|Year : 2018 | Volume
| Issue : 2 | Page : 135-141
Effect of crude apple extract on the cariogenic factors of Streptococcus mutans: An in vitro study
Vabitha Shetty, Nikhitha P Aswath, Amitha M Hegde
Department of Pedodontics and Preventive Dentistry, A B Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India
|Date of Web Publication||2-Jul-2018|
Department of Pedodontics and Preventive Dentistry, A B Shetty Memorial Institute of Dental Sciences, Deralakatte, Mangalore - 575 018, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: The aim was to assess the effects of crude extract of whole apple on the cariogenic properties of Streptococcus mutans by evaluating (1) its growth, (2) its adherence property, and (3) changes caused by acid produced by S. mutans in the presence of sucrose (in vitro). Study Design: Kashmiri Apples (sourced from the local market) were used for the study. Whole apple extracts including the skin were used to obtain undiluted crude apple extract. The growth of S. mutans in the presence of the crude apple extract was evaluated by agar diffusion test as well as direct contact inhibition test. The effects of the crude extract on the adherence of S. mutans was assessed by modified O'Toole method in which the viable cell counts of S. mutans which remained adherent on the microtitre plate were calculated. The effects of the test materials on pH changes caused by S. mutans in the presence of sucrose were assessed using pH meter. Results: Crude extract of the whole apple showed no significant inhibitory effect on the growth of S. mutans while a significant inhibitory activity on the adherence of S. mutans was observed. Furthermore, the significant inhibitory effect on the pH drop was recorded, although pH remained below the critical value of 5.5 at all times. Conclusion: Crude whole apple extract has considerable anti-cariogenic effect on S. mutans and hence can be used as a natural alternative which can aid in the prevention of dental caries.
Keywords: Adhesion, antibacterial, crude whole apple extract, pH, Streptococcus mutans
|How to cite this article:|
Shetty V, Aswath NP, Hegde AM. Effect of crude apple extract on the cariogenic factors of Streptococcus mutans: An in vitro study. J Indian Soc Pedod Prev Dent 2018;36:135-41
|How to cite this URL:|
Shetty V, Aswath NP, Hegde AM. Effect of crude apple extract on the cariogenic factors of Streptococcus mutans: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2018 [cited 2019 Dec 9];36:135-41. Available from: http://www.jisppd.com/text.asp?2018/36/2/135/235689
| Introduction|| |
Dental caries and diseases of the periodontium are among the most common afflictions of oral diseases of humanity. Studies have shown that 57.2% of children in 6 years of age group are affected by dental caries indicating a high prevalence of disease in children.
Among the members of the oral bacterial flora, Streptococcus mutans which belong to streptococci of the mutans group has been confirmed to be a highly cariogenic pathogen in humans. The critical factors responsible for the cariogenicity of this pathogen include its ability to adhere to tooth surfaces, to catabolize carbohydrates and generate acids and to survive at low pH and environmental stresses. The development of highly structured biofilms helps S. mutans to cope with fluctuating environments. A number of surface-associated proteins such as glucosyltransferases (GTFs) (Gtf-B,-C, and-D) help in adhesion and play an important role in the initiation of biofilm formation by S. mutans.
In the current era of preventive dental approach, reduction or inhibition of survival of this pathogenic organism can be one of the best options in combating oral disease. Unfortunately, the economic burden associated with dental diseases is very high and need for care is highest among the poor and underprivileged population. Hence, the deployment of natural substances in dentistry is gaining momentum. Also since the use of commercial antibacterial agents may be restricted by the resistance of microorganisms and associated side effects, great importance is given to natural alternatives for the prevention and decrease of growth and adhesion of microorganisms making the use of natural substitutes the best option to help in outreaching the dental care to all. So far, research has been very active to detect new natural, generally recognized as safe compounds for the successful development of alternative approaches with one goal: To reduce or prevent caries.
