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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.author | 鄭正熙 | zh_TW |
| dc.date.accessioned | 2021-07-01T08:12:26Z | - |
| dc.date.available | 2021-07-01T08:12:26Z | - |
| dc.date.issued | 2000 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75265 | - |
| dc.description.abstract | 麻痺性貝毒（paralytic shellfish poisoning toxin, PSP）是一系列主要由渦鞭毛藻所產之類似毒素，可藉由濾食性貝類的蓄積，而造成人類的誤食引起麻痺性的中毒現象，常奪走人類生命，大多數各國均有食品法規予以可容許含量來管理，由於檢測上的需求，毒素標準品有待製備，又因化學合成產率低，所以需從毒藻的培養來獲取毒素，本研究的目的即在尋求毒藻之最佳培養條件，應用於毒素的純化製備。 根據以往的文獻，台灣株微小亞歷山大藻的適溫範圍內選擇15℃、20℃、25℃及30℃的測試條件，鹽度條件上選擇5ppt、15ppt、25ppt、35ppt等可生長的鹽度範圍，分別在溫度或鹽度單一變因條件下培養20天，於接種後每隔4天記錄細胞數並採取藻細胞樣品進行毒素分析，所有的分析操作前，均經儀器與人為操作部份的精密度分析，以確保分析數據的準確性。 不同溫度培養實驗結果發現，最適合毒藻生長的溫度在20℃-25℃,產毒率、細胞含毒量隨培養溫度下降而增加。在不同鹽度培養實驗結果發現，毒藻生長最適合的鹽度為25ppt，且其產毒率、細胞含毒量均隨著培養液鹽度增加而增加。在本研究的各組溫度及鹽度條件下，均可發現其毒素的組成中的相對量，隨生長期之不同而改變，當接種後於對數生長初期時GTX-2，-3相對量急速升高，而至生長平穩期時，其毒素組成相對量也恢復成接種前的比例。在GTX-4，-1含量比相對下降之際，其細胞毒素的淨產率仍為正值，從這種毒素組成相對量的改變及化學結構上的相關，可以確定GTX-3，-2為GTX-4，-1的前質，當對數生長期時新細胞之GTX-3，-2首先合成，待細胞逐漸成熟後，部份代謝成GTX-4，-l，而造成不同生長期中毒素組成比例的改變。同時在各組不同條件下培養的毒藻，在生長平穩期的毒素組成比例維持恆定，但培養條件不同其間比例亦隨之不同。不同培養條件下，藻類存在不同生長速率μ，隨著生長速率愈高，即較佳的生長條件下其GTX-4，-l的含量比相對提高(r2=0.90）。 由本實驗得到兩種生長處理的模式以獲得較高的毒素生產，一是在最適生長條件（鹽度25ppt、溫度25℃）下，快速達到所需細胞數，持續維持至平穩期時收獲藻細胞，以獲得較單純之藻毒組成（GTX-4，-1相對含量達97％以上），或在隨後之高濃度接種（1:4 接種體積比），並移至較高鹽度35ppt或較低溫度15℃下，於生長初期時收獲藻細胞數，以獲得較高細胞含毒量，但在高鹽情況下，獲得較高之GTX-3，-2含量。在毒素純化方面，依前述實驗結果，以25ppt、25℃條件進行培養，在生長平穩期收藻，經萃取後進行膠濾層析及離子交換層析進行毒素分離純化。膠濾層析各管分液分別測定高效液相層析、螢光點檢驗、pH值，經比較此三種分析方法，以測定pH值方式即可快速提供所需訊息。而在離子交換層析方面，分離策略根據樣品含毒種類不同，以不同濃度醋酸重覆多次梯度沖提，由經膠濾層析處理後樣品含GTX-4，-1，-3，-2分別為0.26,0.87,0.03,0.05mg，經過本實驗重複二次離子交換層析所分離出的單一毒素GTx-4，-2分別得到0.12,3.61×10-2mg，另外得到少量GTx-1，-3分別為1.77 ×10-2,2.06×10-4mg，本研究的分離方式以兩次離子交換層析分離大部份GTX-4，-2，產率分別可達46%,72%。在GTX-1，-3方面，以多次離子交換層析，仍可將此兩種毒素分離。本研究在毒素純化方面，不但實際將四種毒素單離，同時層析上運用的策略，可供日後擴大規模純化工作的參考依據。 | zh_TW |
| dc.description.abstract | Paralytic shellfish poisoning (PSP) toxins which were mainly produced by marine dinoflagellates can be accumulated by bivalves. Because it is strickly harmful to human health, many countries have regulation to protect people from PSP-contaminated shellfish. The PSP toxin standards were finding prepared from culture of toxic dinoflagellates. The goal of this study was the best cultural condition of Alexandrium minutum Halim (AmTK-4) for the highest toxin production and the efficient procedure for purifying individual GTX toxins, Alexandrium minutum Halim (AmTK-4) isolated from TonKong area were cultured under one variable condition, including temperature (15°C, 20°C, 25°C, 30°C) and salinity (5ppt. l5ppt, 25ppt, 35ppt). Algae cells were collected per 4 days and quantitied toxin contents with certified toxin standards by using HPLC-FD system. The temperatures and salinity for optimal growth of A. mintum were 20-25°C and 25ppt, respectively. The toxin production rate and toxin content decreased while cultural temperature decreased. In contrast, while salinity increased, toxin content increased. More over, the relative growing stages, the GTX-2, -3 increased during log phase. It was suggested that the GTX-2, -3 were precursors of GTX-4, -1. Under difference cultural conditions, A. mintum showed different growth rate, μ. The relative percentage of GTX-4, - 1 were higher while growth rate was higher. The mass culture of A. mintum under 25ppt and 25°C, were harvested during stationary phase for toxin production. The ethanol extracts of A. mintum further purified by gel filtration chromatography and ion exchange chromatography. From the mixture of GTX-1, -2, -3, -4 (1.21mg), pure GTX1 (l.77x10-2 mg), GTX2 (3.61xl0-2 mg), GTX3 (2.06x10-4 mg) and GTX4 (0.12 mg) were obtained through two steps of ion exchange chromatography. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-01T08:12:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2000 | en |
