臺灣博碩士論文加值系統

矽藻為海洋環境中重要的初級生產者，富含許多高經濟價值的化合物，目前矽藻中的 fucoxanthin與多醣已被證實具有促進人體健康保健的活性，如何有效率的獲取及分析這些化合物為非常重要的研究課題。
研究中以培養矽藻 Hyalosynedra toxoneides 得知其於五百毫升光生物反應器中10f 培養基具最高的細胞濃度以及 fucoxanthin 產量但有最低的粗多醣產量，分別為0.86 g/L、7.88 mg/L及24.4 mg/L。後續於八公升光生物反應器中進行批次式培養、饋料批次式培養以及半連續式培養。並於 10f 濃度的半連續培養中，發現細胞產量可達1.18 ± 0.15 g/L， fucoxanthin產量為4.45 ± 0.73 mg/L，並且多醣產量為52.4 ± 0.4 mg/L。結果表明此培養方式具有高生化合物之產量。
萃取出之粗色素以膠體管柱層析 (silica gel chromatography) 純化 fucoxanthin後回收率可達73%，並以二乙氨基乙基纖維素膠體 (DEAE cellulose) 進行醣類純化，以五種鹽濃度水溶液 (0 M、0.5 M、1 M、1.5 M、2 M) 進行分離。五種鹽濃度能更細緻地區分出不同帶電性的多醣。本實驗得到各分層的多醣 (Peak 0- 4)，其醣醛酸含量為0-4.3 %，硫化程度達0-4.6 %。並發現不同電荷醣類的生合成情形與培養基濃度有相關性。
本培養實驗得到了高生化合物產量結果，並於未來對萃取出之 fucoxanthin以及多醣進行相關的活性實驗，可進一步提升它們在矽藻培養及保健食品方面的實用性。

Diatoms, key primary producers in marine ecosystems, contain numerous high-value compounds. Specifically, fucoxanthin and polysaccharides in diatoms have been recognized for their health-promoting benefits, making their efficient extraction and analysis a crucial research area.
In this study, I cultured the diatom strain Hyalosynedra toxoneides and discovered that the highest cell and fucoxanthin concentrations, but the lowest crude polysaccharide yield, were achieved using a 10f medium in a 500 mL photobioreactor. These were respectively 0.86 g/L, 7.88 mg/L, and 24.4 mg/L. I subsequently performed batch, fed- batch, and semi-continuous cultivations in an 8 L photobioreactor. During semi-continuous cultivation at a 10f concentration, we found cell yields reached 1.18 ± 0.15 g/L, fucoxanthin yields were 4.45 ± 0.73 mg/L, and polysaccharide yields were 52.4 ± 0.4 mg/L.
Purification processes involving column chromatography and diethylaminoethyl cellulose gel were applied to crude pigment and crude polysaccharides, yielding fucoxanthin recovery rates up to 73%. Diethylaminoethyl cellulose gel separation with five different salt solutions (0 M、0.5 M、1 M、1.5 M、2 M) allowed us to differentiate polysaccharides based on charge more precisely than before. Polysaccharide layers (Peak 0-4) were obtained with uronic acid contents of 0-4.3% and sulfation degrees up to 0-4.6%.
This cultivation experiment yielded high production of biochemical compounds, and future activity experiments on the extracted fucoxanthin and polysaccharides can further enhance their practicality in diatom cultivation and health food applications.

