|Year : 2013 | Volume
| Issue : 2 | Page : 71-78
Role of probiotics on oral health: A randomized, double-blind, placebo-controlled study
Rajan Dhawan1, Shivani Dhawan2
1 Department of Conservative Dentistry, MM College of Dental Sciences and Research, Mullana, Ambala, Haryana, India
2 Department of Perioidontology, MM College of Dental Sciences and Research, Mullana, Ambala, Haryana, India
|Date of Web Publication||11-Feb-2014|
Department of Conservative Dentistry, MM College of Dental Sciences and Research, Mullana, Ambala, Haryana
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim: The aim of this study was to investigate commercially available combined probiotic formulation for its effect on plaque, gingivitis, and salivary Streptococcus mutans levels in subjects with chronic gingivitis. Materials and Methods: A total of 36 subjects were finally enrolled in a double-blind, placebo-controlled, randomized trial. Selected subjects were randomly divided into two groups: Group A (control group) with 17 subjects receiving placebo tablets twice daily and Group B (probiotic group) with 19 subjects receiving probiotic tablets twice daily. The study consisted of two 2-week periods: a 2-week intervention period (T1-T2) and a 2-week posttreatment period (T2-T3). Clinical parameters and bacterial counts of salivary Streptococcus. mutans were evaluated at baseline (T1), at the completion (T2) of medication, and 2 weeks after the medication (T3). Results: On comparative evaluation between the two groups, results indicated that Group B (probiotic group) exhibited statistically significant reduction in Plaque Index, Gingival Index, Calculus Index and Streptococcus. mutans level than Group A (control group) over the entire span of the study. Conclusion: Our results indicated that probiotic could be useful in the improvement/maintenance of oral health.
Clinical Relevance to Interdisciplinary Dentistry
- If left untreated, gingivitis can progress, increasing the risk of tooth loss. Oral administration of probiotics benefits oral health by preventing the growth of harmful microbiota or by modulating mucosal immunity in the oral cavity.
- In this study, probiotics were given orally for 2 weeks to subjects suffering from gingivitis and results showed improvement in oral health.
- Bacteriotherapy in the form of probiotics seems to be a natural way to maintain health and protect oral tissues from disease.
- Efforts have been made in this article to increase the awareness with this aspect of oral disease therapy and encourage the implementation of the concept of "food rather than medicine".
Keywords: Caries, gingivitis, plaque, probiotic, Streptococcus mutans
|How to cite this article:|
Dhawan R, Dhawan S. Role of probiotics on oral health: A randomized, double-blind, placebo-controlled study. J Interdiscip Dentistry 2013;3:71-8
|How to cite this URL:|
Dhawan R, Dhawan S. Role of probiotics on oral health: A randomized, double-blind, placebo-controlled study. J Interdiscip Dentistry [serial online] 2013 [cited 2020 Sep 18];3:71-8. Available from: http://www.jidonline.com/text.asp?2013/3/2/71/126862
| Introduction|| |
Dental plaque, a biofilm of microorganisms on the tooth surface, is an important etiological factor in the development of most common oral diseases - dental caries, gingivitis, and destructive periodontal diseases.  In dental practice, the control of oral inflammation typically involves the use of mechanical or chemical means to remove plaque. Despite the best efforts by dental professionals to sterilize the oral cavity, the oral cavity will inevitably repopulate with oral bacteria. If the unfavorable oral environment persists, so will the disease, and more therapeutic measures will be required in future. Little attention has been paid to the identification of health-associated and potentially beneficial bacterial species that may reside in the oral cavity. Bacteriotherapy in the form of probiotics seems to be a natural way to maintain health and protect oral tissues from disease. This article endeavors to introduce the concept of probiotic in the oral cavity.
The basic principle of probiotic is to use good bacteria to compete against pathogenic bacteria. The term probiotic, which literally means "for life," was first coined in the 1960s by Lilly and Stillwell.  According to the World Health Organization, probiotic bacteria are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host.  The interest in such an alternative way to combat infections is rapidly growing, especially in the light of the frequent use of antibiotics with a subsequent risk for development of resistant strains.
