Anti-Streptococcus mutans and anti-biofilm activities of dextranase and its encapsulation in alginate beads for application in toothpaste

Measurement of dextranase activity

Dextranase can be produced by various microorganisms, including bacteria, yeasts, and filamentous fungi. However, our preliminary results found that fungal dextranase is more effective in degrading dextran than those of other microbes. Hence, the fungal commercial dextranase was selected for use in this study, and it was produced from Chaetomium gracile; dextranase from C. gracile is generally recognized as safe (GRAS) (Virgen-Ortíz et al., 2015) for use in foods. This confirms its safety; therefore, food-grade dextranase was purchased from Mitsubishi chemical foods corporation, Japan. Dextranase activity was measured based on an increasing ratio of the reducing sugar concentration in the reaction with 3,5-dinitrosalicylic acid reagent (Wang et al., 2014). A mixture of 200 mg/mL of commercial dextranase (125 µL) and 20 mg/mL of dextran solution (125 µL) was incubated at 37 °C for 30 min. The reaction was stopped after 30 min by transferring 250 µL aliquots of the enzyme-substrate mix into tubes containing 3,5-dinitrosalicylic acid reagent. Then, the tubes were incubated for 15 min in a boiling water bath, and two mL of distilled water was added. The absorbance of the mixture was measured at 540 nm. One unit of dextranase activity (U) was defined as the amount of enzyme that catalyzed the liberation of one mmol of maltose in 1 min from dextran. In order to assess the possibility of adding commercial dextranase to toothpaste to prevent dental caries, the optimal condition of this enzyme was investigated by varying the pH (3, 4, 5, 6 and 7) and temperature (25, 37, 45 and 55 °C).

Minimal inhibitory concentration (MIC)

The MIC of dextranase against the growth of S. mutans ATCC 25175 was investigated by the broth dilution method. Briefly, S. mutans grown in Tryptic Soy Broth (TSB) was adjusted by the normal saline solution to obtain 10⁶ cells/mL, and then mixed with the serially diluted test of dextranase (144–0.07 unit/g concentrations) in a 96 well-plate, followed by incubation at 37 °C, 5% CO₂ for 24 h. The MIC was determined by spectrophotometric analysis of the growth of S. mutans at 600 nm (Thenmozhi et al., 2009).

Effect of dextranase on biofilm formation of Streptococcus mutans

The effect of sub-MIC (1/2 MIC) of dextranase against biofilm-formation by S. mutans was tested with the modification described by Sato et al. (2018); 100 µL of 3.38 unit/g dextranase enzyme was added into 100 µL of TSB broth containing bacterial suspension at 10⁶ CFU/ml. The 96 well plates were incubated for 24 h at 37 °C and 5% CO₂. After incubation, the biofilm was stained with 0.4% crystal violet for 15 min. The cells were then washed three times with sterile distilled water and air-dried for 60 min. Stained biofilm cells were de-stained using 95% ethanol. Biofilm removal was determined on the basis of visible disruption in biofilm formation and a significant reduction in the readings at OD₅₇₀ nm by comparison with the negative control wells with no S. mutans (Thenmozhi et al., 2009). S. mutans in TSB broth without the addition of dextranase served as the positive control (Table 1). The dextranase enzyme was classified into the following categories based on the OD of bacterial films: non-adherent, weak, moderate, and strong adherent. The cut-off OD for the microtiter-plate test is defined as three standard deviations above the mean OD of the negative control by following the classification according to Stepanović et al. (2000). All tests were carried out three different times and the results were averaged.

OD ≤ ODc (OD in negative control) (non-adherent)

ODc <OD ≤ 2ODc (weakly adherent)

2ODc <OD ≤ 4ODc (moderately adherent)

4ODc <OD (strongly adherent)

Anti-biofilm assay: Confocal Laser Scanning Microscopy (CLSM)

Confocal laser scanning microscopy (CLSM) was used to determine the anti-biofilm activity of dextranase. S. mutans ATCC 25175 was used as a biofilm producer in this study. The experiment was a modification of broth dilution method (Thenmozhi et al., 2009); S. mutans was grown in TSB and adjusted to 10⁶ cells/mL; then, one mL of S. mutans cell suspension was mixed with one mL of sub-MIC (1/2 MIC) dextranase. The biofilms were allowed to grow on 1 cm ×1 cm glass slide placed in 24-well titer plates, followed by incubation at 37 °C and 5% CO₂. After 24 h incubation, the biofilm formed was stained with SYTO^®9 green fluorescent dye (Sigma). The samples were assessed by Nikon Laser Confocal Microscope C1. Images were captured and processed using EZ-C1 version 3.90. The results were compared with the control as without dextranase addition.

