|Year : 2014 | Volume
| Issue : 4 | Page : 219-223
Effect of commercially available probiotic product on the level of salivary microflora in children of West Bengal
Barun Dasgupta1, Shabnam Zahir2
1 Department of Pedodontics and Preventive Dentistry, Hazaribag College Dental Sciences and Research, Jharkhand, India
2 Department of Pedodontics and Preventive Dentistry, Guru Nanak Institute of Dental Science and Research, Panihati, West Bengal, India
|Date of Web Publication||20-Nov-2014|
M\S, Medcico, Maithon More, P.O. Kumardubhi, Dhanbad - 828 203, Jharkhand
Context: Newer concepts and methods are being researched and being implemented every day. One such newest variety of preventive measure is the use of probiotics. Aims: This study tries to explore the effect of commercially available probiotic product on the level of the salivary microflora in children of West Bengal in a specific period. Materials and Methods: The study was of a randomized, placebo-controlled design. The total of 90 children between age group 6 and 14 years were selected at random, as study and control group. The study group was provided with the commercially available probiotic product "Eugi" sachet with milk as a vehicle for "21" days, whereas control group is provided with only plain milk. Salivary concentration of few specific cariogenic bacteria was measured in both study and control group in the beginning and at the interval at 0, 1, 3, and 5 months. Statistical Analysis Used: Average, standard deviation for a varied comparison was calculated. Student's t-test and probability test were performed. Results: The significant difference in between control and study group only existed at 1-month interval and difference again became insignificant in respective intervals ahead. Conclusions: The study concluded that, (1) there was significant reduction of Gram-positive colony (GPC) counts after 1 month in the probiotic consumed group. (2) During the interval of 1 to 3 months, the GPC counts showed increase in a number and they were restored to the baseline concentration which was maintained till the 5 th month. (3) There was no significant variation in GPC count between different age groups at all intervals.
Keywords: Dental caries, Gram-positive colony, probiotics
|How to cite this article:|
Dasgupta B, Zahir S. Effect of commercially available probiotic product on the level of salivary microflora in children of West Bengal. SRM J Res Dent Sci 2014;5:219-23
|How to cite this URL:|
Dasgupta B, Zahir S. Effect of commercially available probiotic product on the level of salivary microflora in children of West Bengal. SRM J Res Dent Sci [serial online] 2014 [cited 2019 Sep 21];5:219-23. Available from: http://www.srmjrds.in/text.asp?2014/5/4/219/145116
| Introduction|| |
Probiotic bacteria by definition are, "live microbial food supplements that may benefit the host by influencing the balance between the many species of the commensal flora both in the oral cavity and the rest of the digestive system."  Probiotic bacteria can provide health benefits to the host by:
- Providing nutrients and cofactors to the host,
- Competing directly with pathogens,
- Interacting with pathogen virulence factors, and
- Stimulating host immune responses (Saier and Mansour 2005). 
Thus, therapeutic targets for probiotics are now widely distributed among infectious and noninfectious diseases. Probiotics have been used to modulate bacterial populations in many regions of the human body either when it has been perceived that there is an imbalance of the normal microbiota allowing unregulated growth of problematic' microorganisms or in order to replenish bacterial populations which play a specific role in a particular ecosystem. Some examples of successful applications of probiotics include the intestinal tract, where the occurrence of certain disease states has been reduced and the prevention of postantibiotic-associated diarrhea, and in the vagina where they have been used to reduce the incidence of vaginosis and vaginitis). 
Disease is always a major limiting factor in the development of any society. Dental caries is the most common infectious oral diseases in humans.  In India, 51.9% in 5-year-old children, 53.8% in 12-year-old children and 63.1% in 15-year-old teenagers are suffering from this disease (National Oral Health Survey and Fluoride Mapping 2004). It is a multifactorial infectious disease with one of the major contributing factor being oral microflora mainly Streptococcus mutans, Streptococcus sanguis, Lactobacillus sp., Actinomyces sp., Actimyces viscosus, Streptococcus sorbinus.
