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ORIGINAL ARTICLE
Year : 2020  |  Volume : 18  |  Issue : 3  |  Page : 193-198

Probiotic potential of daily consumption of homemade curd on dental plaque among schoolchildren: A randomized controlled trial


Departments of Public Health Dentistry, KVG Dental College and Hospital, Sullia, Karnataka, India

Date of Submission10-Dec-2019
Date of Decision21-Jan-2020
Date of Acceptance01-Oct-2020
Date of Web Publication24-Oct-2020

Correspondence Address:
Varsha Manoharan
Department of Public Health Dentistry, KVG Dental College and Hospital, Sullia, Dakshina Kannada, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaphd.jiaphd_127_19

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  Abstract 


Background: Probiotics are live microorganisms having the potential to convert a dysbiotic bacterial environment into a healthy one. Aim: The aim of the study was to assess the probiotic potential of homemade curd on the quantity and quality of dental plaque among schoolchildren. Materials and Methods: This was a randomized controlled trial. A total of 30 schoolchildren were enrolled in this study, 15 participants in each group. The participants in the intervention group were provided with one cup of freshly prepared homemade curd for a period of 30 days. An evaluation was done at baseline, after 30 days, and after a washout period of 14 days which involved both quantitative and qualitative (Streptococcus mutans, Lactobacilli, and anaerobic bacterial counts in colony-forming units [CFUs]/ml) analysis of dental plaque. Results: A statistically significant difference was not observed for quantitative changes in mean plaque score among the interventional and control groups at various stages of evaluation. Qualitative analysis after 30 days yielded a significant reduction in the number of microbial CFUs of S. mutans ([208 (126.5, 268)] vs. [132 (94.5, 156.5)] P < 0.001), lactobacillus ([3 (1, 5)] vs. [2 (1, 2)] P < 0.001), and anaerobic bacteria ([232 (165, 251)] vs. [190 (127.5, 237.5)] P < 0.001) among the intervention group. Gradual recolonization of microorganism was observed following the cessation of curds. Conclusion: This study concludes that homemade curds have a probiotic effect on both aerobic and anaerobic organisms of dental plaque. However, statistically significant changes were not observed for quantitative analysis of dental plaque.

Keywords: Dental plaque, homemade curd, Lactobacillus acidophilus, probiotic potential, probiotics, Streptococcus mutans


How to cite this article:
Manoharan V, Fareed N, Battur H, Praveena J, Ishwar P. Probiotic potential of daily consumption of homemade curd on dental plaque among schoolchildren: A randomized controlled trial. J Indian Assoc Public Health Dent 2020;18:193-8

How to cite this URL:
Manoharan V, Fareed N, Battur H, Praveena J, Ishwar P. Probiotic potential of daily consumption of homemade curd on dental plaque among schoolchildren: A randomized controlled trial. J Indian Assoc Public Health Dent [serial online] 2020 [cited 2020 Nov 27];18:193-8. Available from: https://www.jiaphd.org/text.asp?2020/18/3/193/298998




  Introduction Top


Health is a priceless positive facet of life, maintenance of health for a productive life is of utmost importance. Oral health is an integral part of general health and can be achieved through meticulous oral hygiene. Oral hygiene maintenance is directly related to the control of plaque in the oral cavity.[1] Dental plaque is considered to be a primary etiologic agent for most of the common oral diseases. It is highly specific in nature and its specificity depends on the microorganisms that colonize it.[2] The bulk of the microorganisms that form the biofilm in dental plaque are Streptococcus mutans and other anaerobes and play a major role in the establishment of the early biofilm community though the precise composition varies by location in the mouth.[3]

The rationale for the prevention of oral diseases relies on reducing microbial load in the oral cavity. It has been proved that reductions in the numbers of microorganism in plaque will lead to caries reduction. The application of health-promoting bacteria for therapeutic purposes is one of the strongest emerging fields. Time has come to shift the paradigm of the treatment from specific bacteria elimination to alteration of the bacterial ecology by using probiotics. The Food and Agriculture Organization and the World Health Organization defined probiotics as “Live microorganisms which when administered in adequate amounts confer a health benefit on the host.” These “beneficial bacteria” present in curd and fermented milk products constitute the most important source of probiotics for humans.[1]

Till date, probiotic bacteria have been used as various formulations like milk, yoghurt, tablets, chewing gums, controlled releasing medical devices, and also as mouthrinses.[4] Such bacteria are Generally Regarded as Safe because they can reside in the human body causing no harm and on the other hand, they are the key microorganisms in milk fermentation and food preservation.[1] Curd being a common dietary constituent of the Indian population is readily available, inexpensive, and can be used in several different combinations that are acceptable to the pediatric age group. Daily consumption of this archetypical food seemed to be the most natural way to ingest probiotic bacteria.[5] The present study has thus been undertaken with an aim of assessing the quantitative and qualitative changes on dental plaque among schoolchildren following short-term consumption of probiotic homemade curd.


