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ORIGINAL ARTICLE
Year : 2014  |  Volume : 32  |  Issue : 3  |  Page : 212-219
 

Evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: A pilot study


1 Department of Pedodontics and Preventive Dentistry, Pushpagiri College of Dental Sciences, Perumthuruthy, Kerala, India
2 Department of Pedodontics and Preventive Dentistry, Dr. Dnyandeo Yashwantrao Patil Dental College and Hospital, Navi Mumbai, Maharashtra, India
3 Department of Pedodontics and Preventive Dentistry, Jaipur Dental College, Jaipur, Rajasthan, India
4 Department of Pedodontics and Preventive Dentistry, Rajah Muthiah Dental College and Hospital, Chidambaram, Tamil Nadu, India

Date of Web Publication2-Jul-2014

Correspondence Address:
S Rupesh
Vaikunt, Palace Ward, Alappuzha, Kerala - 688 011
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-4388.135827

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   Abstract 

Background: There has been much debate in the past about whether honey is harmful to the teeth, mostly as part of the debate about raw sugar versus refined sugar and the results have been equivocal. However, what has not been taken into account is that honey varies markedly in the potency of its antibacterial activity. Manuka (Leptospermum scoparium) honey from New Zealand has been found to have substantial levels of non-peroxide antibacterial activity associated with an unidentified phytochemical component, denoted as Unique Manuka Factor (UMF). Aims: Considering the potential antimicrobial effects of manuka honey, the present study attempted to investigate effects of twice daily use of manuka honey with UMF 19.5 on salivary levels of Mutans streptococci in children. Study Design: The investigation was a stratified comparison of two parallel groups of children who either used manuka honey with regular tooth brushing regimen or continued only with regular tooth brushing regimen twice daily under professional supervision for a 21-day period. A total of three salivary samples were taken from each individual at baseline, day 10, and day 21; colony counts of Streptococcus mutans (S. mutans) were determined. All data was subjected to paired T-test and Wilcoxon's signed ranks sum for intra- and intergroup comparisons respectively. Results: Children using manuka honey showed statistically significant reductions in salivary S. mutans after 10 and 21 days. Conclusion: Manuka honey with UMF 19.5 may be considered as an effective adjunctive oral hygiene measure for reducing colony counts in children.


Keywords: Antimicrobial, manuka honey, mutans streptococci, UMF 19.5


How to cite this article:
Rupesh S, Winnier J J, Nayak U A, Rao A P, Reddy N V, Peter J. Evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: A pilot study. J Indian Soc Pedod Prev Dent 2014;32:212-9

How to cite this URL:
Rupesh S, Winnier J J, Nayak U A, Rao A P, Reddy N V, Peter J. Evaluation of the effects of manuka honey on salivary levels of mutans streptococci in children: A pilot study. J Indian Soc Pedod Prev Dent [serial online] 2014 [cited 2017 Jul 25];32:212-9. Available from: http://www.jisppd.com/text.asp?2014/32/3/212/135827



   Introduction Top


Although very well-known as food, honey is not well-recognized as a medicine. Nevertheless, it is one of the oldest medicines known. [1] More recently, honey has been reported to have an inhibitory effect on around 60 species of bacteria including aerobes and anaerobes, Gram positives, and Gram negatives. An antifungal action has also been observed in some yeasts and some species of Aspergillus and Penicillium, as well as all the common dermatophytes. [2]

There has been much debate in the past about whether honey is harmful to the teeth, mostly as part of the debate about raw sugar versus refined sugar. With honey having a high content of fermentable sugars, it could be expected that honey would be cariogenic. Various researchers in the past have found honey to be equally cariogenic as sucrose while some found it to be more cariogenic. [3],[4],[5],[6] Occasionally, the recognition that honey has antibacterial activity has come into this debate, but whenever the antibacterial effect has been investigated, the results have been equivocal. Again, what has not been taken into account is that honey varies markedly in the potency of its antibacterial activity. [7]

