Potential Extract Of Red Algae (Gracilaria verrucosa) from Aceh’s Coast Against Streptococcus mutans

Streptococcus mutans is known as the main agent of dental caries. Characteristics of Streptococcus mutans are a Gram-positive, facultatively anaerobic bacteria with an acidophilic as an advantage characteristic of this bacteria than other cariogenic bacteria. This characteristic gives the ability to survive and thrive in a very low pH atmosphere. Gracilaria verrucosa, the red algae, is one of many marine products of Aceh’s coast, which contains antibacterial compounds such as flavonoids, tannins, and phenols. This research aims to explore the antibacterial potency of red algae extract of Gracilaria verrucosa from Aceh’s coast towards Streptococcus mutans by observing Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). The maceration method was used to produce the extraction of Gracilaria verrucosa with 96% ethanol as solvent. The extract concentration used for this study was 20%, 40%, 60%, 80%, and 100%. The results showed that the growth of S. mutans colonies had started to decrease at a concentration of 20%, i


INTRODUCTION
The province of Aceh which is located at the far west of Indonesia has a territorial area surrounded by the ocean.Seaweed (Gracilaria verrucosa), one of the ocean's natural resources that is advantageous to people, is one such resource.Red algae belonging to the genus Gracilaria verrucosa are a form of seaweed known as agarophytes because they make agar. 1 Algae contain many important metabolite components that can be utilized by the body.The components of seaweed consist of two groups: primary and secondary.
The primary metabolite components are vitamins, minerals, fibers, alginates, and agar.The secondary metabolite component of seaweed has the potential as a producer of various bioactive metabolites with very wide activation as antibacterial, antiviral, anti-fungal, and cytotoxic. 2,3n medicine, recently seaweed was developed as a suture raw material, mixtures in medicines, and so on. 4Meanwhile in dentistry, based on research of Nick Jakubvics (a reference is needed here), the components of seaweed could control bacteria such as Streptococcus mutans (S. mutans) as the main pathogen of dental caries. 5ecause it lives on the dental surface, this bacterium is frequently identified in the human oral cavity after the tooth has emerged.][8][9] The utilization of herbal plants for caries therapy is continuously developed.Previous research, about the antibacterial extract of Glacilaria sp. on the growth of Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella sp showed that Gracilaria sp. could inhibit the growth of these bacteria. 10Studies into the effectiveness of Gracilaria verrucosa extract as an immunostimulant for preventing Pacific white shrimp white spot disease Vannamei Litopenaeus The results showed the potential benefits of implementing Gracilaria verrucosa extract as an immunostimulant. 11here has been no research on the antibacterial potential of red algae species Gracilaria verrucosa against S. mutans.
This study was conducted to test the antibacterial potency of red algae Gracilaria verrucosa extract and to determine Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) extract against S. mutans as the main agent of dental caries.

METHODS
In this study, we used Streptococcus mutans ATCC 31987.Gracilaria verrucosa samples obtained from Pulo Aceh one of the small islands in the province of Aceh producing seaweed.
The maceration method was used to extract Gracilaria verrucosa, as shown below; First, wash the sample and dry it out by not exposing it to direct sunlight for three days.The sample was then cut into 1 cm pieces with scissors and placed in a jar.Then, add ethanol (96%) and macerate for three times 24 hours at room temperature.The solution was filtered every 24 hours to remove the dregs or residue.Finally, all of the filtrate was concentrated using a rotary evaporator set to 40˚C. 10 The extraction results were tested for phytochemicals to determine the active compounds contained therein.
S. mutans were grown in Nutrient Agar (NA) media using the T scratch technique (streak T).The bacterium was inoculated onto a petri disc, which was then placed in an anaerobic jar and incubated at 37°C for 3 x 24 hours. 13Gram staining was done to ensure that S.mutans was not contaminated.S.mutans that has been cultured in NA medium were taken with the ossea needle and put into a test tube containing 5 ml of 0.9% NaCl solution.The test tube was homogenized with a vortex and equalized the turbidity with 0,5 (1.5 x 10 8 CFU/ml) Mc.Farland solution.The next step was making serial dilution of S. mutans. 13,14ix test tubes were produced and labeled to assess the antibacterial properties of Gracilaria verrucosa extract on S. mutans.The following conditions were used to fill each tube with 3.5 ml: Group 1 was a control group that received 0.5 ml of S. mutans suspension and 3.5 ml of distilled water.The positive control group received 0.5 ml of S. mutans suspension and 3.5 ml of Chlorhexidine 2%.Following that 3.5 ml of Gracilaria Available at http://www.jurnal.usk.ac.id/CDJ verrucosa extract concentration of 20% and 0.5 ml of S. mutans suspension were added to the treatment group (P1).P2 was given 3.5 ml of Gracilaria verrucosa extract concentration of 40% and 0.5 ml of S. mutans solution.P3 was given 3.5 ml of Gracilaria verrucosa extract concentration of 60% and 0.5 ml of S. mutans solution.P4 was given 3.5 ml of Gracilaria verrucosa extract concentration of 80% and 0.5 ml of S. mutans suspension.The final treatment group 5 (P5) received 3.5 ml of 100% Gracilaria verrucosa extract and 0.5 ml of S. mutans solution. 12,15ll the tubes were shaken so the solution became homogeneous by using a vortex.Then, followed by taking 0.5 ml of suspension by using Eppendorf pipette from each tube and culturing by spread plate method on Muller Hinton Agar (MHA) medium, Suspension of all groups was flattened with L stem and incubated at anaerobic atmosphere at 37˚ for 3x24 hours in the incubator.Gas packs were used to make anaerobic conditions.The final step of the method was the observation of Streptococcus mutans.It was done by counting the number of S. mutans colonies that grew by using the Colony counter.The Minimum Inhibitory Concentration of Gracilaria verrucosa extract was the lowest concentration that can inhibit S. mutans growth which was seen from the least colony growth rate compared to the negative control while the Minimum Bactericidal Concentration of Gracilaria verrucosa extract was the lowest concentration which was seen from the absent of S. mutans growth at MHA medium compared to Chlorhexidine 2% as positive controls. 14The tests were done on MHA medium and each treatment was repeated three times.

