|Year : 2022 | Volume
| Issue : 2 | Page : 88-92
Antibacterial Activity of Robusta Coffee (Coffea robusta) Husk Extract Against Streptococcus mutans and Lactobacillus acidophilus: In Vitro Study
Arlin R Kusumawardani1, Andi M Machbub1, Rendra C Prasetya2, Nadie Fatimatuzzahro MDSc 2, Tantin Ermawati2
1 Faculty of Dentistry, Jember University, Jember, Indonesia
2 Department of Biomedic, Faculty of Dentistry, Jember University, Jember, Indonesia
|Date of Submission||17-Jun-2022|
|Date of Decision||18-Sep-2022|
|Date of Acceptance||21-Sep-2022|
|Date of Web Publication||10-Jan-2023|
Faculty of Dentistry, Jember University, Kalimantan No 37, Kampus Tegalboto, Jember, Jawa Timur 68121
Source of Support: None, Conflict of Interest: None
Introduction: Caries is a tooth and oral illness caused by Streptococcus mutans and Lactobacillus acidophilus bacteria. The growth of caries-causing bacteria can be controlled by using 0.2% chlorhexidine mouthwash to control plaque, however long-term usage of 0.2% chlorhexidine causes negative effects. Natural components such as robusta coffee (Coffea robusta) husk, which possesses active compounds of polyphenols, flavonoids, alkaloids, tannins, and saponins as alternative antibacterials, can be used to reduce adverse effects. The aim of this research is to assess the antibacterial activity of robusta coffee husk extract against S. mutans and L. acidophilus. Materials and Methods: The robusta coffee husk was extracted using the maceration process with 96% ethanol as the solvent. Antibacterial test is conducted against S. mutans and L. acidophilus using disc diffusion method (Kirby-Baurer) with six treatment groups of extract concentration 250, 500, 750, 1000 mg/mL, positive control (0.2% chlorhexidine), and negative control (aquades steril). The zone of inhibition was measured in millimetres using a digital calliper (mm). The SPSS application was used to examine the calculation findings, which included the Shapiro-Wilk, Levene, One Way ANOVA, and Post Hoc LSD tests. Results: Robusta coffee husk extract at 250, 500, 750, and 1000 mg/mL doses shown bactericidal activity in S. mutans (radical zone) and bacteriostatic activity in L. acidophilus (irradical zone). Conclusion: Robusta coffee husk extract has an antibacterial activity against S. mutans and L. acidophilus. The highest inhibition zone was demonstrated by the 1000 mg/mL concentration of extract.
Keywords: Antibacterial, Robusta coffee husk extract, Streptococcus mutans, lactobacillus acidophilus
|How to cite this article:|
Kusumawardani AR, Machbub AM, Prasetya RC, Fatimatuzzahro N, Ermawati T. Antibacterial Activity of Robusta Coffee (Coffea robusta) Husk Extract Against Streptococcus mutans and Lactobacillus acidophilus: In Vitro Study. J Orofac Sci 2022;14:88-92
|How to cite this URL:|
Kusumawardani AR, Machbub AM, Prasetya RC, Fatimatuzzahro N, Ermawati T. Antibacterial Activity of Robusta Coffee (Coffea robusta) Husk Extract Against Streptococcus mutans and Lactobacillus acidophilus: In Vitro Study. J Orofac Sci [serial online] 2022 [cited 2023 Jun 9];14:88-92. Available from: https://www.jofs.in/text.asp?2022/14/2/88/367441
| Introduction|| |
Caries is one of the most frequent dental and oral illnesses in Indonesia, with a 45.3% incidence prevalence that is increasing year after year. Caries is caused by four interconnected variables: the presence of a substrate in the form of fermentable carbohydrates, host factors, time, and the growth of bacteria (plaque). Streptococcus mutans and Lactobacillus acidophilus are the most common cariogenic bacteria implicated in plaque development.
S. mutans is a cariogenic bacterium capable of fermenting carbohydrates into acids. Bacterial metabolism produces acid, which interacts with calcium on the tooth surface, causing demineralization. Meanwhile, L. acidophilus can create extracellular protein (EPS) and lactic acid, both of which are involved in plaque formation., Furthermore, L. acidophilus has hydrophobic protein s-layers, which increases L. acidophilus capacity to attach to the tooth surface.
Efforts to reduce bacteria accumulation can be made by controlling plaque with antimicrobial mouthwash. One of the ingredients in mouthwash that is considered the gold standard for antiplaque and antigingivitis is 0.2% chlorhexidine. Because it is particularly active against all germs, chlorhexidine is a high-level disinfectant. Long-term and non-adherent usage of chlorhexidine, on the other hand, can induce tooth discoloration, restoration and dorsum of the tongue discoloration, alterations in taste perception, and mucosal erosion. As a result, alternative antibacterials made from natural substances are required since they are easy to get, inexpensive, non-toxic, and have fewer adverse effects than chemicals.
