Heat-killed Lacticaseibacillus rhamnosus GMNL-464 reduces gingivitis through pathobiont co-aggregation in vitro and oral microbiota modulation in a randomized clinical trial

Gingivitis is a prevalent oral inflammatory condition that can progress to periodontitis and contribute to systemic diseases through dysbiotic oral microbiota. Modulating the oral microbiota represents a promising strategy for gingivitis management. This study aimed to identify a heat-killed Lacticaseibacillus rhamnosus strain (GMNL-464) with potent inhibitory activity against gingivitis-associated pathobionts and evaluate its clinical efficacy. Candidate heat-killed L. rhamnosus strains were screened based on their ability to co-aggregate with oral pathobionts. The inhibitory effects on Porphyromonas gingivalis growth, biofilm formation, and P. gingivalis -induced inflammatory responses were then assessed. Whole-genome sequencing was conducted to identify genes potentially involved in co-aggregation. A randomized, double-blind, placebo-controlled, two-period, two-sequence crossover clinical trial (NCT04190485) was performed in patients with mild-to-moderate gingivitis. Eighteen participants were randomly assigned to one of two treatment sequences and used GMNL-464 toothpaste for 4 weeks and placebo toothpaste for 4 weeks, separated by a 1-week washout, over a total study duration of 9 weeks. The planned sample size was 30; all 18 enrolled participants completed both treatment periods, providing 18 observations per treatment condition. The primary endpoint was the gingival index. Secondary endpoints included the plaque index, salivary secretory immunoglobulin A (sIgA), halitosis, a questionnaire assessment, and an analysis of oral microbiota. Changes in the composition of the oral microbiota in dental plaque and gingival crevicular fluid (GCF) were also analyzed. A p-value of less than 0.05 is considered statistically significant. Among 26 candidate strains, heat-killed GMNL-464 exhibited the strongest co-aggregation ability with periodontal pathobionts, including P. gingivalis and Fusobacterium nucleatum , and significantly outperformed Lacticaseibacillus paracasei GMNL-143. GMNL-464 effectively inhibited P. gingivalis growth and biofilm formation, and suppressed P. gingivalis -induced IL-1β and TNF-α production in THP-1 cells. Genomic analysis revealed unique genes encoding cell wall/membrane/envelope-associated proteins potentially contributing to its co-aggregation capacity. Clinically, GMNL-464 toothpaste significantly reduced gingival index scores and decreased the abundance of Porphyromonas spp. and P. gingivalis in dental plaque compared to placebo. Decreasing trends were also observed for Aggregatibacter actinomycetemcomitans , Filifactor spp., and F. nucleatum . Notably, plaque index scores were positively correlated with disease-associated Filifactor spp. and negatively correlated with health-associated Capnocytophaga spp. Heat-killed GMNL-464 is an effective functional ingredient for toothpaste, mitigating gingivitis through modulation of oral pathobionts and host inflammatory responses. These findings highlight its translational potential as a safe and novel strategy for oral health management. ClinicalTrials.gov, NCT04190485. Registered 03 December 2019 Prospectively registered.