## 2 Materials and Methods
### Evaluation of the Antimicrobial Properties of the Developed Toothpastes
This test relies on the diffusion of the test product from the filter paper into the pre-inoculated solidified agar. If the test formulation inhibited or killed the bacteria or fungi, a circular area around the discs, known as the inhibition zone, was formed, demonstrating its inhibitory effect on the microorganisms under test. The microorganisms used for this test along with the nutrient ingredients used for the preparation of their suspensions and the period of incubation are presented in Table 4.  
The discs made of filter paper were processed accordingly before the test in order to remove any background flora. Specifically, all filter discs were immersed in a 70% \(\
u/\
u\) isopropyl alcohol solution and allowed to dry covered, protected from cross-contamination. Standard suspensions of all microorganisms used during the test were prepared and inoculated onto the surface of suitable agar plates to form a uniform layer. 1. Test product: The filter paper discs were impregnated with 25 \(\upmu\)L of the product being tested, which was diluted 50% with water. The discs were placed on the surface of pre-inoculated agar plates. The same procedure was repeated for all microorganisms. 2. Negative test: The filter papers were impregnated with 25 \(\upmu\)L of sterile water. The discs were aseptically placed on the surface of pre-inoculated agar plates. The same procedure was repeated for all the tested microorganisms. 3. Each test product was compared with a negative control product, which did not contain an antimicrobial agent, and with a positive control product, which was a commercially available synthetic toothpaste (Instant Whitening Blue Toothpaste, Frezyderm SA, Athens, Greece), whose antimicrobial action was proven. The composition of this toothpaste is: deionized water, blue covasorb, hydrated silica, Cymenol, 0.32% \(w/w\) sodium fluoride (1450 ppmF). Three independent repetitions were performed for each product and each microorganism to increase accuracy and minimize bias. The evaluation of the antimicrobial activity \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Name** & **Characteristics** & **Incubation Conditions** & **Nutrient Ingredients** \\ \hline \multirow{3}{*}{\begin{tabular}{c} Candida albicans \\ (ATCC 10231) \\ \end{tabular} } & Fungus, one of the main microorganisms of the & & 2 days & \\  & normal oral flora, which, however, can grow, & & 2 days & \\  & multiply easily, and cause fungal infections in the & at 25 \({}^{\circ}\)C & & Sabouraud Dextrose Agar \\  & oral cavity & & \\ \hline \multirow{3}{*}{\
\begin{tabular}{c} Streptococcus mutans \\ (ATCC 25175) \\ \end{tabular} } & Gram-positive, anaerobic, round bacterium (coccus), & & \\  & commonly found in the human oral cavity and & & 2 days & \\  & significantly contributes to the formation of dental & & at 37 \({}^{\circ}\)C & Brain Heart Infusion Agar \\ \hline \multirow{3}{*}{
\begin{tabular}{c} Prevotella intermedia \\ (ATCC 25611) \\ \end{tabular} } & Gram-negative, anaerobic pathogenic bacterium & & & \\  & involved in periodontal infections such as gingivitis & & 2 days & \\  & and periodontitis & & at 37 \({}^{\circ}\)C & Soybean-Casein Digest Agar \\ \hline \hline \end{tabular}
\end{table} 
Table 4: The characteristics, incubation conditions, and nutrient ingredients of the three microorganisms used in the test for evaluating the antimicrobial properties of the toothpastes. of the product was conducted by measuring the diameter of the inhibition zones around the filter discs. Three measurements were taken per paper disc at three different points. The diameter was recorded in mm, and the average value was calculated per disc. The study was conducted using a double-blind design to enhance the objectivity of the research. This implies that neither the researchers conducting the experiments nor the statisticians analyzing the data were aware of the group assignments.