The Effect of Bovine Trypsin on the Adhesion and pH of Dental Plaque Biofilms: An In Vitro Study.

OBJECTIVE: The aim of this study was to investigate the effect of bovine trypsin on the adhesion and pH of dental plaque biofilms. METHODS: A multispecies dental plaque biofilm model and a single-species dental plaque biofilm model were established in vitro. Three groups were tested: (1) blank control group (aseptic ultrapure water); (2) negative control group (1M Tris-HCl buffer, pH = 7.4); and (3) experimental group (bovine trypsin). Adhesion ability was measured using an automatic microplate reader and visualised by confocal laser scanning microscopy (CLSM). The pH was measured using a pH meter. The expression of gtfB, gtfC, and gtfD was analysed using quantitative real-time polymerase chain reaction. RESULTS: Adhesion ability in the experimental group was significantly lower than that in the blank group and the negative control group (P < .05); readhesion ability in the experimental group was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 4 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 2 to 6 hours; P < .05). The decrease in pH was inhibited for a certain period of time (24-hour multispecies biofilms were inhibited from 2 to 8 hours, and the 48- and 72-hour multispecies biofilms were inhibited from 1 to 8 hours; P < .05). Expression levels of gtfB, gtfC, gtfD, and ldh in the experimental group were significantly lower than those in the blank group (P < .05). CONCLUSIONS: Bacterial adhesion, and readhesion, decreasd pH, and expression of adhesion- and acid-related genes by Streptococcus mutans in biofilms could be reduced by bovine trypsin for a certain period of time.

The effect of bovine trypsin on dental biofilm dispersion: an in vitro study.

To investigate the degradation effect of bovine trypsin on multispecies biofilm of caries-related bacteria and provide an experimental foundation for the prevention of dental caries. Standard strains of S. mutans, S. sanguis, S. gordonii, and L. acidophilus were co-cultured to form 24 h, 48 h, and 72 h biofilms. The experimental groups were treated with bovine trypsin for 30 s, 1 min, and 3 min. Morphological observation and quantitative analysis of extracellular polymeric substances (EPS), live bacteria, and dead bacteria were conducted using the confocal laser scanning microscope (CLSM). The morphological changes of EPS and bacteria were also observed using a scanning electron microscope (SEM). When biofilm was treated for 1 min, the minimal inhibitory concentration (MIC) of bovine trypsin to reduce EPS was 0.5 mg/mL in 24 h and 48 h biofilms, and the MIC of bovine trypsin was 2.5 mg/mL in 72 h biofilms (P < 0.05). When biofilm was treated for 3 min, the MIC of bovine trypsin to reduce EPS was 0.25 mg/mL in 24 h and 48 h biofilms, the MIC of bovine trypsin was 1 mg/mL in 72 h biofilm (P < 0.05). The ratio of live-to-dead bacteria in the treatment group was significantly lower than blank group in 24 h, 48 h, and 72 h multispecies biofilms (P < 0.05). Bovine trypsin can destroy multispecies biofilm structure, disperse biofilm and bacteria flora, and reduce the EPS and bacterial biomass in vitro, which are positively correlated with the application time and concentration.

An in vitro study on the degradation of multispecies biofilm of periodontitis-related microorganisms by bovine trypsin.

To investigate the degradation effect of bovine trypsin on multispecies biofilm of periodontitis-related bacteria and to provide an experimental reference for exploring new methods for controlling biofilms of periodontitis-related microorganisms, the multispecies biofilm of periodontitis-related microorganisms was established. Standard strains of Porphyromonas gingivalis, Fusobacterium nucleatum subsp. polymorpha, Actinomyces viscosus, and Aggregatibacter actinomycetemcomitans were co-cultured to form the biofilm. The experimental groups were treated with bovine trypsin, distilled water was applied as the blank control group, and phosphate saline buffer (pH = 7.4) as the negative control group. Morphological observation and quantitative analysis of extracellular polymeric substances (EPS), live bacteria, and dead bacteria were conducted using a laser confocal microscope. The morphological changes of EPS and bacteria were also observed using a scanning electron microscope. The results of morphological observations of modeling were as follows. EPS aggregated as agglomerates, and bacteria flora were wrapped by them, showing a three-dimensional network structure, and channel-like structures were inside the biofilm. Live bacteria were distributed on the surface of the EPS or embedded in them, dead bacteria aggregated between live flora and the bottom layer of biofilms. After being treated with bovine trypsin, the three-dimensional network structure and the channel-like structure disappeared, and the EPS and live and dead bacteria decreased. Quantitative analysis results are as follows. When biofilm was treated for 30 s, 1 min, and 3 min, the minimum effective concentrations of bovine trypsin to reduce EPS were 2 mg/ml (P < 0.05), 0.5 mg/ml (P < 0.05), and 0.25 mg/ml (P < 0.05), respectively. The minimum effective concentrations of bovine trypsin to reduce the live or dead bacteria were 2 mg/ml (P < 0.05), 0.5 mg/ml (P < 0.05), and 0.5 mg/ml (P < 0.05), respectively. There was no significant difference in the ratio of live/dead bacteria after the biofilm was treated for 30 s with bovine trypsin at the concentration of 0.25, 0.5, 1, and 2 mg/ml (P > 0.05), and the minimum effective concentration to reduce the ratio of live bacteria/dead bacteria was 0.25 mg/ml (P < 0.05) after treatment for 1 min and 3 min. Therefore, bovine trypsin can destroy biofilm structure, disperse biofilm and bacteria flora, and reduce the EPS and bacterial biomass, which are positively correlated with the application time and concentration.