  Materials and Methods Top


A randomized, controlled trial was designed and conducted among schoolchildren of Sullia Taluk in Dakshina Kannada district of Karnataka. Ethical clearance was obtained from the Institutional Ethics Committee bearing a registration number IECKVGDCH/SS02/2018-19. This randomized controlled trial is registered at the Clinical Trials Registry – India, bearing a registration number CTRI/2018/12/016709. Permission and approval to conduct the study were obtained from the block education officer. Headmistresses and/headmasters of the included schools were approached, and their cooperation in conducting the study was solicited. Informed consent from the participating children was obtained from the parents through the headmasters/headmistresses.

The sample size was estimated according to the given formula with a mean difference in bacterial counts of 86. Five colony-forming units (CFUs) and standard deviation of 124.04 were obtained from a previous study.[5]



Where,

Z = 2.58 for 99% confidence interval

d = mean difference to be detected

(s)2 = variance.

The calculated sample size was 13.68 subjects per group, an additional 10% of the calculated sample size was added to compensate for sampling loss if any, thus the final sample size accounted for a total of 15 participants per each group, an equal number of participants satisfying the inclusion and exclusion criteria were drawn from another school served as an external control group.

Inclusion criteria

Intervention group

A total of 15 school children aged 12–13 years, with apparently good general health and informed consent, who were willing to abide by our suggestions during the period of study.

Control group

Fifteen schoolchildren selected from another school matched for age, gender, and other clinical parameters who could voluntarily cease the consumption of curds during the period of the study constitute the control group.

Exclusion criteria

Intervention group

Children with dentofacial abnormalities or developmental disorders, history of using any other commercially available probiotic products that may possibly affect the findings of the study and those with a known history of allergy to milk/dairy products.

Control group

Children with dentofacial abnormalities or developmental disorders that could preclude recording of included clinical parameters.

Randomization

A list of schools was obtained from the Block Education Office of Sullia Taluk. All schools were invited to participate in this study; 32 schools agreed to cooperate and participate in the study. One school was chosen as an intervention group and the other one as a control group using a convenient sampling method. The chief investigator and a recorder visited the school, performed the clinical examination. Out of the participants satisfying inclusion/exclusion criteria, 15 children were drawn using a random number table and constituted the final sample [Figure 1].
Figure 1: Schematic representation of the study design. *Curative treatments were given to the participants in both the groups as and when necessary

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Technique of standardization

Investigator calibration

Investigator was trained and calibrated in assessing the clinical parameters and collection of plaque samples. The kappa coefficient value for intra-examiner reliability was 0.89.

Curdling technique

Fifty milliliter of homemade curd with proven probiotic potential from collaborative research was stirred in a stainless steel bowl containing 4 l of pasteurized, boiled, cooled milk and left undisturbed for 12 h in a cool dry place to facilitate curdling.

Calibration of bacterial counts

Every fifth sample was repeated to check the consistency of bacterial counts.

Data collection

Data required for this study were collected on a specially designed structured pro forma comprising two main parts: Part A recorded demographic characteristics and Part B recorded clinical and microbiological parameters.

