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研究生:張謙裕
研究生(外文):Chien-Yu Chang
論文名稱:樟芝菌絲體量產技術開發及相關功能性之探討
論文名稱(外文):Studies on the Mass Production of Antrodia cinnamomea Mycelia and Its Related Biological Activities
指導教授:潘子明潘子明引用關係
指導教授(外文):Tzu-Ming Pan
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:193
中文關鍵詞:樟芝核醣體RNA基因三萜子實體多醣體反應曲面法液體浸沉培養
外文關鍵詞:Antrodia cinnamomearibosomal RNA genestriterpenoidfruiting bodiespolysaccharideresponse surface methodologysubmerged culture
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利用核醣體RNA基因 [包括ITS1、ITS2、5.8S與18S核醣體RNA基因] 的序列為指標,配合特殊引子聚合酶鏈反應 (Polymerase chain reaction)之分子生物學鑑定技術,來鑑別從台灣南部六龜山區所採集之樟芝子實體並經分離與純化後之樟芝菌株,(編號ACTS1與AC0623)是否學名為Antrodia cinnamomea之物種,並與其他的相關菌株包括不同屬之菌株(Antrodiella semisupina, Antrodiella romellii 與 Trametes versicolor)進行序列比對與鑑別,結果證實從子實體分離之樟芝菌株ACTS1與AC0623與食品工業發展研究所生物資源保存及研究中心之菌株BCRC 35396與BCRC 35398應屬同種之相似菌株。本研究以樟芝AC0623菌株為主,利用反應曲面法探討液體浸沉培養之菌絲體生成之最適培養條件,以提高菌體與三萜類的產量,結果指出,搖瓶之液體浸沉培養於28℃,起始pH 5.5及攪拌速率105 rpm下,乾菌體量與三萜類成分分別可增至3.2% (w/w)及31.8 mg/g。本實驗繼續擴大至100-L與700-L槽之培養,根據RSM,經由控制培養基組成及培養條件,於700-L槽之培養,可得到高的三萜類成分(63 mg/g),總多醣含量亦達5.59% (w/w),另一方面,乾菌體量2.0% (w/w)實際上也已經達到工業量產的規模。本研究繼續利用高效液相層析儀,針對樟芝A. cinnamomea AC0623菌株,探討於5公升與100公升醱酵槽培養時,菌體之腺苷、蟲草素等各成分之變化趨勢,同時亦以A. cinnamomea AC0623菌株進行700-L槽培養,經噴霧乾燥處理之乾菌體量,其腺苷、蟲草素及麥角固醇的含量分別為15.70 mg/g、0.29 mg/g與 0.98 mg/g。另從樟芝A. cinnamomea AC0623菌株醱酵菌絲體中萃取多醣體,分析其對人類全血細胞激素的誘導產生量,藉此驗證樟芝多醣的免疫調控功能。醱酵液固液分離後,菌絲體再分別以熱水萃取,萃取液經酒精沉澱,獲得不同的區分,其中區分Ⅰ∼Ⅳ菌絲體熱水可溶區分,明顯誘導全血中IL-6,TNF-α 和IFN-γ等細胞激素的釋放。醱酵液亦獲致同樣的結果,顯示樟芝醱酵不論胞内之菌體或胞外醱酵液均可誘導IL-6 與TNF-α 的釋放。而進一步分析這些區分的多醣,並利用GPC 管柱測定分子量的分佈,得知本試驗經醱酵槽培養之樟芝菌體多醣體含有8.52 × 104 Da。樟芝醱酵菌絲體樣品測定β-D-glucan 含量約0.71%(w/w)。本菌株醱酵菌絲體進行總抗氧化力分析,發現樣品經萃取後使用DMSO 溶劑溶解具有相當高的抗氧化作用。樟芝醱酵液離心之上清液,經試驗證明有抑制tyrosinase 活性的功效,亦即可抑制黑色素的形成,因此在美白產品的應用上具無限的潛力與前景。
本研究並同時對樟芝BCRC 35396 菌株進行醱酵,生理活性與抗發炎之探討,另置於附錄中;BCRC 35396 菌株於醱酵過程中所分泌出的醱酵液代謝物中,含有極為顯著的抗發炎物質,於樟芝醱酵液的劃分過程中,我們得到一具有細胞毒性的成份,經分離純化後再以質譜儀測定其分子量,判斷此具細胞毒性的分子為1-hydroxy-3-isobuty1-4-[4- (3-methy1-2-butenyloxy)- pheny1]-pyrrole-2, 5-dione。
The partial sequences of ribosomal RNA genes, including the internal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA gene, internal transcribed spacer 2 (ITS2), and 18S ribosomal RNA gene, was sequenced and applied to identify the two strains, ACTS1 and AC0623, which were isolated from the fruiting bodies of Antrodia cinnamomea. Comparison of partial nucleotide sequences (ITS1, 5.8S, and ITS2) with those of the strains from different genus (Antrodiella semisupina, Antrodiella romellii and Trametes versicolor) was made. These DNA sequences data demonstrated that there was no difference among ACTS1, AC0623, BCRC 35396 and BCRC 35398. The results suggested that the two strains, ACTS1 and AC0623, isolated from fruiting bodies, and BCRC 35396 and 35398 strains, the original A. cinnamomea strain obtained from Food Industry Research and Development Institute (FIRDI), were the same species.
