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研究生:林佳穎
研究生(外文):Jia-Ying Lin
論文名稱:建立破囊壺藻生物質譜資料庫、分析其高價化合物及其在白蝦養殖上的應用
論文名稱(外文):The Biotyper Database Establishment, High-Value Compound Analyses, and White Shrimp Culture Application of Thraustochytrids
指導教授:廖志中廖志中引用關係
指導教授(外文):Chih-Chuang Liaw
學位類別:碩士
校院名稱:國立中山大學
系所名稱:海洋生物科技暨資源學系研究所
學門:自然科學學門
學類:海洋科學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:中文
論文頁數:135
中文關鍵詞:生物質譜資料庫基質輔助雷射脫附游離飛行時間式質譜儀破囊壺藻ω-3多元不飽和脂肪酸蝦紅素
外文關鍵詞:BiotyperastaxanthinMALDI-TOF MSomega-3 polyunsaturated fatty acidsThraustochytrids
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破囊壺藻(Thraustochytrids),是真核生物域的一種異營性海洋原生生物,廣泛分布於河口及沿近海岸,尤其是紅樹林區域,並且擔任著環境分解者的重要角色。其體內ω-3多元不飽和脂肪酸(PUFA)含量高,使破囊壺藻成為商業化二十二碳六烯酸(DHA)和二十碳五烯酸(EPA)的候選來源。
使用基質輔助雷射脫附游離飛行時間式質譜儀 (matrix-assisted laser desorption ionization time-of-flight, MALDI-TOF MS) 來鑑定藻種,這項技術近年已被廣泛應用在微生物鑑定與研究上,在醫院微生物病原菌種類鑑定具有相當的快速性和高效率,取代繁瑣的DNA定序。目前鑑定破囊壺藻的方法為18S rRNA基因定序,將破囊壺藻的DNA,經過聚合酶連鎖反應 (PCR) 及定序反應獲得藻種的DNA序列,此過程需要耗費大量的人力技術與經費成本且相當耗時。本篇論文利用上述基質輔助雷射脫附游離飛行時間式質譜儀 (matrix-assisted laser desorption ionization time-of-flight, MALDI-TOF MS)開發破囊壺藻的生物質譜資料庫,希望對往後破囊壺藻的鑑種帶來更多的便利性。
我們透過合作實驗室採集台灣本地紅樹林地區的33株破囊壺藻,並分析其 DHA及蝦紅素的含量,其中DHA含量最多的藻株為W1-2(Schizochytrium sp.),而蝦紅素含量最多的藻株為YA7.1(Aurantiochytrium sp.),我們接續以上結果並利用富含DHA及蝦紅素的破囊壺藻開發作為水產蝦類養殖的食品補充劑,結果顯示添加富含DHA的藻粉能使蝦子擁有較大的存活率,而添加富含蝦紅素的藻粉能使蝦子的及體長增加。此外,我們分離並純化破囊壺藻的二次代謝物,也分析了破囊壺藻的諸多生物活性(如抗乙醯膽鹼酶、抗氧化),由結果發現抗乙醯膽鹼酶活性與DHA含量成正比,抗氧化活性與蝦紅素含量成正比。
Thraustochytrids are a group of marine osmoheterotrophic, straminipilan protists that grow in the estuary and coastal, especially in the mangrove area, and play an important role as saprobes. The high content of ω-3 polyunsaturated fatty acids (PUFA) makes thraustochytrids a candidate source for commercial docosahexaenoic acids (DHA) and eicosapentaenoic acid (EPA).
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF MS) can be used on identifying bacterial species. This technique has been widely used in microbial identification and researches in recent years. It is rapid and highly efficient on the identification of microbial pathogens in hospitals to replace that by DNA sequencing. Currently, to identify the species of thraustochytrids is by the 18S rRNA gene sequencing. However, it is manpower, time, and cost-consumed to synthesize multiple copies of DNA sequence of thraustochytrid species by polymerase chain reaction (PCR) and analysis those sequences. Herein, we plan to establish a biotyper database of thraustochytrids by MALDI-TOF MS, which will facilitate the process to identify the thraustochytrids from field collection.
By laboratory cooperation to collect thraustochytrids in the mangrove areas at Taiwan, we quantified the DHA and astaxanthin contents of 33 thraustochytrid species. Among them, the most DHA strain is W1-2 (Schizochytrium sp.) and the most astaxanthin one is YA7.1 (Aurantiochytrium sp.). Following, we fermented those two thraustochytrid strains, W1-2 and YA7.1, as food supplement in shrimp aquaculture. The thraustochytrid rich in DHA can enhance the survival rate of white shrimps; that rich in astaxanthin can increase the body length of white shrimps. Besides, we further evaluated the bioactivities of 33 thraustochytrid species, such as anti-acetylcholinesterase and anti-oxidation activities. Our results indicated that the anti-acetylcholinesterase activity is in direct proportion to their DHA content, meanwhile the antioxidant activity is in direct ratio to their astaxanthin content.
