Clove Oil (Syzygium aromaticum) Edible Film Formulation and Antibacterial Activity Test against Streptococcus mutans

Clove oil contains eugenol as an antibacterial. Meanwhile, products containing clove oil have been widely used as toothpaste and mouthwash. In this study, clove oil was formulated in the form of edible film because it is practical, easy to use, and could be used without water like other oral hygiene preparations. The edible film is a thin layer film made of consumable materials used as a carrier of antibacterial compounds. Clove oil edible film was then formulated with clove oil concentrations of 1%, 1.5%, and 2% and determined for its antimicrobial activity against Streptococcus mutans. Clove oil edible film preparations were evaluated under their physical properties, including friability, drying shrinkage, pH, thickness, and swelling ability. Antibacterial activity testing of clove oil edible film was conducted, employing the blood agar diffusion method against Streptococcus mutans. The physical evaluation of the clove oil edible film showed almost the same physical properties as the comparison (GF). Clove oil edible film test results revealed the greatest inhibition at F1 of 18.6 mm ± 0.577, F2 of 22.3 mm ± 2.081, and F3 of 25.3 mm ± 1.527. According to David and Stout, the inhibition activity of bacteria on F3 was categorized as a very strong group inhibition response. In addition, ANOVA test analysis results uncovered that the concentration of clove oil affected the inhibition of the Streptococcus mutans bacteria with a significance value of 0.000 (p <0.05). Also, Duncan's test exhibited that each concentration of clove oil had a significant difference in the inhibition of Streptococcus mutans bacteria.


INTRODUCTION
Clove oil has secondary metabolites in the form of essential oil with the highest eugenol content so that it has biological activity as an antiseptic and analgesia in the treatment of teeth and mouth, were tested on mouthwash preparations at 0.5%, 0.75%, and 1% clove oil concentrations. It was shown that there was no minimum bactericidal concentration (MBC) at a concentration of 0.5%, so it was concluded that it could inhibit bacterial growth (bacteriostatic). Meanwhile, at concentrations of 0.75% and 1%, clove oil had an MBC value, so that it was denoted to have bacteria-killing activity (bactericidal) 2 .
Moreover, oral hygiene preparations that have been widely circulated are in the form of toothpaste, mouthwash, and edible film as mouth fresheners containing menthol. The edible film is a thin layer made of safefor-consumption materials and used as a wrapper to prevent the process of fat oxidation, organoleptic changes, microbial growth, or absorption of moisture. In addition, edible films function as a barrier against the mass transfer of solutes, as a carrier for additives, and improving the handling of food 3 . The formation of edible films can also prevent the evaporation of eugenol in clove oil since one of its functions is as a barrier to mass transfer. Many studies on edible films have been conducted, including the use of betel leaf extract as an anti-halitosis formulated in the form of edible films 4 and areca seed extract edible films as antibacterial 5 .
On the other hand, Streptococcus mutans is a bacterium capable of attaching to the tooth surface that produces glucosyltransferase enzyme. These enzymes produce insoluble glucans in water and play a role in causing plaque and colonies on the tooth surface 6 . The growth of Streptococcus mutans must be inhibited so that it does not become pathogenic and cause caries by giving antibacterial agents. One way to prevent caries is to limit the plaque formation on the tooth surface, either by preventing its formation or regularly cleaning plaque. Plaque control can be done by mechanical and chemical cleaning of plaque containing antibacterial ingredients, suppressing Streptococcus mutans growth. Based on the description above, the formulation of clove oil in the form of edible film was carried out, which could prevent the evaporation of clove oil compared to the dosage form of toothpaste and mouthwash and tested its antibacterial activity against Streptococcus mutans.

