16S rRNA gene amplification. The primer pair employed for amplification consisted of 27F (5′-AGATGATCATGG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′). For taxonomic assignment, sequence information was analyzed using the National Center for Biotechnology Information (NCBI) database together with EzBioCloud. NCBI GenBank database-based sequence comparison was used to evaluate strain homology. After identification, the isolates were further purified and passaged twice prior to repeat sequencing. The bacterial isolates were cultivated in liquid MRS medium at 37 °C, and growth kinetics were assessed by monitoring OD600 with a microplate reader. To determine viable bacterial counts and examine colony characteristics, cultures were serially diluted, the suspension was evenly plated on MRS agar. Tryptophan Metabolism Analysis Approximately 50 mg of mive fecal samples was mixed with 500 μL methanol for metabolite extraction, followed by supplementation with 20 μL of an internal standard mixture (250 ng/mL). After vortex mixing for 3 min, the samples were incubated at −20 °C for 30 min to facilitate protein precipitation. The mixtures were then centrifuged at 10,000 × g for 10 min at 4 °C, and 250 μL of the resulting supernatant was transferred for a second centrifugation step at 10,000 × g for 5 min at 4 °C. Subsequently, 150 μL of the final supernatant was collected and used for LC–MS analysis.