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Common Oral Diseases and Epidemiology Research Center, Prince of Songkla University, Hat-Yai, 90112, ThailandDepartment of Conservative Dentistry, Prince of Songkla University, Hat-Yai, 90112, Thailand
Common Oral Diseases and Epidemiology Research Center, Prince of Songkla University, Hat-Yai, 90112, ThailandDepartment of Conservative Dentistry, Prince of Songkla University, Hat-Yai, 90112, Thailand
Common Oral Diseases and Epidemiology Research Center, Prince of Songkla University, Hat-Yai, 90112, ThailandDepartment of Stomatology, Faculty of Dentistry, Prince of Songkla University, Hat-Yai, 90112, Thailand
The aims of this study were to evaluate whether short-term consumption of fermented milk containing Lactobacillus rhamnosus SD11 affected levels of oral microbiota in vivo and whether L. rhamnosus SD11 could colonize in the human mouth. We also monitored for potential side effects of the probiotic. The applicability of using L. rhamnosus SD11 compared with Lactobacillus bulgaricus as a starter culture for fermented milk was evaluated. After informed consent, 43 healthy young adults were recruited and randomly assigned to either the probiotic or control group and received fermented milk containing L. rhamnosus SD11 or L. bulgaricus, respectively, once daily for 4 wk. The numbers of mutans streptococci, lactobacilli, and total bacteria in saliva were counted at baseline and then after 4 and 8 wk. An oral examination was performed at baseline and after 8 wk. The persistence of L. rhamnosus SD11 was investigated by DNA fingerprinting using arbitrary primer-PCR. Results demonstrated that statistically significant reductions in mutans streptococci and total bacteria were observed in the probiotic group compared with the control group, and the number of lactobacilli was significantly increased in both groups after receiving fermented milks. Lactobacillus rhamnosus SD11 could be detected (in >80% of subjects) up to 4 wk following cessation of dosing among subjects in the probiotic group. No side effects were reported. Thus, L. rhamnosus SD11 could be used as a starter culture for fermented milk. Daily consumption of L. rhamnosus SD11-containing fermented milk for 4 wk may have beneficial effects on oral health by reducing salivary levels of mutans streptococci. The probiotic was apparently able to colonize the oral cavity for a longer time than previously reported. However, the potential benefits of probiotic L. rhamnosus SD11 on oral health require further evaluation with a larger group of volunteers in a longer-term study.
Probiotics have been used for decades in fermented products such as fermented milk and it is accepted that probiotics can provide a health benefit, such as in the prevention and treatment of gastrointestinal and immunological disorders (
). In our previous studies that screened potential probiotic strains of Lactobacillus strains derived from caries-free subjects, we found that some strains, for example, Lactobacillus paracasei SD1 and Lactobacillus rhamnosus SD11 (previously identified as Lactobacillus fermentum SD11), exhibited strong activity in inhibiting growth of oral pathogens by producing antimicrobial substances (
), it was shown that some strains, especially L. rhamnosus strains, could adhere well to human oral mucosa. Moreover, a reduction of cariogenic pathogens and caries risk has been demonstrated in the clinical trials of L. paracasei SD1 (
). Results confirm that a selection process to obtain a probiotic strain with good properties is needed. No randomized controlled clinical trial has yet explored the effect of L. rhamnosus SD11 on oral microbiota or its safety. Thus, the aims of this preliminary study were to evaluate whether administration of fermented milk containing L. rhamnosus SD11 affects the levels of oral microbiota in vivo and whether L. rhamnosus SD11 could colonize in the human mouth. We also evaluated potential side effects of the probiotic. The applicability of using L. rhamnosus SD11 as a starter culture for fermented milk was compared with use of L. bulgaricus.
