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 Streptococcus mutans biofilms grown in vitro.

Stored S. mutans (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 S. mutans 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 S. mutans biofilms (p < 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 (p < 0.001) compared to the control. Regarding theobromine, 3.125 mg/mL of it significantly increased the growth of S. mutans biofilms. At the concentration of 6.25 mg/mL, theobromine significantly (p < 0.001) inhibited the S. mutans 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 S. mutans biofilm formation.