Bacterial endotoxins and antigens, together with some metabolic by-products produced by the pathogens in the dental biofilm, invariably lead to severe damage of periodontal tissue components.
Streptococcus mutans has been implicated as one of the major and most virulent of the caries-producing microorganisms. , It can colonize the tooth surface and initiate plaque formation by its ability to synthesize extracellular polysaccharides from sucrose, mainly water-insoluble glucan, using the enzyme glucosyltransferase.  Inhibiting the colonization of S. mutans on the tooth surface is believed to prevent the formation of dental plaque and development of dental caries.
The oral cavity has recently been suggested as a relevant target for probiotic applications. In the oral cavity, probiotics adhere to dental tissues as a part of biofilm, acting as a protective lining for oral tissues against oral diseases. Such a biofilm keeps bacterial pathogens off the oral tissues by filling a space which could have served as niche for pathogens in future and competing with the cariogenic bacteria and periodontal pathogens. 
To be able to exert probiotic properties in the oral cavity, however, it is essential for the microorganism to resist the oral environmental conditions, to be able to adhere to saliva-coated surfaces, to colonize and grow in the mouth, and to inhibit oral pathogens. The putative probiotic species also needs to be safe for the host. Kligler and Cohrssen (2008) summarize that significant adverse effects after the use of probiotics are rare. 
The most commonly used and studied probiotics are lactic acid bacteria, in particular Lactobacillus spp. and Bifidobacterium spp. 
To determine the effects of probiotics on oral health promotion, we conducted a double-blind, placebo-controlled, randomized clinical trial in healthy subjects with gingivitis. The specific aim of this study was to investigate the effect of combined probiotic formulation on plaque, gingivitis, calculus, and caries during and after discontinuation of administration.
The objectives of the study were to clinically evaluate the effect of probiotic on
- plaque accumulation
- gingival inflammation
- calculus accumulation, and
by estimating the level of S. mutans in saliva.
| Materials and Methods|| |
The study product (capsule Bifilac-Hp) contains Lactobacillus sporogenes 100 million, Streptococcus faecalis PC 60 million, Clostridium butyrium TO-A 4 million, and Bacillus mesentericus TO-A JPC 2 million. Combination of probiotic strains has been used that may act synergistically and enhance the possibility for permanent installation. This probiotic capsule was tested against a placebo tablet from the manufacturer that contained only microcrystalline cellulose, but was of identical taste, texture, and appearance. The dose was two tablets taken orally every day throughout the test period.
Forty students suffering with chronic gingivitis were recruited from National Dental College and Hospital, Derabassi, India. Their mean age was 21 years with uneven sex distribution (30 F, 10 M).
Subjects were outwardly healthy and further confirmed to meet the following criteria:
- Not currently visiting their dentist
- Not taken antibiotic within last month
- Not using probiotic supplements.
All subjects provided written informed consent, and the study was approved by the research ethics committee constituted by National Dental College and Hospital.
The study was performed as a double-blind, placebo-controlled, randomized trial over a 4-week test period. Subjects selected were divided into two groups, with 20 subjects in each group.
Group A: Control group
Group B: Probiotic group
Subjects in the control group consumed two placebo capsules every day. Those in the probiotic group consumed two capsules containing probiotic (Bifilac-Hp) every day. Subjects in each group were directed to place the capsule in their mouth and swallow it with water. They were also instructed not to change their oral hygiene regimens throughout the test period. Neither professional prophylaxis nor toothbrushing instructions were provided during or before the study period. The study consisted of two 2-week periods: A two-week intervention period (T1-T2) and a 2-week posttreatment period (T2-T3). Clinical parameters and bacterial counts of salivary S. mutans were evaluated at baseline (T1), at the completion (T2) of medication, and 2 weeks after the medication (T3) [Figure 1]. Four subjects were lost due to consumption of antibiotics during the follow-up period. Finally, 36 subjects (17 in the control group and 19 in the test group) were analyzed.