Further study using Antrodia cinnamomea AC0623 for large-scale fermentation was employed. Response surface methodology was used to optimize medium composition for the increment of biomass and triterpenoids production. The results indicated that when a submerged culture in shaking flasks was operated at 28℃, initial pH 5.5 and rotation speed 105 rpm, the biomass and triterpenoids content could be increased to 3.20 % (dry cell weight, w/w) and 31.8 mg/g, respectively. The experiments were further scale-up to 100-L and 700-L fermentors. Higher content of triterpenoids (63 mg/g) and total polysaccharide (5.59%, w/w) were obtained in 700-L fermentation by means of the control of cultural conditions and the modification of medium composition based on the RSM. It is profitable if the biomass reaches 2.0% (w/w), as a result, current study demonstrated a high possibility for commercialization.
The application of high-performance liquid chromatography (HPLC) method for quantitation of adenosine and cordycepin in 5-L and 100-L fermentation from A. cinnamomea AC0623 was also described. The strain A. cinnamomea AC0623 was cultivated in a 700-L fermentor. The contents of adenosine, cordycepin and ergosterol in the whole cell of spray-dried powder were 15.70 mg/g、0.29 mg/g and 0.98 mg/g, respectively. The immunomodulatory effect of the functional polysaccharides of A. cinnamomea AC0623 strain was investigated. The intracellular polysaccharide fraction, Fractions Ⅰ∼Ⅳ, which was prepared by hot water extraction of mycelium followed by ethanol precipitation, induced the release of IL-6, TNF-α and IFN-γ in human whole blood dose-dependently. Same results were obtained by using culture filtrate. The molecular mass distribution of polysaccharide was estimated 8.52 × 104 Da in Fraction Ⅰ, as determined by gel permeation chromatography (GPC) with refractive index detector. The β-D-glucan content was estimated about 0.71% (w/w) in the mycelium. Higher antioxidative activity was also found in DMSO solvent extract of A. cinnamomea AC0623. Cultural filtrate also inhibited the tyrosinase activity, it implied the potential in whitening cosmetic application.
The effects of A. cinnamomea BCRC 35396 on the inhibition of nitric oxide synthesis and cytotoxicity in murine macrophage RAW264.7, was investigated. The result indicated that the extract of. A. cinnamomea BCRC 35396 culture filtrate inhibited lipopolysaccharide-induced nitric oxide production. The cytotoxic compound was isolated and identified by NMR and LC-MS as 1-hydroxy-3-isobuty1-4-[4-(3-methy1-2-butenyloxy)-pheny1]-pyrrole-2, 5-dione.
