Biotransformation of sesaminol triglucoside treated with swine intestinal microorganisms

Sesaminol triglucoside (STG) 為芝麻粕中最主要之木酚素 (lignan)，經生物體代謝後可產生sesaminol而展現良好的抗氧化性；此外亦可代謝生成enterodiol及enterolactone而具有抗乳癌、攝護腺癌等荷爾蒙相關疾病之功效。前人研究發現腸道菌對lignans的代謝為其轉換過程中之必要因素，且服用抗生素會降低受試者血液中enterolactone之濃度，由此可知lignans的代謝深受腸內菌之影響。故本試驗期望以腸道菌對lignans生物轉換之特性，探討腸道菌相於不同之飲食狀態下對於STG生物轉換之影響。試驗以餵食含抗生素飼料之豬隻為一般動物組 (抗生素豬)，而餵食添加黃豆peptide及乳酸菌飼料之豬隻作為健康狀況較佳的動物組 (peptide豬)。試驗中分別取新鮮糞便加入STG進行厭氧發酵，觀測其代謝物含量之變化，並比較兩組豬糞便的菌相平衡關係，接著再以選擇性培養基分離出乳酸菌和產氣莢膜梭菌 (Clostridum perfringens) 進一步探討其對STG代謝之能力。結果顯示peptide豬隻之糞便菌叢，具有快速水解醣基生成sesaminol之能力，其中間代謝產物 (ST-2) 生成量於發酵7天後亦顯著比抗生素組豬隻高。試驗結果中進一步發現將sesaminol代謝為ST-2的微生物對氧氣非常敏感，推測參與此生物轉換之微生物屬於絕對厭氧菌，而非兼性厭氧菌之乳酸菌群。 Peptide豬之糞便型態具有較低的pH值且較濕潤；而菌相檢測結果則顯示兩組別豬隻糞便菌叢均維持有良好的好菌與壞菌之比例關係，其中又以抗生素組糞便中雙叉桿菌 (Bifidobacterium) 及乳酸桿菌 (Lactobacillus) 之菌數顯著高於peptide組別。 STG於分離菌株之代謝試驗結果顯示，乳酸菌及C. perfringens菌群均有切除STG上醣基之能力，但乳酸菌之貢獻效果較弱。綜合以上試驗結果可知，食用不同的飼料配方會影響豬隻糞便菌叢對sesaminol triglucoside (STG) 之生物轉化能力，此結果可能為飲食差異影響其腸內菌相所導致。兩種飼料組別中，以餵飼peptide飼料的豬隻糞便菌叢對STG具有較強的代謝能力，但推測主要可代謝STG之微生物菌群應屬於非乳酸菌之其他糞便菌群。Sesaminol triglucoside (STG) is the major lignan compound in sesame meal. STG can be metabolized to an antioxidant sesaminol in vivo and further into the mammalian lignans, enterodiol and enterolactone, which are reported to protect hormone-dependent diseases. Previous reports showed that intestinal microorginasms action is a key step to metabolize lignans and the intake of antibiotic can affect the conversion of lignans to enterolactone. Therefore we studied the metabolism of STG by the intestinal microorganisms of swines fed with two different kinds of feeds. Feeds containing soy peptide or antibiotic were used in this study, we hypothesize that swine fed with feed containing antibiotic as a normal animal model (antibiotic swine), and swine fed with feed containing soy peptide as a healthier animal model (peptide swine). The fresh swine feces were used as the fermentation medium for STG to monitor its metabolic transformations. We also examine the microorganisms in the feces and used the selective culture of microorganisms such as Lactobacillus, Bifidobacterium, lactic acid bacteria and Clostridium perfringens to study their biotransformation capacity of STG. Our result showed that fecal microorganisms of peptide swine can readily hydrolyze the glucose unit of STG to sesaminol and more ST-2 metabolite was formed than antibiotic swine group. We also discovered that the microorganisms, which convert sesaminol to ST-2 are sensitive to oxygen, might be the absolute anaerobes. Peptide swine feces are lower in pH value and higher in water content than antibiotic swine feces. On the other hand, antibiotic swine feces have more Lactobacillus and Bifidobacterium, but both of two groups have good ratio of good microorganisms and bad microorganisms. All of the selected microorganisms, Lactobacillus, Bifidobacterium, lactic acid bacteria and C. perfringens, can hydrolyze the glucose unit of STG, but the contribution of lactic acid bacteria is less. In conclusion, changes of diet will affect the intestinal microorganisms and therefore affect the metabolism of STG. The microorganisms present in peptide swine feces have stronger ability to metabolize STG, and we postulate that the biotransformation of STG may be due to microorganisms other than lactic acid bcteria present in the feces.