MATERIALS AND METHODS
Bacterial Strains, Culture Conditions, and Fermented Milk Preparation
Lactobacillus rhamnosus SD11, a strain selected from caries-free subjects, was previously identified as L. fermentum SD11 using PCR-RFLP analysis of 16S rRNA gene profiles and protein profiles of SDS-PAGE (
). The identity of the strain was further confirmed as L. rhamnosus SD11 by its 16S rRNA gene sequences, the presence of a specific band at the same level of Lactobacillus rhamnosus GG using denaturing gradient gel electrophoresis with primers of CARP according to
For fermentation, the starter culture L. rhamnosus SD11 strain or a traditional Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) strain (Thermophilic yogurt culture FD-DVS YC-380-Yo-Flex, Chr. Hansen, Hørsholm, Denmark) was cultured as follows. The strain was recovered from storage at −80°C on de Man, Rogosa, and Sharpe (MRS) agar plates and inoculated into 50 mL of MRS broth overnight under anaerobic conditions (80% N2, 10% H2, and 10% CO2) at 37°C. The culture was then added to 450 mL of MRS broth and kept under anaerobic conditions at 37°C for 48 h. Cells were harvested by centrifugation (3,000 × g, 5 min) from the MRS broth and washed 3 times with 0.85% NaCl before being used.
The fermented milks were prepared by the Dairy Home Industries Company, Nakornrachasrima, Thailand, and consisted of fresh milk fermented with L. rhamnosus SD11 or L. bulgaricus as a starter culture for the probiotic and control fermented milks, respectively. Fresh milk (1 L) was inoculated with L. rhamnosus SD11 or L. bulgaricus (1050 cfu/L) and then incubated at 45°C for 6 h. Fermented milks were preserved at 4°C, and were examined for viable counts and pH values using a pH meter (Cyberscan pH 1000, Eutech Instruments Ltd., Singapore) every month for 6 mo.
), in which we first studied L. paracasei SD1. We calculated that there would be an estimated 80% power at the 0.05 level of significance using 2-sided testing. Fifteen participants per group were needed to allow for 30% dropouts. A total sample size of at least 40 participants (20 subjects/group) was judged necessary for this study. In total, 44 subjects provided the consent forms, and 1 subject was excluded due to a history of systemic disease. Thus, the study group comprised 43 healthy nonmedicating adolescents (31 women and 12 men), 20 to 25 yr of age (mean ± SD: 21.86 ± 0.83 yr) who volunteered after giving informed consent.
To be considered for invitation, subjects had to have caries in ≤2 teeth, an absence of periodontal disease, to be nonsmoking, and have daily tooth-brushing habits using a fluoride-containing toothpaste. The exclusion criteria were (a) having habitual consumption of probiotics or xylitol, (b) having systemic antibiotic medication taken within 6 wk, (c) having an allergy to cow milk, lactose intolerance, or severe food allergy, (d) having systemic or severe chronic diseases, or (e) undergoing orthodontic treatment. All subjects underwent professional prophylaxis at the beginning of the study by a clinician.
All subjects were asked to immediately report any adverse side effects and to complete the questionnaire form after 4 wk of fermented milk consumption.
Oral examinations were performed for all subjects at baseline and at the end of the study (after 8 wk). The dental caries status (DMFT; decayed, missing, filled index) was recorded by N. A. according to
The PI evaluates the plaque revealed on the buccal and lingual nonrestored surfaces of the teeth on a scale of 0 to 5, where 0 = no plaque; 1 = isolated flecks of plaque at the gingival margin; 2 = a continuous band of plaque up to 1 mm at the gingival margin; 3 = plaque greater than 1 mm in width and covering up to one-third of the tooth surface; 4 = plaque covering from one- to two-thirds of the tooth surface; and 5 = plaque covering more than two-thirds of the tooth surface. The GI scores the marginal and interproximal tissues separately on the basis of 0 to 3, where 0 = normal gingiva; 1 = mild inflammation, slight change in color and slight edema but no bleeding on probing; 2 = moderate inflammation with redness, edema, and glazing and bleeding on probing; and 3 = severe inflammation, marked redness and edema, and ulceration with tendency to spontaneous bleeding.
Subjects were randomly assigned to the probiotic or control group and drank 100 mL of fermented milk containing probiotic L. rhamnosus SD11 or L. bulgaricus, respectively, once daily for 4 wk under observation by a clinician. The content of the drink (control or probiotic) was unknown to the subjects and to the clinician responsible for the saliva samplings. The study was blinded until the time of statistical calculations.
