Multidimensional characterization of an innovative hybrid phytocystatin for acquired pellicle modulation and initial...

This study presents the design, expression, and characterization of CaneStat, a recombinant hybrid protein combining domains of a sugarcane derived cystatin (CaneCPI-5) and a statherin phosphomimetic peptide (DDSt15).

The protein was expressed in Escherichia coli and evaluated for protease inhibition, cytocompatibility, anti-erosive effects, and antimicrobial activity.

CaneStat and CaneCPI-5 were compared for their ability to inhibit human cathepsins B, K, and L. Residual enzymatic activity did not differ significantly between proteins. Cytotoxicity and viability assays with human gingival fibroblasts demonstrated that CaneStat was non-cytotoxic regardless the concentration. Its anti-erosive potential was assessed in vitro under hydrochloric and citric acid challenges after pellicle formation. Under hydrochloric acid challenge, all CaneStat concentrations reduced enamel hardness loss and showed protective effects comparable to Elmex Erosion Protection®. Under citric acid challenge, CaneStat reduced enamel softening, with greatest protection at 0.025 and 0.1mg/mL. Protection was maintained from the first to the third day. Antimicrobial assays showed moderate inhibitory activity against Streptococcus mutans and Lactobacillus casei, without bactericidal effects or inhibition of Fusobacterium nucleatum, indicating limited direct antimicrobial activity.

CaneStat preserves the key biological properties of its parent molecules and protects enamel against early acid-induced erosion. These findings identify this hybrid protein as a promising candidate for acquired pellicle modulation strategies aimed at strengthening enamel resistance to acidic challenges.

CaneStat may support oral-care products against erosive tooth wear through enhanced enamel resistance under acidic conditions and contribute to protective strategies based on acquired pellicle modulation.