Over the past few decades, many studies have recognized polyphenols as the active constituent responsible for the anticariogenic property of plant-derived food especially fruits.,,,,
Among various fruits apples are one of the most popular fruits in the world. They are rich in antioxidant, phytonutrients, flavonoids, and polyphenolics. Studies show that the antioxidant and anti-inflammatory qualities of apple polyphenols have a wide range of health benefits, including protection of cells against oxidative damage that could cause cancer. However, little is known about the daily intake of these fruit polyphenols or their impact on dental health.
Thus, our study evaluated the effect of crude extract of whole apple on the growth of S. mutans, its effect on adherence of S. mutans to tooth surfaces and also evaluated the pH changes caused by acid produced by S. mutans.
| Materials and Methodology|| |
Preparation of crude whole apple extract
Kashmiri Apples (sourced from the local market), transported to the laboratory were washed using 70% ethyl alcohol followed by distilled water, dried, and cut manually with a sterile knife into small pieces. Whole fruit extracts including the apple skin were obtained using a sterile kitchen-type juicer to obtain undiluted crude apple extract.
Inhibition of Streptococcus mutans
Agar well-diffusion test
A standard strain of S. mutans (MTCC 890) maintained by the Department of Microbiology, K. S. Hegde Medical Academy was used. A suspension of S. mutans was freshly prepared by inoculating the S. mutans strain to Tryptone Soy Broth for 4–6 h and adjusting the turbidity to 0.5 McFarland. This culture was then placed in the centre of brain heart agar plates and spread with the help of a spreader. Wells were punched and 50 μl of the crude apple extract was placed in different petri plates and incubated at 37°C for 24 h. This test was done in triplicate. We, however, observed the absence of any zones of inhibition around any of the wells containing the apple crude extract even after repeating the procedure six times. Therefore, the direct contact test (DCT) was done to further confirm these results.
Direct contact test
A 24 h freshly prepared culture of S. mutans was taken in 5 ml of Tryptone Soy Broth, centrifuged and the supernatant was discarded. The pellet was washed with buffered saline and again centrifuged. The supernatant was again discarded, and the pellet of microorganisms was used for the test. To 200 μl of 100% concentrate of crude apple extract, 20 μl of the inoculums was added in a microwell titer plate. The tests were repeated six times. The plates were incubated for 24 h aerobically at 37°C. After incubation, the loop full of apple extract with the inoculated S. mutans from the microtiter wells were streaked on brain heart agar plates. S. mutans inoculums without exposure to the apple extract were used as the control. The growth of the organism in the control was compared to the growth of the organism exposed to the apple extract.
Effect on adherence of Streptococcus mutans
Isolates of S. mutans strain cultured on a agar plate was inoculated in Tryptone Soy Broth of 3–5 ml for 24 h and incubated at 37°C. The culture was then diluted in the ratio 1:100 using saline. 100 μl of each diluted culture is pipetted into each of four wells in a fresh microtiter plate which has not been tissue culture treated. Crude whole apple extract was then added to the microtiter plate (study group), and S. mutans inoculums without exposure to the apple extract were used as the control. The plates were then covered and incubated for 48 h at 37°C.
After 48 h, the wells were washed six times by adding 100 μl sterile phosphate buffered saline (PBS) per well and then vigorously shaking out the liquid over a waste container to remove planktonic cells. Scissor was used to cut each individual well from the microtiter plate and 100 μl PBS is added to each well. This content is then added to a separate 8-ml tube containing 1.9 ml PBS (for a final liquid volume of 2 ml).
This step is performed so that each sample can be sonicated in an 8-ml tube. Microtiter wells are not suitable for use as sonication vessels, as they are too small. Sonicator microtip was then inserted to sonicate the contents of each tube for 8 s at 40% power. Viable counts of cells were then performed (Phelan, 1996) by plating the resultant suspension on agar medium to enumerate bacteria that were attached to the microtiter well surface. The microbial counts were recorded as colony forming units per ml. The CFU scores were logarithmically (base 10) transformed, and all analyses were performed with the transformed scores.
