Department of Oral Microbiology, School of Dental Medicine, Tsurumi University, Yokohama, Japan.
Research Division of Polymer Functional Materials, Osaka Research Institute of Industrial Science and Technology, Izumi, Japan.
Front Cell Infect Microbiol. 2022 Mar 7;12:804334. doi: 10.3389/fcimb.2022.804334. eCollection 2022.
Periodontal disease develops as a result of oral microbiota in dysbiosis, followed by the growth of periodontal pathogens such as and . 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 ALAL020, which showed the strongest antibacterial activity against and 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 CHON, however the molecular structure remained unknown. Then, structural analysis by NMR revealed CHON 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 and 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 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.
牙周病是由于口腔微生物群落失调导致的，随后牙周病病原体如 和 等开始生长。在出现急性症状时，会使用抗菌剂和消毒剂进行治疗，但是出现耐药菌和过敏问题。近年来，人们已经开展了针对益生菌对牙周炎影响的研究，将其作为牙周病的替代疗法。然而，益生菌抑制牙周病的基本机制尚未得到明确阐明。为了阐明益生菌对牙周病病原体的抑菌机制，我们使用了在 50 株筛选出的乳酸菌菌株中对 和 表现出最强抗菌活性的 ALAL020。鉴定并结构分析了所产生的抗菌物质。在中和 ALAL020 菌株的 MRS 液体培养上清液后，通过凝胶过滤柱层析和反相 HPLC 分离的主要抗菌物质的分子量（m/z）为 226.131。通过 LC-MS 对该低分子量化合物进行分析，揭示其组成为 CHON，但分子结构仍未知。然后，通过 NMR 进行结构分析，发现 CHON 为环状二肽“六氢-7-羟基-3-（2-甲基丙基）吡咯并[1,2-a]吡嗪-1,4-二酮环（Hyp-Leu）”。基于该分析结果，化学合成了环（Hyp-Leu），并测量了其对 和 的抗菌活性。最小抑菌浓度（MIC）为 2.5 g/L，最小杀菌浓度（MBC）小于 5 g/L。此外，在 10 g/L 浓度下进行的上皮组织刺激性试验未显示组织毒性。到目前为止，还没有报道表明这种肽由益生菌产生。此外，这种环状二肽对牙周病细菌的抗菌活性尚未得到证实。本研究的结果可能有助于全面了解针对牙周病细菌的抗菌机制，并有望应用于牙周病的预防。
