臺灣博碩士論文加值系統

雲芝 (Trametes versicolor syn. Coriolus versicolor)為一種珍貴的藥用真菌，其多醣肽具有抗腫瘤、抗癌、保肝、抗氧化及免疫調節等生物活性。目前雲芝多醣肽的來源以液態發酵生產為居多，因液態培養技術可以控制較佳生長條件，並減少人力、空間及縮短生產時間，且得到產物的生物活性與子實體相似，但可防止其受到重金屬之污染。
雲芝菌絲活性與多醣肽之產量會因所使用之培養基種類、濃度而異，生物活性也會因此而有差別，一般以化學培養基培養，但成本較高，因此本研究以台灣本土雲芝菌Trametes versicolor LH1為培養菌株以提高其多醣肽的產量及降低其培養基之成本為前提下，使用農產品培養基替代半化合培養基，以搖瓶探討最適培養碳源及天然氮源來提高多醣肽產量。本實驗結果顯示以4％蔗糖及0.3％花生粉為碳氮源可得到較高之生質量與胞外多醣肽。以5L發酵槽探討最適生長培養條件，結果顯示在25℃、100rpm、1.5vvm下最適合雲芝菌之生長與及有利於多醣肽之產量。以20L發酵槽探討以市售紅砂糖代替試藥級蔗糖做為碳源，發現依然以試藥級蔗糖所得胞外醣肽來的高，但產量差距不大，以經濟效益來看，仍然以紅砂糖所得效益較高。除此之外，以工廠1噸發酵槽培養，發現菌絲較鬆散且較無菌絲球產生，而生質量與胞外醣肽含量明顯提高。
比較農產品培養基所產之胞外多醣肽與半化合培養基所產之胞外多醣肽成分上之差異，實驗結果顯示，不同培養基發酵所得之胞外多醣肽其一般成分、醣肽分子量、單糖組成、耐熱性與官能基，其成分組成與比例上也會不同。以傅立葉紅外線光譜分析結果發現，以兩種培養基所生產雲芝多醣肽皆具有β-1,3葡聚醣官能基之鍵結，因此皆具有生物活性及免疫促進之活性特色。藉由本研究，未來能提供相關保健產品開發與應用之參考。

Trametes versicolor syn. Coriolus versicolor is a valuable medicinal fungi, it’s polysaccharide peptide has been demonstrated having several biological functions, such as anti-tumor, anti-cancer, liver protection, antioxidant power and immune-regulation. At present, the production of batch fermentation of polysaccharide peptide of T. versicolor, the fermentation technological handles the optimum growing condition. In addition, it reduces manpower, space and shortens production time. Base on the technology, the amount is similar to the fruiting bodies. The technology avoids the contamination from heavy metals.
T. versicolor mycelium and polysaccharides of the production activity due to the use of media types, concentration varies and biological activity. It will be a result by the differences. In general, the cost is higher by chemical culture medium, therefore, this study to improve higher production of polysaccharide peptide and lower the cost of the medium under the premise, agricultural products alternative chemical medium, to fit develop the natural carbon and nitrogen source of agricultural product to increase polysaccharide peptide production of T. versicolor LH1. Shake flask culture to explore the optimal carbon and nitrogen to increase the natural production of polysaccharide peptide. The result suggests using 4% sucrose and 0.3% peanut flour as carbon and nitrogen sources, which is available to higher biomass and extracellular polysaccharide peptide. In 5L fermenter culture conditions of optimum culture growth conditions, at 25 ℃, 100 rpm, 1.5vvm, that’s the most suitable conditions to the growth of mycelia T. versicolor and polysaccharide peptide with the production. To plant one ton fermentation tank training. The sterile mycelia have more loose ball, biomass and extracellular polysaccharide peptide is significantly increased.
To compare the agricultural culture medium and chemical culture medium on the extracellular polysaccharide peptide composition differences. The result shows that produced in different medium are different characteristics of extracellular polysaccharide in the general composition, molecular weight, monosaccharide composition, heat resistance, functional groups, composition and proportion. The results showed that extracellular polysaccharide peptide in both culture media produced are a β-1, 3 glucan groups for the bonding with Fourier Transform Infrared Spectroscopy. Are biologically active and therefore promote the activity of the immune characteristics. The result can provide relevant health product development and application of reference in the future.

