Impact of Streptococcus salivarius K12 probiotic on pH and tooth enamel integrity in an in vitro oral biofilm system

Abstract

To evaluate the impact of an early exposure and a single re-administration of the probiotic Streptococcus salivarius K12 on pH dynamics, bacterial viability, and enamel surface hardness in a multispecies cariogenic biofilm over 12 days under the sucrose challenging conditions. Anaerobic co-cultures of five oral streptococcal species ( S. mutans , S. sobrinus , S. sanguinis , S. mitis , and S. gordonii ) were grown with or without S. salivarius K12 on bovine enamel specimens in artificial saliva (AS) in well plates at 37 °C. The static biofilm model was exposed daily to AS with 0.5% sucrose for 8 hours, followed by AS without sucrose for 16 hours. S. salivarius K12 was applied at initial inoculation and re-administered on day 3. Planktonic pH and colony-forming units (CFU/mL) were measured before each medium change. On day 12, enamel Martens hardness (HM), biofilm cell viability (CFU/mL), and extracellular matrix pH (C-SNARF-4 ratiometry) were assessed. Probiotic-only and AS-only conditions served as controls. A single reapplication of S. salivarius K12 to the mixed cariogenic culture significantly reduced acidification by day 3–4 with pH values 5.9 ± 0.26 compared to 4.4 ± 0.06 in culture without probiotic. However, this effect diminished over time. By day 12, no significant differences in HM, extracellular biofilm pH, or CFU/mL were observed between probiotic-treated and untreated cultures. Early exposure and a single re-administration of S. salivarius K12 can temporarily reduce acidification in artificial cariogenic biofilms, however repeated applications is necessary for lasting effects on enamel integrity and biofilm properties.