Clove Oil (Syzygium aromaticum) Edible Film Formula
The edible film formula used referred to the formula made by Harmely et al. (2014) because this formula gave good results in previous studies. Corn starch was dispersed into 20 parts of aqua dest, heated at ± 60ºC, and stirred until a clear gel was formed. HPMC was developed in distilled water, which had added sorbitol and sodium saccharin, and then was stirred at a temperature maintained at ± 60ºC. The two gels were mixed at a temperature of ± 60ºC by adding other additives (nipagin, nipasol, menthol, mint oil, Na metabisulfite, and clove oil) at room temperature; the mixture was stirred homogeneously, then was poured and leveled on the mold (26 x 20 cm). The preparation was dried in the oven at a temperature of 40-50 ºC for 24 hours. Then, the edible film formed was released from the mold and cut into pieces with 2.2 x 3.2 cm.

Organoleptic Examination
The organoleptic examination included visual observations using the five senses on the edible film's shape, smell, taste, and color. This examination was carried out at room temperature (15-30ºC) 7 .

Edible Film Friability Examination
The friability of the edible film was carried out according to the friability test of the tablet, 8,9 utilizing a Roche Friabilator. First, 20 sheets of edible film free of dust (W1) were weighed, then put into the Roche Friabilator. The tool was run for four minutes with a rotation speed of 25 rpm. The 20 edible films were cleaned of dust and weighed again (W2). Edible film friability can be calculated by the formula:

Drying Shrinkage Examination
The porcelain dish was dried in an oven at 105 o C until a constant weight (A) was obtained. Edible film weighed 2g in a porcelain dish (B), then dried in an oven for 2-5 hours until a constant weight was obtained (C). The drying shrinkage was then determined in percent by weight of the sample used 10 .

pH Examination
This examination was performed using the Inolab pH meter. First, this instrument was calibrated employing a buffer of pH 4 and pH 7. The electrodes were rinsed with distilled water and dried. The pH measurement of the clove oil edible film was carried out by dissolving 1g of the edible film in distilled water up to 10 ml. The electrode was immersed in the container, and the number on the pH meter represented the pH value of the clove oil edible film. 7 11 The thickness of the edible film produced was measured utilizing a micrometer with an instrument accuracy of 1 m. Measurements were made at five different places.

Swelling Test
The swelling test was carried out by inserting one sheet of edible film into a glass beaker, then expanded with 10 mL of distilled water. Then, it was determined how long it took the preparation to expand. 12

Antimicrobial Activity Test by Diffusion Method (Kirby Bauer Method)
This method was used to determine the activity of antimicrobial agents. A plate containing an antimicrobial agent was placed on an agar medium on which microorganisms had been grown, which would diffuse into the agar medium. Then, it was incubated at 37ºC for 18-24 hours. The clear zone indicated the presence of inhibition of the growth of microorganisms on the surface of the agar medium 13 .
Clove Oil Antibacterial Activity Test

Sterilization of tools and materials
The tools utilized were washed and dried before being sterilized. Some tools, such as Petri dishes, were wrapped in newspaper, while the mouthpieces, test tubes, and droppers were covered with cotton and then wrapped one by one with newspaper. All tools were sterilized in an autoclave at 121ºC for 15 minutes. Laminar Air Flow was cleaned and then sprayed with 70% ethanol. After that, it was sterilized by turning on a UV lamp for five minutes.

Making Blood Agar Media
The media powder of blood agar base was weighed as much as 4 grams, then dissolved with 100 ml of distilled water and heated to boiling while stirring. Then, it was sterilized by autoclave at 121 o C for 15 minutes. The sterile media was cooled to a temperature of 45-50 o C. Then, 10 mL of fresh sheep blood was added and stirred until smooth. Then, it was poured into a petri dish and waited for it to solidify. Once frozen, the media was ready to use.

Preparation of Test Microbial
Suspension Bacterial colonies were suspended in sterile physiological NaCl solution in sterile test tubes and homogenized by the vortex. Then, the turbidity of the equivalent suspension was measured by standard turbidity UV-Vis Spectrophotometry. Thus, a suspension with a transmittance of 25% at a wavelength of 580nm was obtained 13 .

