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研究生:蘇家暉
研究生(外文):Jia-Huei Su
論文名稱:益生菌相關代謝物對骨骼肌汲取葡萄糖能力之影響
論文名稱(外文):The effect of probiotics derived metabolites on glucose uptake in skeletal muscle cells
指導教授:蔣丙煌蔣丙煌引用關係
指導教授(外文):Been-Huang Chiang
口試委員:潘敏雄蘇南維陳錦樹
口試委員(外文):Min-Hsiung PanNan-Wei SuChin-Shuh Chen
口試日期:2016-07-27
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:中文
論文頁數:109
中文關鍵詞:胰島素阻抗益生菌代謝產物葡萄糖汲取能力骨骼肌
外文關鍵詞:Insulin resistanceProbiotics metabolitesGlucose uptakeSkeletal muscle
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糖尿病(diabetes mellitus, DM)為一複雜的代謝性疾病,並常伴隨著許多併發症的產生,其與胰島素的分泌不足及作用異常有關。其中,第二型糖尿病約佔患者的90%,主要原因是胰島素阻抗(insulin resistance)所引起。因此,如何改善胰島素阻抗被認為是治療糖尿病的重要策略之一。近年來許多研究發現改變腸道菌相的組成,有助於改善肥胖與高血糖的現象,但也越來越多的研究指出腸道菌發酵後所產生的代謝產物(metabolites)可能是另一關鍵因素,並非只是單純改變腸道菌相的結果。因此,本研究採用已呈現胰島素阻抗症狀之L6骨骼肌細胞作為測試平台,針對不同代謝產物進行篩選,篩選出具有刺激骨骼肌細胞葡萄糖汲取能力之三種不同代謝產物,分別為共軛亞麻油酸(conjugated linoleic acid, CLA)、丁酸(butyric acid, BTY)以及亞精胺(spermidine, SPD),並同時篩選出具有生產這些代謝產物能力之菌株,以及透過前驅物亞麻油酸(linoleic acid)及精胺酸(L-arginine)的添加來增加代謝產物的含量。最後,透過三種不同菌株(Bifidobacterium breve、Clostridium butyricum、Bacillus subtilis)的搭配組合,將其應用於黃豆粉固態發酵上,來進行代謝物組合物的發酵生產。結果顯示,經發酵生產後的產品其萃取物可有效提升骨骼肌細胞的葡萄糖汲取能力,可達到控制組 DMSO 的1.67倍,並且也顯著的優於胰島素的表現。因此,未來或許可望透過此產品組合物的應用,來對胰島素阻抗疾病發揮改善的效果。

Diabetes mellitus (DM) is a complex metabolic disease which often accompany with many complications. DM is caused by insufficient insulin secretion or resistance to insulin action on glucose uptake in peripheral tissues. It is estimated that about 90% of DM patients are type II diabetes worldwide and they are caused mainly by insulin resistance. Therefore, how to improve insulin resistance is considered to be one of the most important strategies for treating diabetes. Recently, many reserches have found that the changes in gut microbiota can influence the development of obesity, insulin resistance, and diabetes. Many reports have also indicated that the metabolites derived by gut microbiota may play key role in these functions. In this study, we first chose the palmitic acid -induced insulin resistance in L6 myotubes as the test platform to select effective probiotics metabolites. The results showed that conjugated linoleic acid (CLA), spermidine (SPD) and butyric acid (BTY) were effective metabolites which could increase glucose uptake activity of L6 cells, and the strains that had capacity to produce these selected metabolites were also found. In addition, precursors such as linoleic acid and L-arginine were added to increase the contents of these metabolites. Finally, we used three different probiotics strains to ferment soybean flour to obtain these desired metabolites. We found that the glucose uptake activity of L6 cells treated with the fermented soybean flour extracts was significantly increased to 1.67 times as compared to the control group (the DMSO treated group) and even superior to the results of the positive control group (the insulin treated group). Consequently, the application of the fermented metabolite mixtures might have a potential to improve insulin resistance disorder in the future.