Encapsulation of dextranase enzyme: The study of factors affecting encapsulation to achieve a suitable condition

On the basis of our preliminary work, the independent variables were firstly assessed by Placket Burman design. pH, alginate and calcium chloride concentrations were found as the significant factors on encapsulation efficiency (% EE) and dextranase activity (Dataset S1). Therefore, these three factors were selected as the independent variables tested in the experiment of the Box-Behnken design experiment for achievement of optimal conditions on % EE and dextranase activity as the responses for the combination of the independent variables. Box-Behnken design (BBD) was used to study the three factors affecting encapsulation: pH, calcium chloride concentration and the concentration of sodium alginate. Each of these variables was studied at three different levels (−1, 0, 1) using BBD. The encapsulation of commercial dextranase was designed to have 17 conditions. Dextranase enzyme solution (concentration: 20 mg/ml) and sodium alginate solution (concentration: 0.7%, 0.85%, 1.0%) were mixed at a ratio of 1:1, and the pH of the mixed solution was adjusted (pH: 5, 6, 7). The software package Design Expert, version 10.0 (Stat-Ease Inc., Minneapolis, MN, USA) was used for the experimental design, data analysis and model building. According to analysis of variances (ANOVA) which was applied to assess effects of studied variables, interactions and statistical significance of models) the fitness of the polynomial model equations were expressed by the coefficient of determination R² (Peng et al., 2020).

Three-dimensional response surface plots were drawn to identify the interaction between factors and responses. The Buchi encapsulator was used to prepare the capsules. The nozzle type and size used were single 450 µm, 160 Hz of frequency, 11–15 ml/min of flow rate, 500–600 V of the electrode, 100–150 m Bar of air pressure and 30 min of hardening time. The capsules were dropped into the calcium chloride solution (concentration 10%, 15%, 20%), and the beads were filtered and washed with distilled water 2 times to eliminate excess calcium chloride (Bashari et al., 2013). The characteristics and mean diameter of beads were studied by ZEN lite imaging software of Stereo microscopes (ZEISS Stemi 305).

Encapsulation efficiency (% EE): Protein content in the dextranase enzyme solution and sodium alginate

A quantity of 20 µL of the mixed solution of dextranase enzyme solution (concentration: 20 mg/ml) and sodium alginate (concentration: 0.7%, 0.85%, 1.0%) at a ratio of 1:1 was placed in a microplate. Afterward, 200 µL of Bradford reagent was added into the microplate, and the absorbance of the reaction solution was measured using the UV spectrophotometer at a wavelength of 595 nm. The amount of protein in the mixed solution was calculated (Bashari et al., 2013).

Encapsulation efficiency (% EE): Protein content in calcium chloride solution

A quantity of 20 µL of calcium chloride solution after the encapsulation process was placed in a microplate, followed by the addition of 200 µL of Bradford reagent. The absorbance of the reaction solution was measured using the UV spectrophotometer at a wavelength of 595 nm. The amount of protein in the mixed solution was calculated (Bashari et al., 2013). Also, the encapsulation efficiency was calculated as follows. ${\displaystyle \text{\%}\text{Encapsulation efficiency}=\left(\left(A-B\right)\times 100\right)∕A}$ A = The amount of protein from encapsulation

B = Protein content in calcium chloride solution.

The activity of encapsulated dextranase enzyme in alginate beads

One gram (1g) of beads was grained with 1 g of 0.1 M phosphate buffer saline (PBS, pH 6.0), and 125 µL of bead solution was mixed with 125 µL of dextran solution for 30 min. A quantity of 250 µL of DNS reagent was pipetted into a test tube, and the test tube was immersed in a boiling water bath for 15 min. Then, the test tube was soaked in cool water, and two mL of distilled water was added to dilute the color. A quantity of 200 µL of the reaction solution was pipetted into a microplate. The absorbance was measured with a UV spectrophotometer at a wavelength of 540 nm. The activity of dextranase was calculated as described by Bashari et al. (2013).