Clove Oil Antibacterial Activity
Testing The sterilized blood agar medium was poured into a ±20mL petri dish. After the media solidified, a sterile cotton swab was dipped into the bacterial suspension and then smeared evenly over the media. Sterile disc paper that had been previously dripped with clove oil with concentrations of 1%, 1.5%, 2%, and as a negative control dimethyl sulfoxide (DMSO) was taken using a 10µL micropipette. It was incubated for 24 hours at 37 o C. Then, it was observed for bacterial growth. The inhibition area was also measured, indicated by the appearance of a clear area around the disc utilizing a caliper.

Edible Film Antibacterial Activity
Testing Blood agar media sterilized was poured into a petri dish as much as ± 20 mL. After the media solidified, a 5 mm diameter film sheet was placed on the agar medium and then incubated at 37 o C for ±24 hours. The growth of bacteria was observed, and the inhibition area diameter was measured, indicated by the presence of an area not overgrown by bacteria. Tests were carried out on F1, F2, F3 preparations. F0 was used as a negative control based on the edible film, while (GF®) was employed as a comparison 14 .

Data Analysis
The data on the antibacterial activity of clove oil in edible film preparations were processed by one-way analysis of variation (ANOVA) utilizing the SPSS program. The results will be meaningful if the comparison of inhibition in each formula gives a real and meaningful difference.

RESULTS AND DISCUSSION
This study aimed to formulate a preparation of clove oil (Syzygium aromaticum) in the form of an edible film as a deodorizer and determine its antibacterial activity against Streptococcus mutans. Clove oil contains eugenol as much as 78-98%. The substance is produced from the oil glands on the surface of the clove flower body. In addition, clove oil also contains eugenol acetate, caryophyllene, and other minor compounds, in small amounts 15 . Edible clove oil film was made by casting method on a mold (26 x 20 cm). The edible film formed was removed from the mold and cut into pieces with 2.2 x 3.2 cm (Figure 1).

