臺灣博碩士論文加值系統

雲芝是一種小型多孔真菌，是遍布全世界森林生態中分解死掉或枯萎樹木的重要環節。雲芝不僅作為中藥使用，其發酵菌絲體所產生之多醣體複合物-醣肽也已被開發為「藥品」。本研究主要目的為應用 API-ZYM 多酶套組分析本土發現之雲芝品系 LH-1種菌特性，探討液態培養上清液、菌絲體及平板培養菌落不同代數間酶譜差異之比較、HPLC 指紋圖譜變化、分析野外採集雲芝菌種多酶套組酶譜、評估不同樣品製備處理方式(透析時間、酶處理時間及解凍條件)與不同計算基礎換算對雲芝多醣體含量之影響。此外，分析雲芝市售產品多醣體之分子量與單醣組成之差異。
結果顯示雲芝種菌不同代數培養上清液、菌絲體與菌落應用API-ZYM 多酶套組分析之酶譜活性指數及強弱表現，菌落於酶譜中所顯示之酶種類較培養上清液及菌絲體多樣，且活性指數亦較強，較適於判斷培養菌株代數差異及菌種種菌品管之依據。不同代數培養上清液及菌絲體萃取物所產生之指紋圖譜彼此間並無顯著差異，譜型皆呈現 5 個主要波峰。在譜型判別上，雲芝菌絲體萃取液之分散性較發酵培養液主波峰之集中性更易判斷菌種種菌品質之穩定性。利用 API-ZYM 酶譜中特定酶種類之存在或不存在可做為野外採集雲芝品系間區辨之參考。將萃取液分別以(1)透析法(2)水萃法(3)酒精澱析法等三種方法製備分析樣品，顯示三種樣品製備方法中以「水萃法」所得數據之多醣體含量皆為最高，而以「透析法」與「酒精澱析法」分析不同產品樣品中，所得數據各有高低，以「透析法」分析之多醣體含量較「酒精澱析法」為低。樣品製備處理之透析及酶處理時間與解凍條件，可影響雲芝多醣體分析之結果。材料來源相同但後續雲芝之子實體與菌絲體或發酵液經不同分離及純化單醣含量之比例以及分子量範圍都會不同。
綜合上述結果，API-ZYM 多酶套組可應用於分析雲芝 LH-1品系菌種特性及做為不同世代種菌品管之依據，雲芝市售相關產品以酚硫酸法分析樣品中多醣體含量時，樣品之製備方式顯著影響其分析之結果。

Trametes versicolor (Coriolus versicolor) is a small polypore fungus that is an important part of the forest ecology as a recycler of dead and dying trees in forests throughout the world. Trametes versicolor is not only used as traditional Chinese medicine, the mycelium of Trametes versicolor produced polysaccharide complexes-glycopeptides have also been developed as "pharmaceutical".
The main purpose of this study is as inoculum characteristics of enzyme profile using API-ZYM analysis kit by Trametes versicolor, investigating supernatant of submerged culture, comparing colonies cultured in plate with mycelia in enzyme profile between different generations, finger print, as well as preparation (dialysis, enzyme treatment time and thawing factor) and analysis of polysaccharides from commercial products by Trametes versicolor. Furthermore, analysis polysaccharides of molecular weight and the differences in monosaccharide composition.
Results of show the different Trametes versicolor generations inoculum in submerged culture of supernatant and mycelium colonies, API-ZYM kit multi-enzyme analysis of enzyme profile of activity and the strength of the performance index. Enzyme profile of colonies shows better then submerged culture of supernatant and mycelium, and activity index is also strong, adapted to judge the algebraic difference in culturing fungi strains and quality control of various fungi basis. The supernatant and the mycelium extract with submerged culture produce different algebraic fingerprint, there is no significant difference between them, profile are presented five major peaks. Determining on profiling, compare mycelium extract and submerged culture of supernatant by Trametes versicolor in the dispersion, the main concentrated of the peak is simply judge the stability of species inoculum of quality.
Using API-ZYM enzyme profile can be identified as existence or absence of wild collection among Trametes versicolor strains.The three methods to extract respectively (1) dialysis (2) non-alcoholic precipitation (3) alcohol precipitates analytical sample method, it shows three methods to " non-alcoholic precipitation " of polysaccharide content have the better resulte, and with "dialysis" and " alcohol precipitates" analysis of different product samples, the resulting data have high and low, polysaccharide content in "dialysis" Analysis of comparing "alcohol precipitates Act" is low. Dialysis samples, the time and thawing conditions enzyme treatment may affect the results of analysis of the polysaccharide from Trametes versicolor. The same source material, but different separation and purification will affect the proportion of fruiting bodies and mycelia from Trametes versicolor with monosaccharide content of the fermentation broth and the molecular weight range will be different.
Based on the results, API-ZYM kit can be used to analyze Trametes versicolor LH-1 strain characteristics of different generations and as the basis for quality control of the inoculum, PSK related products commercially available phenol-sulfuric acid method when samples were analyzed for polysaccharide content of the sample pretreatment significantly affect the results of its analysis.

