Epidemiological studies using saliva have revealed relationships between the oral microbiome and many oral and systemic diseases. However, when collecting from a large number of participants such as a large-scale cohort study, the time it takes to collect saliva can be a problem. Mouth-rinsed water, which is water that has been used to rinse the oral cavity, can be used as an alternative method for collecting saliva for oral microbiome analysis because it can be collected in a shorter time than saliva. The purpose of this study was to verify whether mouth-rinsed water is a suitable saliva substitute for analyzing the oral microbiome. We collected samples of mouth-rinsed water, stimulated saliva, unstimulated saliva, and tongue coating from 10 systemic healthy participants, and compared the microbial diversity and composition of the samples using next-generation sequencing of 16S rRNA-encoding genes. The results showed that the microbial diversity of mouth-rinsed water was similar to that of unstimulated and stimulated saliva, and significantly higher than that of tongue-coating samples. The microbial composition at the species level of mouth-rinsed water also showed a very high correlation with the composition of unstimulated and stimulated saliva. These results suggest that the mouth-rinsed water is a suitable collection method instead of saliva for oral microbiome analysis.

Insulin resistance leads to the onset of medical conditions such as type 2 diabetes, and its development is associated with the alteration in the gut microbiota. Although it has been demonstrated that supplementation with prebiotics modulates the gut microbiota, limited evidence is available for effects of prebiotics on insulin resistance, especially for humans. We investigated the prebiotic effect of 1-kestose supplementation on fasting insulin concentration in obesity-prone humans and rats. In the preliminary study using rats, the hyperinsulinemia induced by high-fat diet was suppressed by intake of water with 2% (w/v) 1-kestose. In the clinical study using obese-prone volunteers, the fasting serum insulin level was significantly reduced from 6.5 µU/mL (95% CI, 5.5–7.6) to 5.3 (4.6–6.0) by the 12-week intervention with supplementation of 10 g 1-kestose/day, whereas it was not changed by the intervention with placebo (6.2 µU/mL (5.4–7.1) and 6.5 (5.5–7.6) before and after intervention, respectively). The relative abundance of fecal Bifidobacterium was significantly increased to 0.3244 (SD, 0.1526) in 1-kestose-supplemented participants compared to that in control participants (0.1971 (0.1158)). These results suggest that prebiotic intervention using 1–kestose may potentially ameliorate insulin resistance in overweight humans via the modulation of the gut microbiota. UMIN 000028824.

Saliva includes a substantial amount of biological information, which has enabled us to understand the relationship between oral metabolites and various oral and systemic disorders. However, collecting saliva using a controlled protocol is time-consuming, making saliva an unsuitable analyte in large cohort studies. Mouth-rinsed water (MW), the water used to rinse the mouth, can be collected easily in less time with less difference between subjects than saliva and could be used as an alternative in oral metabolome analyses. In this study, we investigated the potential of MW collection as an efficient alternative to saliva sample collection for oral metabolome profiling. MW, stimulated saliva, and unstimulated saliva were collected from 10 systemically healthy participants. The samples were subjected to metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry, and the types and amounts of metabolites in the samples were compared. Qualitatively, MW contained the same metabolites as unstimulated and stimulated saliva. While the quantity of the metabolites did not drastically change between the sampling methods, all three reflected individual differences, and the features of MW were the same as those of the unstimulated saliva. Overall, these results suggest that MW may be an appropriate alternative to saliva in oral metabolome profile analysis.

Royal jelly (RJ) is a naturally occurring substance synthesized by honeybees and has various health benefits. Herein, we focused on the medium-chain fatty acids (MCFAs) unique to RJ and evaluated their therapeutic efficacy in treating non-alcoholic fatty liver disease (NAFLD). We examined db/m mice that were exclusively fed a normal diet, db/db mice exclusively fed a normal diet, and db/db mice fed varying RJ quantities (0.2, 1, and 5%). RJ improved NAFLD activity scores and decreased gene expression related to fatty acid metabolism, fibrosis, and inflammation in the liver. RJ regulated innate immunity-related inflammatory responses in the small intestine and decreased the expression of genes associated with inflammation and nutrient absorption transporters. RJ increased the number of operational taxonomic units, the abundance of Bacteroides, and seven taxa, including bacteria that produce short-chain fatty acids. RJ increased the concentrations of RJ-related MCFAs (10-hidroxy-2-decenoic acid, 10-hydroxydecanoic acid, 2-decenedioic acid, and sebacic acid) in the serum and liver. These RJ-related MCFAs decreased saturated fatty acid deposition in HepG2 cells and decreased the gene expression associated with fibrosis and fatty acid metabolism. RJ and RJ-related MCFAs improved dysbiosis and regulated the expression of inflammation-, fibrosis-, and nutrient absorption transporter-related genes, thereby preventing NAFLD.