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研究生:謝啟弘
研究生(外文):Chi-Hung Hsieh
論文名稱:不同添加物進行樟芝穀物固態培養生產二次代謝物及其發酵物抗氧化力之影響
論文名稱(外文):The Effect of Additives on the Production of Secondary Metabolites and Antioxidant Properties on Grain Solid-State Culture of Antrodia cinnamomea
指導教授:梁志欽梁志欽引用關係何偉真何偉真引用關係
指導教授(外文):Zeng-Chin LiangWei-Chen Ho
口試委員:石信德吳秋曄
口試委員(外文):Hsin-Der ShihChiu-Yeh Wu
口試日期:2011-07-25
學位類別:碩士
校院名稱:大葉大學
系所名稱:生物資源學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:140
中文關鍵詞:樟芝穀物固態培養多醣體三萜類抗氧化
外文關鍵詞:Antrodia cinnamomeagrain solid-state culturepolysaccharidetriterpenoidantioxidant property
相關次數:
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野生樟芝子實體取得不易,不少單位研發以牛樟木人工栽培樟芝,作為生理活性研究及應付市場的需求,但樟芝生長速度緩慢而導致人工栽培的樟芝供不應求。本篇研究利用不同添加物進行樟芝穀物固態培養,了解不同培養條件 (不同添加物、中草藥及改變培養溫度) 對樟芝二次代謝物產量及其發酵物抗氧化力之影響。
穀物對照組部分,薏仁培養樟芝多醣含量第 30 天最高,為 23.01 mg/g,三萜類含量則是第 45 天最高,為 10.63 %;裸麥培養樟芝第 45 天多醣及三萜類含量最高,分別為 60.80 mg/g 及 10.11 %;小麥培養樟芝第 45 天多醣及三萜類含量最高,分別為 31.48 mg/g 及 0.40 %。
溫度 30 ℃ 部分,多醣體以樟芝於小麥培養第 60 天含量最高為 64.12 mg/g;三萜類以樟芝於裸麥培養第 60 天含量最高為 17.59 %。
添加物部分,多醣體以裸麥添加 0.5 % (w/w) CaCl2 培養樟芝第 30 天含量最高為 68.66 mg/g;三萜類以裸麥添加 0.5 % (w/w) 幾丁聚醣培養樟芝第 60 天含量最高為 16.65 %。
添加不同中草藥部分,多醣體以小麥添加 1 % (w/w) 厚朴培養樟芝第 15 天含量最高為 70.90 mg/g;三萜類以薏仁添加 1% (w/w) 紫蘇培養樟芝第 30 天含量最高為 33.72 %。
清除 DPPH 自由基能力,薏仁添加 0.5 % (w/w) 九層塔培養樟芝第 15 天其發酵物甲醇萃取液濃度 10 mg/ml 清除率最高為 95.47 %;螯合亞鐵離子能力,小麥添加 0.5 % (w/w) 幾丁聚醣培養樟芝第 60 天其發酵物甲醇萃取液濃度 20 mg/ml螯合力最高為 95.75 %;還原力則以裸麥添加 0.5 % (w/w) 荖藤培養樟芝第 60 天發酵物甲醇萃取液濃度 10 mg/ml 最高為 2.957。
Owing to the wild fruiting body is very rare and expensive, some researchers are developing artificial cultivation of Antrodia cinnamomea fruiting bodies using woodblocks of Cinnamomum kanehirae Hayata to meet the demands of tests for physiological function and markets. However, the supply of A. cinnamomea fruiting bodies is unable to meet the demand. The aim of this research is to explore the effect of different additive on the production of secondary metabolites and antioxidant properties of A. cinnamomea by grain solid-state culture. The cultures were under various conditions, and at a period of 30 or 45 days. Grains of Coix lacryma-jobi ,wheat, and pearl barley were used as basic medium respectively. The highest yield of polysaccharide and triterpenes produced by A. cinnamomea cultured on Coix medium were 23.01 mg/ml and 10.63 %, respectively. At 45 day, the highest yield of polysaccharide derived from A. cinnamomea cultured on pearl barley and wheat medium were 60.80 and 31.48 mg/ml, respectively. And the highest yield of triterpenes derived from A. cinnamomea cultured on pearl barley and wheat medium were 10.11 and 0.05 %, respectively. Culturing at 30 ℃ for 60 days, the highest yield of polysaccharide and triterpenes obtained on wheat and pearl barley media of A. cinnamomea were 64.12 mg/ml and 17.59 %, respectively. For different additives on culturing A. cinnamomea, the highest polysaccharide yield was 68.66 mg/ml when 0.5 % (w/w) CaCl2 was added to pearl barley medium at 30 day of culture . And the highest triterpenes obtained was 16.65 %, which was produced at 60 day by A. cinnamomea when 0.5 % (w/w) chitosan was used as additive in pearl barley medium. When Chinese medicinal herbs were added to culture media, the highest polysaccharide yielded 70.90 mg/ml on wheat medium with 1 % (w/w) Magnolia officinalis at 15 day. And the highest triterpenes produced was 33.72 % on pearl barley medium supplemented with 1 % (w/w) Perilla frutescens at 30 day culture of A. cinnamomea. The highest scavenging ability on DPPH radical.of methanolic extract (10 mg/ml) was 95.47 % obtained from culturing A. cinnamomea on Coix medium added with 0.5% (w/w) Ocimum basilicum at 15 day. The highest chelating capability on ferrous ions of methanolic extract (20 mg/ml) was 95.75% derived from adding 0.5 % (w/w) chitosan on wheat medium for culturing A. cinnamomea 60 days. And the highest reducing powers of methanolic extract (10 mg/ml) was 2.957 yielded from A. cinnamomea when culturing on pearl barley medium added with 0.5% (w/w) Piper betle at 60 day.
封面內頁
簽名頁
中文摘要....................................................................................................iii
英文摘要......................................................................................................v
