The measurement of acid production and the formed biofilm pH was performed in 24-well flat bottom plates (Thermo Fisher Scientific). For the group with S. mutans, each dissolved milk (9.9 mL) was mixed with 0.1 mL BHI broth containing S. mutans (10 7 CFU/mL), whereas sterilized BHI broth (0.1 mL) was added to each milk solution instead of the S. mutans suspension in the group without S. mutans. The prepared solutions were then transferred into 24-well plates (2 mL/well) and incubated at 37°C, 5% CO 2 for 24 h. For controls, BHI broth with and without 10% sucrose were the negative and positive controls, respectively.
The pH of the non-incubated solutions in both groups, (i) with and (ii) without S. mutans, was also determined from the leftover solutions. A pH meter (Thermo Fisher Scientific, Orion Star â"¢ A211) with the ST230 pH probe (OHAUS) was used to determine the pH. The starting pH of the milk solutions are shown in online supplementary Table S3.
To determine the biofilm pH after 24-h, the incubated 24-well plates were decanted to remove the culture solutions, and the formed biofilm pH was measured by a STSURF pH probe (OHAUS). Three independent experiments in triplicate were conducted for each solution.
The pH of the non-incubated solutions in both groups, (i) with and (ii) without S. mutans, was also determined from the leftover solutions. A pH meter (Thermo Fisher Scientific, Orion Star â"¢ A211) with the ST230 pH probe (OHAUS) was used to determine the pH. The starting pH of the milk solutions are shown in online supplementary Table S3.
To determine the biofilm pH after 24-h, the incubated 24-well plates were decanted to remove the culture solutions, and the formed biofilm pH was measured by a STSURF pH probe (OHAUS). Three independent experiments in triplicate were conducted for each solution.