目 錄

目錄 I
圖次 VIII
表次 XIII
縮寫表 XIV
中文摘要 XⅥ
英文摘要 XⅧ

第一章 緒論 …………………………………………………………………1
第一節 樟芝之介紹 …………………………………………………… 1
第二節 菇類的機能性 ………………………………………………… 4
2.1 第一級功能—營養成分 …………………………………4
2.2 第二級功能—嗜好特性、食藥性價值 …………………4
2.3 第三級功能—生理活性、醫療價值 ……………………5
2.3.1 TNF-α(腫瘤壞死因子) …………………………… 8
2.3.2 IL-6 (間白素-6) ……………………………………8
2.3.3 IFN-g (干擾素-g) ……………………………………9
第三節 樟芝之成分分析 ……………………………………………10
3.1 一般成分 ………………………………………………10
3.2 多醣體 …………………………………………………10
3.3 三萜類 …………………………………………………13
3.4 腺苷 ……………………………………………………14
第四節 最近國內學者之重要研究成果 ………………………… 15
(一) 樟芝液體醱酵培養研究………………………………………15
(二) 樟芝菌絲體深層培養之研究…………………………………15
(三) 樟芝固體醱酵培養研究………………………………………15
(四) 樟芝菌絲體培養與生理活性成分生成之研究………………15
(五) 樟芝醱酵液之抗腫瘤研究……………………………………16
(六) 樟芝口服急毒性試驗…………………………………………16
(七) 樟芝菌株醱酵液對dimethylnitrosamine 誘發大鼠肝纖維化及腸道的改善效果………………………………………………16
(八) 樟芝對於血糖及血脂質之影響………………………………16
(九) 樟芝培養醱酵濾液保護四氯化碳誘導大鼠急性肝損傷之能力………………………………………………………………17
(十) 樟芝菌絲體之毒性與致突變性之研究………………………17
(十一) 大鼠致畸測試─樟芝醱酵原液凍乾成品 …………………18
(十二) 樟芝之致基因毒性與抗基因毒性評估……………………18
(十三)高通量篩選出樟芝菌絲體抗肝癌先導藥物化合物 ………19
(十四) 樟芝菌絲體保肝及抗B型肝炎病毒………………………19
第五節 研究動機………………………………………………………20

第二章 材料與方法 ………………………………………………………29
第一節 菌株之培養……………………………………………………29
1.1 試驗菌株及來源 ………………………………………29
1.2 培養基組成 ……………………………………………29
1.2.1 菌株保存用培養基…………………………………29
1.2.2 種菌培養基…………………………………………29
第二節 儀器……………………………………………………………30
2.1 培養相關設備 …………………………………………30
2.2 醱酵槽相關設備 ………………………………………30
2.3 萃取及分析相關設備 …………………………………31
2.4 其他 ……………………………………………………33
第三節 藥品……………………………………………………………34
3.1 標準品 …………………………………………………34
3.2 萃取分析溶劑 …………………………………………34
3.3 培養基 …………………………………………………34
3.4 其餘藥品、溶劑皆為試藥級 …………………………34
第四節 從子實體分離樟芝A. cinnamomea菌株之鑑定………… 35
4.1 樟芝子實體之分離 ……………………………………35
4.2 培養條件 ………………………………………………35
4.3 Genomic DNA 萃取 …………………………………35
4.4 rRNA基因之聚合酶鏈反應(PCR) ………………… 37
第五節 利用反應曲面法探討樟芝最適培養條件………………… 38
5.1 菌株 …………………………………………………… 38
5.2 利用反應曲面法探討三角瓶最適培養基組…………38
5.3 槽培養條件 ……………………………………………39
5.4 乾菌體量之測定 ………………………………………39
5.5 殘糖含量測定 (採用HPLC分析法) ………………….39
5.6 胞內總多醣量測定(採用DNS法) ………………… 40
5.7 三萜類總量測定法 …………………………………… 41
第六節 樟芝菌絲體之腺苷 (adenosine),蟲草素 (cordycepin)與麥角固醇 (ergosterol) 成分分析………………………………… 43
6.1 菌株 ……………………………………………………43
6.2 培養條件 ………………………………………………43
6.3 乾菌體量之測定……………………………………… 43
6.4 菌絲體腺苷 (adenosine) 與蟲草素 (cordycepin) 之萃取與分析 …………………………………………………43
6.5 Adenosine與cordycepin 標準品及標準校正曲線之製備 ………………………………………………………44
6.6 菌絲體麥角固醇 (ergosterol) 之萃取與分析…………44
