臺灣博碩士論文加值系統

液態培養是生產靈芝多醣的有效方式，其培養基組成與發酵條件對於菌絲的生長與多醣產量有顯著性的影響，利用豆科基質(黑豆與黃耆)對於靈芝多醣之含量與性質加以探討。在靈芝液態培養樣品來源，分別為三角瓶靜置培養、五公升試驗型發酵槽與兩百公升發酵槽三種。將這些不同來源的多醣中，以水溶性多醣濃度與具有(1,6)分支之(1,3)-β-D glucan為指標探討其多醣性質。在靜置培養樣品結果顯示，培養基中加入豆科發酵基質，明顯增加多醣與(1,3)-β-D glucan產量，未加豆科基質發酵之樣品，則未有明顯的改變。
由五公升試驗型發酵槽與兩百公升發酵槽的結果顯示，許多因子皆會影響多醣與其(1,3)-β-D glucan產量，而培養基中的豆科基質與溫度的影響 (由18到30℃的範圍)比通氣量的影響(由0.25到0.75vvm範圍)更明顯。其多醣含量與多醣中apparent (1,3)-β-D glucan最高可達到322 mg/dL與152 mg/dL，多醣中(1,3)-β-D glucan分子量分布大於85 k Da。在五公升試驗型發酵槽與兩百公升發酵槽中因生產裝置的不同導致其多醣與(1,3)-β-D glucan產量之最適條件也不同，明顯的影響多醣中(1,3)-β-D glucan的含量、分支度與分子量分布。這些實驗結果也指出靈芝中活性多醣性質對於在發酵過程中樣品的監控提供一品管之重要指標參考。

Liquid culture is an efficient method to produce Ganoderma lucidum polysaccharides. Culture media and cultivating conditions have significant effects on mycelium growth and polysaccharide production. The focus of this study was on the effects of using legumes (black soybean and Astragalus membranaceous Bunge) as media and cultivating conditions on producing yield and properties of G. lucidum polysaccharides. Liquid culture samples of G. lucidum mycelium were obtained from three sources: static cultures in flasks and five-liter experimental and two-hundred-liter fermentors. The concentration of water-soluble polysaccharide and (1, 3)-b-D-glucans having (1, 6)-b-glucosyl branches in culture were used as indices to study the differences of polysaccharides from different sources. The results of static culture experiments indicated that the using of combined legume medium significantly increased the production of polysaccharide and the (1, 3)-b-D-glucans but the using of individual legume as medium did not find significant changes. The analytical data from samples produced in five-liter experimental and two-hundred- liter fermentor indicated that multiple factors was affecting the production of polysaccharides and (1, 3)-b-D-glucans and the effects of using legume medium and temperature (in the range of 18°, 24° and 30°) were more significant than aeration (in the range of 0.25~0.75 vvm). The polysaccharide production could reach 322 mg/dL and (1, 3)-b-D-glucans could reach 152 mg/dL. The optimum condition for production polysaccharides and (1, 3)-b-D-glucans differed on five- and two-hundred-liter fermentors as producing apparatus. It was significantly affected by cultivating condition that of content the (1, 3)-b-D-glucan and its degree of branching in polysaccharides as well as molecular weight distribution of polysaccharides. The occurrence of the (1, 3)-b-D-glucans was exclusive in molecular weight larger than 85 kDa. The results indicated that affecting factors on polysaccharide production from G. lucidum mycelia were complicated quality monitoring was necessary for the processing.