摘要 i
Abstract ii
目錄 iii
表目錄 vi
圖目錄 vii
1. 前言 1
2. 研究動機與目的 2
3. 文獻回顧 3
3.1. 矽藻介紹 3
3.2. 矽藻培養 3
3.2.1. 批次 (Batch) 培養 4
3.2.2. 饋料批次 (Fed-batch) 培養 4
3.2.3. 半連續式 (Semi continuous) 培養 4
3.3. 矽藻色素 5
3.4. 矽藻多醣 5
4. 實驗材料與方法 6
4.1. 矽藻簡介 6
4.2. 矽藻培養方法 6
4.2.1 矽藻培養基配製 6
4.2.2. 五百毫升光生物反應器之批次培養 7
4.2.3. 五百毫升光生物反應器之 10f 濃度培養基搭配不同濃度矽酸鹽濃度批次培養 7
4.2.4. 五百毫升光生物反應器之饋料批次培養 7
4.2.5. 八公升光生物反應器培養 7
4.2.6. 八公升光生物反應器之批次式培養 (10f) 8
4.2.7. 八公升光生物反應器之饋料批次式培養 (10f) 8
4.2.8. 八公升光生物反應器之半連續式培養 8
4.2.9矽藻培養之分析 8
4.2.9.1 細胞計數 8
4.2.9.2光細胞密度 (OD750) 9
4.2.9.3 矽酸鹽殘餘量測定 (OD410) 9
4.2.9.4 水質pH 測定 9
4.2.9.5矽藻培養之生物量產率、比生長速率及細胞倍增時間計算 9
4.3. 矽藻之色素及粗多醣萃取 10
4.4 膠體管柱層析法純化分離矽藻粗色素 10
4.5矽藻粗色素中fucoxanthin 之HPLC濃度測定 11
4.6 矽藻水溶性多醣脫除蛋白質 11
4.7 五百毫升光生物反應器批次式培養獲得之矽藻水溶性多醣純化 12
4.8 八公升光生物反應器批次式培養獲得之矽藻水溶性多醣純化 12
4.9 多醣組成分析 12
4.9.1多醣含量測定 12
4.9.2醣醛酸測定 13
4.9.3硫酸鹽測定 13
5. 結果與討論 13
5.1 矽藻培養結果 13
5.1.1 五百毫升光生物反應器批次培養 13
5.1.2 五百毫升光生物反應器 10f 濃度培養基搭配不同濃度矽酸鹽濃度批次培養 14
5.1.3 五百毫升光生物反應器批次饋料培養 15
5.1.4 八公升光生物反應器批次培養 17
5.1.5 八公升光生物反應器饋料批次式培養 17
5.1.6 八公升光生物反應器半連續式培養 18
5.1.7 八公升光生物反應器半連續式培養產量與文獻比較 19
5.2 Fucoxanthin HPLC 分析 19
5.2.1 五百毫升光生物反應器批次式培養之 fucoxanthin HPLC 分析 19
5.2.2 五百毫升光生物反應器不同濃度矽酸鹽培養之 fucoxanthin HPLC 分析 20
5.2.3 五百毫升光生物反應器饋料批次式培養之 fucoxanthin HPLC 分析 20
5.2.4 八公升光生物反應器批次式培養之 fucoxanthin HPLC 分析 20
5.2.5 八公升光生物反應器饋料批次式培養之 fucoxanthin HPLC 分析 20
5.2.6 八公升光生物反應器半連續式培養之 fucoxanthin HPLC 分析 21
5.2.7 純化八公升光生物反應器培養生產 fucoxanthin 產量與文獻分析 21
5.2.8 純化八公升光生物反應器半連續式培養之fucoxanthin 22
5.3 粗多醣 DEAE 陰離子交換樹脂純化 22
5.3.1 五百毫升光生物反應器批次式培養粗多醣除蛋白結果 22
5.3.2 八公升半連續式培養粗多醣除蛋白結果 22
5.4 五百毫升光生物反應器培養獲得之多醣純化 23
5.4.1 f/2 批次式培養收穫粗多醣純化 23
5.4.2 2.5f 批次式培養收穫粗多醣純化 23
5.4.3 5f 批次式培養收穫粗多醣純化 23
5.4.4 10f 批次式培養收穫粗多醣純化 24
5.5 八公升半連續式培養獲得之多醣進行 DEAE 陰離子交換樹脂結果 24
5.5.1 2.5f 培養條件收穫粗多醣純化 24
5.5.2 5f 培養條件收穫粗多醣純化 24
5.5.3 10f 培養條件收穫粗多醣純化 25
5.5.4 三個培養條件收穫粗多醣純化比較 26
6. 結論及未來展望 27
7. 參考文獻 28
結果圖表 32
附錄圖表 64

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