The stability of encapsulated dextranase activity in a toothpaste base

The toothpaste base is a composite of humectant, preservative, sweetening agent, abrasive, coloring agent, and detergent (Grace et al., 2015). As the MIC of dextranase against the growth of S. mutans was 4.50 units/g, the concentration of dextranase was varied by increasing the MIC 10 times, 20 times, and 30 times. These were equivalent to 1%, 2% and 3% alginate beads in the formulas for mixing with toothpaste. It was found that the formulation containing 2% beads with dextranase showed good physical characteristic (Table S1). Besides, the dose selection was considered based on the range of human equivalent dose (HED) for clinical trial safety. Then, the dextranase encapsulation at 2% w/w alginate beads was added to the toothpaste base. The storage experiment followed the guideline of the Thai industrial standards (TIS 45-2552-2009 Toothpastes). Hence, the toothpaste was exposed to accelerated storage condition at 40 ± 2 °C for three months to check the stability of the encapsulated dextranase activity in the toothpaste. Dextranase activity was measured as previously described.

The sensory test

This research focused on the efficiency of anti-biofilm to prevent tooth decay. However, safety is very important to be considered for dental care products, while sensory acceptance by consumers is also important for the possibility to be able to compete in the market. Therefore, the evaluation of the safety and satisfaction of the product is important to gain the basis for improvement and development of toothpaste product. This experiment is a pilot study to assess the satisfaction of toothpaste containing dextranase commercial enzyme encapsulated in alginate. The satisfaction and safety of developed toothpaste was evaluated by 15 volunteers based on the modification of the guidelines of Chowdhury et al. (2013). The ethic of volunteering was approved by the Research Ethics Committee, Faculty of Pharmacy, Chiang Mai University, Thailand with the ethic approval number 05/2562. The research was also submitted to the Thai Clinical Trials Registry (TCTR), a research database for clinical research of Thailand with a study ID: TCTR20200409003 (http://www.clinicaltrials.in.th/).

Inclusion criteria

1. Female or male volunteers aged 20 years or more.

2. Volunteers who agree to participate in the research.

Exclusion criteria

1. Volunteers during pregnancy.

2. People who have a history of cosmetic allergy.

3. People with oral ulcers, those who have taken out teeth or have undergone oral surgery.

Pre-test: oral assessment in volunteers

To make sure that volunteers do not have oral ulcers, their oral cavities were evaluated by a dentist. The result of the evaluation was recorded by the dentist.

Test: volunteers brush their teeth with toothpaste

The volunteers were given toothpaste containing dextranase enzyme and brushed their teeth with the toothpaste for 2 min. They were evaluated during and after brushing.

Post-test: oral assessment in volunteers

To make sure that volunteers do not have irritation after brushing, their oral cavities were evaluated by a dentist. The result of the evaluation was recorded by the dentist (Wattanakrai et al., 2007).

Final-test: assess satisfaction with volunteers

The acceptability of the toothpaste containing encapsulated commercial dextranase was assessed by sensory analysis. Several parameters, including color, odor, flavor, distribution, homogeneity, foaming, clean feeling after use, fresh feeling after use and overall satisfaction, were measured. A 5-point hedonic scale was used to assess the suitability (Graham, Agbenorhevi & Kpodo, 2017).

Measurement of dextranase activity

The activity of commercial dextranase was examined for its optimal pH. The highest dextranase enzyme activity, 4155.09 unit/g, was observed at a pH of 6, followed by pH of 5 and 7, while the minimum activity was observed at pH of 3–4, roughly 1,000 unit/g. The optimal temperature of commercial dextranase activity under the optimal pH of 6 was between 25 and 37 °C with the highest activity of 4,401.71 unit /g, followed by 45 °C. A huge decrease was observed at 55 °C with only 1000 unit/g. Hence, the optimum condition of this enzyme, pH of 6 and 37 °C, was selected to measure its activity in all experiments of this research (Table S2).

Minimal inhibitory concentration (MIC)

The MIC assay is a technique used to determine the lowest concentration of treatments needed to inhibit bacteria. Inhibition was observed after incubation at 37 °C for 24 h. The MIC was read as the lowest concentration of the dextranase enzyme at which there is no visible growth. In determining the OD₆₀₀ values, the dextranase showed positive antibacterial activity against the growth of S. mutans, and its MIC was 4.5 unit/g. This was done along with the determination of the MIC of the positive control (penicillin G) against S. mutans. The MIC observed for penicillin G was 0.24 µg/ml (Table S3).

