CP3365 found to inhibit deterioration of periodontal parameters｜研究開発活動レポート｜研究開発

Periodontal disease (*1) is caused by bacterial infection that leads to inflammation of the gums (gingivitis) and dissolution of the bone supporting the teeth, resulting in loosening of the teeth (periodontitis). Periodontal disease is a major cause of tooth loss. The incidence of periodontal disease increases with age, and about 50% of people in their 40s and 50s are considered to have periodontal disease. In recent years, the relationship between oral health and general health has been elucidated, and research has indicated an association between periodontal disease and diabetes in particular. However, the Japanese public had not shown an adequate understanding of periodontal disease, nor could measures to combat it be described as sufficient. Nevertheless, following reports that the Japanese government had included Specific Consideration of Lifelong Dental Checkups (“Universal Dental Checkups”) in its 2022 Policy Package, the Japanese public’s interest in oral health care has grown. Prior to this trend, we developed a lactic acid bacterium-based postbiotic that helps to improve periodontal parameters (*2). Our research yielded findings that differ from the mechanism by which we initially thought lactic acid bacteria inhibited periodontal disease, so we report those findings here.

Key findings of the study

● The CP3365 strain of Ligilactobacillus salivarius (a species of lactic acid bacteria) was found to have bacteriostatic activity against Porphyromonas gingivalis, which is a bacterium that causes periodontal disease.
● The effects of the CP3365 strain of L. salivarius (denoted here simply as “L. salivarius CP3365”) on the periodontal status of individuals were verified in a double-blinded, randomized, placebo-controlled trial (*3).
● Ingestion of L. salivarius CP3365 by people with good oral health inhibits the deterioration of periodontal parameters.
● An examination of the behavior of oral bacteria revealed that L. salivarius CP3365 inhibited the deterioration of periodontal parameters even in participants in whom P. gingivalis was not present. A detailed examination of oral bacteria revealed that L. salivarius CP3365 maintained periodontal parameters by inhibiting bacteria other than P. gingivalis that are also associated with periodontal disease.

Comments from Asahi researchers

Shinji Sakata

Core Technology Laboratories, Asahi Quality & Innovations, Ltd.

I studied intestinal bacteria from when I was in university until I was a post-doctoral fellow. In the laboratory where I worked at the time, there were many researchers from major dairy manufacturers, some of whom commercially produced the lactic acid bacteria they had researched. I sensed the passion of those researchers and their keen interest in developing products, and I thought to myself that I, too, wanted to have a job where I could provide people with the fruits of my research in the form of new products, and that is what led me to join the Asahi Group.
The mainstream of lactic acid bacteria research involves the use of live bacteria. In contrast, the Asahi Group is pursuing research on the functionality of heat-killed lactic acid bacteria. Unlike live bacteria, there are fewer restrictions on the distribution and processing of heat-killed lactic acid bacteria, and this postbiotic can be used in a wider range of applications. We believe this characteristic will enable us to provide the world with this postbiotic.
Live lactic acid bacteria are known to be effective in preventing periodontal disease, but there have been few studies on heat-killed lactic acid bacteria even in this field. This is because heat-killed lactic acid bacteria were not expected to produce the same antibacterial substances as those produced by live lactic acid bacteria or to display antibacterial activity while still in the oral cavity. I felt that heat-killed lactic acid bacteria had unlimited potential, so I started screening strains of lactic acid bacteria in the belief they might be effective. This belief led to our results: a successful clinical trial of the CP3365 strain of L. salivarius. Looking ahead, I would like to help improve the health of people around the world while pursuing the unlimited potential of lactic acid bacteria.

Comments on the paper by a researcher collaborator

Tsutomu Sugaya

Professor, Hokkaido University Graduate School of Dental Medicine, Division of Oral Medical Science, Division of Oral Health Science

I specialize in periodontal disease with two main goals: to regenerate bone and other tissue lost due to periodontal disease and to stop the progression of periodontal disease. Periodontal disease not only causes tooth loss but is also said to greatly affect conditions such as diabetes, myocardial infarction, cerebral infarction, and dementia. Heavy emphasis has been placed on the prevention and treatment of periodontal disease to help people extend their healthy life spans. The results of this study showed that killed lactic acid bacteria were effective in inhibiting periodontal disease, and the mechanism is presumably by inhibiting the growth of bacteria other than P. gingivalis. Periodontal disease is known to progress as a result of P. gingivalis, but in recent years, we have become aware that other bacteria that coexist with P. gingivalis can reinforce its action, and researchers have begun focusing on the respective roles of all of these bacteria. The results of this study are reasonable because periodontal disease was inhibited by inhibiting the growth of other bacteria that play a supporting role. A mouthwash is sometimes used to inhibit the growth of these oral bacteria, but there are cases in which mouthwashes need to be used with caution, such as in patients who have difficulty swallowing. Thus, we look forward to effectively preventing periodontal disease with safe foods and beverages .
Oral and intestinal bacteria are known to be associated with many diseases, and it is anticipated that food companies will continue their efforts to improve the bacterial flora through food products.

