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研究生:呂寧
研究生(外文):Ning Lu
論文名稱:台灣南部與東部海域沉積物中篩選具抗微生物活性之真菌與放線菌
論文名稱(外文):Screening of fungi and actinomycetes with antimicrobial activity from marine sediments in southern and eastern Taiwan
指導教授:郭傑民
指導教授(外文):Jimmy Kuo
學位類別:碩士
校院名稱:國立東華大學
系所名稱:海洋生物科技研究所
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:2012
畢業學年度:100
論文頁數:76
中文關鍵詞:海洋沉積物二次代謝物海洋真菌放線菌生物活性
外文關鍵詞:marine sedimentsecondary metabolitemarine fungiactinomycetebiological activity
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海洋生態系提供了不同於陸地的生態環境,成為了尋找具潛力活性天然物的重要來源。從台灣東部(台東小港漁港、台東新蘭漁港)及南部(墾丁南灣、屏東大鵬灣)採集到八個沉積物樣本,使用三種不同的前處理方法以及三種不同的選擇性培養基以期望分離出能產出具生物活性的二次代謝物的海洋微生物。總篩菌數為223株,其中5株具有抗菌活性,4株為真菌,1株為放線菌。以16S rRNA或18S rRNA基因片段做親緣分析,結果顯示4株真菌其中1株為Hypocrea屬,其他3株為Aspergillus屬,放線菌則為Streptomyces屬。將這5株菌的粗萃物進行細胞毒殺、生物毒性及抗發炎活性測試,結果顯示有3株菌具有細胞毒殺活性,有2株菌有顯著的生物毒性,有5株菌有不錯的抗發炎活性。從5株菌中挑出活性較佳的菌株(MEA-XG2、MEA-0208-2及OX-0505-6)進行最佳生長條件試驗,結果如下:MEA-XG2培養在25-30°C、培養基碳源為1%澱粉、氮源為0.4% peptone+0.4% yeast extract、鹽度為1.8%,在培養三天後能得到最佳的抗菌活性。MEA-0208-2培養在25-30°C、培養基碳源為1%果糖、氮源為0.4% peptone+0.4% yeast extract、鹽度為1.8-3.5%,在培養七天後能得到最佳的抗菌活性。OX-0505-6培養在25°C、培養基碳源為1%麥芽糖、氮源為0.4% peptone+0.4% yeast extract、鹽度低於5.2%,在培養七天之後可得到最佳的抗菌活性。實驗結果顯示台灣東部及南部海域蘊含豐富生物活性之化合物的潛力。
The marine ecosystem provides a unique ecological environment for diverse microorganisms which could be an important source in searching of important metabolites for industry. In this study, eight marine sediment samples were collected from four different sites in southern (Dapeng Bay and Nanwan Bay in Pingtung) and eastern (Hsinlan Harbor and Siaogang Harbor in Taitung) Taiwan. They were screened for marine microorganism producing secondary metabolites with biological activity. After pretreated sediment samples with three different pretreatment methods, marine microbes were then isolated from these sediments with three selective media by agar-based culture method. A total of 223 isolates were isolated from the sediment samples. Among these isolates, four fungi and one actinomycete showed antimicrobial activity. Phylogenetic analysis of these isolates based on 16S rRNA or 18S rRNA gene sequences indicated that they belong to three genera: Streptomyces (one strain), Hypocrea (one strain) and Aspergillus (three strains). Using crude extracts from fermentation broth, three, five and two isolates were found to have antitumor, anti-inflammatory and biological toxic activity, respectively. Three promising strains with strong biological activity namely MEA-XG2, MEA-0208-2 and OX-0505-6 were selected for further studies. The optimal growth condition for MEA-XG2 was 25 to 30°C, 1% starch as carbon source, 0.4% peptone as well as 0.4% yeast extract as nitrogen source, and 1.8 to 3.5% salinity; the antimicrobial activity was the strongest after three days of cultivation. The optimal growth condition for MEA-0208-2 was 25 to 30°C, with 1% fructose as carbon source, 0.4% peptone as well as 0.4% yeast extract as nitrogen source, and 1.8 to 3.5% salinity; the antimicrobial activity was the strongest after seven days of cultivation. The optimal growth condition for OX-0505-6 was at 25°C, with 1% maltose as carbon source, 0.4% peptone as well as 0.4% yeast extract as nitrogen source, and salinity less than 5.2%; the antimicrobial activity was the strongest after seven days of cultivation. Our study demonstrates that culturable microorganisms from marine sediments in southern and eastern Taiwan have a great potential for the discovery of new biological activity compounds
摘要…………………………………………………………………………………….I
Abstract………………………………………………………………………………..II
目錄…………………………………………………………………………………..III
圖目錄…………………………………………………………………………..……VI
表目錄……………………………………………………………………………....VII
第一章緒論……………………………………………………………………...…….1
1.1前言……………………………………………………………..………..1
1.2海洋天然物………………………………………………………………1
1.3海洋微生物…………………………………………………………..…..2
1.4放線菌……………………………………………………………………2
1.5真菌………………………………………………………………………3
1.6二次代謝物……………….………………………………………..…….5
1.7天然物的生物活性篩選…….……………………………………..…….5
1.8研究目的與動機…………….…………………………………..……….6
第二章實驗材料與方法………………………………………………..…………....11
2.1藥品、儀器及培養基…………………………..…………………...….11
2.1.1藥品………………………………………………….………...….11
2.1.2實驗儀器…………………………………………………….……12
2.1.3生物活性測試菌種………………………………….……………13
2.1.4微生物培養基配製………………………………….……………13
2.2沉積物採樣及來源…………………………………………………..…17