Effects on pH
The pH changes were checked for three groups simultaneously. Group A (positive control) contained 5 ml of only the crude apple extract, Group B (negative control) contained 5 ml of Tryptone Soy Broth and sucrose inoculated with S. mutans, and Group C (study group) contained 5 ml of crude apple extract with S. mutans and sucrose. At first, the baseline pH was first noted for all the three groups without adding the S. mutans, then 2 ml of seed culture of S. mutans was added to Group B and Group C. Change in the pH values was noted thereafter at 1, 10, 20, and 30 min, at 37°C. The data collected was entered into Microsoft excel spreadsheet and analyzed using IBM SPSS Statistics, Version 22 (Armonk, NY: IBM Corp). Descriptive data were presented in the form of mean, median, standard deviation and quartiles. P value <0.05 was considered as statistically significant.
| Results|| |
When inhibition of growth Of S. mutans by direct contact inhibition test was carried out using Mann–Whitney U-test no significant reduction in the CFU/ml was observed [Table 1]. However, when the effect of crude whole apple extract on adherence of S. mutans was calculated using Mann–Whitney U-test a significant reduction in the number of S. mutans was seen [Table 2].
|Table 1: Comparison of growth of Streptococcus mutans in the control plates and the plates containing crude apple extract|
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|Table 2: Effects of crude apple extract on adherence of Streptococcus mutans|
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The pH changes caused due to the acid produced by S. mutans in Group A (crude apple extract only), Group B (S. mutans with sucrose), and Group C (crude apple extract with S. mutans and sucrose), at 1, 10, 20, and 30 min, respectively, was calculated using Friedman test followed by Wilcoxon sign rank test showed that Group A (crude apple extract only) had a base line acidic pH; however, the pH raised with time but remained below critical pH at the end of 30 min [Table 3]. pH changes in Group B (S. mutans with sucrose) showed a gradual drop with time ending at a ph of 5.15 at 30 min which is below the critical pH [Table 4]. In Group C (crude apple extract with S. mutans and sucrose), there was an initial drop in the pH which later had a steady raise. However, the pH was seen to remain well below the critical pH value of about 5.5 at all time [Table 5].
|Table 3: Comparison of pH at different time intervals in Group A (crude apple extract only)|
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Comparison of the changes in the pH values from baseline to 30 min between the groups was compared using Mann–Whitney U-test showed that there was no statistically significant differences between Group A (containing only crude apple extract) and Group C (containing crude apple extract with S. mutans and sucrose). However, statistically significant differences were seen when changes in the pH values obtained in Group C (containing crude apple extract with S. mutans and sucrose) at different time intervals were compared to corresponding pH values obtained in Group B (containing S. mutans and sucrose) [Table 6].
|Table 6: Comparison of change in pH at different and corresponding time intervals in all the groups|
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| Discussion|| |
The plaque biofilm which subjects the teeth and gingival tissues to high concentrations of microbial metabolites has been implicated as an important etiological factor in dental caries. Studies have revealed that S. mutans average from 20% to 40% of the cultivable flora in biofilms removed from carious lesions.S. mutans has unique ability to adhere to tooth surfaces, to catabolize carbohydrates and generate acids and to survive at low pH and environmental stresses which makes it highly cariogenic and pathogenic to oral environment.
The recent era of preventive approach to treat dental caries with the better understanding of participation of the microorganism in oral diseases has prompted an interest in attaining more knowledge toward pathological oral microflora and its susceptibility to antimicrobial agents.
Natural products have been used in many parts of the world to treat various infectious diseases. Many herbal remedies have been used for their anti-inflammatory, anti-bacterial, cytostatic, anti-fungal, and anti-viral activity. The WHO states that almost 80% of the population in the developing countries use traditional medicine to treat a lot of diseases.
Owing to the evidence that diet components not only have a negative influence, as in the case of sucrose, but also a positive one on oral health, as in the case of fluoride, xylitol, and fiber-rich foods, recent research has been directed toward foods that could be protective against oral pathologies and to food components that are able to inhibit oral streptococci growth and/or their virulence traits.