Research Summary

We first selected L. salivarius CP3365 as a strain of lactic acid bacteria that displays bacteriostatic activity against P. gingivalis, which is an oral bacterium known to be involved in the exacerbation of periodontal disease. Next, we conducted a clinical trial of L. salivarius CP3365 in healthy participants and confirmed that it was effective at inhibiting the deterioration of periodontal parameters. That said, we expected P. gingivalis to be involved in the deterioration of periodontal parameters, but P. gingivalis was present in only about 30% of participants, and we found that each parameter deteriorated even in participants in whom P. gingivalis was not present. Thus, we performed a more detailed analysis of the oral flora and found that an increase in F. periodonticum, H. parainfluenzae, and P. pasteri, which are involved in the formation of dental plaque, was associated with the deterioration of periodontal parameters. Therefore, we investigated whether L. salivarius CP3365 actually inhibits the growth of those bacteria and found that it did. This revealed that L. salivarius CP3365 has action that maintains periodontal parameters by inhibiting plaque-forming bacteria.

Results

In an in vitro test (4), L. salivarius* CP3365 significantly reduced the biological activity of P. gingivalis

1-1) We used 28 species of lactic acid bacteria from RIKEN-BRC JCM and our collections to prepare heat-killed bacteria and evaluated their bacteriostatic activity against oral bacteria. Eight species were from the genus Lactobacillus, and 20 were from other genera. In an in vitro test, results indicated that 20 species of lactic acid bacteria outside the genus Lactobacillus significantly reduced the biological activity of P. gingivalis (Figure 1). L. salivarius was selected from among these 20 species because of its consistently high level of activity and growth in MRS broth.

1-2) To select the more promising strain, we evaluated the bacteriostatic activity of six strains of L. salivarius. The bacteriostatic activity of heat-killed Lactobacillus crispatus JCM 1185 ^T against oral bacteria served as a weak or negative control. Results indicated that all L. salivarius strains significantly reduced the biological activity of P. gingivalis (Figure 2). Because L. salivarius CP3365 grew faster than the other L. salivarius strains, it was used in the subsequent trial.

L. salivarius CP3365 prevented the deterioration of periodontal parameters in healthy participants

Participants were males and females between 20 and 70 years of age. Twenty-eight participants in the placebo group and 26 participants in the CP3365 group who met the screening criteria were selected. The study period was 8 weeks, and tablets were taken 3 times a day. At 0, 4, and 8 weeks, a dental hygienist evaluated the parameters of the PCR (*5), BOP (*6), and PPD (*7) as the primary outcomes. Participants were asked to record their self-assessments and oral management.

Table 1 shows the data on participants in the placebo group and the CP3365 group prior to the start of the clinical trial. Participants were assigned to the two groups so that all parameters would be equal. Periodontal parameters were measured. In the placebo group, all parameters significantly deteriorated over the 8 weeks of the trial compared to their levels at week 0. In the CP3365 group, in contrast, there were no changes after week 0, and deterioration was not noted (Figure 3).

After CP3365 or the placebo was ingested for 8 weeks, the relative abundance of the four genera of Porphyromonas (which included P. gingivalis), Neisseria, Fusobacterium, and Haemophilus decreased significantly in the CP3365 group compared to the placebo group.

3-1) After ingestion of CP3365 or the placebo for 8 weeks, the abundance of the genera Porphyromonas and Neisseria in periodontal plaque decreased substantially in the CP3365 group compared to the placebo group. Significant differences in the changes in that abundance were noted (Table 2, Figure 4). There were significant differences between groups in the abundance of the genera Fusobacterium and Haemophilus in saliva. The genus Porphyromonas includes P. gingivalis, so the copy number of P. gingivalis in supragingival plaque and saliva was determined with quantitative PCR. Significant differences in the abundance of P. gingivalis in the CP3365 group and the placebo group were not noted. In addition, P. gingivalis was detected at a low frequency (4 to 12%) in supragingival plaque.

3-2) Representative bacterial species with a high rate of detection and high level of abundance were selected (F. periodonticum, H. parainfluenzae, Neisseria sp., and P. pasteri) from the four genera. Quantitative PCR was used to confirm the abundance of these bacteria in supragingival plaque or saliva, and results confirmed that these bacteria were less abundant in the CP3365 group. To confirm that this was due to the bacteriostatic activity of L. salivarius CP3365, we evaluated the bacteriostatic activity of L. salivarius CP3365 against three species; this excluded Neisseria sp., for which strains were not available. L. salivarius, which includes the CP3365 strain, significantly reduced the rate ofbiological activity of all three species.