Manuka (Leptospermum scoparium) honey from New Zealand has been found to have substantial levels of non-peroxide antibacterial activity (absent in other commonly used table honey) associated with its bioactive phytochemical components. [8] However, not all manuka honey has this non-peroxide antibacterial activity. An easily understood activity rating system-the Unique Manuka Factor (UMF) rating-was devised by the Waikato University's Honey Research Foundation Unit, which is used on the labels of manuka honey. The numbers in the UMF rating indicate the concentration of phenol with the same antibacterial activity as the honey. [7]

Willix et al., studied the sensitivity of wound infecting species to the antibacterial activity of manuka honey and other honey and found that manuka honey inhibited growth of most of the oral microorganisms at concentration of 1.8% (v/v), while other honey caused inhibition at concentrations below 11% (v/v). [9] Thus, the identification of honey with high levels of antibacterial activity in laboratory studies has put new light on the possibility of manuka honey being likely to be non-cariogenic and hence beneficial to oral health. [7],[10]

The effects of the use of such honey may have important dental health implications in terms of prevention of dental caries, especially in children. There is a relative paucity of studies in the dental literature in this regard, Hence, the present study was carried out to determine whether manuka honey with known antibacterial activity rated UMF = 19.5 [Figure 1] and [Figure 2] would be effective in reducing salivary levels of mutans streptococci in children.
Figure 1: Manuka Honey UMF 19.5 used in the study

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Figure 2: Manuka Honey UMF 19.5 used in the study

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   Materials and Methods Top


Volunteers were recruited from a residential school in Chidambaram after a complete oral examination and review of any available medical records. Criteria for patient selection were:

  1. Subjects in the age group of 9-12 years.
  2. At least one decayed tooth present.
  3. Subjects adhering to twice daily tooth brushing routine (using toothbrush and non-fluoridated toothpaste) and practicing no other oral hygiene measures, either professional or home-based, other than the requisites of the research project.
  4. No history of antibiotic usage during the past one month.
  5. No orthodontic appliance worn.
  6. No abscess, draining sinus, cellulitis, or other conditions requiring emergency dental treatment.
  7. Participant co-operation and acceptance of the treatment regimen.
  8. No medical/hereditary condition or long term/recent/current regimen of medication that can affect salivary flow or necessitate diet modification.
  9. No history of adverse reactions to honey.


All school children in the age group of 9-12 years were examined using oral health survey forms, and 30 children were selected based on the selection criteria. Since it was a residential school for boys, female children could not be selected for the present study. The numbers of decayed, missing, and filled surfaces of primary and permanent teeth (dmfs/DMFS) for each child were recorded by a single trained examiner by using portable dental operatories and accepted methods of infection control, according to WHO diagnostic criteria for dental caries. [11] Verbal consent from children and signed consent forms from parents or guardians were obtained after the nature of the study was fully explained. Approval from the concerned institutional ethics committee was obtained for the study.

The allocation of 30 subjects resulted in two balanced groups of fifteen subjects each; Manuka Honey group (MH) and Control Group (CG) that were comparable in terms of gender, age, number of teeth, average dmft/DMFT, and dmfs/DMFS. Each group was constituted of three subgroups of five subjects each, based on the number of decayed coronal tooth surfaces (ds/DS scores):

Subgroup 1 (MH 1, CG 1):

Five subjects each with ds/DS score of 1-5

Subgroup 2 (MH 2, CG 2):

Five subjects each with ds/DS score of 6-10

Subgroup 3 (MH 3, CG 3):

