In vitro Gut Microbiome Fermentation

The in vitro fermentation was performed based on the previous method with minor modifications (Lam et al., 2018 (link)). First, fresh fecal samples were collected from six healthy volunteers (three women and three men, 20–30 years of age) who maintained a regular diet and had not received antibiotics or prebiotic treatment for 3 months. The fecal samples were immediately mixed with sterilized 0.1 M phosphate-buffered saline (pH 7.0) to produce a 10% (w/v) fecal suspension. Then, 2.0 g of peptone, 2.0 g of yeast extract, 0.5 g of cysteine-HCl, 0.1 g of NaCl, 2.0 g of NaHCO₃, 0.04 g of K₂HPO₄, 0.04 g of KH₂PO₄, 0.01 g of MgSO₄ 7H₂O, 0.01 g of CaCl₂ 6H₂O, 0.02 g of hemin, 0.5 g of bile salt, 2.0 ml of Tween 80, 10 μl of vitamin K₁, and 1.0 ml of resazurin 1% (w/v) were dissolved in 1 L ultrapure water to obtain a basic medium. Finally, LDSPs were selected as 1% (w/v) carbon sources, and the medium without carbon sources and 1% (w/v) inulin were used as a blank control (CON) and positive control (INU). The medium was adjusted to pH 7.0 using 1 mol⁻¹ HCl and placed in an anaerobic chamber at 37°C overnight to pre-reduce the media. Here, 1.0 ml of 10% (w/v) fecal slurry was added to a 9.0 ml medium and placed in the 37°C anaerobic chamber and incubated for 0, 6, 12, and 24 h. Then, the samples were collected immediately and stored at −80°C for further analysis.