*3.1. S. mutans Was Promoted by Core Microbiota and the Core + S. Mutans Biofilm Showed Increased Acidogenic Ability*

To have a better consistency of the ECC plaque composition, we developed the core biofilm with an even amount of the selected four genus as well as the Core + *S. mutans* biofilm with 50% of the core microbiota and 50% of the *Streptococcus mutans*. qPCR was used for the analysis of biofilm composition. In the Core group, *V. parvula* was the most dominated species within the biofilm (69.85%, 69.87%, and 66.99%), followed by *L. wadei* (14.24%, 15.59%, and 13.77%), *P. denticola* (11.31%, 12.38%, and 17.36%), and *F. nucleatum* (2.44%, 1.53%, and 1.22%) (Figure 1a). In the Core + *S. mutans* biofilm, *S. mutans* counted for 51.16%, 54.63%, and 63.86% at 24 h, 48 h, and 72 h separately, followed by *V. parvula* (28.53%, 29.59%, and 19.96%); *L. wadei* (16.27%, 9.51%, and 4.75%); *P. denticola* (2.74%, 4.87%, and 7.45%), and *F. nucleatum* (1.30%, 1.40%, and 2.51%) (Figure 1b). In order to detect the influence of the core microbiota on the growth of *S. mutans*, we measured the count of *S. mutans* in the *S. mutans* group and in the Core + *S. mutans* group. *S. mutans* was promoted by the core microbiota and demonstrated a 3.41, 5.17, and 14.63 times increase in CFU counting at 24 h, 48 h, and 72 h compared to single *S. mutans* biofilm (*p* < 0.05) (Figure 1c,d). The pH of the culture medium measured during the experiments was taken as an indirect indicator of the biofilm acidogenicity. We found that the pH of all three groups was under 5, which can serve as the biological initiation of the enamel demineralization. The Core + *S. mutans* group showed the lowest pH at all time points (*p* > 0.05), followed by the *S. mutans* group and the Core group (*p* < 0.05) (Figure 1e).

**Figure 1.** Composition of the biofilm and biofilm pH monitoring. (**a**) Composition of the core biofilm at 24 h, 48 h, and 72 h. (**b**) Composition of the Core + *S. mutans* biofilm at 24 h, 48 h, and 72 h. (**c**) *S. mutans* counting of the Core + *S. mutans* biofilm and *S. mutans* biofilm at 24 h, 48 h, and 72 h (n = 3). (**d**) Ratio of *S. mutans* in Core + *S. mutans*/*S. mutans*. (**e**) Medium pH of the biofilms at 24 h, 48 h, and 72 h (n = 3). Data are expressed as means ± standard errors. \* *p* < 0.05; \*\* *p* < 0.01.
