2.2.1. Medium

The culture medium was based on the modified standard ileal efflux medium (SIEM), as previously described [22]. Per liter, the medium contained the following: 4.5 g NaCl, 2.5 g K2HPO4, 0.45 g CaCl2·2H2O, 0.5 g MgSO4·7H2O, 0.005 g FeSO4·7H2O, 0.05 g ox bile, 0.01 g haemin, 0.4 g cystein, 3 g bactopeptone, and 3 g casein. We then added 1 mL of a vitamin mixture, per liter of the SIEM contained: 1 mg menadione, 2 mg D-biotin, 0.5 mg vitamin B-12, 10 mg pantothenate, 5 mg nicotinamide, 5 mg *para*-aminobenzoic acid, and 4 mg thiamine.

#### 2.2.2. Fecal Inoculum

Fecal samples were obtained from four healthy donors aged 22 to 23 years (mean age 23) who did not have any intestinal diseases and had not been treated with antibiotics for the previous three months. The donors' mean Body Mass Index (BMI) was 26.3 kg/m2 (range: 22.8–35.1). Fecal samples were collected in a centrifuge tube with a sterile phosphatic buffer saline (PBS); then they were swirled and centrifuged (5000× *g*, 10 min). The mixture was diluted with a sterile PBS buffer to 0.1 g/mL to obtain the final fecal inoculum.

### 2.2.3. In Vitro Fermentation

The FOS, PEC, BG, and AX were each mixed with the C3G (CF, CP, CB, and CA groups), and each of the four mixtures was added to the SIEM culture medium as the substrates. The final fermentation system contained a 10 mg/mL concentration of dietary fiber and a 1 mg/mL concentration of C3G. Another experimental group supplemented only with a 1 mg/mL concentration of C3G (C group) was designed. The medium without any dietary fiber or C3G was used as blank control (N group). The mixture (3.6 mL) of medium and fermentation substrate was placed into an anaerobic fermentation tube; then, under anaerobic conditions, we added 0.4 mL of fecal inoculum. The in vitro fermentation was carried out in a vibrating incubator (37 ◦C, 100 rpm), and the fermentation fluids were collected and stored in a refrigerator, at −80 ◦C, after 0, 0.5, 1, 2, 4, 12, and 24 h. Each group was repeated three times in this study.