論文審定書…….………………….…………………………………….………………….….i
謝誌…………………………………………………………………………………………...ii
摘要…………………………………………………………………………...………………iii
Abstract……………………………………………………………………………………….iv
目錄……………………………………………………………………………………….....v
圖目錄…………………………………………………………………………………….....viii
表目錄…………………………………………………………………………………….....xii
壹、 緒論……………………………………………………………………………………1
1.1 前言………………………………………………………………………………….1
1.2 研究動機與目標…………………………………………………………………….3
貳、 文獻回顧…………………………………………………….………………………...4
2.1 破囊壺藻的介紹……………………………………………………….……………4
2.1.1 分類與形態…………………………………………………………………4
2.1.2 生態角色……………………………………………………………………5
2.1.3 應用…………………………………………………………………………6
2.2 β-胡蘿蔔素、蝦紅素、及DHA的重要性……………..………………………….7
2.2.1 β-胡蘿蔔素(β-carotene)…….………..……………………………………..7
2.2.2 蝦紅素(Astaxanthin)….……………………………………………...……..9
2.2.3 DHA二十二碳六烯酸(Docosahexaenoic acid)….………………………..11
2.3 MALDI-TOF MS的簡介………………………………….…………….….……...12
2.4 MALDI Biotyper的介紹與應用………………………………….……………….14
2.5 乙醯膽鹼酶(Acetylcholine Esterase)與其抑制應用…………...…….……………16
2.6 氧化作用與抗發炎…………………………………………………….…………..17
2.7 飼料添加物用於白蝦養殖………………...………………………………………18
參、 材料與方法…………………………………………………………………………..19
3.1 破囊壺藻的培養…………………………………………………………….……...19
3.1.1 藻種來源……………………………..………………………..…....……..19
3.1.2 藻種之水樣處理…………………………………………………...….…..21
3.1.3 藻株初步分離與純化……………………………………………….….....21
3.1.4 藻株之培養與保存……………………………………………………......22
3.1.5 DNA萃取及聚合酶連鎖反應(PCR)…………….……………………….23
3.1.6 類緣關係樹及物種分子鑑定……………………………………………..23
3.2 高單價化合物之分析方法(蝦紅素、DHA、β-胡蘿蔔素)….…………..……….24
3.2.1 實驗器材與藥品……………………………………………...…………...24
3.2.2 培養天數及件………………………………………………...…………...25
3.2.3 最適化萃取方法…………………………………………………………..25
3.2.4 HPLC分析方法………………………………………….………………..27
3.3 MALDI-TOF MS分析…………………………………………..………….….…..29
3.3.1 實驗儀器裝置與藥品………………………………………………....…..29
3.3.2 待測樣品的培養流程與培養時間最適化………………….…..…….…..29
3.3.3 選擇基質溶液與萃取程序……...……………….……….………..……...30
3.3.4 蛋白質/胜肽最適化萃取方法…………...…………………….………….32
3.3.5 使用MALDI-TOF MS進行質譜指紋分析……………...…….….……...33
3.3.6 MALDI-TOF MS質譜圖之參數設定…………………………...………..34
3.4 破囊壺藻用於白蝦養殖之營養補充劑評估………………….…………….…….36
3.4.1 破囊壺藻大量發酵………………………………………………………..36
3.4.2 發酵槽的使用條件………………………………………………………..36
3.4.3 白蝦養殖測試……………………………………………………………..39
3.5 破囊壺藻之主要二次代謝物分離(YA7.1)….…………………………………….40
3.5.1 儀器設備與試藥…………………………………………………………..40
3.5.2 分離流程…………………………………………………………………..41
3.6 抗乙醯膽鹼酶測試(anti-AChE TLC assay)……………………………….………44
3.6.1 實驗原理…………………………………………………………………..44
3.6.2 實驗器材與藥品配製……………………………………………………..45
3.6.3 實驗流程…………………………………………………………………..46
3.7 抗氧化測試(DPPH assay)…..……………………………………………………...48
3.7.1 實驗原理…………………………………………………………………..48
3.7.2 實驗器材與藥品配製……………………………………………………49
3.7.3 實驗流程…………………………………………………………………..49
肆、 結果與討論…………………………………………………………………………..50
4.1 MALDI TOF MS圖譜最佳測試方法……………………………………………...50
4.1.1 藻株培養時間最適化……………………………………………………..50
4.1.2 基質溶液與萃取流程的選擇……………………………………………..51
4.1.3 蛋白質/胜肽最適化破壁萃取…………………………………………..52
4.2 Biotyper軟體鑑定破囊壺藻………………………………………………………55
4.3 破囊壺藻高單價化合物之分析…………………………………………………...67
4.3.1 藻株最適化萃取方式……………………………………………………..67
4.3.2 破囊壺藻高單價化合物之分析結果……………………………………..71
4.4 飼料添加物對於白蝦生長體型及存活率的影響…………….………….….........86
4.5 破囊壺藻之主要二次代謝物分離(YA7.1)….…………………………………….93
4.6 抗乙醯膽鹼酶測試(anti-AChE TLC assay)結果分析………..……………….…104
4.7 抗氧化測試(DPPH assay)結果分析……………………………………………...107
伍、 結論…………………………………………………………………………………109
陸、 參考文獻……………………………………………………………………………111
柒、 附錄……………………………………………………………………………..…..118
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