Saliva samples were taken at baseline (T0), 4 wk (T4), and 8 wk (T8) of the study period using an oral rinse method with 10 mL of phosphate buffer solution (
). The typical colony counts of salivary mutans streptococci and lactobacilli were evaluated using the selective media mitis salivarius bacitracin agar and MRS, respectively. Five to 10 colonies of lactobacilli on MRS plates were collected, purified, and kept at −80°C for monitoring of L. rhamnosus SD11.
For total bacterial counts, salivary samples were cultured on 5% blood brain heart infusion agar for aerobic bacteria and on 5% blood brain heart infusion agar supplemented with vitamin K and hemin for anaerobic bacteria. All plates were incubated under appropriate conditions at 37°C for 24 to 48 h.
Persistence of L. rhamnosus SD11 In Vivo
The persistence of L. rhamnosus SD11 was traced using arbitrary primer (AP)-PCR, with the enterobacterial repetitive intergenic consensus (ERIC) primers ERIC1R: forward (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC2: reverse (5′-AAGTAAGTGACTGG GGTGAGCG-3′). This method is useful for genotyping of Lactobacillus species and a unique DNA fingerprinting pattern is found in each individual subject (
). Thus, DNA fingerprinting was analyzed to demonstrate persistence of L. rhamnosus SD11 among the Lactobacillus strains isolated from saliva of volunteers in both groups.
Samples of DNA of strains were prepared using a Genomic DNA Extraction Kit (RBC Bioscience, Taipei, Taiwan) following the manufacturer's protocol for gram-positive bacteria. The reaction in a 50-μL mixture consisted of 100 ng of DNA template, 1.0 μmol/L (each primer), 1× buffer with 2.0 mmol/L MgCl2, 1.0 U of Taq polymerase, and 0.2 mmol/L of each dNTP. The mixture was subjected to 35 cycles of denaturation at 95°C for 1 min, ramping to 35°C in 3 min, annealing at 35°C for1min, followed by extension at 74°C for 2 min, and a final extension at74°C for 5 min. The PCR products were run on a 7.5% polyacrylamide gel and stained with silver staining.
Analysis of Data
All numerical data are presented as means and standard deviations (means ± SD). The general characteristics of the volunteers between the probiotic and group at T0 (age, pH of saliva, DMFT, GI, and PI) were analyzed using the Mann-Whitney U-test, and sex was analyzed using the chi-squared test. The colony counts of mutans streptococci, lactobacilli, and total bacteria were presented as log10 cfu/mL and the comparison in the same group between T0 and T8 was analyzed using one-way ANOVA followed by the post hoc test (Bonferroni). Analysis between groups at the same time point was conducted using an independent samples t-test. The comparison of parameters (DMFT, GI, and PI) in the same group between T0 and T8 and between the groups at T0 and T8 were analyzed using the Wilcoxon signed-rank test and the Mann-Whitney U-test, respectively. The software package used was SPSS (SPSS Inc., Chicago, IL), and differences were considered significant P < 0.05.
A total 43 subjects (31 women, 12 men; 20 to 25 yr old) fulfilled the criteria, gave informed consent, and were included in the clinical trial. No subjects dropped out during the study, and details of study activities are given in a flowchart (Figure 1). The baseline characteristics for age, sex, salivary pH, bacterial counts, and clinical parameters between control and probiotic groups did not differ (P > 0.05).
The counts of mutans streptococci, lactobacilli, and total bacteria in saliva from T0 to T8 are given in Table 1. We detected no difference between the probiotic and control groups at T0. Total bacterial counts (both aerobic and anaerobic bacteria) and mutans streptococci counts were significantly (P < 0.01) lower in the probiotic group than in the control group at T4 and T8 (subjects consumed the fermented milk until T4). In the probiotic group, the mutans streptococci count was decreased (P = 0.01), whereas lactobacilli counts increased (P = 0.04) at T4 compared with T0. The number of lactobacilli in the control group also increased (P < 0.01) at T4 compared with T0; however, it decreased (P = 0.038) at T8 compared with T4 in the control group.
Table 1Mean counts (log10 cfu/mL) of microbiota of volunteers in both groups at different times
Probiotic and control groups consumed 100 mL/d for 4 wk of fermented milk containing Lactobacillus rhamnosus SD11 or Lactobacillus bulgaricus, respectively, as a starter culture. Saliva samples were collected at baseline (T0), 4 wk (T4), and 8 wk (T8) for bacterial counts.
