乳酸菌548胞外多醣之化學結構與生物活性分析

Abstract

一位古代希臘醫學家，醫學之父，希波克拉底在兩千年前曾經說過:“死亡存在於腸道中”，一語道出了腸道對於健康的重要性。腸道中存在了上百兆個細菌，而這些細菌在腸道中的菌相決定了人體的健康。許多文獻指出益生菌(probiotics) 對於過敏，抗氧化，以及腸道消化都有調節作用，而近年來更在許多研究結果中顯示了腸道菌不只影響了人類生理的健康，也影響了心理健康，就在此時益生菌界多了一個新辭彙“精神益生菌” (psychobiotics)。 本論文是在取得陽明大學生物化學暨分子生物學研究所的研究結果後進行的合作研究，在研究結果內顯示乳酸菌548 (LAB 548) 具有抗發炎的活性，由於此活性也存在於實驗中的熱殺菌對照組中，因此我們猜測活性來源可能來自胞外多醣。針對於此我們對乳酸菌548進行胞外多醣萃取，純化，以及生物活性和化學結構分析。 實驗結果顯示了乳酸菌548胞外多醣的低產率性，免疫活化性以及結構複雜性。乳酸菌548 (LAB 548) 胞外多醣 (EPS) 在常溫下以PBS溶液萃取 (產率約2%)，初萃物再利用凝膠色譜法 (size exclusion chromatography) 與陰離子交換色譜法 (anion exchange chromatography) 分離 (產率約0.125%)。 純化的胞外多醣 (purified EPS) 在老鼠巨噬細胞 (raw264.7) 中進行生物活性分析，比色法 (colorimetry) 之定量結果顯示乳酸菌548胞外多醣可刺激老鼠巨噬細胞raw264.7分泌一氧化氮 (NO) 以及腫瘤壞死因子-α (TNF-α)。而乳酸菌548胞外多醣化學特性在利用氣相層析質譜儀分離之色譜顯示單醣的成分為葡萄醣、半乳醣、N-乙醯基半乳醣胺，以及一個未知酮醣。多醣的鍵結利用甲基化分析法進行鑑定，PMAA在色譜端分離後，質譜端顯示出多醣的鍵結有1-3、1-4、1-6、2-6、3-6、GalNAc1-3之鍵結。多醣之重複單元在核磁共振光譜中顯示此多醣含有11個重複單元 (11個異頭質子) ，並有一個異頭質子之2D-HSQC圖譜顯示出對應碳峰位於化學位移66 ppm之處。 已有許多文獻指出腸道菌能夠調節免疫反應，過敏反應以及發炎反應，更有研究指出腸道菌對於腦部病變，精神疾病也有加劇或減緩作用，雖然腸道菌對於此諸種現象的影響已有科學證據，但對於腸道菌如何影響腸道以及其分子的調控機制還是未知。在此領域更深入的研究，能促使人類的生理與心理健康，或是醫療上的輔助治療的進步。

An ancient Greek doctor, the doctor of western medicine, Hippocrates declared 2000 years ago, “Death sits on the bowels”. The aphorism elucidates that the intestine condition is so relative to human health. Our intestine contains so many bacteria that the number is up to one hundred trillion. These gut bacteria play an important role in human physiological and psychological health. Probiotics can modulate allergy, inflammation, anti-oxidation and intestine digestion. Furthermore, some researches recently elucidated that the probiotic can influence not only physiological condition but also psychological conditions, such as depression, autism and anxiety, etc. Thus, in probiotic field, a new word “psychobiotics’ appears. This dissertation focuses on a probiotic: LAB 548 which was obtained from Nation Yang Ming University, Institute of Biochemistry and Molecular Biology. In their result both living and heat-killed LAB 548 have some anti-inflammatory activities, and thus we suppose that the activities may come from heat-stable exopolysaccharides. Therefore, we attempted to purify the exopolysaccharide from LAB 548 and to analyze the chemical structure and biological activities in this research collaboration. Experimental results revealed the low yields of the exopolysaccharides. Crude exopolysaccharides were extracted into PBS (2% of yield), and sequentially purified by size exclusion chromatography and anion exchange chromatography (0.125% of yield). Purified polysaccharides were subjected to biological activity experiments based on immunomodulatory properties. In the in vitro test, LAB 548 EPS can stimulate the raw 264.7 murine macrophage cells to produce nitric oxide and tumor necrosis factor-α (TNF-α) in a concentration dependent manner. Sugar composition analysis by GC-MS revealed that the polysaccharide was composed of galactose, glucose, N-acetylgalactosamine and an unknown keto sugar. Linkage analysis was done by preparing partial methylated alditol acetates (PMAAs) and analyzed by using GC-MS. The PMAAs analysis revealed that the polysaccharide contained 1-3, 1-4, 1-6, 2-6, 3-6 and GalNAc1-3 linkage. Nuclear Magnetic Resonance (NMR) spectrum of the purified polysaccharide showed eleven signals in the anomeric region indicating that the polysaccharide has eleven repeating units. Furthermore, there was a C1 carbon chemical shift at 66 ppm in 2D-HSQC experiments indicating the presence of one keto sugar. Many research articles have demonstrated that the probiotics can modulate immune, anti-inflammation and allergy condition. Some articles even suggested that the probiotic can also modulate psychological and brain diseases such as parkinosn’s disease, anxiety and depression, etc. Although probiotics have been shown to have some physiological and psychological activities, the molecule mechanisms and how microbiota influence intestine or brain remain unclear. Thus, more researches in this field could potentially improve human physiological, psychological health, or medical adjuvant therapies.