Since there is no consensus about whether starch increases the cariogenic potential of sucrose, we used a validated 3-species biofilm model to evaluate if starch combined with sucrose provokes higher root dentine demineralization than sucrose alone. Biofilms (n = 18) composed by Streptococcus mutans (the most cariogenic bacteria), Actinomces naeslundii (which has amylolytic activity), and Streptococcus gordonii (which binds salivary amylase) were formed on root dentine slabs under exposure 8 ×/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, or a combination of 1% starch and 10% sucrose. Before each treatment, biofilms were pretreated with human whole saliva for 1 min. The pH of the culture medium was measured daily as an indicator of biofilm acidogenicity. After 96 h of growth, the biofilms were collected, and the biomass, bacteria viability, and polysaccharides were analyzed. Dentine demineralization was assessed by surface hardness loss (% SHL). Biofilm bioarchitecture was analyzed using confocal laser scanning microscopy. Treatment with a starch and sucrose combination provoked higher (p = 0.01) dentine demineralization than sucrose alone (% SHL = 53.2 ± 7.0 vs. 43.2 ± 8.7). This was supported by lower pH values (p = 0.007) of the culture medium after daily exposure to the starch and sucrose combination compared with sucrose (4.89 ± 0.29 vs. 5.19 ± 0.32). Microbiological and biochemical findings did not differ between biofilms treated with the combination of starch and sucrose and sucrose alone (p > 0.05). Our findings give support to the hypothesis that a starch and sucrose combination is more cariogenic for root dentine than sucrose alone.

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