- 學位論文
腸道分離菌 Bacillus licheniformis No.TMI-H1221 生理活性代謝物之探討
A study on the physiologically active metabolites of Bacillus licheniformis No.TMI-H1221 originally from human gastrointestinal tract
摘要
地衣芽孢桿菌(Ｂacillus licheniformis)為芽孢桿菌屬(Bacillus spp.)中被視為人體益生菌(Probiotics)的菌種，這些益生菌可藉由表現細胞表面分子或釋出其代謝產物，與腸道細胞相互作用而對腸道形成保護屏障。本研究目的針對地衣芽孢桿菌經由發酵產生的活性代謝產物，進行其成分的追蹤分離純化與分子結構解析，以期了解此菌種在人體腸道中所扮演防疫的功能及其作用機制。 實驗規畫分為：1.發酵最適化條件設計；2.以抗菌活性能力及清除自由基能力篩選發酵條件；3.活性代謝物分離純化；4.活性代謝物性狀分析與鑑定，等四個部分。 實驗中以腸道分離菌株Bacillus licheniformis No. TMI-H1221(簡稱BL菌株)為對象分別進行液態振盪發酵以及液態靜置發酵。發酵產物經抗菌活性以及自由基清除能力試驗篩選結果，確立此菌株最適發酵條件為：1.發酵培養基為 M1培養基，其成分以酵母膏、蛋白腖、葡萄糖為主；2.發酵溫度37℃，以振幅75rpm液態往復振盪發酵3天；3.發酵容積比為1.5L/5L(v/v)三角錐形瓶。 活性代謝物回收流程為：發酵培養液經離心取得上清液後，以ethyl acetate進行酸鹼萃取分層，呈現活性之強酸萃取層再經silica gel 60F管柱液相層析(CLC)進行活性物質分離，可於chloroform: methanol: acetate = 100:0:1沖堤區段中取得粗分之活性畫分。續以silica gel F254薄層液相層析(TLC)分離，以benzene: methanol= 90:10展開後再經刮除及ethyl acetate溶離濃縮可得一活性純化物，命名為BL-A-01。此物質經結構分析鑑定，確認為過去所知的同菌種抗菌代謝物phenylacetic acid。接續上述之沖堤液chloroform: methanol: acetate = 95:5:1分離區段中可得另一粗分活性畫分，此畫分經silica gel F254 TLC分離，以chloroform: methanol: acetic acid = 80:20:1展開後在Rf=0.6處可得一活性純化物，命名為BL-A-02。此物質經HPLC分析後發現為有別於phenylacetic acid之新成分，其純度並以氣相層析(GC)做進一步確認。此外在中弱酸層可另得一無色針狀結晶，此結晶可經溫熱ethyl acetate進行再結晶提純處理，特將其命名為BL-N-01。 針對上述所純化之三個BL菌株代謝物質，檢討其生物及物化特性可知：BL-A-01能抑制Staphylococcus aureus (SA)，methicillin resistant Staphylococcus aureus (MRSA)及Candida albicans (CA)之生長，其紫外最大吸光值(UVmax)為280nm。BL-A-02能抑制SA生長，對MRSA及CA則無抗菌能力，其UVmax為236及278nm。BL-N-01則不具任何抗菌活性，其熔點為198~200℃，UVmax為210nm。本研究由探索人體腸道益生菌Ｂacillus licheniformis發酵代謝產物之角度，針對此菌株活性代謝物之生物特性及分子結構進行解析，以作為其將來在腸道免疫防疫功能及藥物發展研究之重要參考。
並列摘要
Ｂacillus licheniformis, one of the genus of Bacillus, was recognized as a probiotic in human. Probiotics could interact with intestinal cells to develop the barrier effect by presenting cell surface molecules or releasing metabolites. Our study focused on the bioactive metabolites produced byＢacillus licheniformis, by performing the isolation, purification, and structure analysis of the metabolic molecules in order to realize the roles and the mechanism of the strain existing in human intestines. The experimental outline was divided as following: 1. the optimal condition for fermentation, 2. the screening model for fermentation by antimicrobial activity and scavenging activity of free radicals, 3. the isolation and purification of bioactive metabolites, 4. the characterization and identification of bioactive metabolites. In this experiment, Bacillus licheniformis No.TMI-H1221 was selected as the target strain in executing liquid shaking and static fermentation, respectively. The optimal condition for fermentation screened by the antibacterial activity and free radical scavenging ability was: 1. Medium: M1 containing yeast extract, peptone, glucose as the main nutrient source, 2. Fermentation model: temperature 37℃, liquid shaking fermentation with the reciprocal 75 rpm in three days, 3. Fermentation vessel: 1.5L/5L(v/v) in erlenmeyer flask. The recovery procedure of the active metabolites was described as following: after centrifuging the cultural broth, the fermentation supernatant was obtained. The acid-base fractionation to the supernatant was processed successfully with solvent extraction by ethyl acetate. The acidic fraction, being confirmed to be the bioactive fraction, was further burnished by performing column liquid chromatography (CLC, silica gel 60F developed by chloroform: methanol: acetate = 100:0:1) followed by thin layer chromatography (TLC, silica gel F254 developed by benzene: methanol= 90:10). One of the pure compounds, named BL-A-01, was obtained through the process of gel scratch and solvent elution. The compound was identified to be phenylacetic acid, which was also known as one of the antimicrobial metabolites of Ｂacillus licheniformis. According to the isolation procedure described above, the separation condition of chloroform: methanol: acetate = 95:5:1 for CLC in combination with chloroform: methanol: acetic acid = 80:20:1 for TLC was undertaken. By recovering the band existing on Rf =0.6 of the TLC, the bioactive compound, named BL-A-02, was obtained successfully. The result of HPLC analysis indicated that BL-A-02 was a new component different from phenylacetic acid. The purity of the compound was further confirmed by gas chromatography. In addition, a colorless needle crystal was also obtained from the neutral/weakly-acidic fraction. The purity of this compound could be accelerated by re-crystallization with the aid of warm ethyl acetate, thus named it as BL-N-01. The biological and physico-chemical characteristics of the three metabolites were: BL-A-01 was active against Staphylococcus aureus(SA), methicillin resistant Staphylococcus aureus(MRSA), and Candida albicans(CA). The UV spectrum of BL-A-01 showed a maximal absorption at 280 nm. BL-A-02 was active against SA, but not MRSA and CA. The UV spectrum of BL-A-02 showed a maximal absorption at 236 and 278nm. BL-N-01 was inactive to all the test organisms being used in our experiment. The melting point of BL-N-01 was 198~200℃ and the maximal of UV spectrum at 280 nm. Based on the understanding of the biological functions and the molecular structures of the bioactive metabolites derived from Ｂacillus licheniformis, our study may provide a prospective clue in dealing with the functions of Bacillus licheniformis in immune-regulation and disease prevention of the intestinal tract as well as the development of related drugs.