L cysteine hydrochloride

All the stool samples were assessed for the presence of Bifidobacterium. One gram of each stool sample was blended with 9 mL sterile physiological saline. Serial dilution and plating were executed in an anaerobic workstation (AW400TG, Electrotek Scientific Ltd., West Yorkshire, UK). 100 μl of diluent was continuously plated on de Man-Rogosa-Sharpe (MRS) agar plus 0.05% (w/v) L-cysteine hydrochloride (mMRS), 100 mg/L mupirocin (Sangon Biotech Co., Ltd., Shanghai, China) and 50 U/mL nystatin (Sangon Biotech Co., Ltd., Shanghai, China). Agar plates were cultured in the anaerobic workstation flushed with 80% N₂, 10% CO₂, and 10% H₂ at 37°C for 72 h. For each sample, colonies on mMRS plates were counted. Colonies were selected at random and re-streaked onto mMRS agar for purity. The final pure culture was cultured in mMRS broth and preserved in 30% glycerol at −80°C. DNA was extracted from each strain using the Rapid Bacterial Genomic DNA Isolation Kit (Sangon Biotech Co., Ltd., Shanghai, China) and stored at −20°C. Each of the putative Bifidobacterium isolates was identified by a 16S rRNA sequence using the bacterial universal primers (27 F: 5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ and 1492 R: 5ʹ-ACGGCTACCTTGTTACGACTT-3ʹ) by BGI (Shenzhen, China). All the strains were compared with the NCBI BLAST database (http://www.ncbi.nlm.nih.gov/BLAST/) to assign a particular species.

Alginate and its derivatives, including polymannuronate (PM) and polyguluronate (PG), were prepared as previously described [3 (link)]. Tryptone, peptone, yeast extract and Tween 80 were obtained from Sigma (Shanghai, China). Dextran sulfate sodium (DSS) was obtained from MP Biomedicals (Solon, OH, USA). Hemin, agar and L-cysteine hydrochloride were obtained from Sangon Biotech (Shanghai, China) Co., Ltd. (Shanghai, China). All other chemicals of analytical grade were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) unless otherwise specified.

The standard short-chain fatty acids (SCFAs) solutions, including lactate, acetate, propionate, succinate, and butyrate were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Tryptone, peptone, yeast extract, and Tween 80 used for the in vitro anaerobic fermentation experiments were all obtained from Sigma-Aldrich (St. Louis, MO, USA). Dextran sulfate sodium (DSS) was purchased from MP Biomedicals (Solon, OH, USA). DSS was used to induce ulcerative colitis in the colon of C57BL/6J mice.
Hemin, alginate, agar, and L-cysteine hydrochloride were acquired from Sangon Biotech (Shanghai, China). These chemicals were added to the VI medium and were used for the anaerobic culture of the bacterium B. uniformis F18-22. All other chemicals of analytical grade used in the present study were purchased from Sinopharm Chemical (Shanghai, China) unless otherwise specified.