Recently, there has been increasing interest in probiotic control against these oral infections, and a number of clinical trials has been conducted to elucidate the possible impact on oral health. Twetman and Stecksen-Blicks reviewed these trials and concluded that probiotic intervention in childhood may hamper the presence of mutans streptococci in saliva, possibly reducing the risk of dental caries. The oral cavity has only recently been suggested as a relevant target for probiotic applications.  To be able to exert probiotic properties in the oral cavity, however, it is essential for the microorganism to resist the oral environmental conditions and defense mechanisms, to be able to adhere to saliva-coated surfaces, to colonize and grow in the mouth, and to inhibit oral pathogens.
The thinking of this world is changing, so is the modality of treatment plans. Nowadays, prevention seems to be a much feasible option since it is cheap, thus, relieving all socioeconomic groups from the financial burden of cure. As well as, it is painless, so it's attractive enough to pull children toward better oral health. It seems any preventive approach has a better and more retained effect on younger age group than on the older age group, so even replacement of microbial community of the oral cavity from pathogenic to healthy colonies is also better seen in younger children. The best part of probiotic is the adjuvant effect of it, other than causing a positive effect on the oral cavity of children, it also helps in increasing the immunity, and they have well-established effect on gut flora too.
There are very few reported studies on clinical trials on the effect of probiotics on the oral microflora of children. In this present study, the clinical effect of commercially available probiotic on the oral microflora has been evaluated for Bengali population.
| Materials and methods|| |
The study was conducted in a home for underprivileged children, named "save the children home" - Thakurpukur, Kolkata run by government organization. The home maintained a fixed dietary habit and time-table. Children between age group 6 and 14 years were selected at random, both as study and control group. Both the group resided in the home with similar dietary habits. Children were all females, and they were majority of 6-10 years of age. Very few children were of higher age group.
Fifty-two children out of 90 children from the home were selected as the study group, rest 38 children from the same place were selected as a control group. The study protocol was approved by the Ethical Committee at Guru Nanak Institute of Dental Science and Research, Kolkata, West Bengal. All participants were fully informed concerning the objectives and methodology of the study. Informed consent in written was received from the secretary of the study home. Detailed case history of all the children was taken which included their name, age, daily food habit, and brushing habit. The children were asked to chew unflavored paraffin for 45 chews or 1 min and expectorate into a funnel inserted into a graduated cylinder. The collected saliva was then sent to for further microbilogical tests.
Salivary concentration of few specific cariogenic bacteria was measured in both study and control group in the beginning. The study group was provided with the commercially available probiotic product "Eugi" sachet (Wallace Pharmaceuticals) with milk (200 ml) as a vehicle for "21" days, and at the interval at 0, 1, 3, and 5 months, salivary concentration of specific bacteria was determined. The control group was provided with plain milk (200 ml), was also examined at the interval at 0, 1, 3, and 5 months, for salivary concentration of specific bacteria. The difference in the count was tabulated and co-related and compared (One sachet of Eugi contains: Lactobacillus acidophilus 0.24 billion colony forming unit (CFU), Lactobacillus rhamnosus 0.24 billion CFU, Bifidobacterium bifidum 0.24 billion CFU, Bifidobacterium longum 0.24 billion CFU, Saccharomyces boulardii 0.05 billion CFU, Streptococcus thermophilus 0.24 billion CFU, and Fructooligosaccharides 300 mg.). This product is mainly used commercially for gastro-intestinal disorders and antibiotic-associated diarrhea.
The salivary samples were placed immediately into a 1 ml prereduced transport fluid centrifuged tube, which were placed in a thermos flask containing ice cubes. The salivary samples were then transferred to microbiological labs. The sample was incubated in blood agar for a period of 24 h, many colonies of different shapes, sizes, and colors were formed, which were seen under compound microscope. Further, biochemical evaluation of culture media was performed too. The collected data were then statistically analyzed. Average, standard deviation for a varied comparison were calculated. Student's t-test and probability test were performed. Complete analysis was done using SPSS software (version 11) manufactured by SPSS inc.
| Results|| |
The saliva samples were collected from all the groups at a preintervention period and postinterventional intervals of 1, 3, and 5 months. The pre intervention and postintervention levels of Gram-negative colony (GNC) and Gram-positive colony (GPC) were tabulated. All subjects had detectable levels of salivary GPC at baseline (0 month). On each period, the average for the control and experimental group has been compared using Student's t-test.
