*2.8. Rat Caries Model and Caries Scoring*

Rat models of dental caries were established according to previous studies [35]. The experiment was performed on 20 specific pathogen-free female Wister rats. Starting from the age of 20 days, all animals were fed with a cariogenic diet (Keyes 2000) and sterilized water containing 5% *w/v* sucrose ad libitum. For the first three treatment days, the water was supplemented with chloramphenicol, ampicillin, and carbenicillin [1 g/kg]). Then, the animals were screened for *S. mutans* by an oral swab streaked on MSB agar. Furthermore, PCR was performed to detect the four bacteria from the core group.

At the age of 24 days, the rats were randomized into four groups infected for seven consecutive days with core microbiota and *S. mutans* (Group A); *S. mutans* (Group B), core microbiota (Group C), and PBS solution (Group D) (approximately 109 CFU/mL, 0.2 mL/rat). After three weeks, the animals were euthanized by cervical dislocation for Keyes Scoring [36]. Briefly, the teeth of each rat were stained with 0.4% ammonium salt solution for the caries red. Then, the teeth were rinsed, dried, hemisectioned, and finally observed by a stereo microscope. The depth and size of the red ammonium salt solution spread into the teeth represent the severity and impact area of the caries. Five specimens were tested for each group (n = 5).