| dc.description.tableofcontents | 第一章、前言. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 第二章、材料與方法. . . . . . . . . . . . . . . . . . . . . . . . . 7 2.0、藻種培養、毒素萃取與定性定量分析. . . . . . . . . . . . . . . . . . . . . . . 7 2.0.1 藻種的培養. . . . . . . . . . . . . . . . . . . . . . . 7 2.0.2 藻毒的萃取. . . . . . . . . . . . . . . . . . . . . . 7 2.0.3 高效液相層析系統. . . . . . . . . . . . . . . . . . . . 7 2.0.4 毒素的定性定量分析及毒性單位轉換. . . . . . . . . 8 2.0.4.1 毒素標準品. . . . . . . . . . . . . . . . . . . . . . . . . 8 2.0.4.2 毒素的定性分析. . . . . . . . . . . . . . . . . . . . . 8 2.0.4.3 毒素的定量分析. . . . . . . . . . . . . . . . . . . . . . 9 2.0.4.4 毒性單位轉換. . . . . . . . . . . . . . . . . . . . . . 9 2.0.5 比成長率的計算. . . . . . . . . . . . . . . . . . . . . . . . . 10 2.0.6 淨產毒率的計算. . . . . . . . . . . . . . . . . . . . . . . 10 2.1、溫度及鹽度對毒藻產毒效能的影響及毒素組成比例在生長期變化. . . . . . . . 10 2.1.1 不同溫度條件對毒藻生長及產毒影響. . . . . . . . . . 11 2.1.2 不同鹽度條件對毒藻生長及產毒影響. . . . . . . . . 11 2.1.3 毒素組成在緊迫條件下不同生長期中的變化. . . . 11 2.2、藻毒的純化. . . . . . . . . . . . . . . . . . . . 12 2.2.1 藻種的培養與藻細胞收集. . . . . . . . . . . . . . . . . 12 2.2.2 藻毒之萃取. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 2.2.3 膠濾層析分離. . . . . . . . . . . . . . . . . . . . . . . . . 13 2.2.4 離子交換層析分離. . . . . . . . . . . . . . . . . . . . . . . . 13 第三章、結果. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.0、系統及操作精確度確認. . . . . . . . . . . . . . . . . . . . . . 15 3.0.1 系統精確度確認. . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.0.2 操作精確度確認. . . . . . . . . . . . . . . . . . . . . . . . 15 3.1、溫度及鹽度對毒藻產毒的影響及毒素組成比例在生長期變化. . . . . . 15 3.1.1 不同溫度條件下對毒藻生長及產毒影響. . . . . . . . . 15 3.1.2 不同鹽度條件下對毒藻生長及產毒影響. . . . . . . . . 16 3.1.3 毒素組成在緊迫條件下不同生長期中的變化. . . . . 18 3.2、藻毒純化. . . . . . . . . . . . . 18 3.2.1 藻種的培養與藻細胞收集. . . . . . . . . . . . . . . . . . 18 3.2.2 藻毒萃取及毒素分析. . . . . . . . . . . . . . . . . . . . . . 19 3.2.3 膠濾層析分離. . . . . . . . . . . . . . . . . . . . . . . . . . . 19 3.2.4 離子交換層析分離. . . . . . . . . . . . . . . . . . . . . . 20 第四章、討論. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4.1、毒藻產毒效能實驗. . . . . . . . . . . . . . . . . . . . . . . . . 23 4.2、藻毒的純化. . . . . . . . . . . . . . . . . . . . . . . . . 26 第五章、參考文獻. . . . . . . . . . . . . . . . . . . . . . . . . . . 32 附錄. . . . . . . . . . . . . . . . . . . . . . 41 圖. . . . . . . . . . . . . . . . . . . . . . . . . . 44 | |
| dc.language.iso | zh-TW | |
| dc.title | 微小亞歷山大藻AmTK-4藻株之生長與產毒所受溫度及鹽度的影響及其所含麻痺性毒素的純化 | zh_TW |
| dc.title | Temperature and Salinity Effects on the Growth and Toxin Production of Alexandrium minutum Halim AmTK-4, as well as the Isolation of it’s Paralytic Toxins | en |
| dc.date.schoolyear | 89-2 | |
| dc.description.degree | 碩士 | |
| dc.relation.page | 73 | |
| dc.rights.note | 未授權 | |
| dc.contributor.author-dept | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 漁業科學研究所 | zh_TW |
| 顯示於系所單位： | 漁業科學研究所 | |
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