6.7 Ergosterol標準品及標準校正曲線之製備……………45
6.8 700公升槽醱酵液之均質與噴霧燥 …………………45
第七節 樟芝菌絲體生理活性試驗……………………………………46
7.1 菌株 ……………………………………………………46
7.2 培養條件 ………………………………………………46
7.3 菌體量之測定 …………………………………………46
7.4 殘糖量之測定 …………………………………………47
7.5 樟芝菌絲體多醣的萃取 ………………………………47
7.6 樟芝胞內熱水可溶多醣區分 …………………………47
7.7 人體全血細胞激素之測定 ……………………………47
7.8 樣品進行Human IL-6 (eBioscience) 測定之前處理…48
7.8.1 試劑之製備…………………………………………48
7.8.2 前處理………………………………………………49
7.9 樣品進行TNF-α (eBioscience) 測定之前處理 ………50
7.9.1 試劑之製備…………………………………………50
7.9.2 前處理………………………………………………50
7.10 樣品進行IFN-γ (eBioscience) 測定之前處理 ………51
7.10.1 試劑之製備 ………………………………………51
7.10.2 前處理 ……………………………………………51
7.11 多醣類分子量測定……………………………………52
第八節 樟芝抗氧化試驗 ……………………………………………54
8.1 菌株 ……………………………………………………54
8.2培養條件…………………………………………………54
8.3 測試方法 ………………………………………………54
第九節 樟芝美白試驗 ………………………………………………55
9.1 菌株 ……………………………………………………55
9.2 培養條件 ………………………………………………55
9.3 試驗原理 ………………………………………………55
9.4 實驗方法 ………………………………………………55
第十節 樟芝多醣體β-D-glucan分析…………………………………56
10.1菌株 ……………………………………………………56
10.2培養條件 ………………………………………………56
10.3測定1,3-β-D-glucan………………………………56
第十一節 樟芝抗發炎試驗 …………………………………………57
11.1菌株 ……………………………………………………57
11.2培養條件 ………………………………………………57
11.3有效成份分離 …………………………………………57
11.4抗發炎活性測試 ………………………………………57
11.5細胞毒性試驗 …………………………………………57
11.6 Western blotting…………………………………………58
11.7樟芝代謝物細胞毒性成份分離與鑑定……………… 58

第三章 結果與討論 ………………………………………………………64
第一節 樟芝子實體A. cinnamomea菌株之分離與鑑定…………… 64
1.1 樟芝子實體A. cinnamomea菌株之分離………………64
1.2 利用PCR技術進行樟芝A. cinnamomea菌株之鑑定…64
1.3 A. cinnamomea 菌株與其它菌株之DNA序列分析 ………………………………………………………65
第二節 利用反應曲面法探討樟芝最適培養條件 …………………93
2.1 不同碳源對搖瓶培養之影響 …………………………93
2.2 各控制條件其反應性狀模式之建立………………… 93
2.3 根據RSM之最適操作條件 ……………………………93
2.4 醱酵槽培養………………………………………………94
第三節 樟芝菌絲體之腺苷 (adenosine)、 蟲草素 (cordycepin)與麥角固醇 (ergosterol) 成分分析 ……………………………115
3.1樟芝樣品之腺苷 (adenosine) 、蟲草素 (cordycepin) 與麥角固醇 (ergosterol) 層析圖………………………115
3.2 樟芝5公升醱酵槽培養的樣品腺苷、蟲草素與麥角固醇含量分析……………………………………………… 115
3.3樟芝100公升酵槽培養的樣品腺苷、蟲草素與麥角固醇含量分析……………………………………………… 115
3.4樟芝700公升槽培養的樣品腺苷、蟲草素與麥角固醇含量分析………………………………………………… 116
第四節 樟芝菌絲體生理活性試驗…………………………………128
4.1 樟芝AC0623菌株於700公升醱酵槽培養情況………128
4.2 樟芝菌絲體熱水萃取多醣區分對人類全血細胞激素釋放的影響………………………………………………128
4.2.1 IL-6的釋放………………………………………128
4.2.2 TNF-α的釋放……………………………………128
4.2.3 IFN-g的釋放…………………………………… 129
4.3 樟芝醱酵液對人類全血細胞激素的影響……………129
4.3.1 IL-6的釋放………………………………………129
4.3.2 TNF-α的釋放……………………………………129
4.3.3 IFN-g 的釋放……………………………………130
4.4 樟芝菌絲體胞內多醣分子量之分佈…………………130
