Biofouling is a process of surface colonization of microorganisms that adhere to various surfaces, by producing extracellular polymers (polysaccharides and proteins, exopolymers hereafter). Dental plaque is one such example. It often causes serious problems in chemical, medical and pharmaceutical industries. Recently, we demonstrated that some natural phenolic compounds found in plants have an antibiofouling effect on Gram-negative bacteria biofilm formation. In this study, we tested the antibiofouling activities of three natural polyphenols; gallic acid, tannic acid, and quercetin. Ascorbic acid (vitamin C) and salicylic acid were also tested for comparison. Most natural polyphenols are antimicrobial and antioxidant also. To distinguish the antibiofouling effect of the polyphenols from their antimicrobial effects, first antimicrobial activities of the polyphenols were investigated in a separate experimental setting. A plate dilution assay was used to assay the antimicrobial effect of the polyphenols. The concentrations of the polyphenols to be used in antibiofouling tests were determined as minimum inhibitory concentrations (MICs) below which the microorganisms didn’t die.
As these polyphenols are well known as strong antioxidants, it was suggested that the antimicrobial effect may be caused by the polyphenols’ antioxidant effect. To verify the contribution of antioxidant capacity to the antimicrobial effect, DMPD and ABTS decolorizing assays were used to evaluate the antioxidant activities. The results indicate that there are relations between the antimicrobial effect and antioxidant capacity of polyphenols.
The antibiofouling activities of polyphenols were measured using a modified micro-titer plate assay. The polyphenols at the concentrations lesser than the minimum inhibitory concentration (MIC) were observed to reduce the biofilm formation. It was hypothesized that the reduction of biofilm in the presence of the polyphenols was due to the inhibition of exopolymers-producing enzymes by the polyphenols. Glucosyltransferase and Fructosyl transferse enzymes produced by Streptococcus mutans are responsible for exopolymers production, and exopolymers are a major component in biofilm formation. Glucosyltransferases and Fructosyltransferases were separated from a culture medium, and then tested for inhibition by the polyphenols. All the polyphenols were observed to inhibit the enzymes, and when they were inhibited, much less biofilms were formed on a solid surface.
The polyphenols investigated in this study killed the S. mutans via antioxidant activities, and reduced biofilm formation through inhibition of exopolymers-producing enzymes, glucosyltransferase and fructosyltransferase.
It was hypothesized that the exopolymers-producing enzymes were inhibited by the polyphenols, and as a result, exopolymers were not synthesized and no biofilms were formed. Results suggested that these polyphenols were not only good antimicrobials but also very good antioxidants and antifouling agents.