The gingival condition was assessed by gingival index (GI; Loe and Sillness 1963).  Supragingival plaque was scored by plaque index (PI; Sillness and Loe 1964). Both the indices were obtained from Ramford's six teeth (16, 21, 24, 36, 41, and 44 in the FDI two-digit notation system) and considered representative of the whole dentition.  When one of the selected teeth was missing in the oral cavity, parameters were obtained from the adjacent tooth in the same area of the jaw. Calculus surface index (CSI; Ennever, Sturzenberger, and Radike 1961) was used to assess the level of calculus.  This index was obtained by assessment of supragingival calculus on six mandibular anterior teeth.
After the clinical measurements, saliva samples were obtained. The subjects were asked not to eat anything for 1 h before the collection of sample. They were asked to chew sterile cotton swabs to stimulate saliva production. Two to three milliliters of saliva was collected in a sterile disposable container.
Within half an hour of sample collection, culture was done. Using 4-mm-diameter inoculation loop, 10 μl of sample was streaked on Mitis Salivarius Bacitracin (MSB) agar containing 1% potassium tellurite and 10% sucrose.  This medium contains selective agents - crystal violet, potassium tellurite, and bacitracin. These agents inhibit most gram-negative bacilli and most gram-positive bacteria except streptococci. Sucrose is incorporated in 10% concentration, which is utilized as the energy source by streptococci.
MSB plates were incubated in 5% CO 2 candle jar for 24 h at 37˚C. Following incubation, the colonies of S. mutans were identified by gram stain, culture morphology, and biochemical test.
On gram stain: S. mutans appears as gram-positive cocci arranged in short chains.
Biochemical tests: S. mutans is catalase negative and mannitol and sorbitol fermentation positive [Figure 2].
|Figure 2: Biochemical tests of salivary samples for confirmation of Streptococcus mutans. Test tubes showing dark pink as sorbitol and mannitol positive and light pink as sorbitol negative|
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On culture: S. mutans forms raised, convex, undulate, opaque, pale blue colonies with granular frosted glass appearance, sometimes exhibiting glistening bubble on the surface of colony due to excessive synthesis of glucan from sucrose [Figure 3] and [Figure 4].
|Figure 3: Showing growth of Streptococcus mutans on Mitis Salivarius Bacitracin agar|
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|Figure 4: Closer view of MSB agar: Showing convex, pale blue colonies with granular frosted glass appearance of Streptococcus mutans colonies|
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Colony count was done with a magnifying glass and the count of S. mutans was expressed as the number of colony-forming units per milliliter of saliva (CFU/ml).
Semi-quantitation of the number of colonies was done by multiplying the actual colony with 1 × 10 2 .
| Results|| |
All subjects tolerated the surgical procedures well and experienced no postoperative complications, although three subjects in the control group and one subject in the test group were withdrawn from the study during the follow-up period. These subjects took antibiotics during the study protocol and stopped the intake of tablets.
Clinical parameters and bacterial counts of salivary S. mutans were evaluated at baseline (T1), at the completion (T2) of medication, and 2 weeks after the medication (T3). The observations recorded were subjected to statistical analysis. No difference in results was found between male and female subjects.
The mean values of PI [Table 1], GI [Table 2], CSI [Table 3], and S. mutans levels [Table 4] at three points in time were evaluated. The efficacy of the two treatment modalities at T2 and T3 postoperatively was evaluated using paired Student's t-test because the observations at the two points in time were expected to be closely related to each other. The two groups, Group A (control group) and Group B (probiotic group), were then comparatively evaluated over the three time intervals using independent Student's t-test.
|Table 1: Plaque index of Group A (control group) and Group B (probiotic group) (in mm) |
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|Table 2: Gingival index of group A (control group) and Group B (probiotic group) (in mm) |
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|Table 3: Calculus surface index of Group A (control group) and Group B (probiotic group) (in mm) |
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|Table 4: Streptococcus mutans count of Group A (control group) and Group B (probiotic group) (in mm) |
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On analyzing the change in PI [Table 5] and GI [Table 6] of the two groups, it was seen that there had been significant reduction in PI in both the groups at all three points of time.