第五節 樟芝抗氧化試驗……………………………………………143
第六節 樟芝美白試驗………………………………………………146
第七節 β-D-glucan分析……………………………………………149

附錄 Antrodia cinnamomea BCRC 35396與BCRC 35398 菌株相 關之試驗結果………………………………………………… 155

第一節 樟芝菌絲體之腺苷 (adenosine)、蟲草素 (cordycepin)與麥角固醇 (ergosterol) 成分分析………………………………156
1.1 樟芝5公升槽培養的樣品adenosine與cordycepin含量分析……………………………………………………156
1.2 樟芝100公升槽培養的樣品腺苷與蟲草素含量分析156
第二節 樟芝菌絲體生理活性試驗…………………………………161
2.1 樟芝菌絲體熱水萃取多醣區分對人類全血細胞激素釋放的影響………………………………………………161
2.1.1 IL-6的釋放………………………………………161
2.1.2 TNF-α的釋放……………………………………161
2.1.3 IFN-g的釋放 ……………………………………162
2.2 樟芝醱酵液對人類全血細胞激素的影響…………162
2.2.1 IL-6的釋放………………………………………162
2.2.2 TNF-α的釋放……………………………………162
2.2.3 IFN-g的釋放 …………………………………… 163
第三節 抗發炎試驗 ………………………………………………170
3.1 樟芝醱酵液對一氧化氮合成的影響………………… 170
3.2 樟芝代謝物對iNOS與COX-II的影響 ……………170
3.3 樟芝代謝物細胞毒性成份分離與鑑定………………170
第四章 結論 …………………………………………………………177
第五章 參考文獻 …………………………………………………………179




圖 次

圖1-1 樟芝顯微鏡下之菌絲體 ………………………………………… 22
圖1-2 由樟芝子實體萃取物中發現新的三萜類化合物…………………23
圖1-3 楊書威從樟芝子實體萃取物中發現新的三萜類化合物…………24
圖1-4 高曉薇由樟芝分離得到的三個成份………………………………25
圖 1-5 程一華從樟芝分離得到的十三個成份……………………………26
圖2-1 液態培養樟芝菌絲體熱水可溶多醣的區分………………………63
圖 3-1 A. cinnamomea ACTS1菌株從子實體分離,接種於M25洋菜平面培養基一星期後菌落生長之情形 ………………………………70
圖 3-2 A. cinnamomea AC0623菌株從子實體分離,接種於M25洋菜平面培養基三星期後菌落生長之情形 ………………………………71
圖 3-3 A. cinnamomea 菌株ACTS1(A)、AC0623(B)、BCRC 35396(C) 與 BCRC 35398(D) 菌株生長於M25洋菜培養基菌落形態之比較72
圖 3-4 A. cinnamomea 菌株ACTS1(A)、AC0623(B)、BCRC 35396(C) 與 BCRC 35398(D) 生長於PDA洋菜培養基菌落形態之比較……73
圖 3-5 A. cinnamomea 菌株ACTS1(A)、AC0623(B)、BCRC 35396(C) 與 BCRC 35398(D) 生長於OAT洋菜培養基菌落形態之比較……74
圖 3-6 A. cinnamomea 菌株ACTS1(A)、AC0623(B)、BCRC 35396(C) 與 BCRC 35398(D) 生長於SGA洋菜培養基菌落形態之比較……75
圖 3-7 A. cinnamomea 菌株ACTS1(A)、AC0623(B)、BCRC 35396(C) 與 BCRC 35398(D) 生長於NA洋菜培養基菌落形態之比較 ……76
圖 3-8 A. cinnamomea 菌株 ACTS1 (A)、 AC0623 (B)、 BCRC 35396 (C) 與 BCRC 35398 (D) 於顯微鏡下之特徵 (× 400)………………77
圖 3-9 解剖顯微鏡下A. cinnamomea 菌株ACTS1 (A)、AC0623 (B)、BCRC 35396 (C) 與 BCRC 35398 (D) (M25洋菜培養基)之特徵 (× 50)………………………………………………………………78
圖3-10 解剖顯微鏡下A. cinnamomea 菌株 ACTS1 (A)、AC0623 (B)、BCRC 35396 (C) 與 (D) BCRC 35398 (SGA洋菜培養基)之特徵 (× 50)………………………………………………………………79
圖3-11 A. cinnamomea 菌株 ACTS1、AC0623與BCRC 35396、BCRC 35398菌株核醣體RNA基因之PCR擴增產物的比較。以菌株之基因組DNA為模版,分別與4對引子組 (NS1/NS2、 NS3/NS4、 NS5/NS8、 ITS1/ITS4)進行PCR反應,PCR反應後的產物於2.0%洋菜凝膠(agarose gel)進行電泳分析,再以ethidium bromide染色約20分鐘,於紫外燈下觀察結果,100 bp的標準DNA標記(marker)(M)標明於右側 …………………………………80
