DataSheet_4_Limosilactobacillus (Lactobacillus) fermentum ALAL020, a Probiotic Candidate Bacterium, Produces a Cyclic Dipeptide That Suppresses the Periodontal Pathogens Porphyromonas gingivalis and Prevotella intermedia.zip

Periodontal disease develops as a result of oral microbiota in dysbiosis, followed by the growth of periodontal pathogens such as Porphyromonas gingivalis and Prevotella intermedia. In case of acute symptoms, antibacterial agents and disinfectants are administered, however the appearance of drug-resistant bacteria and allergies cause problems. In recent years, studies on the effects of probiotics have been conducted as an alternative therapy for periodontitis. However, the basic mechanism of the inhibitory effect of probiotic bacteria on periodontal disease has not been clearly elucidated. To clarify the antibacterial mechanism of probiotics against periodontal pathogens, we used Limosilactobacillus (Lactobacillus) fermentum ALAL020, which showed the strongest antibacterial activity against P. gingivalis and P. intermedia among 50 screened lactic acid bacteria strains. The antibacterial substances produced were identified and structurally analyzed. After neutralizing the MRS liquid culture supernatant of ALAL020 strain, the molecular weight (m/z) of the main antibacterial substance separated by gel filtration column chromatography and reverse phase HPLC was 226.131. This low molecular weight compound was analyzed by LC-MS and disclosed the composition formula C11H18O3N2, however the molecular structure remained unknown. Then, structural analysis by NMR revealed C11H18O3N2 as the cyclic dipeptide, “hexahydro-7-hydroxy-3- (2-methylpropyl) pyrrolo [1,2-a] pyrazine-1,4-dion cyclo (Hyp-Leu) “. Based on the results of this analysis, cyclo (Hyp-Leu) was chemically synthesized and the antibacterial activity against P. gingivalis and P. intermedia was measured. The minimum inhibitory concentration (MIC) was 2.5 g/L and the minimum bactericidal concentration (MBC) was shown to be less than 5 g/L. In addition, an in vitro epithelial tissue irritation test at 10 g/L showed no tissue toxicity. So far there are no reports of this peptide being produced by probiotic bacteria. Furthermore, antibacterial activity of this cyclic dipeptide against periodontal disease bacteria has not been confirmed. The results of this study might lead to a comprehensive understanding of the antibacterial mechanism against periodontal disease bacteria in future, and are considered applicable for the prevention of periodontal disease.

牙周病之形成源于口腔微生物群失调，进而导致牙龈卟啉单胞菌和中间型普雷沃菌等牙周致病菌的增殖。在急性症状出现时，常施以抗菌剂及消毒剂，然而耐药菌的出现及过敏反应的困扰亦随之而生。近年来，关于益生菌作用的 研究已作为牙周炎的替代疗法展开。然而，益生菌细菌对牙周病抑制作用的根本机制尚未得到明确阐释。为阐明益生菌对牙周致病菌的抗菌机制，本研究采用了具有最强抗菌活性的乳酸杆菌菌株 Limosilactobacillus (Lactobacillus) fermentum ALAL020，该菌株在50种筛选的乳酸杆菌菌株中，对牙龈卟啉单胞菌和中间型普雷沃菌的抗菌活性最为显著。所产生的抗菌物质已被鉴定并进行了结构分析。通过凝胶过滤柱层析和反相高效液相色谱分离，经 ALAL020 菌株的 MRS 液体培养基上清液中和后，主要抗菌物质的质量数（m/z）为 226.131。此低分子量化合物经液相色谱-质谱联用分析，揭示了其分子式为 C11H18O3N2，然而其分子结构尚属未知。随后，通过核磁共振波谱分析，确认了 C11H18O3N2 为环状二肽，即“六氢-7-羟基-3-(2-甲基丙基)吡咯[1,2-a]吡唑-1,4-二酮环（Hyp-Leu）”。基于此分析结果，环状（Hyp-Leu）通过化学合成得到，并对其对牙龈卟啉单胞菌和中间型普雷沃菌的抗菌活性进行了测定。最小抑菌浓度（MIC）为 2.5 g/L，最小杀菌浓度（MBC）则显示低于 5 g/L。此外，在 10 g/L 浓度下进行的体外上皮组织刺激试验显示无组织毒性。迄今为止，尚无报道指出该肽由益生菌细菌产生。此外，该环状二肽对牙周病细菌的抗菌活性亦未得到证实。本研究的结果或许有助于对未来牙周病细菌抗菌机制的综合理解，并被认为是预防牙周病的潜在应用。