Streptococcus mutans form oral biofilms (BFs) and cause dental caries. Roasted green tea (RGT) is prepared by roasting the tea plant, and RGT-specific polyphenols are produced during the roasting process. Catechins, polyphenols in green tea, have BF inhibitory activity against S. mutans; therefore, RGT-specific polyphenols are also expected to have this activity. However, there are few reports on the structural and functional properties of RGT. This study aimed to investigate the inhibitory activity of RGT against S. mutans BF formation and to investigate the active compounds. RGT extract fractionation and BF inhibitory assay were performed. Strong activity was confirmed in the RGT fractions that had medium-high hydrophobicity, were rich in phenolic hydroxyl groups, and lacked catechins. A peak comprising compounds with molecular weights of 918 (mw918) and 1050 (mw1050) was purified from the fraction. Since BF inhibitory activity was confirmed for this peak, these compounds were considered to be part of the active ingredients. The mw918 polyphenol was detected only in RGT and it was thought to be produced during the roasting process. The results of this research will serve as a basis for the future application of RGT as a safe and effective anti-caries agent.

Streptococcus mutans is a major cariogenic organism because of its ability to form biofilms on tooth surfaces. Bacteriocins produced by S. mutans (known as mutacins) are indirect pathogenic factors that play a role in the persistence of this microbe in the oral environment. Nattokinase, a subtilisin-like alkaline serine protease, potently inhibits biofilm formation without affecting S. mutans growth. However, effective strategies utilizing nattokinase to control mutacin production by S. mutans are lacking. In this study, we evaluated the effect of nattokinase on mutacin activity in 46 strains of S. mutans with different mutacin genotypes isolated from the dental plaques of pediatric patients with caries. Nattokinase reduced the activity of mutacin against oral streptococci at a concentration of 1 mg/mL in all clinical isolates. Furthermore, nattokinase reduced the expression of non-lantibiotic mutacin structural genes (nlmABCD) and inactivated the extracellular competence-stimulating peptide involved in comDE activation, which regulates non-lantibiotic mutacin gene expression. These results suggest that nattokinase may reduce the virulence of S. mutans and could potentially be used as a new caries-preventive agent as an alternative to conventional drug treatments.

Our previous study indicated that the water-soluble extract obtained from commercially available natto products inhibited the sucrose-dependent cariogenic biofilm formation of Streptococcus mutans. This inhibitory effect correlated with the protease activity levels of the extract. Subtilisin NAT (nattokinase), a subtilisin-like serine protease, is a dominant protease found in natto products. In this study, we investigated the effect of natto products produced by a subtilisin NAT-deficient mutant (aprN⁻) of the Bacillus subtilis strain NBRC16449 on S. mutans biofilm formation. The mutation decreased the γ-poly glutamic acid (γ-PGA) levels and reduced protease activity in natto products. A significant reduction of the inhibitory effect was observed in the extract from natto products prepared with the aprN⁻ strain. The γ-PGA obtained from the natto products did not affect biofilm formation. We propose that subtilisin NAT plays a major role in biofilm inhibition induced by natto extract.

Natto is a fermented food made from soybeans using Bacillus subtilis natto. B. subtilis is present in honey. In this study, we evaluated the genetic and enzymatic characteristics of B. subtilis obtained from commercial honey and its suitability for natto fermentation. Three bacterial strains isolated from honey samples of different origins were found to be highly homologous to B. subtilis via 16S rDNA analysis. Similar to B. subtilis natto, all three isolates were biotin auxotrophs and possessed two insertion sequences. Genotyping by random amplified polymorphic DNA-polymerase chain reaction and enzyme activity analysis showed that the isolates were genetically different from the commonly used natto strains Miyagino, Takahashi, and Naruse. The three isolates are highly suitable for natto fermentation. These results indicate that honey is a source of B. subtilis natto that is potentially useful in the food industry.

Streptococcus mutans is primarily found in biofilms on tooth surfaces and is associated with the development of dental caries. S. mutans synthesizes water-insoluble glucan (WIG) using sucrose as a substrate, inducing the formation of three-dimensional biofilms. WIG is produced by glucosyltransferases (GTFs) encoded by the tandem and highly homologous gtfB and gtfC genes. Conversely, the homologous recombination of gtfB and gtfC readily happens, producing natural variants without WIG. These WIG⁻ variants are thought to have ecologically pleiotropic functions; however, the molecular basis for their appearance is unclear. This study aimed to determine the sequence of the gtfB–gtfC regions of WIG⁻ variants. We sequenced the gtfB–gtfC regions of 23 WIG⁻ variants derived from S. mutans UA159 and revealed the presence of five alleles and four types of single homologous recombination patterns. Regardless of the allele type, the WIG⁻ variants showed low biofilm formation and GTF activity. To the best of our knowledge, this is the first study to report the presence of alleles in WIG⁻ variants. These findings provide important information for explaining the appearance of mechanisms in WIG⁻ variants.