圖 3-12 ACTS1、 AC0623、 BCRC 35396與 BCRC 35398菌株使用資料庫序列分析進行DNA序列之比較…………………………… 87
圖 3-13 部分核糖體RNA基因(包括部分ITS1、 5.8S rRNA 基因與部分ITS2)之DNA序列比對,包括5株A. cinnamomea 菌株AY378092 (ACTS1)、AY378093 (AC0623)、AY378094 (BCRC 35396)、AY378095 (BCRC 35398)、AJ496402 (BCRC 35716) 及 AF126902 (A. romellii)、AF126903 (A. semisupina) 與 AY309015 (T. versicolor) …………………………………………………………91
圖3-14 演化樹圖譜 ………………………………………………………92
圖 3-15 不同malt extract與peptone濃度對三萜類生成之反應曲面圖 103
圖 3-16 不同malt extract與peptone濃度對菌體生成之反應曲面圖 …104
圖 3-17 Malt extract與peptone濃度0∼3% (yeast extract=3%)對三萜與菌體量生成之contour plot圖………………………………………105
圖 3-18 A. cinnamomea AC0623菌株Hinton三角瓶培養照片…………106
圖 3-19 A. cinnamomea AC0623菌株於5公升醱酵槽培養之生長趨勢107
圖 3-20 A. cinnamomea AC0623菌株5公升醱酵槽培養圖 …………108
圖 3-21 A. cinnamomea AC0623菌株於100公升醱酵槽培養之生長趨勢圖 …………………………………………………………………109
圖 3-22 A. cinnamomea AC0623菌株100公升醱酵槽培養圖 …………110
圖 3-23 A. cinnamomea AC0623菌株100公升醱酵槽培養圖 …………111
圖3-24 A. cinnamomea AC0623菌株於700公升醱酵槽培養之生長趨勢圖 …………………………………………………………………112
圖3-25 A. cinnamomea AC0623菌株於100公升醱酵槽培養之三萜類HPLC層析圖 …………………………………………………… 113
圖 3-26 A. cinnamomea AC0623菌株於100公升醱酵槽培養之三萜類HPLC層析圖………………………………………………………114
圖 3-27 腺苷標準品校正曲線 ……………………………………………117
圖3-28 蟲草素標準品校正曲線 …………………………………………118
圖 3-29 腺苷與蟲草素標準品之HPLC層析圖…………………………119
圖 3-30 樣品中之腺苷與蟲草素之HPLC層析圖 ………………………120
圖3-31 麥角固醇標準品校正曲線……………………………………… 121
圖3-32 麥角固醇標準品層析圖…………………………………………122
圖 3-33 樟芝A. cinnamomea AC0623菌株於5公升醱酵槽培養之生長趨勢 …………………………………………………………………123
圖 3-34 樟芝A. cinnamomea AC0623菌株於100公升醱酵槽培養之生長趨勢 …………………………………………………………………124
圖3-35 麥角固醇樣品層析圖 …………………………………………… 125
圖3-36 A. cinnamomea AC0623於700公升醱酵槽培養之生長趨勢圖 …………………………………………………………………133
圖 3-37 A. cinnamomea AC0623之菌絲體區分對人類全血中IL-6誘導之影響…………………………………………………………………134
圖 3-38 A. cinnamomea AC0623之菌絲體區分對人類全血中TNF-α誘導之影響 ……………………………………………………………135
圖3-39 A. cinnamomea AC0623之菌絲體區分對人類全血中IFN-g誘導之影響 ………………………………………………………………136
圖3-40 A. cinnamomea AC0623之醱酵液對人類全血中IL-6誘導之影響 …………………………………………………………………137
圖 3-41 A. cinnamomea AC0623之醱酵液對人類全血中TNF-α誘導之影響 …………………………………………………………………138
圖3-42 A. cinnamomea AC0623之醱酵液對人類全血中IFN-g誘導之影響 …………………………………………………………………139
圖3-43 P100 (分子量112,000)標準品之膠體滲透層析圖………………140
圖 3-44 GPC 標準品校正曲線…………………………………………141
圖3-45 本試驗樣品多醣體分子量分佈圖………………………………142
圖3-46 樟芝萃取樣品總抗氧化力之比較………………………………145
圖3-47 樟芝美白功能試驗結果…………………………………………148
圖 3-48 β-D-glucan標準曲線圖…………………………………………151
圖 3-49 CR3結構圖由CD11b與CD18兩支鏈所構成…………………153
