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研究生:陳媺蕙
研究生(外文):Mei-Huei Chen
論文名稱:利用裏氏木黴固態發酵玉米胚芽與麩皮之產物對降低類澱粉β-胜肽毒性之功效
論文名稱(外文):Solid fermentation of corn germ and bran by Trichoderma reesei for reducing cytotoxicity of amyloid β peptide.
指導教授:蔣丙煌蔣丙煌引用關係
口試委員:李敏雄陳錦樹周正俊
口試日期:2015-07-29
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:2015
畢業學年度:103
語文別:中文
論文頁數:90
中文關鍵詞:阿茲海默症類澱粉蛋白質玉米胚芽玉米麩皮固態發酵
外文關鍵詞:Alzheimer’s diseaseamyloid-β peptidescorn germcorn bransolid state fermentation
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阿茲海默症主要由類澱粉胜肽(Amyloid β-peptide, Aβ)堆積在腦中形成斑塊,使神經細胞發生氧化與發炎反應,最後導致神經細胞凋亡。近年來許多研究顯示,自然界中的抗氧化劑,例如維生素E、多酚類和類胡蘿蔔素,具有清除自由基和抑制ROS形成之功能,並可修復Aβ產生的發炎與氧化反應,使腦神經細胞受到保護。玉米為世界上重要的穀類作物,研究指出玉米的總酚含量和抗氧化活性皆高於小麥、燕麥和稻米,其富含具抗氧化活性的植化素,主要分布於胚芽與麩皮。玉米胚芽與麩皮為具高活性的抗氧化物,但過去並無文獻探討其對抑制Aβ毒性之功效。另外,日常生活中攝取玉米胚芽主要來自於玉米油,而傳統的製油方式在乾燥胚芽的過程中,經常會使油類發生高度的氧化作用,使胚芽內部的營養物質變質,有研究指出,利用裏氏木黴生產的纖維素酶可將細胞壁分解而釋放油脂,可保有玉米胚芽中的抗氧化物。麩皮除了具有抗氧化物質外,也因富含纖維素可提供微生物良好的生長碳源。因此,本研究目的以裏氏木黴進行固態發酵,產生纖維素酶分解玉米胚芽與麩皮的細胞壁,進而釋放內部的有效物質,接著以細胞平台(IMR-32 cell line)測定發酵液的細胞毒性與抑制Aβ毒性之功效,並用不同溶劑進行萃取以分析有效物質。實驗組別將胚芽與麩皮混和成不同比例,共有五組,分別有胚芽與麩皮比例1:2和2:1,以及水分含量較少的組別1:3、3:1和2:2。結果顯示,各組發酵產物之萃取液皆於濃度100μg/mL以下,細胞存活率達80%以上。在抑制Aβ毒性的能力試驗上,以發酵五天之發酵萃取物的功效最佳,各組在濃度100μg/mL時皆有90%以上之細胞存活率,其中,以胚芽與麩皮的比例3:1細胞存活率達110%,其抑制Aβ毒性之功效最顯著,因此,選此組別進行萃取分析。在不同溶劑萃取發酵液的試驗中,以正己烷、乙酸乙酯、正丁醇和水萃取,結果發現,正丁醇和水層之萃取發酵液,其抑制Aβ毒性效果最好,細胞存活率分別達95%和104%,推測在正丁醇和水層中可萃取出具神經保護功效之物質。將各層萃取液做成分分析,結果顯示正丁醇之總酚含量有507.8 mg/g,而水層萃取液其總酚含量則達324.6 mg/g,推測總酚為功效物質。水層萃取液的總酚含量雖然為次高,但其抑制Aβ毒性之功效為最好,參考前人研究,推測水層中可能含有小分子的碳水化合物,具有清除羥自由基(OH•)的能力。因此,經由裏氏木黴固態發酵玉米胚芽與麩皮之發酵產物,具有預防阿茲海默症的潛力。

