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. 2023 Aug 28;15(1):2250649.
doi: 10.1080/20002297.2023.2250649. eCollection 2023.

Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial

Shinji Sakata  1 , Yukiko Sakamaki  1 , Masahiro Yuki  1 , Tsutomu Sugaya  2 , Tatsuhiko Hirota  1
Affiliations

Screening of heat-killed lactic acid bacteria based on inhibitory activity against oral bacteria and effects of oral administration of heat-killed Ligilactobacillus salivarius CP3365 on periodontal health in healthy participants: a double-blinded, randomized, placebo-controlled trial

Shinji Sakata et al. J Oral Microbiol. .

Abstract

Objectives: The aims of this study were to select heat-killed lactic acid bacteria (HKL) with antibiotic activity and investigate the efficacy of this bacteria in maintaining periodontal parameters in healthy participants.

Materials and methods: An in vitro evaluation was conducted to assess the inhibitory efficacy of lactic acid bacteria against Porphyromonas gingivalis and Fusobacterium nucleatum subsp. nucleatum. The effects of HKL administration on various parameters (plaque control record, bleeding on probing, and probing pocket depth) were assessed in a randomized, placebo-controlled trial. Participants in the test and placebo groups (n = 32) consumed oral tablets containing placebo or HKL daily for 8 weeks. Oral bacteria in supra-plaque and saliva were identified using 16S rRNA gene community profiling analysis.

Results: Heat-killed Ligilactobacillus salivarius CP3365 significantly (p < 0.05) decreased the viability of oral bacteria and was selected for clinical trials. Administration of HKL CP3365 significantly (p < 0.05) inhibited increases in each parameter. No changes in the relative abundance of P. gingivalis or F. nucleatum subsp. nucleatum were detected by HKL CP3365, but the relative abundance of oral bacteria (genera Porphyromonas, Fusobacterium, and Haemophilus) was significantly (p < 0.05) decreased.

Conclusion: HKL CP3365 effectively inhibited oral bacteria growth and was useful for maintaining periodontal health.

Clinical trial registration: [https://www.umin.ac.jp/ctr/index.htm], identifier [UMIN000045656].

Keywords: Bleeding on probing (BOP); Ligilactobacillus salivarius CP3365; dental plaque; heat-killed lactic acid bacteria; probing pocket depth (PPD).

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Conflict of interest statement

S.S., Y. S., M.Y., and T.H. are employees of Asahi Quality and Innovations Ltd., which is affiliated with Asahi Group Holdings Ltd., and they prepared CP3365 and the placebo. T.S receives research consulting fees from Kyowa Trial Co., Ltd.

Figures

Figure 1.
Figure 1.
Bacterial viability measured using WST-8 assay. Porphyromonas gingivalis ATCC 33,277T and the HKLs were co-inoculated into GAM broth and incubated at 37°C for 30 min. The OD460 was then measured at 0 and 120 min. Saline buffer was used as the control. Data are the means of three independent experiments. Significant differences were observed between the control and the HKLs. (*p < 0.05; one-way ANOVA with Dunnett’s post hoc test).
Figure 2.
Figure 2.
Bacterial viability measured using WST-8 assay. Fusobacterium nucleatum subsp. nucleatum JCM 8532T and the HKLs were inoculated into GAM broth and incubated at 37°C for 30 min. The OD460 was then measured at 0 and 120 min. Saline buffer was used as the control. Data are the means of three independent experiments. Significant differences were observed between the control and the HKLs. (*p < 0.05; one-way ANOVA with Dunnett’s post hoc test).
Figure 3.
Figure 3.
Bacterial viability measured using WST-8 assay. Porphyromonas gingivalis ATCC 33,277T (a) and Fusobacterium nucleatum subsp. nucleatum JCM 8532T (b), and the heat-killed L Ligilactobacillus salivarius strains, were inoculated into GAM broth and incubated at 37°C for 30 min. The OD460 was then measured at 0 and 120 min. Saline buffer was used as the control. Heat-killed Lactobacillus crispatus JCM 1185T was the negative or weak positive control. Data are the means of three independent experiments. Significant differences were observed between the control and the HKLs. (*p < 0.05; one-way ANOVA with Dunnett’s post hoc test).
Figure 4.
Figure 4.
Consolidated standards of reporting trials (CONSORT) flow diagram.
Figure 5.
Figure 5.
Approximate relative abundance of > 75% of bacterial genera identified in saliva and supragingival plaque of healthy participants determined by using 16S rRNA gene sequencing.
Figure 6.
Figure 6.
Bacterial viability measured using WST-8 assay. Fusobacterium periodonticum JCM 12,991T (a), Porphyromonas pasteri JCM 30,531T (b), Haemophilus parainfluenzae ATCC33392T (c), and heat-killed Ligilactobacillus salivarius strains were inoculated into GAM broth and incubated at 37°C for 30 min. The OD460 was then measured at 0 and 120 min. Saline buffer was used as the control. Data are the means of three independent experiments. Significant differences were observed between the control and the HKLs. (*p < 0.05; one-way ANOVA with Dunnett’s post hoc test).
Figure 7.
Figure 7.
Stratified analysis of the periodontal parameters in the presence of Porphyromonas gingivalis. the numbers for participants were based on the existence of P. gingivalis in samples at 8 weeks (table S2). (a) PCR: plaque control record; (b) BOP: bleeding on probing; (c) PPD: probing pocket depth. p-values (inter-group) were determined using ANCOVA with adjustment for each baseline value. *: p < 0.05; Δ8 weeks (8 weeks–0 week) were evaluated using the Wilcoxon signed-rank test.
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Grants and funding

This study was funded by Asahi Groups Holdings, Ltd., Japan.

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