Five subjects each with ds/DS score >11

Manuka honey group

Five milliliters manuka honey (UMF 19.5) loaded in a sterile 5-ml syringe (devoid of needle) was deposited on the coronal surfaces of all erupted teeth and the dorsum of the subject's tongue [Figure 3], [Figure 4], [Figure 5]. A trained assistant performed the procedure on all the subjects of this group twice daily-after breakfast in the morning and after dinner-for a 21-day period. After application, the subjects were instructed to hold the honey in the mouth and not swallow it for one minute; they were then asked to expectorate the accumulated saliva and honey at the end of one minute, and not to eat/drink/rinse for 30 minutes thereafter.
Figure 3: The Armamentarium used in the study

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Figure 4: Manuka Honey being deposited using a syringe

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Figure 5: Manuka Honey being deposited using a syringe

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Control group

The subjects of this group performed no treatment regimen other than their normal twice-daily tooth brushing routine.

Three clinical examinations-on day 2, day 10, and day 21-were incorporated into the study design wherein the subjects were questioned and an intra-oral examination was performed to detect any adverse, allergic, or unusual reactions such as desquamation or unpleasant taste. Clinical assessments were performed at the residential school by a single examiner using portable dental operatories and accepted methods of infection control. An assistant coded the study subjects from 1-30 before clinical examination and saliva collection by the examiner to ensure that at no time was the examiner aware of the group assignment of any subject. The data was later decoded at the end of the investigation.

Saliva samples were obtained from each individual initially prior to the start of the experiment to establish baseline mutans streptococci levels. Subsequent samples were obtained on day 10 and day 21, after the start of the experiment [Figure 6]. Samplings were performed unannounced in order to minimize participation effect. At each sampling, paraffin-stimulated whole saliva samples were collected in sterile bottles in the mid-morning with no eating/drinking for two hours prior to the sampling. The child was asked to chew a piece of paraffin wax for two minutes after which the child expectorated the accumulated saliva into the sterile bottle. No transport medium was used as culturing was done within half an hour of collection of samples. [12] The samples were then subjected to microbiological analysis.

Each saliva sample was vortexed vigorously for 30 seconds to ensure a representative mixture throughout the sample prior to the preparation of dilutions and plating. The media used in this study for culturing salivary mutans streptococci was Mitis Salivarius Bacitracin (MSB) agar. [13] 100 μl of the vortexed salivary sample was pipetted out using a standard 100 μl pipette and serial dilutions were prepared. From each of the dilutions, 100 μl was pipetted onto separate agar plates and evenly spread onto the agar surface using sterile spreaders [Figure 7] and [Figure 8]. The preparation of dilutions and agar plating was carried out within an innoculating hood. The plates were then incubated at 37°C for 48 hours under 5-10% CO 2 . To avoid bias, all plates were processed and examined by the same investigator who was unaware of the group assignment of the samples. Colonies of mutans streptococci were identified as round or spherical, raised, convex, black in color, ranging from a pinpoint to pinhead size with a rough surface [Figure 9] and [Figure 10]. The colony count of each plate was recorded and the mean Colony Forming Units (CFU/ml) was determined after multiplying the colony count of each plate with its respective dilution factor. [14]
Figure 6: Collection of saliva samples

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Figure 7: Depositing saliva sample onto MSB agar using micropipettes

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Figure 8: Spreading saliva onto MSB agar surface using sterile spreaders

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Figure 9: Baseline sample

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Figure 10: 21st day post treatment sample of Manuka Honey Group

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All data was entered into a database on Microsoft Excel and analyzed using SPSS software with two-way ANOVA (for overall group mean comparisons), Paired T-test (for intra group comparison of differences between baseline, day 10, and day 21 examinations for salivary mutans streptococci counts) and Wilcoxon's Signed Ranks Sum Test (for inter-group comparisons of salivary mutans streptococci counts).


   Results Top


The sample characteristics of the study population are presented in [Table 1]. The mean salivary mutans streptococci counts of both the groups and their subgroups at baseline, day 10, and day 21 are shown in [Table 2].

Comparison of the differences in salivary mutans streptococci levels between baseline and day 10, baseline and day 21, and day 10 and day 21 are presented in [Table 3].