Study procedure

The study was conducted over a period of 6 weeks, started in the month of December 2018 during which the investigator herself visited the schools with due permission from the headmistress/headmaster and conducted the study according to the protocol, as shown in [Figure 1]. In the first visit, baseline data were obtained which included both quantitative and qualitative assessment of dental plaque. A quantitative assessment of plaque was done using the plaque index (Silness and Loe, 1964).[6] For a qualitative assessment, plaque samples were collected from the buccal surface of the maxillary first permanent molar of all the included participants. Plaque collection was done using sterile toothpicks. The samples were suspended in a previously labeled and coded vials containing 1 ml of reduced transport fluid media and were transported to the Department of Molecular Biology and Immunology Maratha Mandal's NGH Institute of Dental Sciences and Research Center, Belgaum, within 48 h.[7] During the intervention phase, an attendant measured and distributed 150 ml of freshly prepared homemade curd to each participant in the intervention group. They were provided with a stainless steel spoon and instructed to have it. Flavors or sweeteners were not added to them. The consumption was done after 12 h but within 18 h after stirring. Fifty milliliter of curd was preserved every day in a refrigerator for subsequent usage. Consumption was monitored daily by the investigator for a period of 30 days. Soon after the intervention period, the first evaluation was performed and further evaluation was done after a washout period of 14 days.

Statistical analysis

Statistical analysis was performed using IBM SPSS (Statistical Package for Social Sciences) Version 21.0, Chicago, IL.[8] Descriptive in the form of mean, standard deviation, median (with first and third quartiles) for CFUs of bacterial counts, and percentages are presented. Paired t-test and Wilcoxon's signed rank test were performed to compare the intragroup differences in the observed mean and median values. Independent t-test and Mann–Whitney U-test were performed to compare the intergroup differences in the observed mean and median values. P < 0.05 considered as statistically significant.


  Results Top


Analysis of demographic details at baseline revealed an almost similar gender representation among interventional and control groups with a male predominance, there was no statistically significant difference in the quantitative and qualitative analysis of dental plaque, as shown in [Table 1]. A statistically significant difference was not observed for quantitative changes in mean plaque score among the intervention group and control groups at various stages of evaluation, as depicted in [Table 2]. Qualitative analysis of plaque reported a steep reduction in median colony count of S. mutans at the first evaluation ([208 (126.5, 268)] vs. [132 (94.5, 156.5)) P < 0.001) followed by a gradual increase at the final evaluation among intervention group ([208 (126.5, 268)] vs. [188 (118, 238.5)] P < 0.001), whereas a gradual consistent increase was observed among the control group ([185 (170, 237)], [196 (192.5, 263.5)], and [201 (199, 278)]) at various stages of evaluation. Intergroup comparison had shown a statistically significant difference in median bacterial counts among intervention and control groups at the first evaluation ([132 (94.5, 156.5)] ([196 (192.5, 263.5)] P < 0.05) [Table 3].
Table 1: Basic demographic/clinical/microbiological characteristics of the study population at baseline

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Table 2: Comparison of plaque index score among intervention and control groups

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Table 3: Comparison of the number of colonies of Streptococcus mutans among intervention and control groups

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There was a steady reduction in median colony count of Lactobacilli at the first evaluation ([3 (1, 5)] vs. [2 (1, 2)] P < 0.001) succeeded by a gradual increase at the second evaluation among intervention group ([3 (1, 5)] vs. [2 (1.5,3)] P < 0.05), whereas a gradual consistent increase was observed among the control group ([3 (1.5, 4)], [4 (2, 5)], and [4 (3, 5.5)]). In addition, a statistically significant difference in median bacterial counts among intervention and control groups was observed at the first evaluation ([2 (1, 2)]) vs. [4 (2, 5)] P < 0.05), as shown in [Table 4].
Table 4: Comparison of the number of colonies of Lactobacilli among intervention and control groups

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Marked reduction in median colony count of anaerobic bacteria was reported at the first evaluation ([232 (165, 251)] vs. [190 (127.5, 237.5)] P < 0.001) followed by a gradual increase at the second evaluation ([232 (165, 251)] vs. [200 (157.5, 236)] P < 0.05) among intervention group, whereas a gradual steady increase was observed among the control group ([222 (166, 253)], [233 (193.5, 291)], and [233 (204, 291)]). Furthermore, a statistically significant difference in median bacterial counts was reported among intervention and control groups at the first evaluation ([190 (127.5, 237.5)], [233 (193.5, 291)] P < 0.05), as shown in [Table 5].
Table 5: Comparison of the number of colonies of anaerobic bacteria among intervention and control groups

