Cardoso_2026_Int.J.Biol.Macromol__153398

Oral biofilms are common in dogs and are a major cause of periodontal disease with potential systemic effects, largely due to the limited efficacy of conventional oral antiseptics against the extracellular matrix. Enzymatic degradation has emerged as a promising alternative approach that directly targets this structure. This study evaluated the individual and combined activities of four enzymes, lipase from Burkholderia cepacia (BC lipase), lipase from Candida rugosa (CR lipase), alpha-amylase from Bacillus licheniformis, and egg white lysozyme, against monospecies biofilms formed by eight oral-associated bacterial species (Corynebacterium amycolatum, Corynebacterium ulcerans, Streptococcus canis, Enterococcus faecalis, Pasteurella multocida, Moraxella bovis, Escherichia coli, and Stenotrophomonas maltophilia). Biofilm structural changes were analyzed by scanning electron microscopy (SEM), and cytotoxicity was assessed in HaCaT keratinocytes and gingival fibroblasts. BC lipase (40 U/mL) exhibited the broadest antibiofilm activity among the tested enzymes, achieving up to 96% biofilm reduction in S. maltophilia, 87% in E. faecalis, and over 70% in M. bovis and S. canis. Enzymatic treatments were more effective than chlorhexidine for most species. SEM revealed significant reductions in biofilm density and increased colony dispersion, particularly after treatment with BC lipase (40 U/mL). Enzyme combinations showed species-dependent effects, with additive interactions for lipase and antagonistic effects for amylase-containing mixtures. All enzymes maintained high oral cell viability (>97%), whereas chlorhexidine significantly reduced viability. Overall, the enzymes demonstrated the ability to degrade bacterial biofilms without cytotoxic effects, supporting their potential use as green alternatives for controlling canine oral biofilms.