The inter-group comparisons of the salivary mutans streptococci counts at baseline, day 10, and day 21 are presented in [Table 4].
Table 1: Sample characteristics of the study population

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Table 2: The mean and standard deviation values of salivary mutans streptococci counts (log values) at baseline, 10th day, and 21st day of all groups

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Table 3: Comparison of differences between baseline (BL), 10th day and 21st day examination of salivary mutans streptococci using paired T test for variables

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Table 4: Intergroup comparison of salivary mutants streptococci count at Baseline, 10th day and 21stth day using Wilcoxon signed ranks test

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   Discussion Top


The antibacterial property of honey was first recognized by Van Ketel in 1892. [15] Honey, like other saturated sugar syrups and sugar pastes, has an osmolarity sufficient to inhibit bacterial growth. It was earlier assumed that the antibacterial property is entirely due to the osmotic effect of its high sugar content. [16],[17]

Research has been carried out to determine the effect of honey on the growth of cultures of oral bacteria. One study based on seven species of oral streptococci found that the minimum inhibitory concentration of honey for Streptococcus oralis was 12%, for Streptococcus anginosus was 17%, and for Streptococcus gordonii, Streptococcus mutans, Streptococcus salivarius, Streptococcus sanguis, and Streptococcus sobrinus was 25%, which was the same as the minimum inhibitory concentration of sucrose. [18] Another study also found that the minimum inhibitory concentration of honey for Streptococcus mutans was 25%, and for Streptococcus sobrinus was 35%, which also indicated that there was little, if any, antibacterial action in the honey beyond the osmotic effect of its sugar content. The study did, however, find that the salivary bacterial count was reduced by 40% one hour after holding 5 ml of honey in the mouth for 4 minutes; however, this could have been due to a stimulation of flow of saliva washing away the bacteria. [19] Thus, while only few authors showed awareness of honey having antibacterial properties, none took into account the variance in potency of the antibacterial activity of honey, each study using a single unselected honey.

The fact that antibacterial properties of honey are increased when diluted was clearly observed and reported in 1919. [20] The explanation for this apparent paradox came from the finding that honey contains an enzyme that produces hydrogen peroxide when diluted. This agent was referred to as "inhibine" prior to its identification as hydrogen peroxide. [21]

Thus, it is now known that the antibacterial activity is primarily due to hydrogen peroxide formed in a slow-release manner by the enzyme glucose oxidase present in honey, which can vary widely in potency. However, in addition to these, active manuka honey contains non-peroxide antibacterial factors associated with its bioactive phytochemical components (UMF) and is the only honey available for sale (other than its Australian equivalent) that has been tested for its antibacterial activity. [7],[8] Manuka honey contains D glucono δ-lactone, which reduces its pH and exerts natural antibacterial properties rendering it shelf stable. Its low water activity (0.6-0.75) also renders it uninhabitable to most microbes. [22] Methylglyoxal, the aldehyde form of pyruvic acid, has been identified to be the chief antibacterial compound in manuka honey. Apart from the methylglyoxal content, methyl syringate, ortho-methoxyacetophenone, and 3-phenyl lactic acid have been identified as other abundant components in manuka honey. [23] Methylglyoxal is toxic towards pathogens even at low concentrations interrupting cell divisions, arresting growth, and specifically causing the degradation of bacterial DNA. [24] Thus, it can be expected that, although honey may be cariogenic because of its high content of fermentable sugars, with selected honey that have higher levels of antibacterial activity, there is the potential for harm to the teeth to be reduced by inhibition of the cariogenic bacteria. A recent pilot study by English et al. reported manuka honey with UMF 15 to be highly effective in reducing dental plaque and clinical levels of gingivitis. [10] The manuka honey used in the present study had antibacterial activity rated UMF 19.5.

