Fluoride Effects on Biofilm Enzyme Activities

Protein content was used to standardize arginine deiminase (ADS) and urease activity and defined as µmol/min/mg protein (Zheng et al., 2017 (link)). In brief, cells from saliva-derived biofilms were added into a mixture with 50 mM arginine hydrochloride (Sigma-Aldrich Canada, Oakville, Ontario, Canada) and 0.5 mM Tris-maleate buffer (pH 6.0) and then incubated together for 120 min at 37°C to allow ammonia generation. The ammonia production was monitored using Nessler’s reagent (Sigma-Aldrich) based on a standard generated with ammonium sulfate. Simultaneously, protein content was measured using Bradford’s assay and bovine serum albumin was used as standard. Lactic dehydrogenase (LDH) activity was determined using an LDH Activity Assay Kit (Sigma-Aldrich), as per the manufacturer’s guidelines (Zheng et al., 2017 (link)). Further, a deviation between the fluoride group (275 and 1250 ppm NaF) and the control group (0 ppm NaF) for the FR strain and WT was calculated separately. Results were shown as the absolute value of the deviation of enzyme activities compared with corresponding 0 ppm (ΔLDH, ΔADS, and ΔUrease).