2.3沉積物樣本前處理與沉積物所含菌培養…………………………..…17
2.4抗菌活性菌株篩選…………………………………………….……….18
2.4.1具抗菌活性菌株初步篩選(primary screening)..............................18
2.4.2抗菌活性二次篩選(secondary screening) .....................................19
2.5海水需求性測試......................................................................................19
2.6核糖體RNA基因鑑定...........................................................................19
2.6.1萃取菌種之genomic DNA.............................................................19
2.6.2 Ribosomal DNA片段PCR增幅..................................................20
2.6.3 PCR產物純化................................................................................21
2.6.4基因轉殖(cloning) ..........................................................................21
2.6.5轉殖菌落挑選與定序......................................................................22
2.6.6親緣關係分析..................................................................................23
2.7具抗菌活性菌株所產天然物萃取...........................................................24
2.8菌產天然物之生物活性(biological activity) ..........................................24
2.8.1細胞毒殺活性(cytotoxic activity) ..................................................24
2.8.2生物毒殺活性(biological toxicity) .................................................24
2.8.3抗發炎活性(anti-inflammatory activity) ........................................25
2.8.4紙錠擴散抗菌試驗(paper disk-agar diffusion assay) ....................26
2.9抗菌活性物質分泌測試...........................................................................26
2.10初步生長條件及較佳抗菌活性物質生產條件測試.............................26
2.10.1種菌液製備..................................................................................26
2.10.2菌株生長曲線(growth curve)測試..............................................27
2.10.3菌株於不同生長時間下與抗菌活性物質測試..........................27
2.10.4不同溫度對菌株生長及抗菌活性之影響..................................27
2.10.5不同鹽度對菌株生長及抗菌活性之影響..................................28
2.10.6不同碳源對菌株生長及抗菌活性之影響..................................28
2.10.7不同氮源對菌株生長及抗菌活性之影響..................................29
2.11菌種保存.................................................................................................30
2.12統計分析.................................................................................................30
第三章結果..................................................................................................................33
3.1沉積物樣本採集、菌株培養與分離.........................................................33
3.2抗菌活性菌株篩選..................................................................................33
3.2.1具抗菌活性菌株初步篩選.............................................................33
3.2.2抗菌活性二次篩選.........................................................................33
3.3海水需求性測試......................................................................................34
3.4親緣關係分析..........................................................................................34
3.5具抗菌活性菌株所產天然物萃取..........................................................34
3.6微生物天然物之生物活性測試...............................................................34
3.6.1細胞毒殺活性.................................................................................34
3.6.2生物毒殺活性.................................................................................35
3.6.3抗發炎活性.....................................................................................35
3.6.4紙錠擴散抗菌試驗.........................................................................35
3.7抗菌活性物質分泌測試..........................................................................36
3.8菌株初步生長及抗菌活性物質生產最佳條件分析..............................36
3.8.1菌株生長曲線與抗菌活性..............................................................36
3.8.2不同溫度對於菌株生長及抗菌活性之影響..................................37
3.8.3不同鹽度對菌株生長及抗菌活性之影響......................................37
3.8.4不同碳源對菌株生長及抗菌活性之影響.....................................37
3.8.5不同氮源對菌株生長及抗菌活性之影響.....................................38
3.8.6菌株最佳生長與抗菌活性物質生產條件.....................................38
第四章討論..................................................................................................................63
參考文獻......................................................................................................................69
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