3-3) H. parainfluenzae has been detected in early oral biofilm formation (*8). It is considered a critical trigger for oral biofilm formation, producing an adhesin that allows it to aggregate with streptococci and adhere to the pellicle on the tooth surface. The abundance of P. pasteri increases in the middle stage of oral biofilm formation, and P. pasteri is thought to serve as a connecting link between the early and late stages of oral biofilm formation. F. periodonticum has been detected in late oral biofilm, and its adhesin is thought to be associated with the development of oral plaque. These three species were detected and highly abundant in almost all participants in this study, so we hypothesized that these three species of oral bacteria contributed to the deterioration in periodontal parameters in this study. In fact, L. salivarius CP3365 inhibited the activity of each genus (which included the three major bacterial species mentioned earlier), and results confirmed a decrease in their abundance in flora. L. salivarius CP3365 may directly prevent the formation of plaque by inhibiting the activity of various bacteria involved in plaque formation.

L. salivarius CP3365 improved periodontal parameters (ΔPCR, ΔBOP) even in the absence of P. gingivalis

P. gingivalis is a bacterium that causes periodontal disease. However, there were no differences in the abundance of P. gingivalis between the CP3365 group and the placebo group, so we investigated the presence and absence of P. gingivalis and changes in periodontal parameters. Participants were stratified into those with (+) or without (-) P. gingivalis in saliva and then analyzed (Figure 5). Results revealed that ΔPCR, ΔBOP, and ΔPPD deteriorated significantly in the placebo group in which P. gingivalis was absent. There was little change in any of the parameters in the CP3365 group, and differences in those parameters between the groups were noted.

Discussion

Based on these results, most of the exacerbation in periodontal parameters observed in the placebo group was caused by factors independent of P. gingivalis, and L. salivarius CP3365 kept periodontal parameters in the normal range by inhibiting the growth of multiple oral bacteria involved in this process. During the study period, participants were asked to continue brushing their teeth an average of two times per day, which was their normal brushing routine. Nonetheless, all periodontal parameters deteriorated in the placebo group, indicating that completely preventing plaque formation and periodontal disease with toothbrushing alone is difficult. L. salivarius CP3365 may have the potential to serve as a new form of oral care, either in combination with toothbrushing or to help people who have difficulty brushing their teeth for some reason (including infants and the elderly).

About the results of this research

This study was conducted by the researchers at the Core Technology Laboratories in Asahi Quality & Innovations, Ltd. (Head Office: Ibaraki, Japan; President: Yasushi NAGATOMI), responsible for the advanced research functions in the Asahi Group, under the umbrella of the Asahi Group Holdings Ltd. (Head Office: Tokyo, Japan; President: Atsushi KATSUKI) in collaboration with Professor Tsutomu SUGAYA at the Hokkaido University Graduate School of Dental Medicine (then Director General: Kiyohiro HOUKIN). This study was published in the international scientific journal Journal of Oral Microbiology on August 28, 2023. Asahi Quality & Innovations, Ltd. will continue to promote “research and development to provide excellent health materials” and contribute to pursuing customers’ health maintenance and life enrichment.

Original Paper

Sakata et al., Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacterial and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial. Journal of Oral Microbiology 15: 2250649 (2023), 10.1080/20002297.2023.2250649

Glossary

*1 Periodontal disease:
A disease in which the gums (gingiva) and bone (alveolar bone) that support the teeth are destroyed. P. gingivalis is known to exacerbate periodontal disease progression. The first step in the progression of periodontal disease is the accumulation of plaque on the teeth and gums, which causes the gums to swell and bleed easily due to the toxins produced by the bacteria contained in the plaque. As the disease progresses, the gums swell further due to inflammation, and periodontal pockets deepen. Thus, the bacteria in the plaque penetrate deeper into the pockets. Toxins from periodontal pathogens dissolve the bone and tissue supporting the teeth, causing teeth to loosen, the gums to recede, and the teeth to fall out. In addition, periodontopathic bacteria enter blood vessels and attach themselves to various organs, which is known to cause various systemic diseases such as heart disease, stroke, diabetes, and pneumonia.

*2 Periodontal parameters:
These are indicators used to determine the diagnosis of and a treatment plan for periodontal disease. In specific terms, PCR, BOP, and PPD are used as periodontal parameters.

*3 Double-blinded, randomized, placebo-controlled trial:
A trial in which participants are randomly divided into a treatment group and a control group, with neither the participants nor the doctors knowing which group the participants were assigned to. The treatment group is given a new drug, while the control group is given a placebo. The results are compared, and the efficacy of the new drug or treatment is evaluated. This allows the efficacy of the new drug or treatment to be accurately evaluated.

*4 in vitro test:
An evaluation conducted under artificially configured conditions in apparatus such as test tubes and incubators.

*5 Plaque control record (PCR):
An indicator of the state of cleanliness in the mouth. The PCR is a numerical indication of the percentage of plaque that remains after rinsing with a special disclosing [staining] solution.

*6 Bleeding on probing (BOP):
Bleeding when measuring pockets during an examination for periodontal disease.

*7 Probing pocket depth (PPD):
Measurement of the depth of a periodontal pocket.

*8 Oral biofilm formation:
This is the aggregation of microorganisms in the mouth. The film forms on the teeth and in the oral cavity, grows over time, and thickens as it matures, becoming plaque.

CP3365 found to inhibit deterioration of periodontal parameters