|Table 5: Decrease in plaque index of Group A (control group) and Group B (probiotic group) (in mm) |
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|Table 6: Decrease in gingival index of Group A (control group) and Group B (probiotic group) (in mm) |
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Regarding the change in CSI [Table 7], Group A (control group) showed significant reduction in CSI at T2 and T3, but nonsignificant reduction between T2 and T3. In Group B (probiotic group), there had been nonsignificant reduction at T2, and significant reduction at T3 and between T2 and T3.
|Table 7: Decrease in calculus surface index of Group A (control group) and Group B (probiotic group) (in mm) |
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The S. mutans level [Table 8] for both the groups was statistically significant at all the three points of time.
|Table 8: Decrease in S. mutans count of Group A (control group) and Group B (probiotic group) (in mm) |
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On comparative evaluation between the two groups, the results indicated that Group B (probiotic group) exhibited statistically significant reduction in PI [Table 9] and S. mutans level [Table 10] than Group A (control group) over the entire span of the study.
|Table 9: Comparison of mean plaque index scores between Group A (control group) and Group B (probiotic group) |
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|Table 10: Comparison of mean streptococcal mutans counts between Group A (control group) and Group B (probiotic group) |
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Group B (probiotic group) showed statistically highly significant reduction in GI [Table 11] and CSI [Table 12] at T2 and T3 than Group A.
|Table 11: Comparison of mean Gingival Index scores between Group A (control group) and Group B (probiotic group) |
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|Table 12: Comparison of mean calculus surface index scores between Group A (control group) and Group B (probiotic group) |
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| Discussion|| |
The paradigm that oral inflammation is the root cause of not only periodontal disease and caries but also cardiovascular disease, diabetes, arthritis, and many more conditions is rapidly emerging.
Treating the oral infection by reducing the number of pathogenic oral microorganisms and establishing a favorable oral environment to promote predominantly remineralization of tooth structure over time will stop the disease process. 
Probiotic technology represents a breakthrough approach to maintain oral health by utilizing the natural beneficial bacteria commonly found in healthy mouth to provide natural defense against those bacteria thought to be harmful to teeth and gums.
Probiotic may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of salivary pellicle. Probiotic bacteria could modify the protein composition of the pellicle by two different methods, namely, by binding to and by degradation of salivary proteins. 
Probiotics lower the pH so that microorganisms cannot form dental plaque and calculus that cause oral inflammation. Probiotics make an excellent maintenance product since they produce antioxidants. Antioxidants prevent stain and plaque formation by neutralizing the free electrons that are needed for mineral formation.
Polonskaya first described the phenomenon that probiotics such as Lactobacillus acidophilus ains may inhibit the in vitro growth of other bacteria. 
Lactobacilli can produce low-molecular-weight bacteriocins with an inhibitory effect against a wide range of bacterial species including oral streptococci, the causative agents of caries. ,
Fusiform nucleatum has been regarded as a chain microorganism in dental plaque formation, possessing the ability to co-aggregate with the majority of other bacteria in the oral cavity. Lactobacilli co-aggregate with F. nucleatum at nearly 90%, thus modulating the composition of oral biofilms. 
Lactobacilli had been shown to be the strongest inhibitors of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Porphyromonas intermedia, potent microorganisms responsible for periodontal destruction. 
Probiotic microorganisms not only act exclusively on the microbiota but also protect the oral cavity through the promotion of a beneficial host response. They exert effects either by modulating the immunological parameters, epithelial permeability, and bacterial translocation, or by providing bioactive or regulatory metabolites.  The latter effects are appealing for periodontal healthcare because current evidence shows that the destruction of the periodontium is substantially mediated by the host and driven by the bacterial challenge.  Therefore, probiotics might not only suppress the emergence of endogenous pathogens or prevent the superinfection with exogenous pathogens but also protect the oral cavity through the promotion of a beneficial host response. 
Probiotic bacteria or their products (e.g. metabolites, cell wall components, and DNA) can be recognized by host cells such as epithelial cells and immune cells.  Increased phagocytic capacity of macrophages when challenged with L. acidophilus and Lactobacillus casei reported. It is known that probiotics can regulate the expression of phagocytosis receptors in the neutrophils of healthy individuals and enhance natural killer cell activity. ,
A more in-depth study of the molecular mechanisms has revealed that probiotic species could effectively reduce the levels of periodontal inflammation-associated molecules, such as prostaglandin E 2 and interferon-γ, and weaken matrix metalloproteinase activities in saliva. 