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 ...................................................................................... xiv
表目錄 ....................................................................................... xv
1. 前言 ...................................................................................... 1
2. 文獻回顧 .............................................................................. 6
2.1 中藥雲芝 ........................................................................ 6
2.2 雲芝化學組成 ................................................................ 7
2.2.1 一般化學組成 ....................................................... 7
2.2.2 其他化學組成 ...................................................... 7
2.3 多醣體 ............................................................................ 8
2.3.1 多醣體含量 ........................................................... 8
2.3.2 多醣體中單醣組成 ................................................ 8
2.3.3 多醣體分子量 ....................................................... 8
2.3.4 多醣體化學結構 .................................................... 9
2.3.5 醣肽……. ............................................................ 10
2.4 蛋白質 .......................................................................... 10
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2.5 雲芝生物活性 .............................................................. 11
2.6 雲芝非醫藥應用 .......................................................... 12
2.6.1 魚類養殖健康改善 ............................................. 12
2.6.2 植物病害防治 .................................................... 12
2.6.3 生物轉化 ............................................................ 12
2.6.4 分解農業廢棄物塑膠毒物、吸附顆粒及脫色 .. 13
2.7 應用API® ZYM 酶試劑套組於微生物之相關研究 .. 13
2.7.1 API® ZYM 簡介 ................................................. 14
2.7.2 細菌應用 ............................................................ 15
2.7.3 真菌應用 ............................................................ 17
2.8 酚硫酸法 ...................................................................... 18
3. 材料與方法 ........................................................................ 21
3.1 實驗材料 ...................................................................... 21
3.1.1 實驗菌種 ............................................................. 21
3.1.2 基礎培養基 ......................................................... 22
3.1.3 實驗藥品 ............................................................. 22
3.2 實驗儀器 ...................................................................... 23
3.3 菌種培養 ...................................................................... 24
3.4 菌種保存 ...................................................................... 25
3.5 應用API-ZYM 多酶套組分析 .................................... 25
3.6 酚硫酸多醣含量檢測 .................................................. 26
3.7 單醣分析 ..................................................................... 27
3.8 HPLC 指紋圖譜 ........................................................... 27
3.9 分子量分析 .................................................................. 28
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4. 結果與討論 ........................................................................ 29
4.1 雲芝種菌不同代數應用API-ZYM 多酶套組分析之酶
譜表現 ........................................................................ 29
4.1.1 雲芝種菌不同代數培養上清液應用API-ZYM
多酶套組分析之酶譜活性指數表現 ................ 29
4.1.2 雲芝種菌不同代數培養菌絲體應用API-ZYM
多酶套組分析之酶譜活性指數表現 ................ 30
4.1.3 雲芝種菌不同代數培養菌落應用API-ZYM 多
酶套組分析之酶譜活性指數表現 .................... 34
4.1.4 雲芝種菌不同代數培養上清液應用API-ZYM
多酶套組分析之酶譜活性強弱表現 ................ 35
4.1.5 雲芝種菌不同代數培養菌絲體應用API-ZYM
多酶套組分析之酶譜活性強弱表現 ................ 39
4.1.6 雲芝種菌不同代數培養菌落應用API-ZYM 多
酶套組分析之酶譜活性強弱表現 .................... 41
4.1.7 應用API-ZYM 多酶套組比較雲芝種菌發酵培
養液、菌絲體與菌落不同代數酶譜酶平均值活
性指數表現 ....................................................... 43
4.2 雲芝連續培養十代不同代數菌絲體萃取液及發酵培
養液之HPLC 指紋圖譜波峰分析 ............................. 46
4.2.1 雲芝連續培養十代不同代數菌絲體萃取液之
HPLC 指紋圖譜波峰分析 .............................. 46
4.2.2 雲芝發酵培養上清液連續培養十代不同代數之
HPLC 指紋圖 譜與波峰分析 .............. 49
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4.3 應用API-ZYM 多酶套組分析野外採集雲芝菌種培養
之菌落酶譜活性指數與強弱表現 ............................. 51
4.3.1 應用API-ZYM 多酶套組分析野外採集雲芝菌
種培養之菌落酶譜活性指數表現 .................... 51
4.3.2 應用API-ZYM 多酶套組分析野外採集雲芝菌
種培養之菌落酶譜活性強弱表現 .................... 55
4.4 不同樣品製備方法與不同計算基礎換算對不同雲芝
產品中多醣體含量之影響 ......................................... 56
4.4.1 不同樣品製備方法對不同雲芝產品中多醣體含
量之影響 ........................................................... 56
4.4.2 不同計算基礎換算不同雲芝產品中粗多醣體
含量比較 ........................................................... 59
4.4.3 有關Krestin 中多醣體含量 ............................. 61
4.4.4 透析方法時間對雲芝樣品多醣體及蔗糖樣品含
量分析之影響 ................................................... 62
4.4.5 解凍次數對雲芝菌絲體生物質量與多醣體含
量之影響 ........................................................... 66
4.5 雲芝商品多醣體中單醣組成與分子量分析 ............... 68
4.5.1 雲芝商品多醣體中單醣組成 ............................ 68
4.5.2 雲芝商品多醣體分子量分析 ............................ 68
5. 結論 .................................................................................... 72
5.1.種菌液態培養上清液、菌絲體及平板培養菌落不同代
數間酶譜差異之比較................................................... 72
5.2.HPLC 指紋圖譜變化 ................................................... 72
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5.3.野外採集雲芝菌種API-ZYM 多酶套組酶譜之分析 72
5.4.不同雲芝市售產品為樣品，評估不同樣品製備處理方
式(透析時間、酶處理時間及解凍條件)對雲芝多醣體
分析之影響 .................................................................. 73
5.5.測定多醣體之分子量與單醣組成 ............................... 73
參考文獻 ................................................................................... 74

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