ORIGINAL Nagarajappa ARTICLE et al

Effect of Chocobar Ice Cream Containing Bifidobacterium on Salivary Streptococcus mutans and Lactobacilli: A Randomised Controlled Trial Ramesh Nagarajappaa/Hemasha Daryanib/Archana J. Shardac/Kailash Asawac/ Mehak Batrad/Sudhanshu Sanadhyad/Gayathri Rameshe Purpose: To examine the effect of chocobar ice cream containing bifidobacteria on salivary mutans streptococci and lactobacilli. Materials and Methods: A double-blind, randomised controlled trial was conducted with 30 subjects (18 to 22 years of age) divided into 2 groups, test (chocobar ice cream with probiotics) and control (chocobar ice cream without probiotics). The subjects were instructed to eat the allotted chocobar ice cream once daily for 18 days. Saliva samples collected at intervals were cultured on Mitis Salivarius agar and Rogosa agar and examined for salivary mutans streptococci and lactobacilli, respectively. The Mann-Whitney U-test, Friedman and Wilcoxon signed-rank tests were used for statistical analysis. Results: Postingestion in the test group, a statistically significant reduction (p < 0.05) of salivary mutans streptococci was recorded, but a non-significant trend was seen for lactobacilli. Significant differences were was also observed between follow-ups. Conclusion: Short-term daily ingestion of ice cream containing probiotic bifidobacteria may reduce salivary levels of mutans streptococci in young adults. Key words: Bifidobacterium, ice cream, Lactobacillus acidophilus, probiotics, saliva, Streptococcus mutans Oral Health Prev Dent 2015;13:213-218 doi: 10.3290/j.ohpd.a32673

a

Professor and Head, Department of Public Health Dentisty, Rama Dental College and Hospital, Kanpur, Uttar Pradesh, India. Idea, hypothesis, laboratory work, acquisition, analysis and interpretation of data, co-wrote and proofread manuscript.

b

Postgraduate Student, Department of Public Health Dentistry, Pacific Dental College and Hospital, Udaipur, Rajasthan, India. Idea, hypothesis, design, laboratory work, analysis and interpretation of data, co-wrote and proofread manuscript.

c

Senior Lecturer, Department of Public Health Dentistry, Pacific Dental College and Hospital, Udaipur, Rajasthan. Co-wrote and critically revised manuscript, analysed and interpreted data.

d

Postgraduate Student, Department of Public Health Dentistry, Pacific Dental College and Hospital, Udaipur, Rajasthan, India. Study design, analysed and interpreted data, substantially contributed to discussion, co-wrote and proofread manuscript.

e

Reader, Department of Oral and Maxillofacial Pathology, Rama Dental College and Hospital, Kanpur, Uttar Pradesh. Study design, analysed and interpreted data, substantially contributed to discussion, co-wrote and proofread manuscript.

Correspondence: Professor Ramesh Nagarajappa, Department of Public Health Dentistry, Rama Dental College and Hospital, A-1/8 Kanpur 208024, Uttar Pradesh, India. Tel: +91- 962-116-8883, Fax: +91- 512-258-3875. Email: [email protected]

Vol 13, No 3, 2015

Submitted for publication: 12.11.12; accepted for publication: 18.07.13

T

he consumption of bacteria as a part of the diet, mostly in the form of yogurt and other fermented milk products, has increased over the past decade, in part because of the broader availability of these products and partly because of the popular view of such products as healthy foods. Fermented milk products are a common part of the diet in Asia, Europe, parts of Africa and increasingly in North America (Saavedra et al, 2004). Studies have shown beneficial effects of probiotic bacteria, principally lactobacilli and bifidobacteria in conditions such as lactose malabsorption in adults (Onwulata et al, 1989) and children (Montes et al, 1995; Shermak et al, 1995). Probiotic technology represents a breakthrough approach to maintaining oral health by utilising natural beneficial bacteria commonly found in healthy mouths to provide a natural defence against those bacteria thought to be harmful to teeth and gums. In simple words, the basic principle of probiotics is