Effect of dextranase on biofilm formation of S. mutans

An experiment was performed to measure the adherence capacity of the biofilm formed by S. mutans. To confirm the efficacy of dextranase enzyme regarding biofilm removal, as it is one of the ways to prevent dental plaque, the biofilm was dyed with 0.4% crystal violet, and the absorbance was measured with at a wavelength of 570 nm. Biofilm removal was determined based on visible disruption in biofilm formation in the readings compared with the control wells. The summarized results of the microtiter plate tests are presented in Table 1 and Table S4. The medium without S. mutans served as the negative control (ODc = 0.24), which showed non-adherence. The dextranase enzyme showed ODc <0.47 ≤ 2ODc, which was classified as weakly adherent, while the positive control with only S. mutans without dextranase showed strong-adherence.

Anti-biofilm assay: Confocal Laser Scanning Microscopy (CLSM)

Confocal Laser Scanning Microscopy (CLSM) is a popular method to study biofilm structure. It was carried out to confirm whether the synergized effect of dextranase activity can disrupt matured biofilm produced by S. mutans ATCC 25175. The 24 h matured biofilm was exposed to the crude fungal dextranase from P. roquefortii TISTR 3511, and the anti-biofilm activity was analyzed by CLSM after staining with SYTO^®9 green fluorescents. The staining is used to indicate cell viability, as determined by the integrity of the cellular components, including biofilm on the basis of the amount of biofilm formation (Banu et al., 2017; Cerca et al., 2012). The dextranase showed significant reduction in biofilm formation compared with the control with no addition of dextranase, which showed the presence of a dense biofilm produced by S. mutans ATCC 25175 (Fig. 1).

The study of factors affecting encapsulation to achieve a suitable condition

The encapsulation conditions of dextranase enzymes in alginate beads were designed by BBD with 17 conditions of the 3 factors affecting encapsulation: pH, calcium chloride concentration and the concentration of sodium alginate (Table 2; Dataset S2). It was found that Condition No. 14 produced the highest efficiency of enzyme storage (% EE) and enzyme activity. The optimum condition was pH equal to 7, 20% w/v calcium chloride and 0.85% w/v sodium alginate, with the highest activity (18.32 unit/g.bead). Condition No. 14, the most suitable condition for encapsulation, was due to the pH value of 7, which is close to 6, the optimal pH for dextranase. Moreover, the beads from condition No. 14 were regular spherical shape, and the mean diameter of 30 beads was 1.18 ± 0.02 mm (Fig. 2; Table S5).

The efficiency of encapsulation (% EE)

Based on experimental results (actual) and predicted values, Condition No. 14 showed the highest efficiency of enzyme storage (% EE) and enzyme activity, as previously explained. The maximum value of the efficacy of encapsulation (% EE) was equal to 87.17. Quadratic models are the most suitable models for describing the trend of efficiency of encapsulation in the beads (p = 0.0215). The factors that had effect on the efficiency of enzyme storage in the beads showed statistical significance, such as sodium alginate concentration (p = 0.0156) and calcium chloride concentration (p = 0.0356), as shown in Table 3 and Dataset S2.

The F-value of 5.10 showed that this model was significant at a p-value of 0.0215. The lack of fit, F-value of 6.00 and p-value of 0.0581 implied unimportance due to relative pure error. The values of R² and adjusted R² were found to be 0.8677 and 0.6977 respectively, while the lack of significance of the value (p = 0.0581) of lack of fit indicates that the model was fitted with good prediction (Singh, Bajar & Bishnoi, 2017).

From the above experiment, the relationship between factors and responses (% EE) values can be expressed using regression as shown in the following equations. Letters A, B and C are alginate, calcium chloride and pH respectively. ${\displaystyle \text{\%}EE=39.546+13.545A+11.075B-0.470C+5.345AB-13.915AC-2.085BC-14.536AA+9.600BB+22.584CC}$ A response surface plot can be used to analyze the interaction between two factors in relation to the efficiency of enzyme storage (% EE). The shape of a contour plot reflects the intensity of the interaction between the factors to a certain extent (Zhang et al., 2017). The responses are shown in Fig. 3, where red and blue are the maximum and minimum encapsulation efficiencies, respectively.

