Anaerobic Gut Bacteria Cultivation Protocol

Roseburia intestinalis L1-82 (DSMZ14610) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). Bacteroides fragilis (ATCC 25285) and Bacteroides vulgatus were obtained from Ningbo Mingzhou Biotechnology Co., Ltd. (Zhejiang, China). The above bacteria were cultured under anaerobic conditions at 37°C. An anaerobic culture environment was created using Atmosphere Generation Systems (AnaeroJar^™ ASSEMBLY and AnaeroGen^™, OXOID, Thermo Fisher Scientific). The culture medium (Miquel et al., 2015 (link)) was brain heart infusion (BHI) containing 0.5% yeast extract (OXOID), 1 mg/ml cellobiose (Macklin, China), 1 mg/ml maltose (Solarbio, China), 0.5 mg/ml cellobiose (Macklin, China), and 0.5 mg/ml cysteine (Solarbio, China). We established a standard curve to convert the absorbance values at 600 nm (OD600) to CFU values (Supplementary Figure S1). Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Salmonella typhimurium CMCC 50115, Bacillus subtilis DSM 1088, and Bacillus cereus ATCC 11778 were used as controls. These strains were cultured in nutrient broth at 37°C in an aerobic environment.