臺灣博碩士論文加值系統

Ganoderma formosanum又名紫芝，屬於臺灣特有的靈芝品種，近年來已有研究證實其深層培養液所分離出的胞外多醣體具有免疫調節與抗腫瘤的功效；為提高紫芝胞外多醣體的產量，本篇研究以固定化培養的方式- plastic composite support (PCS) 生物反應器作為量產工具，並以抗發炎能力來評估多醣經量產後的活性影響。經最適化結果發現，SF+在四種 (SF+, SY+, SB+ and SR+) PCS中能最有效促進紫芝生長與胞外多醣體的生產；結合培養基碳源的調整和SF+固定化系統的生產，在兩階段醱酵時紫芝胞外多醣的產量已顯著得到提升 ( p < 0.05 )，且連續式生產的實驗中也觀察到重複利用菌體醱酵的情形可達五次之多，如此也反應了PCS生物反應器比起傳統的懸浮培養具有生產週期短、操作便利以及成本低的優勢。在評估多醣體生物活性的實驗中，我們發現經固定化生產的胞外多醣體清除自由基的半抑制濃度較懸浮培養低，且兩者在相同處理濃度下 (125 μg/mL) 抑制RAW 264.7產生發炎因子nitric oxide ( NO ) 的能力亦沒有顯著差異。從單糖組成和β-1,3-glucan的分析結果我們推測其活性來源可能為靈芝活性研究中報導較多的β-glucan和fucose-enriched polysaccharides。

Ganoderma formosanum also named tzu-chih, is an endemic species of lingzhi in Taiwan. Recently, researchers had proved that an extracellular polysaccharides (EPS) purified from submerged culture of G. formosanum could exert immune-modulating and anti-tumor effects. For the purpose of improving G. formosanum EPS, plastic composite support (PCS) immobilization system was developed, also, anti-inflammatory effect was evaluated after the prodution. Among four PCS (SF+, SY+, SB+ and SR+) in selection stage, SF+ immobilized system showed the highest stimulation of EPS and biofilm formation; further in optimization process, SF+ combined with different carbon sources in immobilization system demonstrated a significant improvement of EPS production ( p < 0.05 ) in bi-stage fermentation. During continuous fermentation, 5 repeated-times as many as that of conventional suspended-cell bioreactors was contributed to a shorter producing time, easy manufacturing process and lower cost in PCS bioreactors. Bioactivity evaluation demonstrated that a lower radical scavenging concentration of immobilized group EPS than that of the L/G group, while the anti-inflammatory assay showed no significant difference on inhibiting nitric oxide ( NO ) production with 125 μg/mL EPS treatment in RAW 264.7 cells. Analysis of monosaccharide composition and β-1,3-glucan content depicted that β-glucan and fucose-enriched polysaccharides may contribute to the bioactivity on radical scavenging and anti-inflammation of EPS fermented in suspension and SF+ immobilized system respectively.

目錄
摘要 ii
Abstract iii
目錄 iv
前言 1
第一、文獻回顧 2
第一節、靈芝 2
一、靈芝介紹 2
1.靈芝分類與特性 2
2.紫芝介紹 4
二、活性研究 6
1.靈芝多醣生理活性 6
2.紫芝多醣生理活性 7
三、多醣體研究 12
1. β-glucan 13
2. Fucose-enriched polysaccharides 14
第二節、靈芝屬培養 16
一、培養型態 16
1. 子實體 16
2.菌絲體 17
二、多醣量產策略 18
1.培養基最適化 18
2.兩階段醱酵策略 21
3.固定化培養 25
4.多醣回收條件 27
第三節、發炎反應 28
第二：實驗架構 30
一、實驗目的 30
二、實驗設計 31
三、材料 32
1.實驗菌株 32
2.培養基成分表 32
3.PCS成分表 33
4.藥品 34
四、方法 35
1. 菌株篩選與生長曲線的建立 35
2. PCS製備 35
3. PCS篩選 36
4. 菌株培養 36
5. 胞外多醣回收 37
6. 菌體質量（Biomass）測定 37
7. 總糖測定 37
8. 殘糖測定 38
9. 單糖組成分析 38
10. 鹼溶 β-(1,3;1,6)-D-聚葡萄醣含量與分支度-酵素層析法 39
11. DPPH清除自由基能力之評估 39
12. 紫芝多醣體抗發炎活性之評估 40
13. 統計分析 40
五、結果與討論 41
1. 碳氮源篩選 41
2. G. formosanum ATCC 76538生長曲線的建立 44
3. 起始pH值對G. formosanum ATCC 76538培養之影響 47
4. PCS篩選結果 50
5. 兩階段醱酵( Bi-stage fermentation )策略 55
6. 醱酵多醣體生物活性之評估 57
7.胞外多醣體之結構分析 61
8. 連續式醱酵 63
六、結論與展望 65
參考資料 67

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