Means within a time point (T4 or T8) differ between probiotic and control groups (P < 0.01; independent samples t-test).
a,b Means within a time point (T4 or T8) differ between probiotic and control groups (P < 0.01; independent samples t-test).
A,B Means within a probiotic group differ between T0 and T4 [P < 0.05; one-way ANOVA followed by post hoc test (Bonferroni)].
X,Y Means within control group differ between T4 and T8 [P < 0.05; one-way ANOVA followed by post hoc test (Bonferroni)].
1 Probiotic and control groups consumed 100 mL/d for 4 wk of fermented milk containing Lactobacillus rhamnosus SD11 or Lactobacillus bulgaricus, respectively, as a starter culture. Saliva samples were collected at baseline (T0), 4 wk (T4), and 8 wk (T8) for bacterial counts.
Comparison of clinical parameters of volunteers in both groups between T0 and T8 are shown in Table 2. All clinical parameters (DMFT, GI, and PI) in the probiotic group improved slightly after receiving fermented milk, although this change was not significant (P > 0.05).
Table 2Clinical parameters including gingival index (GI), plaque index (PI), and dental caries status (DMFT; decayed, missing, filled index) of volunteers in groups at different times
Probiotic and control groups consumed 100 mL/d for 4 wk of fermented milk containing Lactobacillus rhamnosus SD11 or Lactobacillus bulgaricus, respectively, as a starter culture. Saliva samples were collected at baseline (T0), 4 wk (T4), and 8 wk (T8) for bacterial counts. Indexes are defined in the Oral Examination section.
0.79 ± 0.38
0.82 ± 0.39
0.66 ± 0.38
0.89 ± 0.42
2.16 ± 0.27
2.14 ± 0.20
2.07 ± 0.45
2.02 ± 0.33
3.0 ± 3.2
3.1 ± 3.0
3.0 ± 3.2
3.1 ± 3.0
1 Probiotic and control groups consumed 100 mL/d for 4 wk of fermented milk containing Lactobacillus rhamnosus SD11 or Lactobacillus bulgaricus, respectively, as a starter culture. Saliva samples were collected at baseline (T0), 4 wk (T4), and 8 wk (T8) for bacterial counts. Indexes are defined in the Oral Examination section.
Lactobacillus rhamnosus SD11 was detected in more than 80% of the subjects in the probiotic group at T8, and examples of the DNA fingerprint profiles of L. rhamnosus SD11 in probiotic subjects are shown in Figure 2. No side effects were reported in any group.
To examine the possibility of using L. rhamnosus SD11 as a starter culture for fermented milk, we monitored viable counts and pH values of fermented milk containing L. bulgaricus or L. rhamnosus SD11 for 6 mo, and the results are shown in Table 3. We detected no difference in viable counts or pH values in the fermented milks over 6 mo; however, the pH of fermented milk containing L. bulgaricus was slightly lower than that of L. rhamnosus SD11 after 1 mo.
Table 3Mean counts (log10 cfu/mL; ± SD) of Lactobacillus strains and pH values (± SD) during 6 mo of storage of fermented milks
Recently, research on probiotics relating to oral health has received a great deal of attention. According to previous studies, probiotic interventions in the oral cavity are considered to exert their effect not only on oral pathogens but also on clinical parameters. Review articles and meta-analysis have shown that probiotic strains in different forms could reduce the number of cariogenic pathogenic mutans streptococci and reduce the risk of dental caries (
). It is important to recognize that the health effects of probiotics can be strain-specific, thus different results may occur from the different strains used.
This is the first study of L. rhamnosus SD11, a strain that originated from the human oral cavity, and its effect in an in vivo study. Our results indicated a significant reducing effect of the L. rhamnosus SD11 on both salivary mutans streptococci and total bacterial counts. This finding is in agreement with previous studies with fermented milks containing lactobacilli or bifidobacteria (
). Fermented milk was chosen in this study because it has a long history of beneficial health effects in humans; for examples, in improving lactose digestion in lactose-intolerant people, affecting intestinal transit time, and stimulating the gut immune system (
). It is also a popular dairy product to deliver adequate numbers of viable probiotic cells to consumers; it is recommended that at least 100 g/d of fermented milk containing at least 106 cfu/mL be consumed regularly to gain a “probiotic effect” (
). Therefore, survival of probiotics during shelf life and until consumption is an important consideration. The probiotic strain being preserved with fermented milk was supported by the present study and other studies, all of which used fermented milk as the carrier for probiotic strains, and all demonstrated that probiotics helped reduce counts of mutans streptococci (
). This indicates that fermented milk containing the probiotic L. rhamnosus SD11 may be an appropriate carrier food for reduction of mutans streptococci. Survival of a probiotic strain in the host as well as in the carrier is generally considered a key feature for probiotics to preserve their health-promoting effects.