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  Discussion Top


Dental plaque is a structurally and functionally organized biofilm. Plaque forms in an ordered way and has a diverse microbial composition that remains relatively stable over time (microbial homeostasis). The predominant species from diseased sites are different from those found in healthy sites, although the putative pathogens can often be detected in low numbers at normal sites. The microbiology of dental caries points toward an initial dominance of acidogenic and later shift toward aciduric bacteria such as mutans streptococci and Lactobacilli, although other species with relevant traits may be involved. S. mutans has been implicated as one of the major and most virulent of caries-producing organisms. Lactobacilli have been implicated in the progression of dental caries, especially in dentin.[5] Strategies to control caries could include inhibition of biofilm development (e.g., prevention of attachment of cariogenic bacteria, manipulation of cell signaling mechanisms, delivery of effective antimicrobials, etc.) or enhancement of the host defenses.[9] Plaque microbiota is considered one of the major risk factors in oral health disease, which, without thorough removal, can determine the progress of caries and periodontal disease, leading to early loss of teeth and consequent functional and esthetic issues. Thus, oral health appears to be strictly correlated to a delicate balance between bacteria within the oral cavity, good oral hygiene care, and a balanced diet.[10]

Probiotics have been with us for as long as people have eaten fermented milk, but their association with health benefits dates only from the turn of the last century.[11] Probiotic may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle. One of the most popular natural foods that contain probiotics is curd.[12] Curd being the staple diet in India has got immense health benefits. This study paved the way to know about the additional oral health benefits of curd. In this study, curd with a proven probiotic potential (obtained from collaborative research conducted in the same department and has isolated Lactobacillus rhamnosus, Lactobacilluscasei, and Lactobacillus paracasei) was used for initial curdling, thus promoting the growth of the same organisms in subsequent curdling. Unlike milk and milk products, curd is semisolid and can be retained in the oral cavity for a longer period of time with a prolonged beneficial effect. Thus, the present study aimed to explore the probiotic potential of daily consumption of homemade curd among children on the quantity and quality of dental plaque for a period of 30 days as stated in the previous literature.[1],[5]

The beneficial effect of probiotic products formulations such as milk, yoghurt, tablets, chewing gums, controlled releasing medical devices, and also as mouthrinses on overall oral health and a few specific bacteria is reported in previous literature; however, no documented studies have been published till date correlating the effect of homemade curd on the quantity of quality of dental plaque.[4],[5],[13],[14],[15],[16]

In the present study, plaques samples were analyzed both for clinical and microbiological parameters aimed at finding the probiotic potential of homemade curd and thus revealed that following the intervention phase, the mean plaque score value in the interventional group was slightly lower as compared to the control group, but this difference was not statistically significant which was in contrary to the study conducted by Karuppaiah et al. where statistically significant reduction in plaque score was observed in the intervention group and the reason remained unclear.[1] A statistically significant difference was observed for all the strains of bacteria included in the study. In accordance with the study conducted by Sudhir et al. where a statistically significant reduction in S. mutans colony counts (P < 0.001) was observed in the probiotic curd group as compared to the regular curd group.[5] A significant reduction in Lactobacilli has not been quoted in any of the published literature, whereas a significant reduction in a few anaerobic bacteria has been observed in the probiotic group.[17],[18] The results of this study should be discussed and interpreted with caution because of paucity in published literature, especially of Lactobacilli and anaerobic bacteria. Moreover, the explanation for the findings and the mechanism of action is not fully clear. Probiotics are reported to inhibit plaque accumulation and maintain good oral hygiene by microbial replacement because of the direct contact with the oral tissues and biofilm.[1] Our study has not found a quantitative reduction of plaque, although a qualitative beneficial change was observed. Most antimicrobial strategies are effective only as long as the patient is on the treatment. Following the cessation of the intervention, the oral bacterial counts rapidly return to the original counts.[5] Similarly, in this study after a washout period of 14 days, gradual recolonization of the studied organisms was observed. Recolonization implies that probiotic bacteria are not able to colonize on a permanent basis in the mouth. Oral ecology will revert back after discontinuation of intake. In fact in this study, consumption of homemade curd among children on a regular basis was proved to be beneficial. Significant findings obtained in our study are still uncertain and any definitive conclusion should be avoided at this stage. Nonetheless, the current findings pave the way for upcoming studies to assess the possible effects of homemade curds on oral ecology.

