Effect of bovine milk fermented with Lactobacillus rhamnosus L8020 on periodontal disease in individuals with intellectual disability: a randomized clinical trial

Abstract Studies on the oral health of individuals with intellectual disability (ID) have identified problems that include a high prevalence of periodontal disease. The use of probiotics to treat periodontal disease has been the focus of considerable research, and bovine milk fermented with Lactobacillus rhamnosus L8020 (L8020 yogurt) has been shown to reduce the oral prevalence of four periodontal pathogens. Objective The aim of this randomized, double-blind, placebo-controlled trial was to compare the effects of L8020 yogurt (test group) with those of placebo yogurt (placebo group) on the papillary-marginal-attached (PMA) index, gingival index (GI), and probing depth (PD) in 23 individuals with ID. Methodology All patients were required to consume the allocated yogurt after breakfast for 90 days. PMA index and GI scores as well as PDs were assessed before the start of yogurt consumption (baseline), after 45 and 90 days of consumption, and 30 days after the cessation of consumption. Student’s t-test, Mann–Whitney U test or Fisher’s exact test was used for inter-group comparisons, and the mixed effect model of repeated measurements was used for data analysis. Results The decrease in PMA index score was significantly greater in the test group than in the placebo group (p<0.001). The GI score also decreased during the study, with a tendency for greater decrease in the test group. Furthermore, decreases in PD between baseline, 45 and 90 days tended to be greater in the test group than in the placebo group. Conclusion These results suggest that regular consumption of bovine milk fermented with L. rhamnosus L8020 can lower the risk of periodontal disease in individuals with ID.


Introduction
Intellectual disability (ID) is defined as subaverage general intellectual function and is associated with impaired adaptive behavior. 1 Many researchers have reported that individuals with ID have worse oral hygiene and higher rates of dental disease than ordinary individuals. 2-4 Reported oral healthrelated problems in individuals with ID include poor oral hygiene, progressed dental caries, and a high prevalence of periodontal disease. 5 The oral health status of these patients is affected by their patterns of cognition, developmental age, 6 and other factors such as age, the personality and compliance of the caregiver, and the presence of underlying diseases, including physical disabilities. 2 Individuals with ID experience difficulty in understanding the importance of oral health and tend to co-operate less for oral cleaning procedures. Because maintenance of a healthy oral environment in patients with ID is not easy, 7 available measures for the prevention of oral disease and improvement of oral hygiene in these individuals are limited, 8 although necessary. 9 The Food and Agriculture Organization/World Health Organization defines probiotics as live microorganisms that confer a health benefit to the host (i.e. improve the microbial balance in the intestinal tract) when consumed in adequate amounts (in food or as dietary supplements). 10,11 The use of probiotics for the treatment of periodontal disease has been the focus of considerable research. 12,13 Organisms commonly associated with periodontal disease include Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, Dialister pneumosintes, Campylobacter rectus, Fusobacterium spp, Selenomonas sputigena, Parvimonas micra, and spirochetes such as Treponema denticola. 14 Nikawa,et al. 15 (2004)

demonstrated that yogurt containing
Lactobacillus rhamnosus L8020 (L8020 yogurt), which has been detected as a normal strain of bacteria in the oral cavity, 16 significantly lowered the risk of dental caries, suggesting that daily consumption of this probiotic organism may be useful in the prevention of caries. L. rhamnosus L8020 is also known to be active against a broad spectrum of bacteria that cause periodontal disease. 16 Specifically, L8020 yogurt has been shown to decrease the oral carriage of four known periodontal pathogens, namely P. gingivalis, P. intermedia, T. forsythia, and Fusobacterium spp. 16 Although mechanical removal of plaque biofilm by scaling and root planing is an essential aspect of periodontal therapy, the complete removal of plaqueretentive factors is not always possible, 17 particularly for individuals with ID, who are mostly noncompliant with dental treatment. The use of probiotics to promote oral health has been reported in a few studies, 18,19 with the probiotics used being mostly derived from the intestine. However, data on the effects of probiotic bacteria on periodontal pathogens are limited. 16 The aim of this randomized controlled trial was to determine the effect of L8020 yogurt on the papillary-marginalattached (PMA) index, 20

