*2.2. Sampling and Microbial DNA Extraction*

Saliva samples were collected by chewing paraffin wax for 5 min. Collected samples were kept on ice. Upon transporting them to the laboratory, samples were frozen at −20 ◦C until DNA extraction. Saliva samples were centrifuged at 3000 rpm for 10 min. The Maxwell 16 LEV Blood DNA Kit (Promega KK, Tokyo, Japan) was used for DNA extraction. The NanoDrop ND-2000 (Thermo Fisher Scientific KK, Tokyo, Japan) was used for the measurement of DNA concentration. DNA degradation was visually checked by electrophoresis on a 1% agarose gel using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific KK, Tokyo, Japan). The inclusion criteria for DNA samples subjected to further analysis were as follows: concentration > 20 ng/μL, volume ≥ 20 μL, A260/<sup>280</sup> ≥ 1.8, and A260/<sup>230</sup> >1.5 [32,37].
