L tryptophan

Anaerobic cultivation was performed according to the instructions of the Leibniz Institute DSMZ [79 ]. The Hungate anaerobic culture tube and Coy anaerobic chamber (Coy Laboratory Products, USA) were used in this experiment. BP01R2 was precultured in M9 minimal medium (248510; Becton Dickinson, USA) for 24 h, and then washed three times using the same method before use as an inoculant. The inoculant density was set at OD₆₀₀ = 0.01; the cultivation volume was 5 ml, and the culture was carried out by shaking at 120 rpm and 37°C. M9 minimal medium supplemented with 2 mM MgSO₄ (131-00405; FUJIFILM Wako, Japan), 0.1 mM CaCl₂ (21 075; Sigma-Aldrich, USA), 10 mM NaNO₃ (195-02545; FUJIFILM Wako, Japan), and 0.01% L-tryptophan (A10230; Alfa Aesar, USA) was prepared as a general incubation medium. Extra glucose was tested as an organic carbon resource at concentrations (w/v) of 0.4%, 0.2%, 0.04%, and 0%; the tube headspace gas was composed of either sterile air or 90% H₂ plus 10% CO₂ (N₂). Growth curves were measured at OD₅₉₅ using the PARADIG detection platform (Beckman, USA) or a Sunrise absorbance microplate reader (Tecan, Austria).

Chlorpropamide (CLP, >97% purity), tolbutamide (TLB, >97% purity), ascorbic acid (ASC, >99.0% purity), citric acid (CIT, ≥99.5% purity), p-aminobenzoic acid (PABA, 99.0% purity), tromethamine (TRIS, >99.0% purity), Triton^® X-100, thiazolyl blue tetrazolium (MTT), dimethyl sulfoxide (DMSO), and Dubelcco’s phosphate buffered saline, trypan-blue solution were acquired from Sigma-Aldrich (St. Louis, MO, USA). L-(+)-arginine (ARG, ≥98% purity) was purchased from Acros Organics (Fair Lawn, NJ, USA), L-tryptophan (TRY, 99% purity) from Alfa Aesar (Kandel, Germany), and malic acid (MAL, >99.5% purity) from Merck (Darmstadt, Germany). The water used was ultra-pure grade (resistivity of 18.2 MΩ·cm), purified by a Heal Force ultra-pure water system (Shanghai, China). All chemicals were used without further purification.
Dulbecco’s modified Eagle’s medium (DMEM) with Glutamax and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, trypsin-EDTA (1×), penicillin–streptomycin (PenStrep), inactivated fetal bovine serum (FBS), and non-essential amino acids were purchased from Gibco by Life Technologies (New York, NY, USA. A lactate dehydrogenase (LDH) cytotoxicity detection kit was obtained from Takara Bio Inc. (Shiga, Japan). Murine fibroblast (L929) and intestinal epithelial (Caco-2) cell lines were acquired from the European Collection of Authenticated Cell Cultures (ECACC, Salisbury, UK).

IAA production of each PGP isolate was determined using Salkowski’s reagent^{47 (link)}. Inoculum (100 µL) of each isolate was added into 5 mL sterile TSB medium supplemented with 1 g L⁻¹l-tryptophan (Acros Organics). The tubes were done in triplicate and then incubated at 30 °C, 150 rpm for 48 h. Aliquots of 1 mL of supernatant were mixed with 2 mL of Salkowski’s reagent^{47 (link)}, before being incubated in the darkness for 30 min. The appearance of red coloration was used to quantify IAA production. The sample was read at 530 nm. A standard curve was constructed using IAA (Acros Organics).