Antimicrobial and anti-caries effects of a novel cystatin from sugarcane on saliva-derived multi-species biofilms

Abstract

This study evaluated the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of a new cystatin derived from sugarcane (CaneCPI-5). Microcosm biofilm was produced on bovine enamel specimens (4 x 4 mm; n=48) from a mixture of human saliva and McBain saliva at the first 8 h. From this moment until the end of the experiment, the enamel specimens were exposed to lsaMcBain saliva containing 0.2% sucrose and, once a day, they were treated with the test solutions for 1 min. This treatment was performed for 5 days. The solutions evaluated were: PBS (negative control), 0.12% chlorhexidine (positive control), 0.1 mg/ml CaneCPI-5 and 1.0 mg/ml CaneCPI-5. The biofilm viability was determined by fluorescence using confocal microscopy and the enamel demineralization was quantified using transverse microradiography (TMR). The data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests for biofilm and enamel, respectively (p<0.05). With respect to the antimicrobial effect, all treatment solutions significantly reduced the biofilm viability compared with PBS. The best antimicrobial effect was found for 1.0 mg/ml CaneCPI-5 (82.37±10.01% dead bacteria) that significantly differed from 0.12% chlorhexidine (73.13±15.07% dead bacteria). For the anti-caries effect, only 0.12% chlorhexidine (ΔZ: 2610, 1683-4343) performed significantly better than PBS (ΔZ: 8030, 7213-9115), but 0.12% chlorhexidine did not significantly differ from 0.1 mg/ml Cane-CPI-5. Under this experimental model, CaneCPI-5 significantly reduced the biofilm viability, but this effect was not reflected on its anti-caries potential.