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Student Number 88321043
Author Z-Yi Wu(吳姿宜)
Author's Email Address No Public.
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Department Chemical and Materials Engineering
Year 2000
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title 探討不同培養方式對猴頭菇抗氧化與抗腫瘤性質的影響
Date of Defense 2001-07-16
Page Count 83
Keyword
  • 抗氧化
  • 抗腫瘤
  • 猴頭菇
  • Abstract 文獻指出人體老化、病變與體內氧化還原平衡失調有關,科學家證實自由基(free radical)與多種疾病的產生有密切的關係,他能引起動脈粥狀硬化、糖尿病、癌症、發炎反應、老化等疾病。近年來發現許多的菇類(如舞菇、猴頭菇、金針菇、香菇、雲芝和赤芝等)以中極性甲醇萃取評估其具有抗氧化性質的成分,菇類的抗氧化性更值得去探討。猴頭菇在中國是著名的藥用真菌之一,其生理活性成分豐富,據文獻記載,猴頭菇子實體中的抗氧化物質以酚類物質所佔的比率最高,加上有許多的研究指出酚類物質具有相當強的抗氧化力與抗腫瘤能力,以茶多酚為例,能抗低密度脂蛋白氧化作用、有抗衰老作用、對抗紫外線及放射線致癌作用、抗突變等作用。
    本研究將探討不同萃取溶液(甲醇、乙醇、丙酮)、不同菌種(猴頭王、中特猴、CCRC 36431、CCRC 36431)、不同培養方式(固態培養、搖瓶培養、發酵槽培養)對猴頭菇子實體萃取酚類物質能力或菌絲體的菌體產率、總酚產率及其清除DPPH自由基的影響進行比較,以選出較佳的培養條件與分析方法。然後在以最適條件進行改變培養基實驗,在培養基中添加有利物質-綠茶多酚,探討添加綠茶多酚是否能增加猴頭菇抗氧化、及抗腫瘤性質。
    實驗結果發現,猴頭菇的酚類物質萃取溶液以乙醇較為適合,菌種篩選部份則以猴頭王生長速率最快,萃取出來的酚類物質含量及清除自由基能力最強,品質最好,而培養方式則以2L bubble column發酵槽效率最好,菌絲體萃取液酚類物質產率(1.12mg/g.day)遠比子實體含量高,約為子實體的16.4倍,且其菌絲體清除DPPH自由基能力(86.8%)約為子實體的2.3倍。在培養基中添加不同量綠茶多酚部份,隨著綠茶多酚添加量的增加,猴頭菇的生長速率、菌體轉化率、總酚產率、水溶性酚類物質之抗腫瘤能力有增加的趨勢,以添加3.3g綠茶多酚為例,他的生長速率為0.0364 1/hr,約為control組的2倍,總酚產率為0.113 mg/g-hr為control組的2.3倍。探討水溶性酚類物質對不同癌細胞的抑制能力部份,發現菌絲體所得的腫瘤抑制率較子實體抑制能力高,隨著綠茶多酚添加量的增加,其抑制能力有增加的趨勢。以胃癌為例,添加3.3g綠茶多酚所得菌體水溶性酚類濃度為7.5ppm,胃癌細胞的存活率為35.8%較control組的存活率72.6%高。
    Table of Content 圖索引………………………………………………………………….Ⅰ
    表索引………………………………………………………………….Ⅳ
    第一章前言…………………………………………………………….1
    1.1.研究背景……………………………………………………………1
    1.2.研究目的……………………………………………………………2
    第二章文獻回顧……………………………………………………….4
    2.1.菇類的介紹…………………………………………………………4
    2.2.猴頭菇的介紹………………………………………………………4
    2.2.1.商業化產品……………………………………………………5
    2.2.2.生理活性物質…………………………………………………6
    2.2.2.1.多醣類(polysaccharides)…………………………….7
    2.2.2.2. HeLa細胞增殖的抑制物……………………………….8
    2.2.2.3神經成長因子(NGF)合成的促進物質.…………………10
    2.3.抗氧化物質……………………………………………………….11
    2.3.1.自由基形成的原因………………………………………….12
    2.3.2.自由基致病機理…………………………………………….15
    2.3.3.抗氧化劑的作用…………………………………………….16
    2.3.4.抗氧化物質………………………………………………….18
    2.3.4.1人工合成抗氧化劑………………………………………18
    2.3.4.2.天然抗氧化劑………………………………………….19
    2.4.茶多酚介紹……………………………………………………….20
    2.5.萃取方法介紹…………………………………………………….24