The results of this clinical trial showed that all three MH subgroups exhibited significant reductions in salivary mutans streptococci count when baseline values were compared with post treatment values after day 10 and day 21. These results showed that there is a definite decrease in the salivary mutans streptococci levels even within 10 days of regular use of manuka honey irrespective of the baseline salivary mutans streptococci count. The control groups, however, did not show any significant change, when day 10 and day 21 values were compared with baseline values. An interesting observation of the present study was that the Control Group subgroup 3 (CG 3) demonstrated a significant reduction in bacterial counts from baseline to the day 10. But this trend did not seem to last till the end after 21 days. This initial decrease in the values in CG 3 may possibly be explained by the Hawthorne effect or Participation effect. The Hawthorne effect refers to changes in the behavior of subjects that occur solely as a function of participation in an experiment, seen in the initial stages of clinical trials. [25]

Intergroup comparisons revealed that there were no significant differences in salivary mutans streptococci levels between the MH group and the CG (and their respective subgroups) at baseline. This implies that both groups (and their respective subgroups) were statistically equivalent before the start of treatment. It was observed that, after 10 days and 21 days, all MG subgroups showed statistically significant differences over the CG subgroups. Thus, in the present study, the use of manuka honey emerged as an effective adjunctive oral hygiene measure in reducing colony counts.

Colonization of tooth surfaces by bacteria is an important etiological factor in most of the common oral diseases, namely dental caries, gingivitis, and destructive periodontal disease. [26] However, it has also been well-established that increased bacterial growth on the tongue is the reason for increased numbers of bacteria in the saliva. The oral surfaces are colonized by over 500 bacterial species and tongue has the largest bacterial load of any oral tissue and makes the greatest contribution to the bacteria found in saliva. [27] Hence, the dorsum of the tongue was also included in the treatment regimen in the MH group. The study subjects were required to keep the honey in the oral cavity for not more than one minute in order to rule out the possibility of salivary bacterial count reductions by stimulation of flow of saliva washing away the bacteria.

The present study was conducted in a residential school for boys; hence, female subjects could not be selected. However, epidemiological studies in caries prevalence have not shown any significant difference in the caries susceptibility of boys and girls at an average age. [28]

Since the present study was conducted in a residential school, all participants consumed the same diet during the period of investigation. Thus, as diet (an important factor in dental caries) was controlled in the present study, manuka honey was given the best chance of demonstrating its efficacy against Streptococcus mutans.

Again, age is a critical factor in subject selection for many reasons, of which the most important is the number of tooth surfaces at risk. Subjects with a mean age of approximately 11 years were chosen since they were entering a period of high caries activity with many permanent teeth erupting. [29]

Subjects with either rampant tooth decay (subgroup 3) or very minimal caries activity (subgroup 1) were included in the study, as it was important to see if the protocol remained effective for different baselines of salivary mutans streptococci.

For several decades, the principal cariogenic microorganisms were the lactobacilli that were the focus of caries studies. More recently, it was found that the DMF index is only weakly linked to salivary lactobacilli and is independent of plaque lactobacilli, that carious dentin is responsible for the salivary hyper-contamination with lactobacilli, and that the lactobacillus count could hardly be a predictive test. [30] Thus, lactobacilli have been replaced as the cariogenic microorganisms par preference and mutans streptococci have taken their place. Because of their relationship to the disease, the evaluation of mutans streptococci concentrations in saliva aids the diagnosis of caries activity. [12]

In the present study, sampling was done with no eating or drinking for two hours prior, as saliva samples collected immediately after food could interrupt the microbial levels of the oral environment. [31] Because the results of the tests performed on unstimulated saliva are less reliable than those performed on stimulated secretion, paraffin-stimulated whole saliva samples were collected. Chewing helps to wash out bacteria from tooth surfaces and mix them with the secretion. The stimulation of flow rate also minimizes intra-individual variations of secretion flow. [12]