圖 3-50 藉引導iC3b所調理的酵母菌來活化嗜中性白血球表面CR3細胞 毒性之作用機制 …………………………………………………154
圖 3-51 樟芝A. cinnamomea BCRC 35396菌株於5公升醱酵槽培養之生長趨勢 ……………………………………………………………157
圖 3-52 樟芝A. cinnamomea BCRC 35398菌株於5公升醱酵槽培養之生長趨勢 ……………………………………………………………158
圖 3-53 樟芝A. cinnamomea BCRC 35396菌株於100公升醱酵槽培養之生長趨勢 ……………………………………………………………159
圖 3-54 樟芝A. cinnamomea BCRC 35398菌株於100公升醱酵槽培養之生長趨勢 …………………………………………………………160
圖 3-55 A. cinnamomea BCRC 35396菌絲體對人類全血中IL-6誘導之影響 …………………………………………………………………164
圖 3-56 A. cinnamomea BCRC 35396菌絲體對人類全血中TNF-α誘導之影響 ………………………………………………………………165
圖 3-57 A. cinnamomea BCRC 35396菌絲體對人類全血中IFN-γ誘導之影響……………………………………………………………… 166
圖 3-58 A. cinnamomea BCRC 35396醱酵液對人類全血中IL-6誘導之影響 …………………………………………………………………167
圖 3-59 A. cinnamomea BCRC 35396醱酵液對人類全血中TNF-α誘導之影響 ………………………………………………………………168
圖 3-60 A. cinnamomea BCRC 35396醱酵液對人類全血中IFN-γ誘導之影響 ……………………………………………………………169
圖 3-61 Murine macrophage RAW 264.7 由 LPS誘導NO合成之抑制作用 …………………………………………………………………172
圖3-62 細胞與樟芝A. cinnamomea BCRC 35396醱酵液作用之毒性分析…………………………………………………………………173
圖3-63 LPS活化Raw 264.7 macrophage的iNOS之Western blot 分析…………………………………………………………………174
圖3-64 LPS活化Raw 264.7 macrophage的COX-II之Western blot 分析…………………………………………………………………175
圖3-65 由樟芝A. cinnamomea BCRC 35396醱酵液分離之細胞毒性化合物 1-Hydroxy-3-isobutyl-4-[4-(3-methyl-2-butenyloxy)-phenyl]-pyrrole-2,5-dione…………………………………………………………176



表 次

表1-1 菇類與其他食品營養價值的比較…………………………………27
表1-2 樟芝一般成分 (%) ………………………………………………28
表 2-1 本試驗所使用之A. cinnamomea 菌株及其來源…………………59
表2-2 本試驗所使用之引子………………………………………………60
表2-3 三變數-三階層反應曲面設計之操作條件變數及階層……………61
表2-4 三變數-三階層因子設計及實驗數據………………………………62
表3-1 A. cinnamomea菌株於不同培養基菌落生長及直徑長度之比較…68
表3-2 四株A. cinnamomea 菌株ACTS1 (AY378092)、 AC0623 (AY378093)、 BCRC 35396 (AY378094)、 及 BCRC 35398 (AY378095) 部分核糖體RNA基因之DNA序列的相似性(%)…69
表3-3 不同碳源對A. cinnamomea AC0623菌株之菌體量與三萜類生成量的影響………………………………………………………………98
表3-4 不同培養條件生產A. cinnamomea AC0623菌株之菌體與三萜類之變異數分析…………………………………………………………99
表3-5 A. cinnamomea AC0623菌株於搖瓶之不同培養條件對菌體與三萜類生成量之影響………………………………………………… 100
表3-6 A. cinnamomea AC0623菌株培養於不同醱酵槽之菌體量、三萜類與總多醣生成量之比較………………………………………… 101
表3-7 市售各廠牌與自製樟芝之三萜類與總多醣含量分析比較………102
表3-8 A. cinnamomea AC0623菌株於700公升槽培養醱酵液經不同乾燥方式處理後之菌體其腺苷、蟲草素與麥角固醇含量之比較 …………………………………………………………………126
表3-9 市售各廠牌與自製樟芝之腺苷、蟲草素與麥角固醇含量分析比較 …………………………………………………………………127
表3-10 各種不同的食物中1,3-b-Glucan含量之比較……………………152
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