The result and observation of the study were calculated in three main parameters:
- Comparison of GPC/GNC study and control group all age group at different time intervals.
- Comparison of GNC/GPC at different time intervals among both study and control groups.
[Table 1]a-c and Charts 1 [Additional file 1]a-c below show comparison between the control and study groups at intervals of 1 month, 3 months, and 5 months in the different age groups, which concludes that in all age group the significant difference between control and study group only existed at 1-month interval and difference again became insignificant in respective intervals ahead.
[Table 2]a and b show the changes in the average counts (GPC) from the period to period, that is, at baseline to 1month, 1 month to 3 months, and 3 months to 5 months, for each age group in the study group and control group, respectively.
In the study group, it could be noted that for each age group, the average counts for GPC highly decreased from baseline to 1 month period (P < 0.001 in all cases which shows they were highly significant). Again after 1 month, the average counts for GPC count increased from 1 to 3 month period (P < 0.001 in most cases). Even in the period between 3 and 5 months, the average was also significant (P < 0.001 in most cases but four samples had P < 0.01)
Whereas in the control group at 1 month interval, for GPC counts all the changes are not significant (P > 0.05). In between 1 month to 3 months period, there have been an increase in average counts in all the age groups as seen in base to 1 month period. GPC count in 6-8 years did not increase significantly (P > 0.05). But in all other cases, an increase in averages is significant (P < 0.001 except in three samples P < 0.05). In 3 months to 5 months period, all the increases in averages (in all age group for GPC) are not significant.
| Discussion|| |
Human body is a reservoir of diverse ecology of microbes. Their role forms part of normal, healthy human physiology, however, if number of microbes grow beyond their typical ranges or if microbes populate in typical areas of the body (such as through poor hygiene or injury), disease can result. Oral microflora population are not static. They change in response to the age, hormonal status, diet, and overall health of an individual.  The exact composition of species will vary among individuals and over time, in the same individual. It is now estimated that there are more than 700 different types of microbes that can be isolated from the mouth, but >50% of these cannot currently be grown in pure culture in the laboratory. ,
Out of many oral microbial diseases seen in children, dental caries is the most prevalent. Dental caries is viewed as a consequence of an imbalance in the resident microflora.  Mutan streptococci are the principal etiological agents of dental caries which generally consist of S. mutans, S. sorbinus, Streptococcus cricetus, Streptococcus ferrus, Streptococcus ratti, Streptococcus macacae, and Streptococcus downie. Mutans streptococci appear in the mouth after teeth have erupted as they need solid surfaces to colonize. The second most predominant species were seen to be Lactobacillus. They are cariogenic, highly acidogenic organisms, however, has low affinity for tooth surfaces. 
The present study has tried to see the influence of probiotic on salivary oral microflora. This study sees, the mean difference in count of GPC (S. mutan) in a specific period in children aged 6-14 years by giving them commercially available probiotic powders with milk. They were instructed to swipe their whole mouth with the milk before drinking it down. In the present study, the commercially available probiotic product we used were predomonantly of Bifidobacterium and Lactobacillus species which includes L. acidophilus, L. rhamnosus, B. bifidum, B. longum. Twetman, and Stecksén-Blicks suggested that Bifidobacterium and Lactobacillus were the most important species of bacteria in giving positive response in a reduction of pathogenic bacteria in oral ecology and also suggested that posttreatment retention of these species were much higher than other strains of bacteria used as an probiotic agent. 
Nase et al. were the first to test a dietary Lactobacillus strain, L. rhamnosus GG, on its caries inhibiting ability in vivo. The probiotic milk showed just a very moderate tendency to reduce S. mutans levels, which were semi-quantitatively detected in a pooled saliva-plaque sample using a diagnostic test. The probiotic product used in this study also contained L. rhamnous as its one of the constituent. 