誌謝........................................................................................................vii
目錄.........................................................................................................ix
圖目錄......................................................................................................xii
表目錄.......................................................................................................xv

第一章 前言...................................................................................................1
第二章 文獻回顧...............................................................................................4
第一節 樟芝概述........................................................................................4
一、樟芝的分類.......................................................................................4
二、樟芝的形態特徵...................................................................................4
三、樟芝的生物活性成分................................................................................5
(一) 三萜類.....................................................................................6
(二) 多醣體.....................................................................................9
第二節 自由基.........................................................................................11
一、自由基的種類....................................................................................11
二、自由基來源與疾病關...............................................................................16
第三節 抗氧化力.......................................................................................19
一、清除 α , α-Diphenyl-β-picrylhydrazyl (DPPH) 自由基能力之測定.....................................19
二、螯合亞鐵離子....................................................................................19
三、還原力.........................................................................................20
四、總抗氧化力......................................................................................20
五、清除超氧陰離子能力測定...........................................................................21
第四節 固態發酵培養....................................................................................22
一、固態發酵培養基質.................................................................................22
二、添加物對樟芝發酵培養之影響........................................................................23
第三章 材料方法..............................................................................................26
第一節 實驗材料.......................................................................................26
一、菌株...........................................................................................26
二、藥品...........................................................................................26
第二節 實驗方法.......................................................................................27
一、菌種保存與活化..................................................................................27
二、液態菌種製備....................................................................................27
三、不同穀物固態培養.................................................................................28
四、總多醣含量測定..................................................................................28
五、三萜類含量測定..................................................................................29
六、抗氧化力測定....................................................................................30
七、統計分析........................................................................................32
第四章 結果與討論.............................................................................................33
第一節 總多醣含量測定................................................................................33
一、薏仁添加不同添加物及在不同溫度進行樟芝固態培養之總多糖含量..........................................33
二、裸麥添加不同添加物及在不同溫度進行樟芝固態培養之總多糖含............................................37
三、小麥添加不同添加物及在不同溫度進行樟.芝固態培養之總多糖含量.........................................41
第二節 三萜類含量測定................................................................................45
一、薏仁添加不同添加物及在不同溫度進行樟芝固態培養之三萜類類含量........................................45
二、裸麥添加不同添加物及在不同溫度進行樟芝固態培養之三萜類含量..........................................47
三、小麥添加不同添加物及在不同溫度進行樟芝固態培養之三萜類含量..........................................49
第三節 抗氧化力測定..................................................................................52