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to use good bacteria to compete against pathogenic bacteria (Harini and Anegundi, 2010). Probiotics are defined as live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (FAO/WHO, 2001) (Hasslof et al, 2010). Probiotics have been used for decades in fermented products, but the potential use of probiotics as a nutritional medical therapy has not been formally acknowledged (Flichy-Fernandez et al, 2010). The most commonly used strains belong to the genera Lactobacillus and Bifidobacterium, commonly found in the oral cavity, including carious lesions (Reddy et al, 2011). It is generally thought that bifidobacteria have combined local and systemic effects involving adhesion, coaggregation, competitive inhibition, production of organic acids and bacteriocin-like compounds and immune modulation (Teughels et al, 2008). Clinical studies have indicated that lactobacilli and bifi dobacteria have some promise for prevention of caries. The tendency towards a decreased number of mutans streptococci in the saliva seems to be independent of the product or strain used; however, such an effect has not been observed in all studies. The discrepancies between results cannot be explained only by the use of different probiotic strains, as different results have also been obtained using the same strains (Haukioja, 2010). Lactobacilli associated with periodontal health were found to inhibit the growth of certain periodontal pathogens. It was proposed that they may reduce the levels of these pathogens on the tongue, which constitutes a major reservoir for their transmission, and thereby indirectly reduce the colonisation of subgingival plaque by periodontal pathogens (Koduganti et al, 2011). Currently available information on oral probiotic uses, such as therapy for oral cavity disorders including tooth decay or periodontal disease, is limited. Hence, the present study was conducted with the objective of examining whether or not the consumption of chocobar ice cream containing bifidobacteria/Bifantis (B. infantis 35624) affects the salivary levels of mutans streptococci and lactobacilli in young adults.

MATERIALS AND METHODS Subjects and study design A double-blind, placebo-controlled, parallel-group, randomised controlled trial was carried out to investigate the effect of chocobar ice cream (AMUL;

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Anand, India) containing bifidobacteria on salivary mutans streptococci and lactobacilli counts among 30 volunteers who were orally and systemically healthy non-medicating dental students of the Pacific Dental College and Hospital, Udaipur, India, aged 18 to 22 years (19 females, 11 males). The exclusion criteria were subjects with: habitual consumption of probiotics or xylitol chewing gums, systemic antibiotic medication within the previous 6 weeks, recent repeated topical fluoride treatments and using oral hygiene aids other than routine toothbrushing. We applied blinding to group allocation, for data collection and data entry. Our research was conducted in full accordance with the World Medical Association Declaration of Helsinki. The study protocol was reviewed and approved by the Institutional Ethics Committee. Written informed consent was obtained from all the study participants after explaining the nature and purpose of this research. A pilot study was conducted on 10 subjects to assess the feasibility, sample size of the study and to become familiar with the laboratory procedure of salivary analysis. Based on the results of pilot study, a sample size of 15 participants for each group was obtained using a mean difference of 1.03 x 106 CFU/ml saliva (± 7.1 x 106), 80% power and a significance level of 5%.

Methodology The baseline data were collected on 24 October 2011 and the outcome data after 18 days. All participants attended three consultations (baseline, after 1 h and follow-up). To safeguard against bias, the investigator who obtained outcome measurements was not informed of the group assignment, which ensured strict blinding. In the first consultation, all subjects were seen at 9:00 AM and collection of baseline saliva was performed. Prior to saliva collection, patients were kept seated for 5 min, relaxed and silent. After a thorough rinse with water and an initial swallow, unstimulated saliva was collected by participants allowing the saliva to flow over a period of 5 min into a graduated test tube by tilting their heads forward. The average flow rate of collected saliva was 0.3 ml/min. Following this procedure, the participants (n = 30) were randomly allocated to test (chocobar ice cream with probiotics) and control (chocobar ice cream without probiotics) groups consisting of 15 subjects in each by another investigator (Fig 1) us-

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Dental students of Pacific Dental College and Hospital, Udaipur were selected for the study

Enrollment

Assessed for eligibility (n=42)

Excluded (n=12) Did not meet eligibility criteria (n=8) Declined to participate (n=4)

Allocation

Allocated to experimental group – Probiotic (n=15)

Allocated to control group – Non-probiotic (n=15)

Follow-up

Loss to follow-up (n=0)

Loss to follow-up (n=0)

Analysis

Randomised (n=30)

Analysed (n=15)

Analysed (n=15)

Fig 1  Flow-chart of the inclusion of subjects.