In the present study, the beneficial impact of probiotic bacteria is well established by statistically significant reduction observed in PI and the level of S. mutans in Group B (probiotic group) as compared to Group A (control group). , The observed improvement in clinical status may be attributed to the reduced levels of cariogenic as well as periodontal pathogens and effective colonization of the probiotic bacteria within the oral cavity. Significant reduction was also observed in GI and CSI in Group B (probiotic group) as compared to Group A (control group) at T2 and T3. Nonsignificant change between T2 and T3 signifies that improvement in gingival condition tends to remain stable after cessation of probiotic intake. Residence time of probiotics in the oral cavity after treatment withdrawal is not yet known. The results do not suggest that a permanent installation can take place in persons with established microflora. , But the mechanism of action of probiotics suggests that they do not permanently colonize their host; even repeated daily use of probiotic over a long period of time will support its increased level in the oral cavity. Since it seems unlikely that probiotics have any significant residual effect after discontinuation of intake, daily intake seems to be a prerequisite for potential action. ,
| Conclusions|| |
Probiotics used for the management of oral disease may reduce the cost of conventional therapy and prevention programs. The idea of replacing harmful microorganisms with non-harmful, inactivated, or genetically modified bacteria is attractive. With the focus on disease prevention and optimal health for all ages, the potential for probiotics' use is enormous. Efforts should be made to increase the awareness of the general dental practitioners about this aspect of oral disease therapy and encourage the implementation of the concept of "food rather than medicine." The existence of probiotics in the indigenous oral microflora of humans warrants exploration because these bacteria offer the advantage of being perfectly adapted to the oral ecosystem. Much more scientific developments are needed to have a better understanding of these tiny forms of lives in order to broaden their potential applications.
| References|| |
|1.||Rupesh S, Winnier JJ, Nayak UA, Rao AP, Reddy NV. Comparative evaluation of the effects of an alum containing mouthrinse and a saturated saline rinse on the salivary levels of Streptococcus mutans. J Indian Soc Pedod Prev Dent 2010;28:138-44. |
|2.||Senok AC, Ismaeel AY, Botta GA. Probiotics: Facts and myths. Clin Microbial Infect 2005;11:958-66. |
|3.||Food and Agriculture organization and World Health Organisation Expert Consultation. Evaluation of health and nutritional properties of powder milk and live lactic acid bacteria. 2001, Available from: http//www.who.int/food safety/fsmanagement/en/probioticguideline.Pdf [Last accessed date 24 Oct, 2012]. |
|4.||Bowden GH. Which bacteria are cariogenic in man in markers of high and low risk groups and individuals for Dental Caries. In: Johnson NW, Editor. Cambridge Univ Press; 1991. p. 266-86. |
|5.||Loesche WJ, Rowan J, Straffon HL, Loos PJ. Association of Streptococcus mutans with human dental decay. Infect Immun 1975;11:1252-60. |
|6.||Selvan AK, Singh RC, Prabhu T. Antibacterial activity of bee propolis against clinical strains of streptococcus mutans and synergism with chlorhexidine. Int J Pharm Stud Res 2011;2:85-90. |
|7.||Cornelli EM, Guggenheim B, Stingele F, Neeser JR. Selection of dairy bacterial strains as probiotics for oral health. Eur J Oral Sci 2002;110:218-24. |
|8.||Kligler B, Cohrssen A. Probiotics. Am Fam Physician 2008;78:1073-8. |