RESULTS
The results of S. mutans culture on Trypticase Soy with Sucrose and Bacitracin (TYS20B) as selective media and Gram staining are shown in Figures 1 and 2. The colonies that grew on selective media, notably TYS20B, resembled S. mutans colonies in the form of round-shaped colonies with flat edges, yellowish-white hue, and an overwhelming yeast-like odor, as shown in the figure above.Meanwhile, the Gram staining technique was used to identify S. mutans, and the colonies were purple in color, coccusshaped (round), and organized in a chain, as shown in Figure 2.

Figure 2. Gram Staining of S. mutans
The result of this test showed the growth of S. mutans colonies after being divided by their dilution rate (10 -4 ) then obtained the highest average number of colony growth in the treatment group with the concentration of 20% was 9,95 x 10 6 CFU/ml and the least colony growth was in the concentration of 100% that was 5,4 x 10 5 CFU/ml, whereas in the positive control group (CHX 2%) there was no growth of bacterial colonies and in the negative control group (distilled water) there was bacterial colonies growth that was 2,95 x 10 7 CFU/ml.

DISCUSSION
The Gracilaria verrucosa extraction results of phytochemical tests showed that the red algae extract of Gracilaria verrucosa contained steroid, terpenoid, tannin, and phenolic compounds.It also showed that the red algae Gracilaria verrucosa extract did not contain alkaloids, saponins, or flavonoid compounds.This might be due to several natural factors such as environmental conditions including light, water, temperature or salinity, life phase, age, geographic location, and season. 17,18The temperature factors could affect the photosynthesis process which then influenced the compound level in plants. 17,8he season factor influenced was when high rainfall which could cause the plants to get less sunlight intake.9][20] This might be a consideration in antibacterial activity not only associated with a single compound but also could be attributed to several compounds and metabolites combination. 19he dilution method was used in this research because this method could be used to measure Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).The results of the antibacterial potency test of Gracilaria verrucosa extract against S. mutans showed that Gracilaria verrucosa extract was able to inhibit the growth of S. mutans.This was proved by decreasing number of S. mutans colonies grown in each treatment concentration when compared to the negative controls.The growth of S. mutans decreased with the high concentration of Gracilaria verrucosa extract.The higher the concentration, the more chemical compounds there are.The presence of tannins in Gracilaria verrucosa causes an antibacterial effect, which damages the cell membranes of S. mutans.Terpenoids in the extract also cause cell wall damage by reacting with porins, which are transmembrane proteins on the outer membrane of the bacterial cell wall, generating strong polymeric linkages that cause porin damage.This is aggravated by the presence of steroid-induced liposome leakage.The activity of these active chemicals damages the cell wall of S. mutans, preventing the bacteria from carrying out its duties.The higher the concentration, the more chemical compounds there are.The presence of tannins in Gracilaria verrucosa causes an antibacterial effect, which damages the cell membranes of S. mutans.Terpenoids in the extract also cause cell wall damage by reacting with porins, which are transmembrane proteins on the outer membrane of the bacterial cell wall, generating strong polymeric linkages that cause porin damage.This is aggravated by the presence of steroid-induced liposome leakage.][18] In this study, the minimum inhibitory concentration (MIC) was 20%, whereas the minimum bactericidal concentration (MBC) was not reached since S. mutans.grew even at the highest concentration of 100%.This could be because the Gracilaria verrucosa extract lacks flavonoids.The study by Almeida (2011)  stated that the hydroxyl groups present in the flavonoid compound structure could cause changes in organic components and transport of nutrients which eventually resulted in toxic effects on bacteria. 18rom the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), the extract of Gracilaria verrucosa against S. mutans as the main agent of dental caries can be concluded that the extraction of red algae Gracilaria verrucosa had antibacterial potency which could inhibit the growth of S. mutans.The Minimum Inhibitory Concentration (MIC) of red algae extract of Gracilaria verrucosa against S. mutans was 20% without Minimum Bactericidal Concentration (MBC).

CONCLUSION
Based on the result of this research concluded that Gracilaria verrucosa extract has antibacterial potential against S. mutans with MIC at a concentration of 20% whereas MBC is not found.

CONFLICT OF INTEREST
no conflict of interest.

Figure 1 .
Figure 1.Colonies of S. mutans on TYS20B media

Table 1 .
the number of S. mutans colonies after being tested by Gracilaria verrucosa extract.