臺灣博碩士論文加值系統

Stigmastane-3,6-dione被發現有許多生物活性，但取得方式耗費時間、
金錢，而且獲得的量也少，所以想利用菰黑穗菌去大量生產stigmastane-3,6-dione，實驗第一部分是使用反應曲面法來找尋菰黑穗菌大量生產stigmastane-3,6-dione的條件。菰黑穗菌已被報導可以抗骨質疏鬆以及其抗骨質疏鬆物質也被確定是stigmastane-3,6-dione。其中改變因子有培養溫度、培養轉速及起始pH值，結果得到在溫度27.93℃、轉速119.41 rpm及起始pH值5.01時，stigmastane-3,6-dione可達到最大濃度53.4 mM。
本來想使用酒粕液去刺激菰黑穗菌去大量生產stigmastane-3,6-dione，但是產量不但沒有提升，而且還下降，但是意外發現培養液變成黏稠狀，推測有大量的成分A產生，故本實驗第二部分使用酒粕液去刺激菰黑穗菌生產大量的成分A-1，發現酒粕液 : PDB培養基= 15 : 85 時，成分A-1有最大的產率，將此成分A-1進行化學結構及生物活性的分析，發現組成單糖可能是葡萄糖及甘露糖，而鍵結是以1-6鍵結最多、1-3鍵結第三多，而抗發炎實驗則是添加到100µg/ml的成分A-1開始有效果且添加越多效果越強，但EC50的結果是超過800µg/ml，可能是結構組成差異，造成抗發炎效果不明顯。

Stigmastane-3 ,6-dione have been found in many biological activities, but it takes a lot of time and money to acquire just a small amount of it. In this study, we have utilized Ustilago Esculenta to produce a numerous amount of stigmastane-3,6-dione. The first part of the experiment was to use what we called the Response Surface Methodology for finding the optimal condition in producing stigmastane-3,6-dione with the use of Ustilago Esculenta. Ustilago Esculenta has been reported to slow down the osteoporotic process, which has been believed to be induced by stigmastane-3, 6-dione. Also, there are many factors that come into play in producing stigmastane-3 ,6-dione such as the incubation temperature, rotational speed of the culture and the initial pH values. Under these conditions, the maximal concentration of stigmastane-3 ,6-dione has been reached and it was 53.4 mM, at the temperature of 27.93℃, the rotational speed of 119.41 rpm and the initial pH of 5.01. Originally, it was our intention to use distilled grain liquid to stimulate Ustilago Esculenta for the growth of stigmastane-3 ,6-dione; as a result, the yield has become significantly decreased. However, from this experiment, we have found out the culture medium has become viscous, assuming that there was a tremendous production of composition A-1 because of that. Therefore, the second part of the experiment was to use distiller grain liquid to stimulate Ustilago Esculenta to produce large amount of composition A-1. Found in distillers grains liquid: PDB medium = 15 : 85., the composition A-1 have the greatest yield. As a result, the analysis of the chemical structures and biological activities of composition A-1 has been done, the composition of monosaccharides could possibly be glucose and mannose with maximum amount of 1-6 bonding, followed by1-3 bonding. The anti-inflammatory study has a positive effect when added with 100µg/ml composition A-1, and a stronger effect with a larger dose. But, the EC50 result was over 800µg/ml, due to different in composition and structure, resulting the anti-inflammatory effect was not obvious.