The major constituent which is responsible for the anticariogenic property of plant-derived food especially fruits is polyphenols. The effects of polyphenols have been surveyed through both in vitro studies and in vivo studies in animals and humans investigating its effect against mutans streptococci.
Apple is not only most commonly consumed fruit by the population but also ranked the second for the total concentration of phenolic compounds; its pulp contains catechin, procyanidin, caffeic acid, and chlorogenic acid among other components. The skin contains all the aforementioned substances as well as flavonoids, not present in pulp, such as quercetin glycosides and cyanidin glycosides.
Most importantly, apples have the highest portion of free phenolics when compared to other fruits. This means that these compounds are not bound to other compounds in the fruit, and may be more available for eventual absorption hence increasing the beneficial effect of polyphenols compared to other fruits.
However, most researchers have studied the effect polyphenols extracted from the apple on the activity of S. mutans. However, in our study, we have used the whole apple as several researchers have reported that the total phenolic and flavnoid content of apple peel was superior than pulp fractions.
Agar diffusion test is a very common test done to evaluate the antimicrobial effect on certain organisms. Here, antimicrobial components to be tested diffuse through the agar medium containing the organism and the width of the zone of inhibition was read. Hence, this method was chosen to evaluate the antimicrobial effect of crude whole apple extract on S. mutans. The results of our study revealed that crude whole apple extract did not show any inhibitory activity (as evidenced by the absence of zones of inhibition) when subjected to the agar diffusion test. The absence of diffusion areas around the test materials suggested that the active components were not diffusible in the agar, which could be attributed to their larger particle size or there was no antibacterial effect of apple on S. mutans. Therefore, we decided to use the DCT to further confirm the absence of antimicrobial activity.
The DCT, used by Gomes et al., 2004, and Anumula et al., 2012, is a valuable in vitro assay to study antimicrobial properties of solid materials. It enables direct contact between the microorganisms and the whole apple extract and thereby measures the effect of the extract on microbial growth.
In our study, we observed no significant differences in the microbial growth between control group and study group with apple extract (P > 0.05). This demonstrated that crude extract of the whole apple had no inhibitory effect on the growth of S. mutans.
The result obtained was consistent with a study done by Yanagida et al. who investigated the inhibitory effects of apple polyphenols and related compounds on the synthesis of water-insoluble glucans and on the growth of mutans streptococci. In spite of using the Apple Polyphenols (APP) extract which presumably has a smaller particle size, it was observed that there was no substantial effect on the growth of Streptococcus sobrinus 671 or S. mutans MT8148 on TTY-agar plates under aerobic conditions and no substantial effects on the growth of these bacteria on Mitis-Salivarius agar under anaerobic conditions also. Most studies have used extracted polyphenols (APP). However, in our study, crude apple extract was used both of which showed no antibacterial effect on S. mutans.
However, a study conducted by Abhay and Dinnimath tested the effect of alcoholic extract of apple peel on Staphylococcus aureus, Enterococcus faecalis, S. mutans, and Porphyromonas gingivalis. They found good antimicrobial activity against S. mutans with a MIC of 62.5 mg/ml.
This difference in results might be attributed to the type of apple extract used. There might be better solubility of apple compound in alcoholic extract, or maybe the alcohol in itself may alter the effect.
Another most important virulent property of S. mutans is their ability to adhere and maintain themselves on selected host tissues. The mechanism of adherence of S. mutans on the tooth is by the production of extracellular glucans from sucrose through the action of GTFs enzyme.
In our study, we evaluated the effect of apple crude extract on adherence property of S. mutans using a method described by Merritt et al. The number of S. mutans cells adhering after exposure to apple extract were calculated by direct enumeration of S. mutans on agar plate and counting the viable cells. A significant decrease in the viable cell count of S. muatans was observed in our study after exposure to crude apple extract (P < 0.05). The process by which apple polyphenols inhibit the adherence of S. mutans was studied by Yanagida et al. who demonstrated that apple polyphenols have inhibitory effect on synthesis of water-insoluble glucans by GTF of S. mutans and on the sucrose-dependent adherence of the bacterial cells. We found no other study had demonstrated the effect of crude apple extract on the adherence property of S. mutans.
