# Antibacterial Effects of _Paeonia lactiflora_ Extract on Oral Microcosm Biofilms
## 4 Discussion
These compounds inhibit oral biofilm maturation by disrupting EPS synthesis and damaging bacterial membranes [45,46]. This effect was also observed in the fluorescence images of the oral microcosm biofilms obtained using the QLF-D camera (Figure 3). QLF-D utilizes 405 nm blue light and a specialized filter to detect endogenous porphyrins and EPSs accumulated within mature oral biofilms as red fluorescence [47,48]. A significant positive correlation has been established between the red fluorescence intensity of oral biofilms and their maturity [47]. In this study, QLF-D was used to monitor and quantify the maturation of oral microcosm biofilms. Red fluorescence intensity in the PL group was lower than that in the DW group but similar to that in the CHX group. These findings suggest that PL extract effectively inhibits the maturation of oral microcosm biofilms, exhibiting antibacterial efficacy comparable with that of CHX. In the analysis of bacterial viability within the oral microcosm biofilm, the PL group exhibited bacterial viability similar to that in the CHX group and significantly lower than that in the DW group (Table 2, Figure 4). These results are consistent with those of a prior study that reported growth-inhibitory effects of PL extract on oral pathogenic bacteria, including _S. mutans_, _Streptococcus sanguinis_, _P. gingivalis_, and _Fusobacterium nucleatum_[49]. However, that study evaluated the antibacterial effects on a single bacterial strain. Oral biofilms are composed of a complex microbial environment with interactions among hundreds of bacterial species [50]. Thus, if specific strains are used to assess antibacterial effects, the actual oral microbial ecosystem cannot be fully replicated. The present study evaluated the antibacterial effects of PL extract using an oral microcosm biofilm model that more accurately reflects the actual oral microbiota. This approach is expected to enhance the clinical relevance of this study compared with that of the previous study. Aciduric bacteria were selected in our study because of their critical role in variogenic oral biofilms; they thrive in acidic environments, which are key factors in dental caries development and ecological shifts within oral biofilms [51]. The analysis of the aciduric  
Figure 4: Representative confocal laser scanning micrographs (10 \(\times\) magnification) of cells stained with green and red dyes. The intensity of green represents live bacteria stained with SYTO-9, and the intensity of red represents dead bacteria stained with propidium iodide. bacterial count revealed that PL extract exhibited antibacterial effects comparable with those of CHX (Table 3). Aciduric bacteria can survive in acidic oral environments and contribute to dental caries [51]. Compared with planktonic cells, _S. mutans_, one of the most representative aciduric bacteria, reportedly demonstrated enhanced acid tolerance when residing within oral biofilms [51; 52; 53]. The results of studies evaluating the antibacterial effects of PL extract on aciduric bacteria, such as _S. mutans_, _Bifidobacterium adolescentis_, and _Lactobacillus acidophilus_, are solely based on comparisons with planktonic cells [25; 29]. The key significance of the present study lies in its demonstration that PL extract has antibacterial effects comparable with those of CHX against aciduric bacteria cultured within an oral microcosm biofilm. This study had some limitations. First, saliva collected from a single donor was used to form the oral microcosm biofilms. The microbial species present in the saliva vary among individuals [54]. However, ecological pressures exert a greater influence on oral biofilm pathogenicity than do these microbial compositional differences [55]. Consequently, oral biofilms formed using saliva from a single donor or multiple donors exhibit similar cariogenic activity when environmental conditions are consistently controlled [56; 57]. Therefore, using saliva from a single donor was sufficient to assess oral biofilm responses to environmental shifts.