游若篍Yu, Roch-Chui臺灣大學:食品科技研究所王姿以Wang, Tzu-YiTzu-YiWang2010-05-112018-06-292010-05-112018-06-292009U0001-1008200915280800http://ntur.lib.ntu.edu.tw//handle/246246/182207人體腸胃道菌相種類複雜，包含益菌如雙叉桿菌(bifidobacteria)及乳酸桿菌(lactobacilli)及壞菌如產氣莢膜梭菌(Clostridium perfringens)等，腸道好壞菌相之平衡對人體健康扮演重要角色。許多文獻指出攝食益生質(prebiotics)能選擇性促進腸道好菌生長，進而改善宿主腸道健康，菊醣是益生質的一種，體外及體內之研究皆顯示可促進腸道中雙叉桿菌生長。本研究探討攝食菊醣對20位22~30歲健康成人腸道菌相、糞便水分含量及pH值之影響，並評估攝食菊醣降低糞便萃取液對HT-29及Caco-2細胞所造成之細胞毒性及基因毒性之效果。試驗分成四個時期，第一期為飲食調整期，第二及第三期為攝食前期及攝食後期，第四期為排空期。驗結果顯示攝食菊醣後，實驗組糞便中之雙叉桿菌菌數由第一期之9.46 log CFU/g顯著增加到第二期之10.39 log CFU/g (p < 0.01)及第三期之10.90 log CFU/g (p < 0.01)，控制組變化不大，且實驗組之第二及第三期雙叉桿菌菌數與控制組之第二及第三期比較，均有顯著差異 (p < 0.01)。實驗組糞便中之產氣莢膜梭菌菌數由第一期之7.36 log CFU/g顯著降低至第二期之6.93 log CFU/g (p < 0.05) 及第三期之6.60 log CFU/g (p < 0.01)，與控制組第二及第三期比較皆有顯著差異(p < 0.01)。試驗期間實驗組與控制組糞便水分含量分別為75.5~77.7%及77.3~78.6%，兩組間無顯著差異 (p > 0.05)；實驗組糞便pH值由第一期之6.60顯著降低至第二期之6.14及第三期之5.96 (p < 0.01)，而控制組pH值在試驗之四個時期無顯著變化 (p > 0.05)，約為6.40~6.62。試驗前後受試者身高、體重、BMI值及血液生化值均無顯著變化 (p > 0.05)，顯示本試驗對受試者無有不良影響。胞毒性試驗結果顯示，攝食菊醣可降低實驗組受試者糞便萃取液對HT-29細胞及Caco-2細胞株之毒性 (p < 0.05)，而控制組則無差異 (p > 0.05)。基因毒性部份，實驗組攝食菊醣後，其糞便萃取液對HT-29及Caco-2細胞之基因毒性亦顯著下降 (p < 0.05)。綜合上述，本試驗確認攝食菊醣可顯著增加腸道中益菌數，減少壞菌數，降低糞便萃取液對腸道細胞之毒性，可改善腸道健康。鍵字：腸道菌相、菊醣、雙叉桿菌、產氣莢膜梭菌、細胞毒性、基因毒性The gastrointestinal tract of human is a complex ecosystem which contains beneficial bacteria such as bifidobacteria and lactobacilli and pathogenic bacteria such as C. perfringens. The balance between beneficial bacteria and harmful pathogenic bacteria plays a key role in normal gut function and human health. Many researches indicate that the administration of prebiotics can modulate the intestinal microflora. Inulin, a kind of prebiotics, can selectively stimulate the growth of bifidobacteria. The aim of this study is to evaluate the effects of inulin intake on the change of intestinal microflora, fecal water content and fecal pH value in human subjects and to investigate fecal cytotoxicity and genotoxcity towards HT-29 and Caco-2 cells. Results indicated that inulin intake significantly increased the fecal bifidobacteria of treatment group from 9.46 log CFU/g in period 1 to 10.39 log CFU/g in period 2 and 10.90 log CFU/g in period 3 (p < 0.01), and were both higher than control group in period 2 and 3 (p < 0.01). Fecal C. perfringens also decreased significantly in treatment group from 7.36 log CFU/g in period 1 to 6.93 log CFU/g in period 2 (p < 0.05) and 6.60 log CFU/g in period 3 (p < 0.01), and were both lower than control group in period 2 and 3 (p < 0.01). During the experiment, fecal water contents were 75.5~77.7 % in treatment group and 77.3~78.6 % in control group without significant difference (p > 0.05). Fecal pH value of control group was 6.40~6.62 without significant difference (p > 0.05). However, in treatment group, the pH value significantly decreased from 6.60 in period 1 to 6.14 and 5.96 in period 2 and 3 after inulin intake (p<0.01). No differences were found for the height, weight, BMI and serum biochemical values of 20 subjects before and after the experiment (p > 0.05). MTT assay showed that inulin intake significantly decreased the fecal water cytotoxicity of treatment group toward HT-29 and Caco-2 cells (p < 0.05), but not in control group. Results from comet assay revealed that after inulin consumption, the genotoxicity of fecal water in treatment group significantly decreased (p < 0.05). To conclude the above, this study confirmed that inulin intake could increase the beneficial bacteria bifidobacteria but decrease harmful bacterial C. perfringens in human intestinal tract, and could decrease the toxicity of fecal water and therefore improve human intestinal health.ey words: intestinal