The Inhibition of Streptococcus mutans Biofilms following Exposure to Different Chocolate Ingredients

This project aimed to investigate the anticariogenic effect of four chocolate ingredients (polyphenol, theobromine, cacao, and flavanol) against <i>Streptococcus mutans</i> biofilms grown <i>in vitro</i>.Stored <i>S. mutans</i> (UA 159) was transferred to Brain Heart Infusion (BHI) broth and incubated in aerobic incubator for 24 hours at 37°C in 5% CO₂. Following this, 190 µL of each ingredient concentration (0.78-200 mg/mL) and 10 µL of the culture were added to a 96-well plate and incubated for 24 hours at 37°C in 5% CO₂. Then, biofilms were fixed, stained with crystal violet, and analyzed for formation using a spectrophotometer. Control groups included negative control with only <i>S. mutans</i> and sterility control with BHI media.One-way analysis of variance and Tukey tests analyzed the data.Flavonoid at the 6.25 to 25 mg/mL concentrations reduced the <i>S. mutans</i> biofilms (<i>p</i> < 0.001) by 5- to 33-fold. Meanwhile, 50 mg/mL concentrations and higher completely eradicated biofilm growth. Similarly, cocoa concentrations ranging between 12.5 and 200 mg/mL revealed massive antibiofilm action from a 22-fold reduction at 12.5 mg/mL to complete biofilm eradication at 200 mg/mL. Polyphenol was the only ingredient showing biofilm inhibition at all concentrations ranging from almost 10-fold reduction to complete biofilm eradication, which were all significant (<i>p</i> < 0.001) compared to the control. Regarding theobromine, 3.125 mg/mL of it significantly increased the growth of <i>S. mutans</i> biofilms. At the concentration of 6.25 mg/mL, theobromine significantly (<i>p</i> < 0.001) inhibited the <i>S. mutans</i> biofilms by 3.35-fold. While at the range of 25 to 200 mg/mL, theobromine resulted in a reduction between 11-fold and complete biofilm eradication.The findings suggest that flavonoid, cacao, polyphenol, and theobromine may serve as effective adjuncts in preventing dental caries by inhibiting <i>S. mutans</i> biofilm formation.