The effects of exposure to O2- and HOCl-nanobubble water on human salivary microbiota

Abstract

Nanobubbles of gas remain dissolved in water for longer periods than ordinary bubbles, and exhibit unique physicochemical and biological properties. As a result, nanobubble water (NBW) is finding widespread use many applications, such as cleaning in the industry and purification of lake water. The ozone NBW (O₃-NBW), in particular, has been used in clinical dentistry; however, it has several disadvantages, including the instability of ozone, which is spontaneously converted to molecular oxygen (O₃ to O₂), and its broad range of antibacterial activity, which can disrupt the oral microbiota. Therefore, the use of NBW in dental medicine requires greater evaluation. Here, we examined the effects of oxygen and hypochlorite NBW (O₂-NBW and HOCl-NBW, respectively) on the microbiota in human saliva in 16 male patients (35-75 years old; median: 53.5 years) using multiple assays, including next generation sequencing analysis. 16S rRNA gene sequencing revealed no significant changes in both alpha-diversity and beta-diversity. Principal Coordinate Analysis (PCoA) revealed two subclusters in both unweighted and weighted UniFrac distances. Overall, the results revealed that HOCl-NBW exposure of saliva may lead to inhibition or delay in oral biofilm formation while maintaining the balance of the oral microbiome. These results can lead to the development of a novel type of mouthrinse for prevention of oral infectious diseases.

Figure 1

Beta-diversity of unweighted UniFraq distance (A) and weighted UniFraq distance (B). Colored dots indicate individual sample groups: Black: Control; red: O₂-NBW; green: HOCl-NBW. Colored circles indicate groups exposed to NBW; Black: Control; Red: O₂-NBW; Green: HOCl-NBW.

Figure 2

Results of cluster analysis of relative abundance in oral microbiome (N = 16). (A) Unweighted cluster. (B) Weighted cluster. Stratified cluster analyses were performed according to the Ward method based on the results of PCoA. Numbers indicate sample ID. Clustering was performed using the Ward method with Euclidian Distance.

Figure 3

Scatter plots and correlation coefficient tests in CL3 group (N = 9). Spearman’s rank correlation coefficient. Alternative hypothesis: true ρ is greater than 0. The significance level was set at alpha = 0.05. (A) Spearman’s rank correlation coefficient − 0.0667 (P = 0.58). (B) Spearman’s rank correlation coefficient 0.653 (P = 0.028). (C) Spearman’s rank correlation coefficient 0.131(P = 0.37).