中文摘要 ........................................................................................................................I文摘要 ……………………………………………………………………………...II錄 ............................................................................................................................ IV次 ..........................................................................................................................VIII次 ..............................................................................................................................X、 前言 ......................................................................................................................1、 文獻回顧 .............................................................................................................2、 芝麻簡介 2一)、 芝麻的基本介紹及應用 ................................................................2二)、 芝麻與芝麻木酚素(lignans)的生理活性 .....................................3三)、 Sesaminol triglucoside之生物活性與代謝 ..................................7、 木酚素(Lignans)經動物體及腸內菌之代謝 9一)、 飲食中木酚素代謝的相關研究 ...................................................9二)、 腸內菌對木酚素之代謝 .............................................................13、 腸內微生物與宿主間的關係 17一)、 人與豬的腸內微生物菌群簡介 ..................................................18二)、 腸內菌存在對宿主腸道之功用 ..................................................21三)、 影響腸道菌相的因素 ..................................................................23、 服用抗生素對木酚素代謝的影響 27、 研究目的與實驗設計 .........................................................................................30、 研究目的 30、 實驗架構 31、 實驗流程 33、 材料與方法 .........................................................................................................38、 實驗材料 38、 試驗動物分組 38一)、 一般含抗生素飼料組豬隻 (Antibiotic豬) ................................38二)、 乳酸菌及peptide添加之飼料組豬隻 (Peptide豬) ..................40、 化學藥品與溶劑 40一)、 標準品 ..........................................................................................40二)、 藥品、試劑 .................................................................................42三)、 溶劑 ..............................................................................................42四)、 層析材料 ......................................................................................42五)、 微生物培養基及稀釋液 .............................................................43六)、 其他器材 ......................................................................................45、 儀器設備 45一)、 一般儀器設備 ..............................................................................45二)、 化學分析相關儀器設備 ..............................................................46三)、 微生物培養相關器材設備 ..........................................................47、 實驗方法 48一)、 Sesaminol triglucoside (STG) 樣品製備與純度分析 ................48二)、 豬大腸不同區段微生物菌群對STG代謝能力之預試驗方法 50. 豬大腸各區段之採樣方法 50. 腸內菌發酵及發酵液中STG代謝物之萃取 51. 腸內菌發酵之STG代謝產物分析方法 53三)、 糞便發酵試驗方法 ......................................................................54. 糞便檢體之收集 54. 糞便發酵及發酵液萃取 54. STG經糞便發酵之代謝產物分析 56四)、 糞便菌相檢測 .............................................................................57. 培養基及厭氧稀釋液之配製 57. 糞便檢體之收集 61. 糞便中產氣莢膜梭菌 (C. perfringens) 之菌數測定 61. 糞便中雙叉桿菌 (Bifidobacterium)、乳酸桿菌 (Lactobacillus) 及乳酸菌群 (Lactic acid bacteria) 之菌數測定 61. 糞便水含量及pH質測定 62五)、 乳酸菌及產氣莢膜梭菌對STG代謝能力試驗方法 ................62. 菌群培養與繼代 62. 產氣莢膜梭菌、雙叉桿菌、乳酸桿菌及乳酸菌群對STG代謝能力測定 63. 發酵液中產氣莢膜梭菌、雙叉桿菌、乳酸桿菌及乳酸菌群之菌數測定 64、 統計分析 64、 結果與討論 ........................................................................................................65、 Sesaminol triglucoside樣品製備與純度分析 65、 豬大腸不同區段菌群對STG代謝能力之預試驗 67、 糞便菌叢對sesaminol triglucoside (STG) 之代謝能力試驗 70一)、 豬隻糞便發酵液中STG代謝產物之定性分析 ........................70二)、 豬隻糞便發酵液中STG代謝產物之定量分析 ........................771. 高效能液相層析系統分析方法之確效 .....................................77. STG及其代謝物於糞便發酵液萃取之回收率 77. 兩種豬糞便發酵液中STG及其代謝物生成量隨時間之變化 80. STG於微厭氧環境下代謝物生成之變化 87、 糞便菌相檢測 89一)、 糞便中產氣莢膜梭菌及乳酸菌之菌數測定 ..............................89二)、 糞便水含量及pH質測定 ...........................................................93、 乳酸菌及產氣莢膜梭菌對STG代謝能力試驗 96. 各時間點發酵液中菌數變化情形 96. 各時間點發酵液中STG代謝物濃度檢測 96、 結論 ...................................................................................................................100、 後續研究發展建議 ...........................................................................................101、 參考文獻 ...........................................................................................................102、 附錄 ...................................................................................................................11