總目錄
中文摘要……………………………………………………………I
英文摘要……………………………………………………………II
總目錄………………………………………………………………IV
表目錄………………………………………………………………Ⅸ
圖目錄………………………………………………………………Ⅹ
壹、前……………………………………………………………….1
貳、文獻回顧……………………………………………………….2
一、靈芝………………………………………………………….2
(一)靈芝特徵介紹……………………………………………2
(二)靈芝生物分類……………………………………………3
(三)靈芝生物活性功能………………………………………4
1.免疫調節…………………………………………………4
2.抗腫瘤……………………………………………………5
3.抗菌作用…………………………………………………5
4.降血脂……………………………………………………5
5.降血糖……………………………………………………6
6.降血壓……………………………………………………6
7.抗發炎……………………………………………………6
8.抑制潰瘍(antiulcer)…………………………………7
9.抗血小板凝集………………………………………….7
10.抗氧化..………………………………………………7
11.抑制多胺類(polyamine)形成……………………….7
12.抗血管新生(antiangiogenesis)……………………8
13.調控細胞週期…………………………………………8
(四)靈芝活性成分……………………………………………9
1.多醣……………………………………………………9
a.同質多醣…………………………………………10
b.異質多醣…………………………………………11
c.蛋白多醣…………………………………………11
2.三萜類……………………………………………….16
3.蛋白質……………………………………………….16
4.其他成份…………………………………………….17
(五)靈芝栽培方式……………………………………………18
1.子實體栽培……………………………………………18
a.太空包栽培.……………………………………….18
b.段木栽培……………………………………………18
2.液態培養………………………………………………18
(六)影響靈芝生長因子………………………………………19
1.溫度.……………………………………………………19
2.pH值.……………………………………………………19
3.接種量………………………………………………….19
4通氣量.………………………………………………….19
5.轉速…………………………………………………….20
6.碳源…………………………………………………….20
7.氮源…………………………………………………….20
8.碳/氮比…………………………………………………20
二、影響(1,3)-β-D glucan性質…………………………………21
(一)生理活性……………………………………………………21
1.分子量……………………………………………………21
2.分支度……………………………………………………23
3.構形………………………………………………………25
(二)(1,3)-β-D glucan生合成……………………………….26
三、以aniline blue螢光染色法檢測 (1,3)-β-D glucan…….28
四、豆科基質介紹…………………………………………………30
(一)黃耆………………………………………………………….30
(二)黑豆………………………………………………………….31
參、材料與方法…………………………………………………….36
一、材料……………………………………………………………36
二、化學藥品與試劑………………………………………………36
三、靈芝液態培養樣品製備………………………………………37
(一)液態發酵培養基…………………………………………….37
(二)豆科培養基質的處理……………………………………….38
(三)三角瓶靜置液態發酵培養……………………………………38
(四)五公升試驗型發酵槽及兩百公升發酵槽菌絲體液態培養製備
…………….........................................39
四、多醣樣品置製備…………………………………………………40
(一)靈芝菌絲體液態發酵培養………………………………….40
1.靜置菌絲體液態發酵培養樣品………………………….40
2.試驗型五公升發酵槽與兩百公升發酵槽液態發酵培養品40
五、分析方法……………………………………………………....40
(一)固型物乾重………………………………………………….40
(二)醣量分析…………………………………………………….40
1.總醣量與多醣量之測定-Phenol-sulfuric acid method.40
2.(1,3)-β-D-glucan之定量-Aniline blue method……….41
a.樣品檢測………………………………………..41
b.標準曲線建立……………………………......41
(三)蛋白質含量之測定—Folin-Lowry method………………..42
(四) Uronic acid含量之測定—m-hydroxydiphenyl method..42
(五)(1,3)-β-D-glucan含量與分支度-酵素-層析法…………..42
(六)分子量分布-膠體過濾層析法……………………………….43
(七)單醣組成之測定………………………………………………44
伍、結果與討論………………………………………………………45
一、豆科基質之組成…………………………………………………45
(一)黑豆與黃耆熱水萃取物組成…………………………………45
(二)黑豆及黃耆單醣組成…………………………………………46
(三)黑豆與黃耆多醣分子量分布…………………………………47
二、靜置液態培養菌絲體多醣性質…………………………………51
(一)靈芝靜置液態培養醣含量及組成成…………………………51
(二) (1,3)-β-D-glucan含量與分支度………………………….54
(三)多醣分子量分布………………………………………………58
三、發酵槽生產菌絲體多醣之性質…………………………………63
(一) 五公升試驗型發酵槽……………………………………….63
1.發酵液沉降多醣中醣含量與組成………………………….63
a.溫度影響………………………………….....63
b.通氣量影響…………………………………….65
c.接菌量影響…………………………………….67
d.兩階段影響…………………………………….69
2. (1,3)-β-D-glucan含量與分支度………………………..72
3.多醣分子量分布…………………………………………….74
a.溫度影響……………………………………….74
b.通氣量影響…………………………………….77
c.接菌量影響…………………………………….80
d.兩階段影響…………………………………….83
(二) 兩百公升發酵槽……………………………………………….88
1.發酵液沉降多醣中醣含量與組成……………………….88
2.(1,3)-β-D-glucan含量與分支度……………………….90
3.多醣分子量分布………………………………………….92
陸、結論………………………………………………………………96
柒、參考文獻…………………………………………………………98

