Protease and Betaine for Canine Oral Health: From Biofilm Disruption to Oral Microbiome Evaluation

Periodontal disease (PD) is highly prevalent in dogs and is associated with oral biofilms that promote chronic inflammation and tissue damage. Disrupting biofilms may therefore support canine oral health. This study evaluated protease (subtilisin) and betaine using complementary in vitro and in vivo approaches. Porphyromonas gingivalis, Porphyromonas gulae, and Streptococcus mutans were used as model oral pathogens in biofilm inhibition, biofilm removal, and planktonic growth assays. An 8-day canine pilot study assessed the tolerability of a dental chew containing protease and betaine and its effects on the oral microbiome. In vitro data were analyzed using standard least-squares models, and microbiome responses using diversity and differential abundance analyses. Protease reduced biofilm formation by P. gingivalis and S. mutans (p < 0.0001) and removed established biofilms (p < 0.05), while showing limited effects on planktonic growth. Betaine responses varied by bacterial strain and assay type and did not significantly interact with protease. The chew showed good physiological tolerability, although not all dogs consumed it completely. Microbiome analysis revealed limited treatment effects, with no differences in alpha diversity between groups. Overall, protease was the main driver of biofilm-related responses, supporting further investigation in canine oral care products; however, clinical periodontal outcomes, inflammatory biomarkers, and direct evidence of betaine-mediated tissue protection were not assessed.