A glucan production test was conducted by modifying the methods used in previous studies (44 (link), 45 (link)). A dilution of S. mutans cultured overnight (OD595 of 0.01) in BHI medium containing 10 μM sucrose was incubated at 37°C for 24 h with shaking (200 rpm). The supernatant of incubated S. mutans obtained by centrifugation at 4°C for 30 min was precipitated with solid ammonium sulfate (Sigma-Aldrich). After agitation at 4°C for 1 h and centrifugation at 10,000 × g at 4°C for 30 min, the precipitate was diluted with 10 mM potassium phosphate buffer (pH 6.0). The extracted crude GTFs were stored at −80°C.
To measure the effects of raffinose treatment on water-insoluble glucan production, mixtures of GTFs treated with raffinose (0 to 1,000 μM) and 20 μL of 0.0625 M potassium phosphate buffer (pH 6.5) were reacted with 12.5 μg/L sucrose and 0.25 μg/L sodium azide (Sigma-Aldrich) and then incubated at 37°C for 24 h. Precipitated water-insoluble glucans were dispersed using a sonicator (VCX 750; SONICS, Newtown, CT, USA) for 4 cycles of 5 s of operation and 5 s of pause, at a frequency of 3.5 Hz. The glucan production was estimated by measuring the OD550 using a UV-visible spectrophotometer.
To measure the effects of raffinose treatment on water-insoluble glucan production, mixtures of GTFs treated with raffinose (0 to 1,000 μM) and 20 μL of 0.0625 M potassium phosphate buffer (pH 6.5) were reacted with 12.5 μg/L sucrose and 0.25 μg/L sodium azide (Sigma-Aldrich) and then incubated at 37°C for 24 h. Precipitated water-insoluble glucans were dispersed using a sonicator (VCX 750; SONICS, Newtown, CT, USA) for 4 cycles of 5 s of operation and 5 s of pause, at a frequency of 3.5 Hz. The glucan production was estimated by measuring the OD550 using a UV-visible spectrophotometer.