The vehicle by which probiotics are ingested or delivered in the oral cavity can, however, influence the cariogenic potential and the oral colonization of a probiotic. The most commonly used dietary lactobacilli are consumed in milk products (e.g. fermented milk drink, yoghurt, or cheese). When lactic acid bacteria are being consumed in milk products, the buffer capacity of the milk will decrease the production of acid.  The presence of calcium, calcium lactate, and other organic and inorganic compounds in milk are anticariogenic and reduce the colonization of pathogens. Most of the probiotic intervention studies for caries prevention use dietary probiotics that are often employed for the prevention of gastrointestinal pathologies. The present study too used milk as a vehicle of transport for the probiotic. The study group samples were even instructed to swish their mouth with the milk before drinking. 
The present study finally suggested that short-term consumption of milk containing probiotic product may help in reducing the GPC count present in oral ecology which are the sole pathogen behind major oral diseases such as dental caries, halitosis, and gingival enlargement. The present study also suggests that may be the postretention time period of probiotic is very less in the oral cavity if administered for a short period of time. Further, elaborate study has to be done about the effect of commercially available probiotic on a larger population with more specificity about the microbial community of saliva.
From the findings in the present study, we can conclude that:
- There was significant reduction of GPC counts after 1 month in the probiotic consumed group.
- During the interval of 1-3 months, the GPC counts showed increase in a number and they were restored to the baseline concentration which was maintained till the 5 th month.
- There was no significant variation in GPC count between different age groups at all intervals.
A study in a larger and diverse population ahead will give a better and broader picture in finding the role of the probiotic product in altering the oral microflora colonies.
| References|| |
Saier MH Jr, Mansour NM. Probiotics and prebiotics in human health. J Mol Microbiol Biotechnol 2005;10:22-5.
Reid G, Charbonneau D, Erb J, Kochanowski B, Beuerman D, Poehner R, et al.
Oral use of Lactobacillus rhamnosus
GR-1 and L. Fermentum RC-14 significantly alters vaginal flora: Randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 2003;35:131-4.
Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria; Food and Agriculture Organization of the United Nations, WHO; Oct. 2001.
Caglar E, Kargul B, Tanboga I. Bacteriotherapy and probiotics′ role on oral health. Oral Dis 2005;11:131-7.
Twetman S, Stecksén-Blicks C. Probiotics and oral health effects in children. Int J Paediatr Dent 2008;18:3-10.
Young VB, Britton RA, Schmidt TM. The human microbiome and infectious diseases: Beyond koch. Interdiscip Perspect Infect Dis 2008;2008:296873.
Marsh PD. Microbiology of dental plaque biofilms and their role in oral health and caries. Dent Clin North Am 2010;54:441-54.
Philip D, Roberts MC. Mechanisms of antibiotic resistance in oral bacteria. APUA Newsl 1997;15:14-6.
Miller WD. Micro-Organisms of the Human Mouth. Philadelphia: SS White; 1890.
Teughels W, Van Essche M, Sliepen I, Quirynen M. Probiotics and oral healthcare. Periodontol 2000;48:111-47.
C Stecksén Blicks. Effect of Long-Term Consumption of Milk Supplemented with Probiotic Lactobacilli and Fluoride on Dental Caries and General Health in Preschool Children: A Cluster-Randomized Study. Caries Research 2009;43:374-381.
Hatakka K, Nase L et al
. Effect of long term consumption of probiotic milk on infections in children attending day care centres: Double blind, randomised trial. Br Med J 2001;322:1327.
Ahola AJ, Yli-Knuuttila H, Suomalainen T, Poussa T, Ahlström A, Meurman JH, et al.
Short-term consumption of probiotic-containing cheese and its effect on dental caries risk factors. Arch Oral Biol 2002;47:799-804.
Montalto M, Vastola M, Marigo L, Covino M, Graziosetto R, Curigliano V, et al.
Probiotic treatment increases salivary counts of lactobacilli: A double-blind, randomized, controlled study. Digestion 2004;69:53-6.
[Table 1], [Table 2]