ing a random-number table. Everyone was supplied with the chocobar ice cream (wrapper removed) containing either probiotic bifidobacteria or a control chocobar ice cream without viable bacteria; the ice-cream bars had a similar colour, taste and consistency. Each test chocobar ice cream, with a net weight of 42 g and net volume of 60 ml, contained bifidobacteria as a probiotic organism (106/ ml). The participants were instructed to eat the chocobar ice cream and the same procedure of salivary sample collection was repeated 1 h after consumption. The participants were not allowed to eat or drink anything during this period. The subjects were further instructed to report to the department every morning at 10:00 for consumption of ice cream. The experimental period comprised

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18 days. Subjects were encouraged to maintain their normal oral hygiene habits and to continue brushing their teeth twice a day during the study period. They followed these guidelines to improve standardisation of data collection. At the end of the study period, each participant underwent the final follow-up collection of saliva samples on the 18th day after ingesting the ice cream. Neither local nor systemic side effects were noted in the two groups.

Microbial evaluation The counts of salivary mutans streptococci and lactobacilli were evaluated. The selectivity of MSB media was achieved by raising the sucrose concentra-

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Table 1 Comparison of mean Streptococcus mutans and lactobacilli count between test and control groups Groups (n=30)

Baseline Test (n=15)

Control (n=15)

Streptococcus mutans

Time of assessment

Mean ± SD

Lactobacillus

F value

6

7

6

7

p-value

Mean ± SD 5

8.1648x10 ± 1.5240x10

7.6869x10 ± 1.9961x10 22.6279

3.0557x105 ± 7.0893x105

0.001*

After 1 h

6.5127x10 ± 1.9890x10

Follow-up

2.19x105 ± 7.1572x105**

2.6736x105 ± 3.5070x105

Baseline

3.4277x106 ± 6.6035x106

6.0562x105 ± 1.6344x106

After 1 h

4.4958x106 ± 9.7630x106

Follow-up

5.2119x106 ± 1.0006x106

0.5833

0.565

F-value

p-value

0.2534

0.778

0.4495

0.642

5

2.8664x105 ± 6.0742x105 1.9718x105 ± 3.1973x105

Test applied: Friedman’s test (between regimes), *p < 0.05 statistically significant. Test applied: Mann-Whitney U-test (between test and control group), ** p < 0.05 statistically significant.

Table 2 Comparison of mean rank of Streptococcus mutans and lactobacillus count within regimes among test and control groups Streptococcus mutans Groups (n=30) Test (n=15)

Control (n=15)

Lactobacillus

Time of assessment

Mean positive rank

Mean negative rank

p-value

Mean positive rank

Mean negative rank

p-value

Baseline and 1 h

8.25

7.91

0.0730

5.50

9.67

0.1354

1 h and follow-up

0.00

8.00

0.0003*

9.00

7.33

0.7615

Baseline and follow-up

2.00

8.43

0.0002*

7.14

8.75

0.5995

Baseline and 1 h

8.57

7.50

1.0000

5.83

9.44

0.1688

1 h and follow-up

7.90

8.20

0.3028

8.86

7.25

0.9341

Baseline and follow-up

9.25

6.57

0.4543

7.67

8.22

0.4543

Test applied: Wilcoxon signed rank test (within regimes), *p < 0.05 statistically significant.

tion in the original media (MS) from 5% to 20% and by addition of bacitracin. Saliva was inoculated on a petri dish containing Mitis salivarius bacitracin agar and Rogosa agar with selective agar media for mutans streptococci and lactobacilli, respectively (Ly et al, 2008). The laboratory procedure was meticulously followed and Robertson Meat Broth was used as transport media to prevent contamination of samples. The slides were cultivated at 37°C for 48 h. The colonies were identified by morphology with the aid of a stereomicroscope at 10X magnification and the density of the colony forming units (CFU/ml of saliva) was calculated.

Statistical analysis The data obtained were analysed using SPSS (Statistical Package for Social Sciences) version 11.5

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(SPSS; Chicago, IL, USA). Non-parametric statistical tests were used, as the Kolmogorov-Smirnov test for normality showed the variables to have a non-normal distribution. Comparisons of mean bacterial scores between and within the regimens were performed using the Friedman test and the Wilcoxon signed-rank test, respectively. The comparison between test and control groups was performed using the Mann-Whitney U-test. A p-value ≤ 0.05 was considered statistically significant.