Within the impediment like a short duration of the study trial, the results of this study point out that short-term daily ingestion of probiotic bacteria conveyed by means of homemade curd in the diet reduced the microbial burden of plaque. The potential confounder that we were unable to eliminate was children's daily oral hygiene practices. Therefore, an obvious reduction of microbial load in dental plaque would notionally imply a reduced risk of dental caries, but this must be confirmed in future clinical trials. Consequently, if the consumption of homemade curd is promoted in the regular diet by modifying the dietary patterns, it would help in improving the oral wellbeing by diminishing the plaque microflora. Due to the scarcity of studies utilizing homemade curd, an unequivocal conclusion on the possible mechanism of action in lessening cariogenic microorganisms cannot be drawn from the present investigation. Further studies with higher sample size, longer duration trials, and evidence-based research need to be conducted to elucidate the effectiveness of regular consumption of homemade curd on oral ecology.


  Conclusion Top


With an expanding worldwide issue of antibiotic resistance, adding to inadequate treatment of microbial infections, probiotic bacteria in homemade curd by means of natural therapy seems to be an inventive method for the aversion of dental caries. Regular intake of homemade curd containing probiotic bacteria can be considered as a reasonable, simple, and safe strategy for preventing dental caries.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Karuppaiah RM, Shankar S, Raj SK, Ramesh K, Prakash R, Kruthika M. Evaluation of the efficacy of probiotics in plaque reduction and gingival health maintenance among school children-A randomized control trial. J Int Oral Health 2013;5:33-7.  Back to cited text no. 1
    
2.
Pocha SR, Kumar DK, Dhanya M, Sudhakar K. Effects of sodium fluoride solution, chlorhexidine gel and fluoride varnish on the microbiology of dental plaque–A randomised controlled trial. J Indian Assoc Public Health Dent 2018;16:109.  Back to cited text no. 2
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Kolenbrander PE. Oral microbial communities: biofilms, interactions, and genetic systems. Annu Rev Microbiol 2000;54:413-37.  Back to cited text no. 3
    
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Thakkar PK, Imranulla M, Kumar PN, Prashant GM, Sakeenabi B, Sushanth VH. Effect of probiotic mouthrinse on dental plaque accumulation: A randomized controlled trial. Dent Med Res 2013;1:7.  Back to cited text no. 4
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Sudhir R, Praveen P, Anantharaj A, Venkataraghavan K. Assessment of the effect of probiotic curd consumption on salivary pH and Streptococcus mutans counts. Niger Med J 2012;53:135-9.  Back to cited text no. 5
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6.
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Hsu KL, Osgood RC, Cutter GR, Childers NK. Variability of two plaque sampling methods in quantitation of Streptococcus mutans. Caries Res 2010;44:160-4.  Back to cited text no. 7
    
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Marsh PD. Dental plaque as a biofilm and a microbial community–implications for health and disease. BMC Oral Health 2006;6:S14.  Back to cited text no. 9
    
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Farronato G, Giannini L, Nolet F, Galbiati G, Garagiola U, Maspero C. Qualitative and quantitative assessment of plaque bacteria in pediatric patients, patients undergoing orthodontic treatment, combined orthdontic-surgical treatment and implant-prosthetic rehabilitation. Minerva Stomatol 2014;63:167-78.  Back to cited text no. 10
    
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Anuradha S, Rajeshwari K. Probiotics in health and disease. J Indian Acad Clin Med 2005;6:67-72.  Back to cited text no. 11
    
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Parihar AS, Katoch V. Probiotics in oral health: A review. J Adv Med Dent Sci Res 2015;3:57.  Back to cited text no. 12
    
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Becirovic A, Abdi-Dezfuli JF, Hansen MF, Lie SA, Vasstrand EN, Bolstad AI. The effects of a probiotic milk drink on bacterial composition in the supra- and subgingival biofilm: A pilot study. Benef Microbes 2018;9:865-74.  Back to cited text no. 13
    
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Caglar E, Cildir SK, Ergeneli S, Sandalli N, Twetman S. Salivary mutans streptococci and Lactobacilli levels after ingestion of the probiotic bacterium Lactobacillus reuteri ATCC 55730 by straws or tablets. Acta Odontol Scand 2006;64:314-8.  Back to cited text no. 14
    
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Nadelman P, Magno MB, Masterson D, da Cruz AG, Maia LC. Are dairy products containing probiotics beneficial for oral health? A systematic review and meta-analysis. Clin Oral Investig 2018;22:2763-85.  Back to cited text no. 18
    


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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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