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Studies on the development of a mouthwash based on arginine hydrolases and urease

Grant number: 18/21225-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): January 01, 2019
Effective date (End): December 31, 2019
Field of knowledge:Physical Sciences and Mathematics - Chemistry
Principal Investigator:Augusto Etchegaray Junior
Grantee:Adeline Leonello da Silva
Home Institution: Pró-Reitoria de Pesquisa e Pós-Graduação. Pontifícia Universidade Católica de Campinas (PUC-CAMP). Campinas , SP, Brazil

Abstract

Caries prevention is carried out by the change in food habbits and by personal hygiene. The currrent hygiene products control the formation and accumulation of biofilm and use the remineralizing properties of fluoride. There are evidences that patients with caries resistance present higher levels of enzymes responsable for the production of ammonia on dental plaque, such as arginine deiminase and urease. Since the lowering in plaque pH is a determining factor for demineralization, the local production of ammonia may be a strategy to lower its incidence. Mouthrinses have an important role in caries prevention for patients with hihgh susceptibility, such as those with xerostomia. In the present project we propose the production of a mouthwash based on enzymes that contribute to raise the biofilms' pH and, consequently exert an inhibitory activity over the growth and/or adhesion of Streptococcus mutans, by pH enhancement. The strategy involves the concomitant utilization of the enzymes arginase from Bacillus sp. and urease from Canavalia ensiformis, besides also the addition of arginine deiminase. Arginase produces ornithine and urea, using arginine as substrate; urease produces ammonia from urea. In the same way as arginine deiminase also produces ammonia from arginine. Therefore, the presence of these enzymes, for instance, if attached to acquired pellicle, after hyalinization with mouthrinse, may contribute to raise the pH of dental plaque, in the presence or arginine. Thus, the aim of this project is to propose a novel mouthwash containing arginase and urease and/or arginine deiminase to act on arginine, as to prevent the growth or adhesion of S. mutans. Experiments will involve the cultivation of S. mutans on BHI medium, in the presence of the substrate arginine and the enzymes arginase/urease/arginine deiminase. Initially we will screen for the most significant variables using factorial design including arginine, arginase, urease, arginine deiminase, manganese and fluoride. Bacterial growth will be evaluated by disk diffusion assay, liquid culture, adhesion on glass tube and lactic acid production. It is expected that the results will allow the identification and contribution of arginase/urease/arginine deiminase to inhibit S. mutans and neutralize the produced lactic acid. From statistical analysis we would like to select the best composition of arginase/urease/arginine deiminase and arginine, in the presence or absence of fluoride to suggest a mouthwash formulation. Considering the existance of dentifrices containing arginine in the formulation, this strategy permits and potentially benefits from the use of a dentifrice containing arginine and fluoride, followed by a mouthwash containing arginine hydrolases and urease. Considering the extraction of arginase from Bacillus biomass, this project presents a strategy to add value for residues from biosurfactant production.