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研究生:游玉萍
研究生(外文):Yu-ping Yu
論文名稱:天貝對腸道菌相及結腸癌前病灶的影響
論文名稱(外文):Effects of the Tempeh on Intestinal Microbiota and Colon Precancerous Lesions in Rats
指導教授:鍾雲琴
指導教授(外文):Yun-Chin Chung
學位類別:碩士
校院名稱:靜宜大學
系所名稱:食品營養研究所
學門:醫藥衛生學門
學類:營養學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:108
中文關鍵詞:異常腺窩短鏈脂肪酸盲腸菌相天貝
外文關鍵詞:tempehcecal microbiotaaberrant crypt focishort chain fatty acid
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天貝為印尼傳統發酵產品,具有抗氧化、降低膽固醇、抑制特定癌症發生及抗菌等活性。本實驗目的是藉由測定天貝對微生物生長的影響、對動物腫瘤細胞之毒殺作用及對老鼠腸道菌相及異常腺窩病灶(aberrant crypt foci,ACF) 數量的改變,以評估天貝的腸道功能。微生物方面將以濾紙盤擴散試驗(paper disc diffusion test) 評估天貝對病原菌 (包含Salmonella typhimurium,Staphylococcus aureus,Listera Monocytogenes,Psuedomonas Aeruginosa,Propionibacterium freudenreichii及 Escherichia coli) 生長的影響;細胞毒性方面則以MTT方法 (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay) 評估天貝對人類腸癌細胞 (Caco-2) 腫瘤細胞株生長的影響。動物體內試驗則以1,2-Dimethylhydrazine Dihydrochloride (DMH)誘發SD (Sprague Dawley)公鼠產生ACF後,每日分別管灌 300 mg/kg BW或600 mg/kg BW天貝或黃豆,12週後測定其腸道菌相、肝臟抗氧化物質濃度及抗氧化活性與結腸ACF數量。結果顯示天貝能夠抑制L. monocytogenes及大腸腺癌細胞的生長。動物實驗結果發現以皮下注射DMH誘發SD大鼠產生ACF後,每日給予300或600 mg/kg BW的黃豆或天貝並不會影響其體重及攝食量。黃豆或天貝的攝取可降低腸內C.perfringens數量;餵食黃豆後能夠增加老鼠肝臟SOD活性,但餵食天貝則無此功效;餵食黃豆及天貝對腸道脂肪酸含量及pH值並無顯著影響 (P > 0.05)。黃豆與天貝皆能顯著抑制ACF產生 (P < 0.05),300 mg/kg BW的黃豆及 600 mg/kg BW的天貝皆可降低近50 %的ACF總數;其中天貝在 600 mg/kg BW於含有4個以上腺窩的ACF具最佳抑制效果。因此,黃豆與天貝能夠降低腸內ACF產生量可能是因為其可增加SOD活性或是能夠抑制腸道Clostridium perfringens生長,減少動物體內β-Glucuronidase量,進而抑制ACF產生。
Tempeh is a traditional fermentative food in Indonesia. The bio-functions of tempeh including antioxidant activity, cholesterol lowing, preventing progress of several cancers and antimicrobial activity. This study aimed to evaluate the antimicrobial activity and cytotoxicity toward mammalian cancer cell of water extract of tempeh; furthermore, the effect of tempeh on the alteration of cecal microbiota and also their inhibitory effect on the precancerous colon lesions (aberrant crypt foci, ACF) in male SD (Sprague Dawley) rats were assessed. The paper disc diffusion test was performed to assess the effect of water extract of tempeh on the growth of pathogenic bacteria, including Salmonella typhimurium, Staphylococcus aureus, Listera monocytogenes, Psuedomonas aeruginosa, Propionibacterium freudenreichii and Escherichia coli. Cytotoxicity was detected by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay) assay using human Caco-2 colon adenocarcinoma cell. Effect of tempeh on anticoloncancer was evaluated in SD (Sprague Dawley) rats. After induced ACF by 1, 2-Dimethylhydrazine Dihydrochloride (DMH), rats were gavaged with either soybean or tempeh at a daily dosage of 300 mg/kg BW or 600 mg/kg BW for 12 weeks. At the end of experiment, colons were collected and analyzed for cecal microbiota, cecal pH, short chain fatty acid composition, ACF formation and crypt multiplicity; on the other hand, blood biochemical parameters and antioxidant activitues were determined as well. The results of this study showed that water extract of tempeh could inhibit the growth of L. Monocytogenes and Caco-2 cell. Oral administration of soybean or tempeh did not affect on body weight and food intake in SD rats. Both soybean and tempeh could decrease the number of C. perfugens in cecal content compared to DMH-induced group. SD rats were gavaged with 300 mg/kg BW soybean could increase superoxide dismutase activity; however, this phenomenon did not appear in the rats fed with equal dosage of tempeh. Both soybean and tempeh significantly reduced the number of aberrant crypt foci (ACF) in the colon of DMH-treated rat (p&lt;0.05). Oral administration of 300 mg/kg BW soybean or 600 mg/kg BW tempeh could reduce 50 % of tatol number of ACF. Especially, daily intake tempeh 600 mg/kg BW could reduce number of ACF which was composed by more than 4 crypts. These results suggested that soybean and tempeh supplementation may be beneficial to inhibit colon cancer by elevating the SOD activity of liver tissue or lowering β-Glucuronidase activity in cecal through inhibiting the growth of C.perfringens.
