2.3. Stage II, the Validation Experiment

In vitro validation. B. pseudolongum was inoculated into MRS agar medium (agar 20 g, glucose 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, C₆H₅O₇(NH₄)₃ 2 g, Tween-801 mL, CH₃COONa 5 g, K₂HPO₄ 2 g, MgSO₄ 0.58 g, MnSO₄ 0.25 g, water 1 L) as the control and JSRS agar medium (agar 20 g, JSRS 20 g, peptone 10 g, beef extract 10 g, yeast extract 5 g, C₆H₅O₇(NH₄)₃ 2 g, Tween-80 1 mL, CH₃COONa 5 g, K₂HPO₄ 2 g, MgSO₄ 0.58 g, MnSO₄ 0.25 g, water 1 L) anaerobic culture for 48 h, and the total number of microbial colonies were calculated. To determine the extent of starch utilization by B. pseudolongum, the plate was treated with iodine plus potassium iodide solution, and diameter of the colony and transparent circle was calculated.

In vivo validation. After 2 weeks of acclimatization on an NFD to the laboratory environment, mice (n = 40) were randomly subdivided equally into 4 groups [NFD, (n = 10); TR (HFD was fed for the first 3 weeks for making the obese mice model, then, 90% NFD plus 10% JSRS plus 8Log CFU B. pseudolongum infusions were done later, n = 10); HFD (n = 10); PR (90% HFD plus 10% JSRS plus 8Log CFU B. pseudolongum infusions, n = 10)] and were treated for 3 weeks to make the nutritionally obese mice model. After 3 weeks, three mice from each group were euthanized to determine abdominal fat accumulation. The rest of the mice were continued on the respective dietary treatment for another 3 weeks. The NFD and HFD groups were gavaged with an equal volume of normal saline as controls (Figure 1B). Body weight was recorded weekly. Feces were collected weekly and stored at −80 °C. After 8 weeks of intervention, mice were subjected to a 16-h fast and then were euthanized. Livers and abdominal fat were excised and weighed quickly. Then, livers were washed by phosphate buffer saline and processed for further analysis.