Methodology Study design
Twenty-three outpatients with ID were enrolled in this randomized, double-blind, placebo-controlled trial.
The patients were randomly allocated to a L8020 yogurt group (n=12) or a placebo yogurt group (n=11, after one patient withdrew consent before randomization; Figure 1). All patients were required to consume 80 g of the allocated yogurt after breakfast in the morning for 90 days. During this time, they were instructed to refrain from toothbrushing or consuming any food or beverage for 1 h after consumption of the yogurt. The patients were assessed at baseline (before the start of daily yogurt consumption), after 45 and 90 days of yogurt consumption, and 30 days after the cessation of yogurt consumption (120 days after the start of consumption). Simple randomization and centralized allocation methods were used. A random number    Results Written informed consent was obtained for 24 patients before randomization. Consent was withdrawn for one patient who was not accustomed to eating breakfast every day. Twenty-three patients completed the study after randomization to the test (n=12) and placebo (n=11) groups and were consequently included in our analysis (Figure 1). There were no significant differences in the baseline characteristics Data are expressed as means ± standard errors of the mean *p<0.05, **p<0.01 vs. time 0; †p<0.05, ‡p<0.01 vs. placebo p=0.050; Figure 2D].

Discussion
In this study, we found that regular consumption of bovine milk fermented with L. rhamnosus L8020 (L8020 yogurt) can lower the risk of periodontal disease in individuals with ID.
The number of individuals with ID is increasing worldwide, including Japan, because of population growth, increased reporting and more accurate methods for detection and diagnosis. 24  In our study, there was a decrease in PD after 90 days of continuous yogurt consumption in both experimental groups. We speculate that there may be two explanations for the decrease in PD in the patients who consumed the placebo yogurt that did not contain L. rhamnosus L8020. First, all study participants were instructed to refrain from toothbrushing or consuming any food or beverage for 1 h after consumption of the allocated yogurt in order to maximize the amount of time for which the probiotic bacteria in the L8020 yogurt remained in the oral cavity. Also, the participants' guardians or caregivers were instructed to brush their teeth at a specific time in the morning before yogurt consumption, so daily toothbrushing was performed for all participants in both study groups, including those who were not accustomed to it. Second, Lactobacillus strains are commonly used probiotics, 16 and it is possible that Lactobacillus spp. contained in the placebo yogurt also influenced periodontal bacteria. Further research using nextgeneration sequencing is necessary to elucidate the changes that occur in the oral microbiota in response to continuous consumption of yogurt containing different types of probiotics.
This study has some potential limitations, including the limited observation period of 120 days, the relatively short yogurt consumption period of 90 days, and the small sample size. As this study was a preliminary rather than a confirmation study investigating the clinical effect of L8020 yogurt, there was no previous information with which to determine an adequate sample size. On the basis of a similar previous study, 29 we calculated that 15 individuals with ID who were otherwise healthy would be required to ensure an adequate sample size. However, our trial was performed on patients with severe ID, and we were thus limited in the recruitment of participants who met the study criteria. In particular, among the 1109 outpatients who regularly visit our office in the Hiroshima area, it was extremely difficult to find individuals with severe ID who were available for irregular visits and repeated dental examinations. test group and -4.1±5.3 in the placebo group, meaning that our sample size was appropriate for determining differences between the two groups with a power of 80.2%. The preliminary results presented here remain to be confirmed in additional subjects in a future study.
Although probiotics in the microbiota of the human oral cavity, if compatible with the ecosystem, are likely to be effective in preventing periodontal disease, 29 the minimum duration of consumption before the development of changes in the oral microbiota remains unclear. The difference in PD between baseline and 45 days and between baseline and 90 days in our test group indicates that PD decreases with an increase in the duration of yogurt consumption; however, it remains unknown whether long-term consumption of L8020 yogurt has additional benefits or a ceiling effect. Therefore, toothbrushing remains the most important preventive and therapeutic strategy for periodontal disease, and probiotics should only be used as a supplementary measure. Patients, parents, and caregivers should be warned that the consumption of L8020 yogurt alone cannot replace toothbrushing as a measure for the maintenance of oral health.

Conclusions
In conclusion, the consumption of bovine milkderived L. rhamnosus L8020 yogurt for 90 days resulted in significantly greater changes in the PMA index than the consumption of placebo yogurt for the same duration; moreover, it tended to decrease GI score and PD. Our findings suggest that L. rhamnosus L8020 may be a useful probiotic organism when included in products that are ingested daily. However, further investigation is needed to elucidate the effect of L. rhamnosus L8020 on the oral microbiota and clarify the limits of the effects of this probiotic organism on periodontal disease.

Ethical approval
The study protocol was approved by the Ethical