封面內頁
簽名頁
中文摘要 iii
英文摘要 v
誌謝 vii
目錄 viii
圖目錄 xii
表目錄 xiv

1. 前言 1
2. 文獻回顧 2
2.1雲芝簡介 2
2.1.1雲芝的分類 2
2.1.2雲芝的型態特徵 2
2.1.3分佈及生態環境 4
2.2雲芝多醣肽 6
2.2.1雲芝多醣肽化學特性 7
2.3雲芝菌的培養 8
2.3.1發酵工程 8
2.3.2液態培養 11
2.3.2雲芝菌菌絲體液態培養之培養基 13
2.4雲芝菌液態培養條件 14
2.5雲芝菌菌絲形態之影響因子 15
2.6胞外多醣肽的回收 18
2.7花生的營養成分 19
2.8試藥級蔗糖、冰糖、白砂糖以及紅砂糖之差別 20
3. 材料與方法 24
3.1實驗材料 24
3.1.1實驗菌種 24
3.1.2實驗藥品 24
3.1.3儀器設備 25
3.2實驗流程與研究目的 26
3.3雲芝菌株培養 27
3.3.1菌種保存與更新 27
3.3.2母株培養 27
3.3.3液態培養 27
3.4不同培養基組成對Trametes versicolor LH1生產胞外多醣肽之探討 31
3.4.1不同碳源對胞外多醣肽生產之影響 31
3.4.2不同天然氮源對胞外多醣肽生產之影響 31
3.5五公升發酵槽液態發酵最適生產條件之探討 31
3.5.1不同溫度對雲芝胞外多醣肽之影響 31
3.5.2不同攪拌速率對雲芝胞外多醣肽之影響 32
3.5.3不同通氣量對雲芝胞外多醣肽之影響 32
3.6二十公升發酵槽液態發酵探討蔗糖與紅砂糖對於胞外多醣肽產量之影響 32
3.7由三角錐形瓶放大培養至1頓發酵槽之產程探討 33
3.8分析方法 33
3.8.1菌絲生物質量之製備與測定 33
3.8.2胞外多醣肽之製備與分析 33
3.8.3酚-硫酸法分析總醣 33
3.9基本成分分析 34
3.9.1含水量測定 34
3.9.2灰分測定 34
3.9.3粗蛋白測定 35
3.9.4粗脂肪測定 35
3.10胞外多醣肽分子量分佈測定 36
3.10.1標準曲線配置 36
3.11胞外多醣肽之單糖組成分析 37
3.11.1標準曲線配置 38
3.12發酵液中胞外多醣肽耐熱性試驗 38
3.13傅立葉紅外線光譜儀試驗(Fourier Transform Infrared Spectroscopy，FTIR) 38
4. 結果與討論 39
4.1三角錐形瓶最適培養碳源及天然氮源之探討 39
4.1.1添加不同碳源對雲芝菌絲體、胞外多醣肽產量之影響 39
4.1.2添加不同天然氮源對雲芝菌絲體、胞外多醣肽之影響 41
4.2五公升發酵槽最適培養條件之探討 42
4.2.1不同培養溫度對雲芝菌絲體與胞外多醣肽之影響 42
4.2.2不同攪拌速率對菌絲體與胞外多醣肽之探討 49
4.2.3不同進氣量對雲芝菌絲體與胞外多醣肽產量之探討 55
4.3試藥級蔗糖、冰糖、白砂糖以及紅砂糖成本之差異性 61
4.4二十公升發酵槽液態發酵探討蔗糖與紅砂糖對胞外多醣肽產量之影響 61
4.5由三角錐形瓶放大培養至1噸發酵槽之產程探討 64
4.6農產品培養基與半化合培養基所產胞外多醣肽成分組成上之差異性 68
4.6.1半化合培養基與農產品培養基所產之胞外多醣肽基本成分分析之比較 68
4.6.2半化合培養基與農產品培養基所產之胞外多醣肽分子量分佈 70
4.6.3半化合培養基與農產品培養基所產之胞外多醣肽單糖組成分析 71
4.7發酵液中胞外多醣肽耐熱性試驗 79
4.8以FTIR分析PSK、PSP及農產品培養基與半化合培養基所產胞外多醣肽之官能基 81
5. 結論. 84
參考文獻 85