一、清除 DPPH 自由基能力.........................................................................52
二、螯合亞鐵離子能力測定..........................................................................62
三、還原力測定...................................................................................71
第五章 結論..................................................................................................81
參考文獻.....................................................................................................83
附錄.........................................................................................................90

圖目錄
圖 1、牛樟芝之顯微特徵。........................................................................................5
圖 2、從樟芝子實體分離出的三萜類化合物。..........................................................................7
圖 3、從樟芝子實體分離出的三萜類化合物。..........................................................................8
圖 4、使用X-射線繞射分析 β-1,3-D-葡聚醣三股螺旋結晶構形。.........................................................10
圖 5、主要的活性氧種類。.......................................................................................12
圖 6、哺乳類動物細胞中氧和氮自由基產物及其他反應種類。.............................................................14
圖 7、脂質過氧化過程。.........................................................................................15
圖 8、組織損害引起氧化壓力的原因(A)及氧化壓力對人類疾病的關係(B)。..................................................17
圖 9、實驗架構圖。............................................................................................25
圖 10、樟芝於不同溫度及添加物的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的清除 DPPH 自由基能力。............53
圖 11、樟芝於不同溫度及添加物的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的清除 DPPH 自由基能力。............55
圖 13、25℃下樟芝於添加不同中草藥的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的清除 DPPH 自由基能力。.........57
圖 14、25℃下樟芝於添加不同中草藥的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的清 DPPH 自由基能力。..........58
圖 15、25℃下樟芝於添加不同中草藥的小麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的清除 DPPH 自由基能力。.........60
圖 16、樟芝於不同溫度及添加物的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。................63
圖 17、樟芝於不同溫度及添加物的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。................64
圖 18、樟芝於不同溫度及添加物的小麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。................65
圖 19、25℃下樟芝於添加不同中草藥的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。.............67
圖 20、25℃下樟芝於添加不同中草藥的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。.............68
圖 21、25℃下樟芝於添加不同中草藥的小麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的螯合亞鐵離子能力。.............70
圖 22、樟芝於不同溫度及添加物的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.........................72
圖 23、樟芝於不同溫度及添加物的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.........................73
圖 24、樟芝於不同溫度及添加物的小麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.........................74
圖 25、25℃下樟芝於添加不同中草藥的薏仁培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.....................76
圖 26、25℃下樟芝於添加不同中草藥的裸麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.....................77
圖 27、25℃下樟芝於添加不同中草藥的小麥培養基培養 60 天其發酵物甲醇萃取液濃度 10 mg/ml 的還原力。.....................78

表目錄
表一、氧化對人體的可能的傷害....................................................................................18
表二、不同溫度下薏仁培養樟芝多醣產量的變化........................................................................34
表三、25℃下薏仁添加不同添加物培養樟芝多醣產量的變化...............................................................35
表四、25℃下薏仁添加不同比例中草藥培養樟芝多醣產量的變化...........................................................36
表五、在不同溫度下裸麥培養樟芝多醣產量的.........................................................................38
表六、25℃下裸麥添加不同添加物培養樟芝多醣產量的變化...............................................................39
表七、25℃下裸麥添加不同比例中草藥培養樟芝多醣產量的變化...........................................................40
表八、在不同溫度下小麥培養樟芝多醣產量的變化......................................................................42
表九、25℃下小麥添加不同添加物培養樟芝多醣產量的變化...............................................................43
表十、25℃下小麥添加不同比例中草藥培養樟芝多醣產量的變化….........................................................44
表十一、薏仁添加不同添加物及不同溫度下培養樟芝三萜類含量的變化......................................................46
表十二、裸麥添加不同添加物及在不同溫度下培養樟芝三萜類含量的變化....................................................48
表十三、小麥添加不同添加物及在不同溫度下培養樟芝三萜類含量的變化....................................................51

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