【圖目錄】 V
【表目錄】 VI
中文摘要 VII
英文摘要 IX
第一章、前言 1
第二章、文獻回顧 2
壹、結腸癌之癌前病變-異常腺窩病灶 2
一、結腸癌 2
二、異常腺窩病灶 4
貳、氧化壓力與癌症 8
一、氧化壓力與癌症的關係 8
二、抗氧化物質對腸癌的預防效果 10
A.覆盆子 11
B.蘋果 11
C.水楊酸 11
D.綠茶 12
E.薑 13
F.白藜蘆醇 13
参、腸道益生菌對健康之影響 14
一、改善腹瀉 15
二、降低膽固醇 16
三、改善發炎性大腸疾病 16
四、降低胃潰瘍發生率 16
五、增進乳糖代謝 17
六、預防癌症發生 18
七、短鏈脂肪酸與腸道健康 19
八、改善腸道菌相功能評估 19
肆、天貝 20
一、天貝簡介 20
二、天貝製程及發酵過程中成分的變化 20
A.製程 20
B.發酵過程中成分的變化 22
三、生理活性 23
A.易消化 23
B.抗氧化 24
C.抗菌 25
D.其他 26
第三章、材料與方法 28
壹、實驗架構. 28
貳、實驗材料 29
一、樣品 29
二、實驗動物 29
三、儀器 29
四、藥品 30
参、實驗方法 32
一、樣品製備 32
A.天貝製備 32
B.天貝水萃物製備 32
二、體外實驗 33
A.天貝對微生物生長的影響 33
B.天貝對腸癌細胞生長的影響 37
三、動物餵食實驗 40
A.動物餵食 40
B. ACF計數 43
C.腸內菌相評估 44
D.抗氧化物質測定 50
E.糞便脂肪酸之測定 62
四、統計分析 63
第四章、結果與討論 64
壹、樣品製備 64
貳、體外實驗 64
一、天貝對微生物生長的影響 64
二、天貝對腸癌細胞生長的影響 65
参、動物實驗 68
一、體重、攝食、血液生化值 68
二、腸道菌相 79
三、短鏈脂肪酸 81
四、ACF計數 84
五、餵食黃豆或天貝對DMH誘發之大鼠體內抗氧化能力的影響 88
第五章、結論 91
第六章、參考文獻 92

【圖目錄】
圖1.歷年結腸直腸癌死亡人數 2
圖2.歷年結腸直腸癌死亡率 2
圖3.正常及過量熱量攝取的比較 4
圖4.異常腺窩病灶之形成過程 5
圖5.DMH的致癌機制 6
圖6.ACF的外觀 8
圖7.氧化壓力存在對癌症產生之影響 9
圖8.腸道表皮正常細胞受到自由基作用癌化之過程 10
圖9.腸道菌相與腸道環境的關係 14
圖10.天貝製程 21
圖11.烹煮過的黃豆及天貝萃取物對ETEC K88 (ID 1000) 黏附至小腸絨毛的影響 26
圖12.實驗架構 28
圖13.林慶福博士改良之天貝發酵法 33
圖14.以MTT assay 評估天貝水萃物對人類結腸癌細胞(Caco-2)活性的影響 39
圖15.動物試驗流程 42
圖16. ACF末段結腸染色步驟 44
圖17.各培養基的菌落 49
圖18.不同細胞量Caco-2細胞於DMEM培養基培養 96小時之生長曲線 66
圖19.天貝對Caco-2細胞生長的影響 67
圖20. SD大鼠餵食毒性試驗控制組(未誘發ACF且未餵食樣品)之臟器病理切片。對照組臟器無明顯組織病理變化(H&E stain, 400x)。72
圖21.大鼠餵食毒性試驗DMH誘發組(未餵食樣品)之臟器病理切片。誘發ACF組臟器無明顯組織病理變化(H&E stain, 400x)。 73
圖22.大鼠餵食毒性試驗經胃管餵食高劑量黃豆組之臟器病理切片。各臟器無明顯組織病理變化(H&E stain, 400x) 74
圖23.大鼠餵食毒性試驗經胃管餵食高劑量天貝組之臟器病理切片。各臟器無明顯組織病理變化(H&E stain, 400x) 75
圖24.染色後的結腸組織 84
圖25.黃豆及天貝對SD大鼠肝臟中抗氧化物質及酵素麩胱甘肽、超氧歧化酶、麩胱甘肽還原酶、晚期氧化蛋白、過氧化氫酶、麩胱甘肽過氧化酶之影響 90

【表目錄】
表一 異常腺窩之特徵 7
表二 影響ACF形成之因子 7
表三 益生菌與幽門螺旋桿菌感染 17
表四 以 Rhizopus microsporus接種在cowpea flour後,經浸泡、烹煮發酵後其中胰蛋白酶抑制劑含量的改變 23
表五 在不同培養基之天貝酶抑菌活性 25
表六 餵食天貝或黃豆後其腹瀉發生狀況。 27
表七 使用藥品 30
表八 測試菌株適用培養基 36
表九 抑制圈大小分級 36
表十 天貝及天貝水萃物對微生物生長的影響 65
表十一 SD大鼠經胃管餵食黃豆或天貝12週之體重變化及攝食量 70
表十二 SD大鼠經胃管餵食黃豆或天貝12週後相對器官重 (g/100g BW) 71
表十三 不同組間實驗動物之盲腸、盲腸壁、盲腸內容物重、酸鹼值及糞便水分含量 77
表十四 SD大鼠經胃管餵食黃豆或天貝12週後血清生化值 78
表十五 不同組別之盲腸內容物微生物菌相 82
表十六 連續胃管餵食黃豆或天貝12週對SD大鼠盲腸內容物短鏈脂肪酸之變化 83
表十七 黃豆或天貝對以DMH誘發產生異常腺窩病灶之影響 87
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