圖目錄

圖2.1 雲芝子實體 5
圖2.2 菇類多醣肽(polysaccharide-peptides)的分子結構 9
圖2.3 雲芝胞外多醣肽的化學結構 10
圖2.4 蔗糖結構 22
圖3.1 實驗流程圖1 28
圖3.2 實驗流程圖2 29
圖4.1 添加不同碳源於雲芝LH1培養基中對菌絲體、胞外多醣肽之影響 40
圖4.2 添加不同天然氮源於雲芝LH1培養基中對菌絲體、胞外多醣肽之影響 43
圖4.3 以5L發酵槽在25℃下培養雲芝LH1之發酵產程 44
圖4.4 以5L發酵槽在28℃下培養雲芝LH1之發酵產程 45
圖4.5 以5L發酵槽在30℃下培養雲芝LH1之發酵產程 46
圖4.6 不同培養溫度對雲芝LH1生質量之影響 47
圖4.7 不同培養溫度對雲芝LH1胞外多醣肽之影響 48
圖4.8 以5L發酵槽在50rpm下培養雲芝LH1之發酵產程 50
圖4.9 以5L發酵槽在100rpm下培養雲芝LH1之發酵產程 51
圖4.10 以5L發酵槽在150rpm下培養雲芝LH1之發酵產程 52
圖4.11 在不同轉速培養下對雲芝LH1生質量之影響 53
圖4.12 在不同轉速培養下對雲芝LH1胞外多醣肽之影響 54
圖4.13 以5L發酵槽在0.5vvm下培養雲芝LH1之發酵產程 56
圖4.14 以5L發酵槽在1vvm下培養雲芝LH1之發酵產程 57
圖4.15 以5L發酵槽在1.5vvm下培養雲芝LH1之發酵產程 58
圖4.16 在不同通氣量培養下對雲芝LH1生質量之影響 59
圖4.17 在不同通氣量培養下對雲芝LH1胞外多醣肽之影響 60
圖4.19 以試藥級蔗糖與紅砂糖為碳源對雲芝LH1胞外多醣肽之影響 63
圖4.20 雲芝LH1放大產程生質量之變化 65
圖4.21 雲芝LH1放大產程胞外多醣肽之變化 66
圖4.22 雲芝LH1發酵液低溫濃縮後之結果 67
圖4.23 分子量標準曲線 72
圖4.24 以半化合培養基培養雲芝LH1所產胞外多醣肽(PS-EPS)分子量 73
圖4.25 以農產品培養基培養雲芝LH1所產胞外多醣肽(PB-EPS)分子量 75
圖4.26 商業滅菌對不同培養基培養雲芝LH1所產胞外多醣肽之影響 80
圖4.27 β-1,3-Glucan、PSK、PB-EPS、PS-EPS之FTIR圖譜分析 83

表目錄

表2.1 花生營養成分表 21
表2.2 不同精製程度之蔗糖 23
表3.1 液態培養基組成 30
表4.1 雲芝LH1胞外多醣肽一般組成 69
表4.2 以半化合培養基培養雲芝菌所產胞外多醣肽(PS-EPS)分子量 74
表4.3 以農產品培養基培養雲芝LH1所產胞外多醣肽(PB-EPS)分子量 76
表4.4 PS-EPS與PB-EPS之單糖組成含量 78

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