Among previously studied Lactobacillus rhamnosus strains, L. rhamnosus GG and L. rhamnosus LC 705 were commonly strains used in clinical trials for dental caries prevention (
); both are industrial strains that have been used routinely for decades as adjunct starter cultures in dairy products. To our knowledge, these strains and traditional fermented milk starters (Lactobacillus bulgaricus) have non-oral human origins; therefore, they may not have the ability to adhere to oral epithelial cells. It has been suggested that a “probiotic must be taken in sufficient amounts on a daily basis, since probiotic strains do not permanently colonize the oral cavity or intestines” (
showed that counts of the probiotic LGG (a strain isolated from the intestinal tract of a healthy human) were increased during the consumption period; however, counts were not maintained following cessation of consumption. This might be interpreted as a probiotic failure in bacterial competition for its colonization. The present study showed that lactobacilli increased in both groups during the intervention trial (T4), which was expected because subjects in both groups were consuming fermented milks containing lactobacilli. However, the counts of lactobacilli were slightly higher in the probiotic group than in the control group. A significant decrease in lactobacilli count was found in the control group at T8 compared with T4. This implied that L. rhamnosus SD11, originating from human oral cavity, may be better able to adhere or compete for colonization in the oral cavity than L. bulgaricus. Lactobacillus rhamnosus SD11 persisted in the probiotic group (>80% of subjects) up to 4 wk following cessation of dosing, whereas L. rhamnosus GG could be detected in only 3.7% of subjects after 7 d of intervention (
). These findings indicate that L. rhamnosus SD11 may be an appropriate probiotic strain for oral the cavity because of its ability to adhere to human oral mucosa.
The reason for the effect of probiotic L. rhamnosus SD11 on decreasing mutans streptococci and total bacteria counts is not fully known but might result from a combination of the strain's abilities to produce bacteriocin (
). In the current study, no significant change in clinical parameters (GI, PI, and DMFT) was found in either group. This was expected because the study was conducted over a short time and all subjects underwent professional prophylaxis at the beginning of the study. However, the short trial was sufficient to monitor the change in oral microbiota—the primary outcome. A study lasting at least 1 yr would be required to monitor the progression of dental caries, especially in permanent teeth, and that is planned for our further study.
No side or adverse effects were reported during the trial, indicating the safety of the strain used. However, it should be noted that the sample size was limited, subjects were healthy young adults, and the study was conducted with a short intervention time.
Although not the main purpose of this evaluation, we also examined the possibility of using L. rhamnosus SD11 as a starter culture for fermented milk. Results showed no significant differences in terms of viable lactobacilli counts and pH values for L. rhamnosus SD11 compared with L. bulgaricus. After 6 mo of storage, the fermented milk met the quality requirement of viable lactobacilli, having more than 106 cfu/mL (
). Results indicate that L. rhamnosus SD11 could be used as a starter culture for fermented milk.
Lactobacillus rhamnosus SD11 has potential as a good probiotic strain. The daily consumption of fermented milk containing L. rhamnosus SD11 for 4 wk may have beneficial effects on oral health by reducing the salivary levels of mutans streptococci, and L. rhamnosus SD11 was apparently able to colonize the oral cavity longer than reported previously. However, the potential benefits of probiotic L. rhamnosus SD11 for oral health require further studies with a larger group of volunteers in a longer-term study.
This work was supported by the National Research Council of Thailand (Bangkok; DEN580217M) and the Prince of Songkla University research fund, Super clusters (Hat Yai, Thailand; DEN600729S).
Effect of chlorhexidine pretreatment on the stability of salivary lactobacilli probiotic in six- to twelve-year-old children: A randomized controlled trial.