    2.6.液態發酵………………………………………………………….25
    2.7.動物細胞簡介…………………………………………………….25
    第三章材料與方法……………………………………………………27
    3.1.藥品與儀器……………………………………………………….27
    3.1.1.藥品………………………………………………………….27
    3.1.2.分析儀器…………………………………………………….28
    3.2.細胞株與菌種…………………………………………………….29
    3.3.論文架構………………………………………………………….31
    3.4.材料:可分子實體與菌絲體兩種…………………………………32
    3.4.1.猴頭菇子實體……………………………………………….32
    3.4.2.猴頭菇菌絲體……………………………………………….33
    3.4.3.液態培養…………………………………………………….34
    3.4.3.1.菌種保存……………………………………………….34
    3.4.3.2.菌種活化……………………………………………….34
    3.4.3.3.搖瓶培養……………………………………………….35
    3.4.3.4.發酵槽培養…………………………………………….35
    3.5.分析方法………………………………………………………….36
    3.5.1.酚類物質萃取流程………………………………………….36
    3.5.2.茶多酚萃取方法…………………………………………….39
    3.5.3.葡萄糖含量測定…………………………………………….39
    3.5.4.茶多酚含量測定…………………………………………….39
    3.5.5.總酚類化合物測定………………………………………….40
    3.5.6.捕抓1,1-二苯基-2-苦味併基(DPPH•)自由基能力之測定.40
    3.6.酚類物質抗腫瘤能力測定……………………………………….41
    3.6.1.細胞株保存………………………………………………….41
    3.6.2.細胞株解凍………………………………………………….42
    3.6.3.動物細胞繼代培養………………………………………….42
    3.6.4.動物細胞培養液組成……………………………………….42
    3.6.5.MTT 細胞活性染色法(MTT assay)………………………..44
    第四章 結果與討論…….……………………………………………46
    4.1子實體最適萃取溶液之探討………………………………………46
    4.1.1.不同萃取溶液之萃取率、總酚含量的測定…………………46
    4.1.2.清除DPPH自由基的比較……………………………………47
    4.2.子實體萃取之水溶性酚類物質其抗腫瘤能力之探討………….49
    4.3.篩選菌種………………………………………………………….51
    4.4.不同培養方式的探討(子實體、搖瓶、發酵槽)……………….55
    4.5.添加不同量茶多酚對猴頭菇發酵槽發酵的影響……………….57
    4.5.1.觀察添加不同量茶多酚其發酵液顏色之變化情形……….57
    4.5.2.觀察添加不同量茶多酚其菌絲體顏色之變化情形……….59
    4.5.3.添加不同濃度之綠茶多酚對猴頭菇液態發酵結果
    總整理……………………………………………………….61
    4.5.4.添加不同量茶多酚其發酵液中OD 420隨時間的
    變化情形…………………………………………………….63
    4.5.5.添加不同量茶多酚其發酵液中總酚含量隨時間的
    變化情形…………………………………………………….66
    4.5.6.添加不同量茶多酚發酵槽所得菌體之總酚含量與清除自由基能力……………………………………………………….68
    4.5.7.添加不同量茶多酚發酵槽所得菌體之水溶性酚類物質的抗腫瘤能力…………………………………………………….70
    4.5.7.1.添加0g綠茶多酚發酵槽菌體所得水溶性酚類物質對不同癌細胞的抑制能力………………………………….70
    4.5.7.2.添加1.3g綠茶多酚發酵槽菌體所得水溶性酚類物質對不同癌細胞的抑制能力……………………………….71
    4.5.7.3.添加3.3g綠茶多酚發酵槽菌體所得水溶性酚類物質對不同癌細胞的抑制能力……………………………….72
    4.5.7.4.猴頭菇子實體與添加不同濃度茶多酚發酵所得菌體其酚類物質抗腫瘤能力的比較………………………….73
    第五章結論與建議………………………………………………….76
    5.1.結論……………………………………………………………….76
    5.2.建議……………………………………………………………….77
    參考文獻……………………………………………………………….79
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