microflora, inulin, bifidobacteria, Clostridium perfringens, cytotoxicity, genotoxicity目錄 頁次誌...................................................i要...................................................iii文摘要...............................................iv錄...................................................v目錄.................................................viii目錄.................................................ix、前言...............................................1、文獻整理...........................................2、人體消化道菌群分布.................................2一） 人體腸道菌相之簡介 ............................2二） 影響腸道菌相之因素............................7、腸道菌相與人體健康.................................10一） 腸道菌相之功效................................10二） 腸內物質的組成................................12三） 腸道細菌代謝造成的毒性影響....................12、糞便毒性與大腸癌...................................18一）糞便之毒性.......................................18二）大腸癌...........................................18、測定大腸功能之方法.................................19一）簡介.............................................19二）基因毒性試驗法--慧星試驗 (Comet assay) .........19、雙叉桿菌之簡介.....................................22一） 雙叉桿菌的介紹................................22二） 雙叉桿菌的營養機能及健康功效..................22、益生質之簡介.......................................25一） 益生質........................................25二） 菊醣類寡醣介紹................................25三） 菊醣類寡醣之健康功效..........................27、材料與方法.........................................29、實驗架構...........................................29、實驗材料...........................................33一）菊醣膳食纖維.....................................33二）試驗細胞株.......................................33三）藥品.............................................33四）器材.............................................35、實驗方法...........................................36一） 人體試驗之設計................................36. 受試者........................................36. 試驗設計......................................37. 飲食控制與飲食紀錄............................38二） 人體試驗之方法................................38. 培養基........................................38. 取樣及微生物檢測..............................39. 糞便檢體水分含量測定..........................40. 糞便檢體pH值測定..............................40三） 細胞株的活化、繼代培養與保存..................40四） 糞便萃取液之製備..............................41五） MTT assay.....................................42六） 糞便檢體萃取液對HT-29及Caco-2之細胞毒性試驗...42七） 糞便檢體萃取液對HT-29及Caco-2之基因毒性試驗...43、統計分析...........................................44、結果與討論.........................................45、試驗期間受試者身高體重、血液生化值及每日營養素平均攝取量之變化.................................................45、攝食菊醣對受試者腸道中雙叉桿菌之影響...............46、攝食菊醣對受試者腸道中產氣莢膜梭菌之影響...........47、攝食菊醣對受試者腸道中雙叉桿菌及產氣莢膜梭菌菌數對數比值之影響.................................................49、攝食菊醣對受試者糞便中水分含量及pH值之影響.........50、攝食菊醣之受試者糞便萃取液對HT-29及Caco-2細胞株之細胞毒性試驗.................................................51、攝食菊醣之受試者糞便萃取液對HT-29及Caco-2細胞株之基因毒性試驗.................................................54、結論...............................................57、參考文獻...........................................77錄...................................................88錄一、受試者基本資料.................................88錄二、糞便性質與生活習慣問卷.........................89錄三、飲食習慣問卷...................................91錄四、人體試驗後測問卷...............................92錄五、飲食紀錄範例...................................94錄六、臺灣保健食品學會同意臨床試驗證明書—菊醣（中文）...................................................95錄七、臺灣保健食品學會同意臨床試驗證明書—菊醣（英文）...................................................96application/pdf3516798 bytesapplication/pdfen-US腸道菌相菊醣雙叉桿菌產氣莢膜梭菌細胞毒性基因毒性intestinal microflorainulinbifidobacteriaClostridium perfringenscytotoxicitygenotoxicity[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/182207/1/ntu-98-R96641005-1.pdf