謝誌 i
中文摘要 ii
Abstract iii
目錄 iv
圖目錄 vii
表目錄 x

第一章、文獻回顧 1
第一節、糖尿病 1
1.1 糖尿病基本介紹 1
1.2 糖尿病的分類 1
第二節、胰島素阻抗 3
2.1 胰島素阻抗(insulin resistance) 3
2.2 胰島素阻抗原因 4
2.3 棕櫚酸誘導骨骼肌胰島素阻抗 5
第三節、葡萄糖轉運與分子訊息的傳遞 7
3.1 葡萄糖的轉運作用 7
3.2 骨骼肌汲取葡萄糖之相關訊息傳遞路徑 8
第四節、乳酸菌與胰島素阻抗之關聯 10
4.1 乳酸菌與胰島素阻抗 10
4.2 乳酸菌及其相關腸內代謝產物 12
第五節、代謝產物特性及與胰島素阻抗之關聯 15
5.1 共軛亞麻油酸(conjugated linoleic acid) 15
5.2 多元胺(polyamine) 19
5.3 短鏈脂肪酸(short-chain fatty acid) 22
第二章、實驗目的與架構 25
第一節、實驗目的 25
第二節、實驗架構 26
2.1 乳酸菌相關代謝物之葡萄糖汲取活性測試 26
2.2 菌種篩選與相關代謝物之生產 27
2.3.1 納豆菌利用黃豆組合發酵生產相關之代謝物 28
2.3.2 乳酸及丁酸菌利用黃豆組合發酵生產相關之代謝物 29
2.3.3 乳酸及丁酸菌利用黃豆組合發酵生產相關之代謝物 30
第三章、材料與方法 31
第一節、實驗材料 31
1.1 發酵基質 31
1.2 發酵菌種 31
1.3 細胞株 31
第二節、實驗藥品 32
第三節、實驗儀器 34
第四節、實驗方法 35
4.1 黃豆粉的製備 35
4.2 菌株的保存與活化 35
4.3 發酵樣品的製備 36
4.4 發酵樣品成份分析 36
4.5 乳酸菌生產CLA能力之篩選 42
4.6 乳酸菌與丁酸菌共培養生產丁酸能力之篩選 43
4.7 納豆菌生產亞精胺能力之篩選 44
4.8 發酵完後樣品之萃取與分析 44
4.9 骨骼肌細胞之培養 45
第四章、結果與討論 50
第一節、骨骼肌細胞平台之建立 50
1.1 骨骼肌細胞之分化 50
1.2 以棕櫚酸誘導骨骼肌細胞呈胰島素阻抗狀態之葡萄糖汲取能力 51
第二節、益生菌相關代謝產物之篩選 53
2.1 ω-3脂肪酸與共軛亞麻油酸刺激細胞汲取葡萄糖能力之影響 53
2.2 短鏈脂肪酸刺激細胞汲取葡萄糖能力之影響 56
2.3 多元胺刺激細胞汲取葡萄糖能力之影響 59
第三節、代謝物間互補效應 61
3.1 共軛亞麻油酸與ω-3次亞麻油酸共處理之效果 61
3.2 共軛亞麻油酸與ω-3次亞麻油酸及亞精胺共處理之效果 65
3.3 共軛亞麻油酸、ω-3次亞麻油酸、亞精胺及丁酸共處理效果 67
第四節、有效代謝物之發酵生產 69
4.1 生產共軛亞麻油酸之乳酸菌菌種篩選與前驅物添加之影響 69
4.2 乳酸菌與丁酸菌共培養生產丁酸能力之評估 73
4.3 生產亞精胺之納豆菌菌種篩選與前驅物添加之影響 77
第五節、代謝物之組合發酵生產 82
5.1 黃豆粉經乳酸菌、丁酸菌共培養及納豆菌發酵後其水份含量及pH值 之變化 82
5.2 黃豆粉經納豆菌發酵與有無添加精胺酸發酵對其亞精胺含量之變化 87
5.3 黃豆粉經乳酸菌、丁酸菌共發酵及亞麻油酸添加與否對其丁酸含量之變化 89
5.4 黃豆粉經乳酸菌、丁酸菌共發酵及有無添加亞麻油酸發酵對其共軛亞麻油酸 91
5.5 黃豆粉發酵後組合萃取物對刺激細胞汲取葡萄糖能力之影響 93
第五章、結論 96
第六章、參考文獻 97



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