The coffee plant is one of the natural components that is empirically utilized as an antibacterial. Coffee plants, particularly the robusta type, have been proven to have antiinflammatory and antioxidant effects in addition to being antibacterial. Robusta coffee is a plantation crop that is widely grown in Jember City, Indonesia. The mass production of coffee generates trash in the form of high robusta coffee husk. Robusta coffee husk includes various active components, including polyphenols, flavonoids, alkaloids, tannins, and saponins, all of which have antimicrobial properties.
Previous studies demonstrated that robusta coffee husk extract can suppress the growth of Staphylococcus aureus and Escherichia More Details coli bacteria. However, no research has been conducted on the inhibitory impact of robusta coffee husk extract on S. mutans and L. acidophilus.
| Materials and Methods|| |
Ethical approval for this study (0076/KKEP/FKG-UGM/EC/2021) was provided by the Ethical Committee of Medical Research at the Faculty of Dentistry in Gadjah Mada University, Yogyakarta, on September 21, 2021. This is an experimental laboratory study with a post-test-only control group design. This study has six treatment groups: robusta coffee husk extract concentration 250, 500, 750, 1000 mg/mL, positive control (0.2% chlorhexidine), and negative control (Aquades steril).
Blender (Philips, Holland), stir bar (Pyrex, Japan), filter paper (No Brand), digital scale (Scale SF400, China), test tube (Iwaki Pyrex, Japan), rotary evaporator (Heidolph, Germany), vortex (Labinco L46, the Netherlands), non-insulated petridish 100 mm (Iwaki Pyrex, Japan), oven (Binder, Germany), incubator (LabTech, Indonesia), spectrophotometer (Nikrom, Indonesia).
Robusta coffee husk extract, S. mutans and L. acidophilus bacteria, Mueller Hinton Agar (MHA) (HiMedia, India), Mueller Hinton Broth (MHB) (HiMedia, India), Aquades steril (Onelab, Indonesia), 96% ethanol (No Brand), 0.2% chlorhexidine (Minosep Minorock, Indonesia).
Preparation of robusta coffee husk extract
The maceration process was employed to Robusta coffee husk samples from Banjarsengon Village, Jember City, Indonesia. Robusta coffee husk was washed and dried in the sun before being dried in an oven at 50°C for 24 hours. The rind of dried and crushed robusta coffee fruit is weighed to 800 g and steeped in 96% ethanol solvent in a 1:5 ratio for 3 × 24 hours. The maceration results were filtered through fine filter paper and concentrated for 12 hours in a rotary evaporator.
Preparation concentration of robusta coffee husk extract
The extract was concentrated by placing the robusta coffee husk extract in a test tube containing 1 mL of aquades steril and homogenizing it with a vortex.
S. mutans ATCC 25175 and L. acidophilus ATCC 4356 were the microorganisms used. One dose of bacterial culture was placed in a tube containing 2 mL of Muller Hinton Broth (MHB) liquid medium, and the suspension was vibrated using a vortex and adjusted for turbidity to the 0.5 McFarland standard.
The disc diffusion method was used in this study’s antibacterial test (Kirby-Bauer). Each suspension of S. mutans and L. acidophilus was streaked (zig-zag scratched) onto the surface of Mueller Hinton Agar (MHA) media with a cotton swab. Using a micropipette, solutions of each concentration of robusta coffee husk extract, positive control (0.2 % chlorhexidine), and negative control (aquades steril) were poured onto disc paper and left to infuse. The disc paper was placed on the surface of the S. mutans or L. acidophilus inoculated MHA media, then the petridish was closed and placed in an incubator at 37°C for 24 hours. The restricted growth of S. mutans or L. acidophilus is seen in the clear zone surrounding the paper disc. The diameter of the circle was used to calculate the circular inhibition zone, while the long and short diameters were added together and divided by two to get the elliptical inhibition zone.