Dental caries is caused by oral microorganisms through metabolizing carbohydrate, producing acid, and demineralizing the tooth surface. Acidogenicity is an important cariogenic property of S. mutans. In the presence of fermentable carbohydrates S. mutans utilizes the glycolytic pathway and produce lactate, formate, acetate, and ethanol as fermentation products. Thus, the reduction of acid production is a logical approach to prevent dental caries. Therefore, in our study, the effect of crude extract of the whole apple was evaluated for its effect on the pH changes brought about by S. mutans in the presence of sucrose, a fermentable sugar was studied.
In our study, Group B (containing S. mutans and the broth) had a baseline median pH of 7.2. On introduction of fermentable sugar, sucrose the pH gradually reduced to 5.15 at the end of 30 min. This reduction in pH below the critical pH follows the well-established Stephens curve  which demonstrates the decrease in the pH of dental plaque response to its exposure to fermentable carbohydrate. The decrease in the pH was rapid, reaching a minimum of 5.15 approximately at the end of 30 min.
Apple is an acidic fruit, at an average, the pH of crude apple extract was found to be between 3 and 4. In our study, we found the baseline pH of crude apple extract (Group A) to be 4.27. We observed a significant increase in the pH of crude apple extract from a baseline pH of 4.27–4.75 at the end of 30 min (P < 0.05). However, at all intervals, the pH remained always below the critical pH of 5.5.
This is in accordance with a study done by Babsky et al. wherein he pointed out that the slight increase in the pH might be partly due to copolymerization of organic acids with products of the browning reactions, Lewis et al. also suggested that organic acids can react with reducing sugars to produce brown pigments.
When S. mutans and sucrose were added to the crude apple extract (Group C), we recorded a baseline pH of 4.27. There was an initial drop in the pH to 4.22 after 1 min. Thereafter, there was a steady, significant increase in the pH up to the end of 30 min to 4.87 (P < 0.05). However, throughout our study, the pH remained below the critical pH of 5.5.
From our study, we deduce that crude whole apple extract did not allow further reduction in the pH even after the addition of S. mutans and sucrose; however, pH remained slightly below the critical pH of 5.5 at the end of 30 min. A study done by Suck-Young found that the polyphenols from unripe apple did not have effect on the acid production by S. mutans.
A study compared the effects of pH changes and trace elements of various fruit juices on enamel erosion showed that even though apple juice was acidic in pH, it did not show any erosive effect on enamel due to the fluoride and phosphorus trace elements present in them. Hence, apple juice was considered to be strongly cariostatic and was recommended for consumption.
The results of the present study showed no significant effect on the growth of S. mutans. However, we found strong inhibitory effect on the adherence property of S. mutans. We also observed that crude whole apple extract had the ability to significantly inhibit the drop in pH. However, the increase in the pH did not rise above the critical pH of about 5.5 which is significant in preventing demineralization of tooth surfaces. Hence, whether the extract can actually prevent enamel demineralization awaits further evaluation.
The anticariogenic effect of crude extract of whole apple demonstrated in our study reinforces the already known benefits of apple on general and oral health due to its considerable antioxidant capacity and high phenolic content, thus making this fruit even more healthier and giving credence to the popular statement “Apple-natures own toothbrush.”
However, further in vivo studies need to be conducted to access the activity of apple in the oral cavity. Furthermore, different types of extracts need to be tested both in vivo and in vitro which could be applied for future commercial use.
| Conclusion|| |
Results of the in vitro evaluation of crude extract of whole apple showed:
- No significant inhibitory effect on the growth of S. mutans
- A significant inhibitory activity on the adherence of S. mutans
- A significant inhibitory effect on the pH drop, although pH remained below the critical value of 5.5 at all times.
We conclude that crude whole apple extract had a considerable anticariogenic effect on S. mutans.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]