RESULTS The mean values of salivary Streptococcus mutans and lactobacilli count were compared at baseline, 1 h after ice-cream consumption and follow-up (Tables 1 and 2). In the test group, the mean value of Streptococcus mutans had decreased significantly (p < 0.05) from baseline (8.1648 x 106) to the fol-

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low-up period (2.19 x 106). The baseline mean values for the test and control groups did not differ statistically significantly, but the follow-up mean values for the test group (2.19 x 106) were found to be significantly (p < 0.05) lower than those of the control group (5.2119 × 106). An increase in the mean S. mutans count from baseline (3.4277 × 106) to follow-up (5.2119 × 106) was observed in the control group. Regarding salivary lactobacilli, a detectable decrease in count was observed in both test (7.6869 × 105 to 2.6736 × 105) and control groups (6.0562 × 105 to 1.9718 × 105). These observations were not statistically significant. When the mean ranks of S. mutans were compared and the Wilcoxon signed-rank test was applied within the regimens, a significant difference was found between baseline and follow-up and also between the ‘1 h after’ and ‘follow-up’ regimens (p < 0.05). The results within regimens for the control group did not differ significantly. Furthermore, the Lactobacillus count among both the test and control groups was also not significant.

DISCUSSION The present study is unique, as the probiotic chocobar ice cream administered in this study had not been examined previously. The intervention was built on evidence supporting the use of probiotic products in improving oral health. In the oral cavity, bifidobacteria are quite acidogenic and may play a role in deep dentin caries progression rather than in the early enamel demineralisation (Hasslof et al, 2010). Probiotics can create a biofilm, acting as a protective coating for oral tissues against oral diseases. Reddy et al (2010) reported that such a biofilm can keep bacterial pathogens off oral tissues by filling space which pathogens would otherwise invade in the absence of the biofilm and by competing with cariogenic bacteria and periodontal pathogens. The present finding that ice cream could serve as a vehicle for probiotics was interesting. Ice cream, with its natural content of casein, calcium and phosphorus, is a dairy product that can be stored for a long time without any significant decrease in the number of viable probiotic cells (Bibby et al, 1980; Hekmat and McMahon, 1992). For example, the shelf life of the present ice cream was 24 months. The product was well accepted by all participants and no one in the study group could detect the difference between the active and control ice cream. Thus, there are reasons for believ-

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ing that probiotic ice cream could be a suitable product also for young children. During the period of test product consumption, the mean value of mutans streptococci counts dropped continuously, despite the sugar content of the ice cream. A caries-inhibiting effect of the probiotic organisms may have counteracted the caries-enhancing influence of the sugar, resulting in a reduction of the mutans streptococci count. The opposite was observed in the control group, which is probably related to the high sugar content and absence of probiotic organisms in the control chocobar. Recently, a study by Cildir et al (2012) demonstrated similar results, that one daily dose of lactobacilli fails to reduce the salivary MS counts in children. In contrast, two recent trials on the consumption of L. rhamnosus LB21 in milk did not reveal any decrease in salivary or plaque MS counts (Stecksen-Blicks et al, 2009; Lexner et al, 2010). However, Näse et al (2001) reported a reduced caries incidence in children consuming probiotic L. rhamnosus GG-enriched milk vs a control group of children drinking milk without probiotic enrichment. Similar findings were observed by Ahola et al (2002) after administering the same probiotic. Nikawa et al (2004) showed that cow’s milk fermented with Lactobacillus reuteri was effective against S. mutans, resulting in a reduced risk for tooth decay. This study had several strengths. For instance, unstimulated saliva was collected from each participant, as the analysis of unstimulated saliva is more sensitive than analysis of stimulated saliva (Kaufman and Lamster, 2002). Moreover, stimulated whole saliva is less suitable for diagnostic applications because the foreign substances used to stimulate saliva tend to modulate salivary content and generally stimulate the water phase of saliva secretion, resulting in a dilution in the concentration of microbial count (Miller et al, 2010). In addition, the randomised controlled trial design meant that many of the confounders were taken into account. The study was adequately powered to detect a mean difference in number of bacteria between the groups. Further, sucrose causes S. mutans to form colonies with a characteristic morphology which facilitates its identification, while the other microfloras are suppressed by the high sucrose concentration. S. salivarius, which forms large, slimy colonies that can overgrow the culture dish, is inhibited by bacitracin (Schaeken et al, 1986). These factors improved the external validity; hence, the results can be generalised. However, the data must be interpreted with caution, as no long-term

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studies are available on the effect of bifidobacteria on the oral microflora. In the future, crossover trials should be conducted.

