01

Inhibitory effect against Porphylomonas gingivalis

I've been asked whether "is yogurt suppressing dental caries no longer available?" from my friends belong to Japan Dental Association and/or juniors' dentists.
To discover novel lactobacilli, we and students form Biology Technology Research club Bi-tech (Advisor Prof. Hiroki Nikawa) , and isolated 42 strains of lactobacilli from saliva provided by the volunteers (n=13) who had no caries-treatment experience. We then found out a lactobacillus which has a strong inhibitory activity against P. gingivalis.

02

Inhibitory effect against mutans streptococci

We have picked up the isolates with high antimicrobial activity against both S. mutans and S.sobirinus, among the strains with strong antimicrobial activity against P.gingivalis.

03

Inhibitory effect on C.albicans GDH18 strain

By investigating antimicrobial action of L8020 against C. albicans, we determined that acid is not the main cause for the effect.

04

Effects of bovine milk fermented with Lactobacillus rhamnosus L8020 (8020 yogurt) and placebo-fermented milk (placebo yogurt) on Streptococcus mutans and Streptococcus sobrinus in vitro

Streptococcus thermophilus was added to a large amount of the frozen stock culture of Lactobacillus rhamnosus L8020 to prepare the 8020 yogurt. The placebo yogurt was prepared with Lactobacillus bulgaricus and Streptococcus thermophilus, which are widely used in fermented milk products in Japan. Standard fermentation candidates were used. The 8020 yogurt contained approximately 1.5 • 10⁹ lactic acid bacteria in total, including 1.4–2.5 • 10⁸ L8020 cells. The placebo yogurt contained approximately 1.5 • 10⁹ lactic acid bacteria. The final pH values of the 8020 yogurt and placebo yogurt were 4.35 and 4.37, respectively.

The inhibition assay, using each yogurt variety, was conducted as follows: 500 lL of TSBY was dispensed into each well of the multiwell tissue culture plates (NunclonR Delta; Nunc, Kamstrup, Denmark). An intercell (pore size 0.22 lm; Kurabo, Osaka, Japan), into which 50 mg (49.96 ± 0.07 mg) of either 8020 yogurt or placebo yogurt was placed, was then inserted into the well. Subse- quently, 50 lL streptococcal suspension was dispensed into each well of the multiwell tissue culture plates and incubated for 24 h at 37°C. The control intercell con- tained the same volume of TSBY. After incubation, the amount of ATP of streptococci grown in the well of the multiwell tissue culture plates was quantified using a bio- luminescence apparatus.

The assays were carried out on at least two indepen- dent occasions, with quadruplicate samples on each occasion. All the numerical data obtained were analyzed by ANOVA and Tukey's multiple range test at a 1% level.

* The vertical axis of the graph shows the amount of ATP（pmol/well）

Inhibitory effects of 8020 yogurt in vitro
As shown in Figure, the 8020 yogurt demonstrated a significant inhibitory effect against either S. sobrinus B13 (ANOVA, P < 0.01) or S. mutans Ingbritt (ANOVA, P < 0.01), whereas the placebo yogurt showed no effects (P > 0.05).

* "Placebo" is an ordinary yoghurt that does not contain L8020 bacteria.

05

Effects of bovine milk fermented with Lactobacillus rhamnosus L8020 (8020 yogurt) and placebo-fermented milk (placebo yogurt) on the oral carriage of mutans streptococci and periodontal pathogens

A total of 50 healthy participants (age: 21.3 ± 1.7; 12 males and 38 female) were divided into two groups. No participant had active caries, symptoms of gingivitis or periodontal disease, smoking habit, or was on any medica- tion. This group did not include individuals who had tooth decay, a tooth under treatment, a tooth with restoratives (other than class I or class V restorations), and/or missing teeth. The saliva collections were carried out at least 2 h after lunch. The participants were asked to chew a piece of paraffin for 10 min, and the saliva produced was expectorated into a plastic container. The number of mutans streptococci was determined with conventional viable counts. Periodontal pathogens released in saliva, such as Pg, Prevotella intermedia (Pi), Tannerella forsythia (Tf), and Fusobacterium spp. (Fuso), were determined semiquantitatively using real-time poly- merase chain reaction by BML (Kawagoe, Japan).

A cohort of equal standing, in terms of oral health, comprised students at Hiroshima University. The partici- pants in the first group consumed a cup (128 g) of pla- cebo yogurt at lunchtime (12.00–13.00 hours) daily for a period of 2 weeks, and then consumed a cup of 8020 yogurt at lunchtime daily for another 2 weeks after the 3-week interval. The participants in the second group consumed the 8020 yogurt at lunchtime daily for 2 weeks, and then the placebo yogurt at lunchtime daily for another 2 weeks after the 3-week interval. Before and after consuming each yogurt variety, oral carriage levels of mutans streptococci and periodontal pathogens were determined, as described earlier. Both the participants and investigators were unaware of which yogurt contained Lactobacillus rhamnosus L8020 throughout the study. The data were analyzed by Wilco- xon test at a 1% level.

Double blind (cohort study)

For Streptococcus mutans, count the number of viable organism like last time and for the number of P.gingivalis, use CPR by invader technology.
* Both are commissioned analysis by BML,INC.

Changes in the amount of Streptococcus mutans in the oral cavity

Changes in the amount of P.gingivalis in the oral cavity

Results

Consuming the 8020 yogurt significantly reduced the oral carriage of mutans streptococci in each group (P < 0.01; Figure 5); these effects were not observed with the pla- cebo yogurt (P > 0.05).

The effects of the 8020 yogurt on the oral carriage of Pg are shown in Figure 6. The placebo yogurt did not ignificantly reduce the oral carriage of Pg (Wilcoxon t-test, P > 0.05), whereas the 8020 yogurt did (Wilcoxon t-test, P < 0.01), although the frequency of the oral car- riage of Pg was very low for eight of the 50 participants.

The frequency of the oral carriage of Pi was low for 24 of the 50 participants, and the 8020 yogurt significantly reduced the oral carriage of Pi. (Wilcoxon t-test, P < 0.01; Figure 7), but this phenomenon was not observed with the Placebo Yogurt (Wilcoxon t-test, P > 0.05). In the present study, Tf was recovered from all partici- pants, and the oral carriage of Tf was significantly reduced by the 8020 yogurt (Wilcoxon t-test, P < 0.01; Figure 8), but not by the placebo yogurt (P > 0.05).

Fuso was recovered from all the participants, and the oral carriage of Fuso was significantly reduced by the 8020 yogurt (Wilcoxon t-test, P < 0.01; Figure 9), but not by the placebo yogurt (P > 0.05).

Throughout the intervention, no symptoms of the occurrence of dental erosion or oral health problems were observed in the participants.

06

Ethical approval and informed consent

The protocols of the saliva sampling and cohort study using yogurt was approved by ethical authorities at Hiro- shima University. Informed consent was obtained from all the participants after explanation of both the merits and demerits of the protocol and samples used for the experiment. Because the yogurt included a high concen- tration of lactobacilli, which is known as a risk factor for dental caries, we examined the occurrence of dental ero- sion or oral health problems of each participant twice a week during the periods of the cohort tests to prevent other side-effects. All participants instructed not to take other products containing Lactobacillus or other pharma- ceutical lactic acid bacteria for 1 week prior to and throughout the intervention.