目次
摘要…………………………………………………………………………………….I
Abstract……………………………………………………………………………..II
目次………………………………………………………………………………… .IV
圖目次……………………………………………………………………………...VII
表目次……………………………………………………………………………IX
第一章、緒論
1.1 引言………………………………………………………………………………1
第二章、文獻回顧
2.1 骨質疏鬆症………………………………………………………………………2
2.2 茭白筍……………………………………………………………………………3
2.3 菰黑穗菌…………………………………………………………………………4
2.4 Stigmastane-3-6-dione…………………………………………………………..5
2.5 反應曲面法……………………………………………………………………6
2.6 多醣體…………………………………………………………………………9
第三章、菰黑穗菌發酵液成分分析之探討 : 以反應曲面法生產stigmastane-3,6-dione
3.1 實驗背景及目的………………………………………………………………14
3.2 實驗儀器………………………………………………………………………15
3.3 實驗藥品………………………………………………………………………16
3.4 實驗方法
3.4.1 搖瓶培養測試………………………………………………………16
3.4.2 固態直接培養………………………………………………………17
3.4.3 液態培養後再固態培養…………………………………………….17
3.4.4 搖瓶培養 ……………………………………………………………17
3.4.5 Stigmastane-3-6-dione萃取………………………………………….17
3.4.6 Stigmastane-3-6-dione 分析………………………………………...17
3.4.7 Stigmastane-3-6-dione 校正曲線…………………………………...18
3.4.8 Stigmastane-3-6-dione 質譜分析…………………………………...18
3.4.9 培養時間對於stigmastane-3-6-dione產率之影響…………………18
3.4.10 孢子數對於stigmastane-3-6-dione產率之影響…………………..18
3.4.11 培養溫度對於stigmastane-3-6-dione產率之影響………………...19
3.4.12 培養轉速對於stigmastane-3-6-dione產率之影響………………..19
3.4.13 起始pH對於stigmastane-3-6-dione產率之影響…………………19
3.4.14 反應曲面法…………………………………………………………19
3.5實驗結果與討論
3.5.1 Stigmastane-3-6-dione 的分析鑑定………………………………21
3.5.2 菌液中stigmastane-3-6-dione 的分析鑑定………………………23
3.5.3 直接塗盤與液態培養放大之stigmastane-3-6-dione產率比較……25
3.5.4 培養天數對stigmastane-3-6-dione產率之影響……………………27
3.5.5 培養孢子數對stigmastane-3-6-dione產率之影響………………….28
3.5.6 培養溫度對於stigmastane-3-6-dione產率之影響………………….29
3.5.7 培養轉數對於stigmastane-3-6-dione產率之影響………………….30
3.5.8 起始pH值對於stigmastane-3-6-dione產率之影響………………31
3.5.9 反面曲面法…………………………………………………………32
3.6 實驗結論………………………………………………………………………34
3.7 未來展望………………………………………………………………………34
第四章、菰黑穗菌發酵液成分分析之探討 : 以酒粕液刺激生產成分A-1之結構與生化活性的研究
4.1 實驗背景及目的………………………………………………………………35
4.2 實驗儀器……………………………………………………………………..36
4.3 實驗藥品……………………………………………………………………..37
4.4 實驗方法
4.4.1 元素分析……………………………………………………………38
4.4.2 氨基酸組成成分分析………………………………………………38
4.4.3 成分A-1的生產……………………………………………………38
4.4.4 成分A-1的萃取……………………………………………………39
4.4.5 單醣鑑定……………………………………………………………39
4.4.6 甲基化………………………………………………………………39
4.4.7 成分A-1紅外線光譜結構鑑定…………………….……………….40
4.4.8 抗發炎實驗…………………………………………………………40
4.5結果與討論
4.5.1 酒粕液添加物對stigmastane-3-6-dione產率影響及成分分析………41
4.5.2酒粕液添加量與成分A-1產量……………………………………….42
4.5.3 FT-IR對成分A-1結構鑑定…………………………………………43
4.5.4 單糖組成測定……………………………………………..…………44
4.5.5 結構鍵結的鑑定………………….………………………….………45
4.5.6 抗發炎反應實驗……………………………………………………..47
4.6 實驗結論………………………………………………………………………48
4.7 未來展望………………………………………………………………………48
參考文獻……………………………………………………………………49
附錄…………………………………………………………………………………59
圖目次
圖一、正常骨骼與骨質疏鬆骨骼比較………………………..………………………2
圖二、內含菰黑穗菌的筊白筍………………………………………………………..4
圖三、Stigmastane-3-6-dione的結構………………………………………………….5
圖四、逐次尋找最佳化點……………………………………………………………6
圖五、β-1→3、β-1→6-D-葡聚醣結晶結構………………………………………12
圖六、標準品之高效能液相層析圖…………………………………………………21
圖七、標準品之正離子一次質譜圖…………………………………………………22
圖八、標準品之二次質譜圖…………………………………………………………22
圖九、菌液之高效能液相層析圖……………………………………………………23
圖十、菌液42分鐘波峰之正離子一次質譜圖…………...…………………………24
圖十一、菌液42分鐘波峰之二次質譜圖………………………….………………24
圖十二、不同天數下兩組的stigmastane-3-6-dione的產量比較圖………………25
圖十三、不同起始孢子數下兩組的stigmastane-3-6-dione的產量比較圖………25
圖十四、不同培養溫度下兩組的stigmastane-3-6-dione的產量比較圖…………26
圖十五、不同培養轉速下兩組的stigmastane-3-6-dione的產量比較圖…………26
圖十六、不同起始pH值下兩組的stigmastane-3-6-dione的產量比較圖………26
圖十七、培養天數對stigmastane-3-6-dione產率比較……………………...……27
圖十八、起始孢子數對stigmastane-3-6-dione產率比較圖………………………28
圖十九、培養溫度對stigmastane-3-6-dione產率比較圖…………………...……29
圖二十、培養轉速對stigmastane-3-6-dione產率比較圖………………….………30
圖二十一、起始pH值對stigmastane-3-6-dione產率比較圖………….……………31
圖二十二、變數因子對產率影響的相關性圖………………………………………32
圖二十三、反應曲面圖，x軸為轉速，y軸為溫度，z軸為stigmastane-3-6-dione濃度……………………………………………………………………………….….33
圖二十四、反應曲面圖，x軸為pH，y軸為溫度，z軸為stigmastane-3-6-dione濃度…………………………………………………………………………………..33
圖二十五、反應曲面圖，x軸為pH，y軸為轉速，z軸為stigmastane-3-6-dione濃度…………………………………………………………………………………..33
圖二十六、酒粕液添加物培養菌液之高效能液相層析圖………..………………..41
圖二十七、添加不同比例酒粕液產生成分A-1重量比較圖………………………42
圖二十八、成分A-1之紅外線光譜圖………………………………………………43
圖二十九、乙醯化成分A-1之氣相層析圖…………………………………………44
圖三十、成分A-1甲基衍生化之氣相層析圖………………………………………45
圖三十一、成分A-1甲基化氣相層析於16.92分鐘波峰之質譜圖……………..…46
圖三十二、成分A-1甲基化氣相層析於15.99分鐘波峰之質譜圖………………..46
圖三十三、成分A-1甲基化氣相層析於17.80分鐘波峰之質譜………………….46
圖三十四、添加成分A-1抗發炎效果比較圖………………………………………47
表目次
表一、變數因子的選擇步驟模式……………………………………………………7
表二、pH 、溫度 、轉速之中心混成模式之實驗設計表…………………………20

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