The Statistical Package for the Social Sciences 25.0 software was used to analyze the data (SPSS for Macbook; SPSS, Chicago, IL, USA). The acquired data were checked for normality with Shapiro-Wilk and homogeneity with Levene (P > 0.05). The One Way ANOVA parametric test was employed for all treatment groups, followed by the Post Hoc LSD test to determine the significant differences in each group (P < 0.05).
| Results|| |
[Figure 1] shows the results of the antibacterial power test of robusta coffee husk extract against S. mutans (a) and L. acidophilus (b). Robusta coffee husk extract inhibits S. mutans by forming a radical zone and an irradical zone against L. acidophilus. A radical inhibition zone was formed by the positive control (0.2% chlorhexidine), but not by the negative control (Aquades steril) (0 mm).
|Figure 1 Antibacterial activity against S. mutans (a) and L. acidophilus (b). K250: 250 mg/mL extract concentration; K500: 500 mg/mL extract concentration; K750: 750 mg/mL extract concentration; K1000: 1000 mg/mL extract concentration; K+: positive control; K−: negative control.|
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The diameter of the inhibition zone revealed that the robusta coffee husk extract at 1000 mg/mL resulted in the biggest inhibition zone against S. mutans of 10.75 [Table 1] and L. acidophilus of 15.45 mm [Table 2] and reduced as the husk extract concentration decreased.
The Saphiro-Wilk and Levene tests for robusta coffee husk extract’s antibacterial efficacy against S. mutans and L. acidophilus yielded (P > 0.05), indicating that the findings were normally distributed and homogeneous. The P value for the One Way ANOVA and Post Hoc LSD tests was 0.000, indicating that there is a significant difference in the average diameter of the inhibition zone across groups.
| Discussion|| |
By measuring the inhibition zone produced around the paper disc, an antibacterial test was performed to measure the response of microbe growth to an antimicrobial material. There are two sorts of inhibition zones: radical zones and irradical zones. The radical zone is a clean zone around the paper disc that contains no bacterial growth (bactericidal). Meanwhile, the irradical zone is a region where bacteria are just inhibited rather than killed (bacteriostatic), indicated by presence of unclear or equal boundaries between the bacterial colony and a clear zone containing bacteria.
The antibacterial activity of robusta coffee husk extract against S. mutans and L. acidophilus differed, as shown by the type of inhibitory zone created. Bactericidal activity is possessed by S. mutans, but bacteriostatic activity is possessed by L. acidophilus. This variability in antibacterial activity is hypothesized to be produced by diverse microbe species, which can impact the sensitivity of each microorganism. This demonstrates that S. mutans is a type of bacteria that is sensitive to the antibacterial properties of robusta coffee husk extract, whereas L. acidophilus is not.
The average diameter of the inhibition zone revealed that the concentration of robsuta coffee husk extract increased the diameter of the inhibition zone generated. The concentration of the active ingredient in the robusta coffee husk is hypothesized to influence the difference in the value of this inhibitory zone. The stronger the inhibitory reaction, the higher the concentration of the extract, because the extract contains more bioactive components.
The active chemicals found in robusta coffee husk extract are assumed to be responsible for its antibacterial activity against S. mutans and L. acidophilus. These active substances include polyphenols, flavonoids, alkaloids, tannins, and saponins. Polyphenols act as a poison in the protoplasm, causing the porin molecule to degrade. Damage to the porin allows polyphenolic chemicals to enter, reducing the permeability of the bacterial cell wall, and inhibiting or even killing bacterial development.
Flavonoids have an antibacterial effect in three ways: inhibit nucleic acid synthesis, inhibit cytoplasmic membrane function, and inhibit energy metabolism by preventing bacteria from using oxygen, which disrupts the process of macromolecular biosynthesis and bacterial metabolite absorption.
Alkaloids act as antibacterials by reducing the synthesis of nucleic acids in bacterial cells by blocking the action of dihydrofolate reductase, a key enzyme in the creation of amino acids, RNA, and bacterial DNA. As a result, bacteria become inactive and die.
Tannins have the ability to inactivate bacterial adhesins by binding to adhesin proteins, resulting in an incomplete bacterial cell wall layer, preventing bacteria from attaching to the host, and causing lysis owing to high osmotic pressure.
While saponins serve as antibacterials by lowering surface tension, increasing cell wall permeability, and allowing the cytoplasm to leak and intracellular chemicals to be released, bacteria are unable to metabolize in order to grow and reproduce.
A radical zone was formed by the positive control (0.2% chlorhexidine) against both S. mutans and L. acidophilus. This implies that the antimicrobial test technique followed the implementation procedure. While the negative control (Aquades steril) did not produce an inhibitory zone, this indicates that the solvent used to make the extract concentration had no effect on the active ingredient content.
| Conclusion|| |
Robusta coffee husk extract has an antibacterial activity. Its bactericidal activity (radical zone) against S. mutans and bacteriostatic (irradical zone) against L. acidophilus at doses of 250, 500, 750, and 1000 mg/mL. The highest inhibition zone was demonstrated by 1000 mg/mL concentration of extract. The lower the concentration, the less its antibacterial effect.
The authors thank the biomedical and microbiological laboratory technician at the University of Jember, Faculty of Dentistry for their assistance with this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]