Since, in contrast to most other bacteria, mutans streptococci can grow in an environment with a high sucrose concentration and are resistant to a particular antibiotic bacitracin, the most commonly used selective medium, MSB agar was used for incubation in the present study. [13] The technique adopted for agar plating and colony counting was similar to that suggested by Wan et al. [14]

Our study was a novel attempt designed to simulate a realistic home regimen in which the subjects used the honey for one minute twice daily while continuing their normal twice-daily tooth brushing routine. In this context, it is noteworthy that the reductions in salivary mutans streptococci in our study occurred in addition to the effects of daily tooth brushing. Since the study was completed without observing any indications of negative effects resulting from the use of manuka honey and was well accepted by the children who participated in the experiment, the honey appears safe for long term oral use. Furthermore, it may be considered worthy of further study for potential applications in the practice of preventive dentistry and dental hygiene.

Though the present study demonstrated the efficacy of manuka honey (with antibacterial activity rated UMF 19.5) in reducing the salivary mutans streptococci levels, the degree to which the inhibition of cariogenic bacteria occurs in the oral cavity would have to be determined by feeding experiments. Also, future research directed towards production of candy using honey with high antibacterial activity in place of sugar, may play a potential role in caries prevention, especially in children.


   Conclusion Top


The Manuka Honey subgroups (MH 1, MH 2, and MH 3) showed statistically significant reductions in salivary mutans streptococci levels on comparison of the data obtained at baseline, day 10, and day 21. The Control Group subgroups (CG 1, CG 2, and CG 3) did not exhibit any significant reduction in salivary mutans streptococci levels. Thus, in the present study, the use of manuka honey emerged as an effective adjunctive oral hygiene measure in reducing colony counts.


   Acknowledgement Top


Ms. Arularasi Aparna (Microbiologist, Rajah Muthiah Dental College & Hospital) and Dr. Oommen Aju Jacob (Former Principal, Pushpagiri College of Dental Sciences) are thanked for the expertise rendered in the microbiological procedures and for the invaluable suggestions during manuscript preparation respectively.

 
   References Top

1.Zumla A, Lulat A. Honey - a remedy rediscovered. J R Soc Med 1989;82:384-5.  Back to cited text no. 1
    
2.Molan PC. The antibacterial activity of honey. 1. The nature of the antibacterial activity. Bee World 1992;73:5-28.  Back to cited text no. 2
    
3.Rells GK, Nizel AE. Cariogenicity of honey. J Am Dent Assoc 1973;87:29.  Back to cited text no. 3
    
4.Shannon IL, Edmonds EJ, Madsen KO. Honey: Sugar content and cariogenicity. ASDC J Dent Child 1979;46:29-33.  Back to cited text no. 4
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5.Wakeman EJ, Smith JK, Zepplin M, Sarles WB, Phillips PH. Microorganisms associated with dental caries in the cotton rat. J Dent Res 1948;27:489-92.  Back to cited text no. 5
    
6.König KG. Caries induced in laboratory rats. Post-eruptive effect of sucrose and of bread of different degrees of refinement. Br Dent J 1967;123:585-9.  Back to cited text no. 6
    
7.Molan PC. The antibacterial activity of honey. 2. Variation in the potency of the antibacterial activity. Bee World 1992;73:59-76.  Back to cited text no. 7
    
8.Allen KL, Molan PC, Reid GM. A survey of antibacterial activity of some New Zealand honeys. J Pharm Pharmacol 1991;43:817-22.  Back to cited text no. 8
    
9.Willix DJ, Molan PC, Harfoot CG. A comparison of the sensitivity of wound-infecting species of bacteria to the antibacterial activity of manuka honey and other honey. J Appl Bacteriol 1992;73:388-94.  Back to cited text no. 9
    
10.English HK, Pack AR, Molan PC. The effects of manuka honey on plaque and gingivitis: A pilot study. J Int Acad Periodontol 2004;6:63-7.  Back to cited text no. 10
    
