## 4 Discussion  
Generally, the resulting oral microbial sequences were estimated using QIIME 2 TM, which revealed the phyla Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Fusionbacteria, TM7, and Spirochaetes were observed predominantly in healthy subjects rather than Synergistetes, SR1, and Tenericutes. We previously reported that the use of DMW for weeks reduced the PI in healthy subjects without affecting the PBD, GI, and BOP values. The use of DMW and DNMW for four weeks reduced the PI in dental caries subjects [52]. The current study revealed the impact of DMW and DNMW (for 30 days) on the oral microbial community in healthy subjects. Mouthwashes with chemicals are common among the general population due to their ease of use, reduced plaque biofilm, and breath-freshening impact. Although industrialized nations utilize various oral hygiene products to achieve oral hygiene, appropriate scientific proof must be available to back up such claims [53; 54; 55]. The minimal inhibitory concentration is also usually quoted as the primary indicator of the potential efficacy of plaque control agents. Oral care products often use the appropriate concentration, which can effectively help control plaque and gingivitis [56]. Dextran-degrading enzymes, commonly called dextranases, form a diverse group of different carbohydrates and transferases. They specifically hydrolyze (1\(\rightarrow\)6)-\(\alpha\)-glucosidaselinkages in dextrans. Dextrans could diffuse into the biofilm, while most particles were excluded and inhibited the growth of _S. mutans_[57]. Nisin is an antimicrobial peptide that disturbs the bacterial cell membrane [58; 59]. Nisin inhibited the growth of oral bacteria and the development of biofilms by preventing cell attachment and proliferation, as reported earlier [60]. In this study, dextranase showed the MIC of 4.5 unit/g against _S. mutans_. The nisin shows the inhibitory effect against all the tested microbes (_P. gingivalis_, _A. actinomycetemitants_, _T. forysthia_, and _S. mutans_) (Table 2). We selected _S. mutans_ as the key microbial candidate to test the antibiofilm activity of the DMW (Formulation I) and DNMW (Formulation II), and it showed the inhibition against the biofilm formation (Figure 1). Formulation I and II were stable for three months at 4 \({}^{\circ}\)C, room temperature, and 45 \({}^{\circ}\)C (Tables 3 and 4). As per the results, the formation of plaque in the gingival sulcus and the development of periodontal pathogenesis might reduce significantly during the test period (Table 5). The DMW and DNMW have a closer effect on the oral health of healthy volunteers. However, DNMW reduces the PBD (Table 5). Moreover, on tested parameters, the impacts were insignificant (Table 6) among the mouthwash formulations, indicating that both DMW and DNMW exhibited a similar protective role. Current methods for identifying and quantifying bacteria in dental biofilms, including genome sequencing, have shown a considerably more complicated ecology than previously thought [61; 62]. The genera _Catonella_, _Centipeda_, _Freitbacterium_, _Rhizobium_, _Ochrobactrum_, _Mogibacterium_, _Actinomyces_, _Streptococcus_, _Rothia_, _Selenomonas_, _and Veillonella_ were detected as major dental caries pathogens [63; 64]. _Atopobium_, _Bacteroides_, _Cryptobacterium_, _Lactobacillus_, _Pseudomonas_, _Mogibacterium_, _Ochrobactrum_, _Rhizobium_, _Centipeda_, _Camrylobacter_, _Alloprevotella_, _Megashipara_, and _Mycoplasma_ were the suspected pathogens of dental caries [65; 66; 67]. [PERSON] et al. reported that the common microorganisms implicated in oral disease are _S. mutans and L. acidophilus_[68]. Here, the results of our study showed that DMW completely eradicated the genera _Stenotrophomonas_, _Serratia_, _Citrobacter_, _Enterococcus_, _Achromobacter Deflia_, _Chromobacterium_, _Klebsiella_, _Stenotrophomonas_, and _Psychrobacter_ in the post-treated samples of G1.