Figure 1. Preparation of clove oil edible film
Description: P = Comparative Preparation Formula "GF ® " F0 = Edible film base formula with 0% clove oil concentration F1 = Edible film base formula with 1% clove oil concentration F2 = Edible film base formula with 1.5% clove oil concentration F3 = Edible film base formula with 2% clove oil concentration The edible clove oil film formed was evaluated for physical properties and activities against Streptococcus mutans. Evaluation of the clove oil edible film physical properties was then compared to the circulating physical properties (GF). The organoleptic examination of the clove oil edible film was carried out for six weeks. Every week, the organoleptic examination results of edible films showed no change in shape, smell, taste, and color. It indicates that the edible film preparation was stable during storage. In addition, the friability examination of edible films utilizing the "Roche Friabilator" friability test equipment did not appear to have changed shape or broken the edible film during the test. However, there was a reduction in the dosage weight caused by the edible film experiencing friction during testing. The % friability test results of the edible film also revealed that the % friability of edible film F3 (1.17%) was higher than the comparison (1.06%). This friability test describes the resistance of the preparation surface to the friction experienced during packaging, shipping, and storage. 16 The results of the edible film drying shrinkage examination uncovered that the highest drying shrinkage was shown in the formula F0, which was 13.91%, whereas the lowest was displayed in the comparison preparation, which was 12.69%. Meanwhile, according to previous research, the percentage of good drying shrinkage for edible films was less than 9.29%. 17 Moreover, the pH evaluation of the edible film was performed for six weeks. The test results exposed that the pH changed every week but still met the physiological pH range of the mouth. Weekly changes in pH values might be caused by environmental factors, such as temperature, storage, and the sensitivity of the pH meter. The pH value of the resulting edible film must be in the pH range of the oral cavity, which ranges from 5.5 to 7.918 18 , so as not to irritate the oral mucosa when the preparation is consumed.
Next, the thickness of the edible film was examined employing a screw micrometer with an accuracy of 0.01 mm at five different places, and the results were then averaged. The thickness difference occurred due to clove oil concentration differences in each preparation, where F3 was the thickest compared to F1, F2, and F0. The measuring results of the edible film thickness in all formulas met the requirements of a good thickness of the edible film, which was <0.25 mm. 19 This thickness test was carried out to see that the edible film produced is thin but not easily torn, which is an indicator of uniformity and quality control of the edible film.
Further, in the swelling test, the mean swelling time results of the edible film were obtained. The measurement results disclosed that F1, F2, and F3 required a longer time for the edible film to swell because it contained clove oil, which is difficult to dissolve in water with different concentrations. It is suspected that the mechanism of film destruction is through swelling and wicking mechanisms. The swelling process is that after the film is in contact with water, water penetration occurs due to capillarization (wicking) so that the film swells. Meanwhile, the wicking mechanism is the presence of water drawn by the disintegrant, where water will penetrate the film pores. As a result, the bonds between the particles become weak, and the film swells. 11 Furthermore, the antibacterial activity testing of clove oil edible film against Streptococcus mutans bacteria was conducted using the agar diffusion method. This method is most commonly used to determine the activity of the test material against bacteria. The 24-hour incubation was carried out to give time for bacteria to multiply rapidly and carry out metabolic activities. The results showed that after 24 hours of incubation, all clove oil groups' concentrations could inhibit the bacteria Streptococcus mutans growth. The higher the concentration, the higher the zone of inhibition. It aligns with Pelczer and Chan's opinion that the higher the concentration of an antibacterial agent, the stronger the antibacterial activity. 20 In this study, the test bacteria were suspended in a 0.9% physiological NaCl solution because the physiological NaCl solution was an isotonic environment for the test bacteria. The suspension was homogenized by vortex, and the turbidity was measured utilizing a UV-Vis spectrophotometer with a wavelength of 580 nm and transmitter 25%, describing the optimal cell density for antibacterial activity testing. 13 From the antibacterial activity test analysis, homogeneous and normal data were obtained, which showed that the data obtained were parametric, followed by one-way ANOVA statistics utilizing SPSS 16 and Duncan's test. The results of the one-way ANOVA statistical test revealed the P-value (<0.005). It signifies a significant difference between all formulas. The difference in the concentration of clove oil at a 2% concentration did not show a significant difference with a 1.5% concentration but showed greater inhibition at a 2% concentration. In the edible film preparation, between the comparisons of the GF® edible film, a significant difference was shown with the edible film preparation in varying concentrations. However, on 1.5% clove oil edible film, it did not show a significant difference with 1% and 2% edible film, but 2% clove oil edible film had greater antibacterial inhibition. Based on the response table of bacterial growth inhibition according to Davis and Stout, the inhibition classification was divided into four categories: very strong = 20 mm, strong = 10-20 mm, moderate = 5-10 mm, and weak = 5 mm. From the results obtained for each clove oil edible film formula, F1 = 18.6 mm, F2 = 22.3 mm, and F3 = 25.3 mm. It was categorized into a very strong bacterial growth inhibition response. According to a study, 21 factors that affect the size of the inhibition zone include the organism sensitivity, the culture medium, the incubation conditions, and the rate of agar diffusion. Meanwhile, the factors that influence the rate of agar diffusion consist of the microorganism concentration, the medium composition, the incubation temperature, the incubation time, and the pH value of the medium. In this regard, some antibacterials work well under acidic conditions and others under alkaline conditions.

CONCLUSION
Clove oil with a concentration of 1% (F1), 1.5% (F2), and 2% (F3) can be formulated in the form of edible film. From the physical evaluation results, almost the same results as the comparison of GF® in the form of edible film containing menthol were obtained. In addition, the antibacterial activity test results showed significant differences between F1, F2, and F3. Of the three formulas containing clove oil, F3 gave the greatest inhibitory power of 25.3mm (p<0.05) and was included in the very strong bacterial growth inhibition response.