11.Ismail. Criteria for detection of dental caries. J Dent Res 2004;83:C60.  Back to cited text no. 11
    
12.Brambilla E, Garcia-Godoy F, Strohmenger L. Principles of diagnosis and treatment of high-caries-risk subjects. Dent Clin North Am 2000;44:507-40.  Back to cited text no. 12
    
13.Gold OG, Jordan HV, Van Houte J. A selective medium for Streptococcus mutans. Arch Oral Biol 1973;18:1357-64.  Back to cited text no. 13
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14.Wan AK, Seow WK, Walsh LJ, Bird PS. Comparison of five selective media for the growth and enumeration of Streptococcus mutans. Aust Dent J 2002;47:21-6.  Back to cited text no. 14
    
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17.Tovey FI. Honey and healing. J R Soc Med 1991;84:447.  Back to cited text no. 17
    
18.Basson NJ, du Toit IJ, Grobler SR. Antibacterial action of honey on oral streptococci. J Dent Assoc S Afr 1994;49:339-41.  Back to cited text no. 18
    
19.Steinberg D, Kaine G, Gedalia I. Antibacterial effect of propolis and honey on oral bacteria. Am J Dent 1996;9:236-9.  Back to cited text no. 19
    
20.Sackett WG. Honey as a carrier of intestinal diseases. Bull Colorado State Univ Agric Exp Stn 1919;252:1-18.  Back to cited text no. 20
    
21.White JW Jr, Subers MH, Schepartz AI. The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochem Biophys Acta 1963;73:57-70.  Back to cited text no. 21
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22.Oddo LP, Heard TA, Rodriguez-Malaver A, Perez RA, Fernandez-Muino M, Sancho MT, et al. Composition and antioxidant activity of Trigona cabonaria honey from Australia. J Med Food 2008;11:789-94.  Back to cited text no. 22
    
23.Daher S, Gulacar FO. Analysis of phenolic and other aromatic compounds in honey by solid-phase microextraction followed by gas chromatography-mass spectrometry. J Agric Food Chem 2008;56:5775-80.  Back to cited text no. 23
    
24.Bhandary S, Chaki S, Mukherjee S, Das S, Mukherjee S, Chaudhri K, et al. Degradation of bacterial DNA by a natural antimicrobial agent with the help of a biomimetic membrane system. Indian J Exp Biol 2012;50:491-6.  Back to cited text no. 24
    
25.Putt MS, Kleber CJ, Smith CE. Evaluation of an alum-containing mouthrinse in children for plaque and gingivitis inhibition during 4 weeks of supervised use. Pediatr Dent 1996;18:139-44.  Back to cited text no. 25
    
26.Axelsson P, Kristofferson K, Karlsson R, Bratthall D. A 30-month longitudinal study of the effects of some oral hygiene measures on Streptococcus mutans and approximal dental caries. J Dent Res 1987;66:761-5.  Back to cited text no. 26
    
27.Loeshe WJ, Kazor C. Microbiology and treatment of halitosis. Periodontol 2000. 2002;28:256-79.  Back to cited text no. 27
    
28.Finn SB. Clinical Pedodontics. 4 th ed. Philadelphia: W. B. Saunders Company; 1999. p. 454-74.  Back to cited text no. 28
    
29.Kleber CJ, Putt MS, Smith CE, Gish CW. Effect of supervised use of an alum mouthrinse on dental caries incidence in caries-susceptible children: A pilot study. ASDC J Dent Child 1996;63:393-402.  Back to cited text no. 29
    
30.Nancy J, Dorignac G. Lactobacilli from the dentin and saliva in children. J Clin Pediatr Dent 1992;16:107-11.  Back to cited text no. 30
    
31.Wyne AH, Guile EE. Caries activity indicators. A review. Indian J Dent Res 1993;4:39-46.  Back to cited text no. 31
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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