CONCLUSION The findings of this study on the effect of daily intake of ice cream containing probiotic bifidobacteria showed a reduction in the levels of caries-associated mutans streptococci in saliva. Further studies are needed to clarify whether or not this can be applied effectively at the population level, in addition to validating this approach as an alternative strategy for the prevention of enamel demineralisation.

ACKNOWLEDGEMENTS The authors would like to thank the study volunteers for their participation and kind cooperation throughout the study.

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9. Kaufman E, Lamster IB. The diagnostic applications of saliva – a review. Crit Rev Oral Biol Med 2002;13:197–212. 10. Koduganti RR, Sandeep N, Guduguntla S, Chandana Gorthi VS. Probiotics and prebiotics in periodontal therapy. Indian J Dent Res 2011;22:324–330. 11. Lexner MO, Blomqvist S, Dahlén G, Twetman S. Microbiological profiles in saliva and supragingival plaque from caries-active adolescents before and after a short-term daily intake of milk supplemented with probiotic bacteria – a pilot study. Oral Health Prev Dent 2010;8:383–388. 12. Ly KA, Riedy CA, Milgrom P, Rothen M, Roberts MC, Zhou L. Xylitol gummy bear snacks: a school-based randomized clinical trial.BMC Oral Health 2008;25:8-20. 13. Miller CS, Foley JD, Bailey AL, Campell CL, Humphries RL, Christodoulides N, et al. Current developments in salivary diagnostics. Biomark Med 2010;4:171–189. 14. Montes RG, Bayless TM, Saavedra JM, Perman JA. Effect of milks inoculated with Lactobacillus acidophilus or a yogurt starter culture in lactose-maldigesting children. J Dairy Sci 1995;78:1657–1664. 15. Näse L, Hatakka K, Savilahti E, Saxelin M, Pönkä A, Poussa T, et al. Effect of long-term consumption of a probiotic bacterium, Lactobacillus rhamnosus GG, in milk on dental caries and caries risk in children. Caries Res 2001;35:412–420. 16. Nikawa H, Makihira S, Fukushima H, Nishimura H, Ozaki Y, Ishida K, et al. Lactobacillus reuteri in bovine milk fermented decreases the oral carriage of mutans streptococci. Int J Food Microbiol 2004;95:219–223. 17. Onwulata CI, Rao DR, Vankineni P. Relative efficiency of yogurt, sweet acidophilus milk, hydrolyzed-lactose milk, and a commercial lactase tablet in alleviating lactose maldigestion. Am J Clin Nutr 1989;49:1233–1237. 18. Reddy JJ, Sampathkumar N, Aradhya S. Probiotics in dentistry: review of the current status. Rev Clín Pesq Odontol 2010;6:261–267. 19. Reddy VN, Rao AP, Mohan G, Raja RK. Probiotic lactobacilli and oral health. Annals Essences Dent 2011;3:100–103. 20. Saavedra JM, Abi-Hanna A, Moore N, Yolken RH. Longterm consumption of infant formula containing live probiotic bacteria: tolerance and safety. Am J Clin Nutr 2004;79:261–267. 21. Schaeken MJ, van der Hoeven JS, Franken HC. Comparative recovery of Streptococcus mutans on five isolation media, including a new simple selective medium. J Dent Res 1986;65:906–908. 22. Shermak MA, Saavedra JM, Jackson TL, Huang SS, Bayless TM, Perman JA. Effect of yogurt on symptoms and kinetics of hydrogen production in lactose-malabsorbing children. Am J Clin Nutr 1995;62:1003–1006. 23. Stecksén-Blicks C, Sjöström I, Twetman S. Effect of longterm consumption of milk supplemented with probiotic lactobacilli and fluoride on dental caries and general health in preschool children: a cluster-randomized study. Caries Res 2009;43:374–381. 24. Teughels W, Van Essche M, Sliepen I, Quirynen M. Probiotics and oral healthcare. Periodontol 2000 2008;48:111–147.

Oral Health & Preventive Dentistry

Effect of Chocobar Ice Cream Containing Bifidobacterium on Salivary Streptococcus mutans and Lactobacilli: A Randomised Controlled Trial.

To examine the effect of chocobar ice cream containing bifidobacteria on salivary mutans streptococci and lactobacilli...
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