|9.||Shimauchi H, Mayanagi G, Nakaya S, Minamibuchi M, Yto Y, Yamaki K, et al. Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: A randomized doubleblind, placebo-controlled study. J Clin Periodontol 2008;35:897-905. |
|10.||Ramford SP. Indices for prevalence and incidence of periodontal disease. J Periodontol 1959;30:51-9. |
|11.||Silness J, Loe H. Periodontal disease in pregnancy. II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;24:747-59. |
|12.||Soben P. Essentials of Preventive and Community Dentistry, 3 rd ed, Arya Publishing House, New Delhi 2006. p. 144-5. |
|13.||Gold OG, Jordan HV, Van Houte J. A selective medium for Streptococcus mutans. Arch Oral Microbiol 1973;18:1357-64. |
|14.||Haukioja A, Loimaranta V, Tenovuo J. Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro. Oral Microbial Immunol 2008;23:336-43. |
|15.||Polonskaya MS. Antibiotic from acidophilus. Mikrobiologiia 1952;21:303-10. |
|16.||Ishihara K, Miyakawa H, Takazoe I, Kawai Y. Growth inhibition of streptococcus mutans by cellular extracts of human intestinal lactic acid bacteria. Infect Immun 1985;49:692-4. |
|17.||Silva M, Jacobus NV, Deneke C, Gorbach SL. Antimicrobial substance from a human Lactobacillus strain. Antimicrob Agents Chemother 1987;31:1231-3. |
|18.||Stamatova I, Karo K, Meurman JH. In vitro evaluation of antimicrobial activity of putative probiotic lactobacilli against oral pathogens. Int J Probiotics Prebiotics 2008;2;225-32. |
|19.||Koll-Klais P, Mandar R, Leiber E, Marcotte H, Hammarstrom L, Mikelsaar M. Oral Lactobacilli in chronic Periodontitis and periodontal health: Species composition and antimicrobial activity. Oral Microbiol Immunol 2005;20:354-61. |
|20.||Riccia DN, Bizzini F, Perilli MG, Polimeni A, Trinchieri V, Amicosante G, et al. Anti-inflammatory effects of L. brevis (DC2) on periodontal disease. Oral Dis 2007;13:376-85. |
|21.||de Vrese, M, Schrezenmeir J. Probiotics, Prebiotics And Synbiotics. Adv Biochem Eng Biotechnol 2008;111:1-66. |
|22.||Sanz M, Quirynen M. Advances in the aetiology of periodontitis. J Clin Periodontol 2005;32 Suppl 6:54-6. |
|23.||Roberts FA, Darveau RP. Beneficial bacteria of the periodontium. Periodontol 2000 2002;30:40-50. |
|24.||Oelschlaeger TA. Mechanisms of probiotic actions - A review. Int J Med Microbiol 2010;300:57-62. |
|25.||Pelto L, Isolauri E, Lilius EM, Nuutila J, Salminen S. Probiotic bacteria down-regulate the milk-induced inflammatory response in milk-hypersensitive subjects but have an immunostimulatory effect in healthy subjects. Clin Exp Allergy 1998;28:1474-9. |
|26.||Takeda K, Suzuki T, Shimada SI, Shida K, Nanno M, Okumura K. Interleukin-112 is involved in the enhancement of human naturalkiller cell activity by Lactobacillus casei Shirota. Clin Exp Immunol 2006;146:109-15. |
|27.||Caglar E, Kavaloglu S, Eurgeneli S, Sandalli N, Twetman S. Salivary mutans streptococci and lactobacilli levels after ingestion of the probiotic bacterium Lactobacillus reuteri ATCC 55730 by straws and tablets. Acta Odontol Scand 2006;64:314-8. |
|28.||Caglar E, Kavaloglu SC, Kuscu OO, Sandalli N, Holgerson PL, Twetman S. Effect of chewing gums containing xylitol and probiotic bacteria on salivary mutans streptococci and lactobacilli. Clin Oral Investig 2007;11:425-9. |
|29.||Meurman JH, Antila H, Salminen S. Recovery of Lactobacillus strain GG (ATCC 53103) from saliva of healthy volunteers after consumption of yoghurt prepared with the bacterium. Microb Ecol Health Dis 1994;7:295-8. |
|30.||Meurman JH. Probiotics: Do they have a role in oral medicine and dentistry? Eur J Oral Sci 2005;113:188-96. |
|31.||Nikawa H, Makihira S, Fukushima H, Nishimura H, Ozaki Y, Ishida K. Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci. Int J Food Microbiol 2004;95:219-23. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]