精神益生菌植物乳桿菌 PS128 所產次級代謝物探索

Abstract

近年來研究顯示，腸道菌群的多樣性和穩定性與宿主的健康或疾病息息相關。腸–腦軸 (gut-brain axis) 在重度憂鬱症、自閉症譜系障礙 (簡稱自閉症) 等多種精神疾患中扮演關鍵角色。先前研究表明， Lactiplantibacillus plantarum PS128有助於改善部分自閉症兒童的行為問題，並具潛在抗憂鬱效果。 為釐清L. plantarum PS128之代謝物之化學成分，本研究將L. plantarum PS128於MRS肉湯培養基中培養16小時後的上清液，以液-液分配法進行極性分割，得到正己烷、乙酸乙酯、正丁醇及水可溶部分，以固相萃取進行初步分離與富集，並利用LC-MS進行分析，最終鎖定具未知代謝物的乙酸乙酯層，其LC-ESI-MS 分析結果顯示[M+Na]^+偽分子峰為m/z 1,278.7，推估分子量為 1,256 Da。以逆相管柱層析技術進一步分離與純化，並使用NMR技術進行結構鑑定，結果顯示該代謝物為非核糖體多肽類似物，由三種胺基酸組成並連接一條長碳鏈，顯示該代謝物為一種脂肽。然儘管嘗試調整流動相之沖提比例及變更固定相之材質，仍未能完全去除其中之雜質。 此外，本研究同時針對L. plantarum PS128菌體內代謝物之萃取方法進行優化比較，比較不同的胞內代謝物萃取及淬滅方法。碘化丙啶染色及ATP含量測定結果顯示，常見之預冷有機溶劑萃滅法會破壞細胞膜，導致大量胞內代謝物的外洩，因此進行非靶向代謝體學時應採用不淬滅的處理方式，以最大化胞內代謝物的保留效果；此外，採用甲醇/乙腈/水混合溶劑萃取具有較廣泛的代謝物覆蓋範圍與更佳的整體再現性，更適合作為非靶向代謝體學研究的首選方法。同時，本研究嘗試解決MRS肉湯培養基內之Tween 80對LC-MS分析所造成的離子化競爭問題，發現油酸取代之MRS肉湯培養基能成功支持L. plantarum PS128生長，為後續相關研究提供新方向。 綜合上述，本研究對L. plantarum PS128的菌體外代謝物進行分離和鑑定並初步建立利用資訊技術及搭配低解析質譜儀以更有效率的尋找微生物產的次級代謝物，同時對L. plantarum PS128的菌體內代謝物的萃取進行方法優化。

Recent studies have demonstrated that the diversity and stability of the gut microbiota are closely associated with host health and disease. The gut-brain axis plays a critical role in various psychiatric disorders, including major depressive disorder (MDD) and autism spectrum disorder (ASD). Previous research has shown that Lactiplantibacillus plantarum PS128 can alleviate behavioral problems in some children with ASD and exhibits potential antidepressant effects. To elucidate the chemical composition of metabolites produced by L. plantarum PS128, the culture supernatant obtained after 16 h of cultivation in MRS broth was subjected to liquid-liquid extraction to fractionate into Hexanes, EtOAc, n-Butanol, and "H" _"2" "O" fractions. Preliminary enrichment was performed using solid-phase extraction (SPE), followed by LC-MS analysis, which identified the EtOAc fraction as containing unknown metabolites of interest. LC-ESI-MS analysis revealed a pseudomolecular ion peak at m/z 1,278.7 [M+Na]^+, corresponding to a calculated molecular weight of 1,256 Da. Further purification was achieved by reversed-phase column chromatography, and the structure was elucidated using NMR spectroscopy. The results indicate that this metabolite is a nonribosomal peptide-like compound composed of three amino acids linked to a long carbon chain, suggesting that it is a lipopeptide. However, despite attempts to adjust the mobile phase composition and the stationary phase, the co-eluting impurities could not be removed. In parallel, the extraction methods for intracellular metabolites were systematically optimized and compared. Propidium iodide staining and ATP quantification indicated that conventional cold organic solvent quenching disrupted the cell membrane, leading to extensive intracellular metabolite leakage. Thus, non-quenching approaches should be adopted in untargeted metabolomics to maximize the intracellular metabolite retention. Moreover, extraction with a MeOH/ACN/ "H" _"2" "O" mixture provided broader metabolite coverage and superior reproducibility, making it the preferred method for untargeted metabolomics studies. This study also addressed the ionization competition caused by Tween 80 in MRS broth. Replacing Tween 80 with oleic acid successfully supported the growth of L. plantarum PS128, thereby offering a new direction for future investigations. In conclusion, this work reports the isolation and structural elucidation of extracellular metabolites of L. plantarum PS128, and preliminary establishes an efficient strategy combining informatics with low-resolution mass spectrometry for the discovery of microbial secondary metabolites. In addition, optimized methodologies for the extraction of intracellular metabolites of L. plantarum PS128 were developed.