The interaction between the calcium chloride concentration (%w/v) and sodium alginate concentration (% w/v) (Fig. 3A) was indicated by the p value = 0.4050, while the interaction between pH and sodium alginate concentration (% w/v) (Fig. 3B) was indicated by the p value = 0.0544. For the interaction between pH and calcium chloride concentration (% w/v) (Fig. 3C), it was indicated by the p value = 0.7398. The interactions (Figs. 3A–3C) showed no significance; however, the optimum condition was considered as the maximum encapsulation efficiency, which was found at pH of 7, 20% w/v calcium chloride and 0.85% w/v sodium alginate.

The activity of dextranase enzyme encapsulated in alginate beads

Condition No. 14 also produced the highest enzyme activity, which was 18.32 unit /g. bead. The statistical analysis of the activity of the enzyme trapped in the alginate beads for all 17 conditions found that the quadratic model was the most suitable model for describing the activity of encapsulated enzyme, p = 0.0236. The factors that were statistically significant for effect on enzyme activity were the concentrations of calcium chloride and alginate, as shown in Table 4 and Dataset S2.

The F-value of 4.93 showed that this model was significant at a p-value of 0.0236. The lack of fit, F-value of 5.66 and p-value of 0.0637 implied unimportance due to relative pure error. In addition, the values of R² and adjusted R² were found to be 0.8637 and 0.6885 respectively, while the lack of significance of the value (p = 0.0637) of lack of fit indicates that the model was fitted with good prediction (Singh, Bajar & Bishnoi, 2017).

From the above experiment, the relationship between factors and responses (dextranase activity) values can be expressed using regression, as shown in the following equations. Letters A, B and C are alginate, calcium chloride and pH respectively. ${\displaystyle Dextranaseactivity=8.246+2.836A+2.376B-0.073C+1.195AB-2.994AC-0.498BC-3.208AA+2.037BB+4.765CC}$ Figures 4A–4C shows the response surface plots for the interactions between various factors, where red and blue are the maximum and minimum dextranase enzyme activities of encapsulation respectively. In addition, the interaction between the calcium chloride concentration (% w/v) and sodium alginate concentration (% w/v) (Fig. 4A) was indicated by the p-value = 0.3913. The interaction between pH and sodium alginate concentration (% w/v) (Fig. 4B) was indicated by the p-value = 0.0560, and the interaction between pH and calcium chloride concentration (% w/v) (Fig. 4C) was indicated by the p-value = 0.7150. The interactions between various factors (Figs. 4A–4C) showed no significance.

Considering the two responses (encapsulation efficiency and trapped dextranase activity in alginate beads), it was found that the optimum condition was the same as a pH equal to 7, 20% w/v calcium chloride concentration and 0.85% w/v sodium alginate concentration. Therefore, this condition was selected for producing dextranase beads for further study of the stability of the enzyme in oral care product (toothpaste).

The stability of encapsulated dextranase activity in a toothpaste base

A 2% alginate beads containing dextranase enzyme was investigated to determine the stability of dextranase activity in toothpaste formulation at baseline and after 3 months of testing. The data was analyzed by the t-test statistic, and it was found that the dextranase activity in the beads was not significantly different (p-value = 0.1423) after storage of the product at 40 ± 2 °C for 3 months (Table 5; Table S6). In addition, the characteristics of the toothpaste did not change after 3 months compared to baseline. Generally, the stability of the toothpaste was still according to the properties of good toothpaste, i.e., no change of appearance based on color, odor, viscosity, pH and homogeneous.

The satisfaction of toothpaste

The volunteers were selected with inclusion and exclusion criteria. The experiment was conducted under the supervision of an expert dentist. The demographic data of volunteers indicated that there were 15 volunteers, 5 males and 10 females. The average age was 35 years, and all the volunteers did not have a wound in the oral cavity. Then, they were given brush and toothpaste containing beads with commercial dextranase enzyme to brush once for 2 min for assessment, and their oral cavities were evaluated by the dentist again. The result showed that they did not have irritation after brushing.

Regarding the sensory satisfaction of the toothpaste containing alginate beads with dextranase enzyme, most volunteers chose “like slightly” (3 points) for all parameters: color, odor, flavor, the distribution of toothpaste, homogeneity of ingredient, the amount of toothpaste foaming while brushing, freshness, cleanness and overall satisfaction (Fig. 5; Table S7). The volunteers preferred the ideal toothpaste (near 4 points).