The major hallmarks of Alzheimer’s disease (AD) is the accumulation of amyloid-β peptides (Aβ). Aβ would promote pro-inflammatory responses and activate neurotoxic pathways, leading to dysfunction and death of brain cells. Recently studies showed that the nature antioxidants, such as vitamin E, polyphenols and carotenoids, could protect neurons from inflammation and oxidation triggered by Aβ. Corn is the important cereal in the world. Studies indicate that the content of polyphenols and antioxidant activity in corn are higher than wheat, oats, and rice, and mainly distribute in the corn germ and bran. However, there were very few studies about the corn germ and bran on reducing cytotoxicity of amyloid β peptide. In addition, the dietary source of corn germ is mainly from corn oil. In the traditional way for making oil, the drying process would destroy the nutrient in corn germ. Recently, some studies indicate that enzymes producing form Trichoderma reesei not only could decompose the cell wall to release oil, but also have much more effective compounds in the milder process. And corn brans are rich in cellulose to provide T. reesei grow. In this study, we used Trichoderma reesei to decompose the corn germ and bran’s cell wall, and then released the effective compounds during the solid fermentation state. The human neuroblastoma (IMR-32) and amyloid-β peptides was used as in vitro model to evaluate the bioactivity of the fermentation product. The effective compounds were extracted from the fermentation product by different solvents. We mixed corn germ and bran in different ratio and there were six sample, such as 1:2, 1:3, 2:1, 3:1 and 2:2. Results showed that the dose below 100μg/mL would not exert cytotoxic effect on the human neuroblastoma. The ethanol extracts of 5 day fermented products could reduce the cytotoxic of Aβ best. Because 3:1 sample had the significant inhibit effect on cytotoxic of Aβ, we used it for further extraction. We use n-hexane, ethyl acetate, n-butanol and water for extraction. Results showed that the layer of n-butanol and water had the significant effect on reducing the cytotoxic of Aβ. We thought the layer of n-butanol and water had the protective compounds for neurons. Results of the extraction analysis showed that the layer of n-butanol had highest total phenolic contents about 507.8 mg/g, but the layer of water had lower total phenolic contents about 324.6 mg/g. Maybe the bioactivity of the fermentation product are phenolic compound in corn. In addition, previous studies show that the layer of water may have small molecule carbohydrates, and they could scavenge the hydroxyl radicals. To sum up, corn germ and bran fermented by T. reesei have potential neuroprotective effect on AD.

目錄
第一章、緒論 1
第二章、文獻回顧 3
第一節、阿茲海默症(Alzheimer''s disease)的相關介紹 3
2-1-1. 症狀與病理特徵(Symptoms and Pathology) 3
2-1-2. 成因(Etiology) 9
2-1-3. 治療方式 12
第二節、玉米 14
2-2-1. 介紹 14
2-2-2. 結構與化學組成 14
2-2-3. 玉米油 18
2-2-4. 功效物質 20
2-2-4-4. 阿魏酸阿拉伯糖 22
第三節、裏氏木黴(Trichoderma reesei) 25
2-3-1. 介紹 25
2-3-2. 纖維素酶與半纖維素酶 25
第四節、固態發酵 28
2-5-1. 固態發酵(Solid State Fermentation , SSF)的介紹 28
2-5-2. 固態發酵的優缺點 28
2-5-3. 固態發酵的應用 29
第五節、IMR-32細胞株 32
2-5-1.介紹 32
第三章、實驗目的與設計 34
第一節、實驗目的 34
第二節、實驗架構 35
3-2-1. 裏氏木黴固態發酵 35
3-2-2. 細胞平台的建立與驗證 36
第四章、材料與方法 37
第一節、實驗材料 37
4-1-1.細胞來源與型態 37
4-1-2.微生物菌種 37
4-1-3.藥品試劑 37
第二節、實驗儀器 38
第三節、實驗方法 40
4-3-1. 樣品發酵 40
4-3-3. 細胞實驗 41
4-3-4. 成分分析 43
第五章、結果與討論 46
第一節、發酵過程變化 46
5-1-1.pH值 46
5-1-2.還原糖含量分析 50
第二節、細胞實驗 55
5-2-1.發酵物之細胞毒性測試 55
5-2-2.發酵物對Aβ之毒性測試 64
5-2-3.分層萃取液對Aβ之毒性測試 74
5-2-4.分層萃取液成分分析-總酚含量分析 77
第六章、結論與未來展望 81
第七章、參考文獻 83




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