表目錄
表一、靈芝中生物活性成分…………………………………………12
表二、靈芝中多醣特徵及生理活性…………………………………15
表三、比較靈芝與其它真菌的(1,3)-β-D glucan 分支度與抑制老鼠肉瘤細胞之活性………………………………………………………………………24
表四、靈芝靜置液態培養基組成……………………………………38
表五、黃耆與黑豆萃取物醣類含量分析……………………………46
表六、黃耆與黑豆熱水萃之粗多醣單醣組成………………………46
表七、靈芝靜置培養醣含量與組成…………………………………53
表八、靜置培養靈芝多醣中(1, 3)-β-D-glucan的含量及分支度..57
表九、靜置培養多醣分子量分布區分比例………………………….62
表十、五公升試驗型發酵槽中靈芝發酵液醣類含量分析(1)………70
表十一、五公升試驗型發酵槽中靈芝發酵液醣類含量分析(2)……71
表十二、五公升試驗型發酵槽中靈芝多醣(1,3)-β-D gluacn的含量及分支度
…………………………………………………………………………73
表十三、五公升試驗型發酵槽中靈芝多醣分子量分布區分比例…87
表十四、兩百公升發酵槽中靈芝發酵液醣類含量分析……………89
表十五、兩百公升發酵槽中靈芝多醣(1,3)-β-D gluacn的含量及分 支度....................................................91
表十六、兩百公升發酵槽中靈芝多醣分子量分布區分比例………95


圖目錄
圖一、靈芝中多醣結構………………………………………………13
圖二、β-D glucan生物反應修飾劑:可能的免疫機制…………….14
圖三、裂褶菌多醣(Schizophyllan)分子量、構形與腫瘤抑制率相關性………………………………………………………………………22
圖四、真菌中(1,3)-β-D glucan生合成途徑………………………27
圖五、Aniline blue主要成分結構…………………………………28
圖六、Sirofluor的結構…………………………………………….29
圖七、大豆中異黃酮結構……………………………………………32
圖八、花靑素結構……………………………………………………34
圖九、黃耆熱水萃取多醣分子量分布………………………………47
圖十、黑豆熱水萃取多醣之分子量分布……………………………48
圖十一、五種標準單糖樣品於HPAEC-PAD層析圖譜……………….49
圖十二、 (A)黃耆與(B)黑豆單醣組成HPAEC-PAD層析圖譜………50
圖十三、(1,3)-β-D glucanase經過酵素水解產物之HPAEC分析圖譜 …………………………………………………………………………56
圖十四、靜置液態培養靈芝菌絲體熱水萃取之多醣分子量分布…60
圖十五、溫度對於靈芝發酵液中固形物、多醣及多醣中的(1,3)-β-D-glucans變化量………………………………………………………64
圖十六、通氣量對於靈芝發酵液中固形物、多醣及多醣中的(1,3)-β-D- glucans變化量圖………………………………………………66
圖十七、接菌量對於靈芝發酵液中固形物、多醣及多醣中的(1,3)-β-D- glucans變化量圖………………………………………………68
圖十八、比較不同溫度對於靈芝多醣中分子量分布的影響(A)18℃ (B)24℃(C)30℃………………………………………………………75
圖十九、比較不同通氣量對於靈芝多醣中分子量分布的影響(A)0.25vvm
(B)0.50vvm(C)0.75vvm………………………………………………78
圖二十、比較不同接菌量對於靈芝多醣分子量分布影響(A)0.1%(B)1%(C)10%...............................................81
圖二十一、比較兩階段發酵對靈芝多醣分子量分布影響(A)GL-E control
(B)GL-E two stage……………………………………………………84
圖二十二、比較兩階段發酵(發酵五天，在18℃靜置6天)對靈芝多醣分子量分布影響(A)GL-E-30-75-18℃………………………………85
圖二十三、在兩百公升發酵槽中溫度對於靈芝多醣分子量分布的影響.......................................................93

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