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The ampicillin/erythromycin-resistant AIEC LF82, isolated from a chronic ileal lesion of a CD patient [32 (link)], was used as the AIEC reference strain. AIEC was grown in a nutrient broth at 37 °C under aerobic conditions upon inoculating 1% from a frozen stock stored at −80 °C with 20% (v/v) of glycerol.
Two media were used during fecal batch incubations. In Test 1, the background medium consisted of 5.2 g/L of K₂HPO₄, 16.3 g/L of KH₂PO₄, 2.0 g/L of NaHCO₃ (Chem-lab NV, Zedelgem, Belgium), 2.0 g/L of Yeast Extract (Oxoid, Aalst, Belgium), 2.0 g/L of pepton (Oxoid, Aalst, Belgium), 1.0 g/L of mucin (Carl Roth, Karlsruhe, Germany), 0.5 g/L of L-cystein, and 2.0 mL/L of Tween80. In Test 2, the concentrated background medium consisted of 7.6 g/L of K₂HPO₄, 23.9 g/L of KH₂PO₄, 2.9 g/L of NaHCO₃ (Chem-lab NV, Zedelgem, Belgium), 2.9 g/L of Yeast Extract (Oxoid, Aalst, Belgium), 2.9 g/L of pepton (Oxoid, Aalst, Belgium), 1.5 g/L of mucin (Carl Roth, Karlsruhe, Germany), 0.7 g/L of L-cystein, and 2.9 mL/L of Tween80.

Short-term colonic incubations were performed as described in [31 (link)]. Briefly, fresh fecal material from a healthy human donor (f, 26y) was collected and after preparation of an anaerobic fecal slurry inoculated at 10 vol% in a sugar-depleted nutritional medium containing 5.2 g/L K₂HPO₄, 16.3 g/L KH₂PO₄, 2.0 g/L NaHCO₃ (Chem-lab NV, Zedelgem, Belgium), 2.0 g/L yeast extract, 2.0 g/L pepton (Oxoid, Aalst, Belgium), 1.0 g/L mucin (Carl Roth, Karlsruhe, Germany), 0.5 g/L L-cystein and 2.0 mL/L Tween80 (Sigma–Aldrich, Bornem, Belgium). Test products (bpRG-I and cRG-I) were dosed at 5 g/L and reactors were anaerobically incubated at 37 °C for 48 h. The base medium with no addition of test product and inoculated by the fecal slurry was used as the negative control (blank). All experiments were performed in technical triplicate.

Short-term colonic fermentations were performed as described recently [18 (link)]. Briefly, colonic background medium containing 5.2 g/L K₂HPO₄, 16.3 g/L KH₂PO₄, 2.0 g/L NaHCO₃ (Chem-lab NV, Zedelgem, Belgium), 2.0 g/L Yeast Extract, 2.0 g/L pepton (Oxoid, Aalst, Belgium), 1.0 g/L mucin (Carl Roth, Karlsruhe, Germany), 0.5 g/L L-cystein, and 2.0 mL/L Tween80 (Sigma-Aldrich, Bornem, Belgium) was added to incubation reactors (90 vol%), already containing the correct amount of the test products for obtaining a final concentration of 0 g/L (Blank) or 5 g/L (for both AADE and FOS), respectively. The reactors were sealed and anaerobiosis was obtained by flushing with N₂. Subsequently, fresh fecal material of a healthy human donor (no history of antibiotic use in the six months preceding the study) was collected (according to the ethical approval of the University Hospital Ghent with reference number B670201836585; 06/08/2018). After preparation of an anaerobic fecal slurry, this was inoculated at 10 vol% in the aforementioned medium. All incubations were performed in biological triplicate for 48 h at 37 °C under anaerobic conditions with continuous shaking (90 rpm).