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系統識別號 U0026-0108201915112700
論文名稱(中文) 三種藍綠菌在不同生長階段中產生BMAA濃度之測定
論文名稱(英文) Determination of BMAA in Three Cyanobacterial Strains Under Different Growth Phases
校院名稱 成功大學
系所名稱(中) 環境工程學系
系所名稱(英) Department of Environmental Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 蔣寶玉
研究生(英文) Deborah Kezia Yudisaputra
學號 P56067029
學位類別 碩士
語文別 英文
論文頁數 99頁
口試委員 指導教授-林財富
口試委員-黃良銘
口試委員-周佩欣
口試委員-陳女菀如
中文關鍵字 β-甲胺基-L-丙胺酸(BMAA)  2,4-二胺基丁酸(DAB)  微囊藻  魚腥藻  柱孢藻  生長期  液相層析串聯質譜(LC-MS / MS) 
英文關鍵字 β-methylamino-L-alanine (BMAA)  2,4-diaminobutyric acid (DAB)  Microcystis aeruginosa  Anabaena circninalis  Cylindrospermopsis raciborskii  growth phase  Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) 
學科別分類
中文摘要 BMAA(β-甲基胺基-L-丙氨酸)及其同分異物DAB(2,4-二胺基丁酸),均是由藍細菌產生、與疾病相關的非蛋白質胺基酸。本研究擬建立藻體內BMAA及DAB分析方法,以確定藍綠菌細胞中的總、游離、可溶及與蛋白質結合態,及細胞外水溶性的BMAA濃度。細胞內BMAA係以蛋白質沉澱法、細胞外則以固相萃取(SPE)法進行前處理。樣品分析則以酰胺柱通過親水相互作用液相層析(HILIC)分離、並使用氘標記的內標(d3-BMAA),通過串聯質譜儀(MS / MS)進行檢測。 研究中探討三種藍綠菌菌株,包括銅綠微囊藻,捲曲魚腥藻,及柱孢藻,在不同生長階段下BMAA的進行BMAA濃度分析。研究結果顯示,在不同生長階段的藍綠菌實驗室培養菌株和環境樣品中,均能檢測到細胞內BMAA,濃度分別為3.76-16.64μg/g和9.66-14.45 μg/g;細胞外濃度則在0.0103-0.0446 μg/L間。此外,本研究亦確認DAB存在於一些藍綠菌樣品中。
英文摘要 BMAA (β-methylamino-L-alanine) and its most common structural isomer DAB (2,4- diaminobutyric acid) produced by cyanobacteria, are non-proteinogenic amino acids that have been associated with neurodegenerative diseases. Protein precipitation method was developed to determine the intracellular BMAA concentration in total, free, soluble and protein-bound BMAA fraction remained in cyanobacterial cells, while solid phase extraction (SPE) method was carried out to determine the extracellular free BMAA fraction remained in the supernatant. This method is based on direct analysis of the underivatized molecule, using an amide column for separation by Hydrophilic Interaction Liquid Chromatography (HILIC) and detection by tandem mass spectrometry (MS/MS) using a deuterium labeled internal standard (d3-BMAA). BMAA extraction was carried out in three cyanobacterial strains which are Microcystis aeruginosa PCC7820, Anabaena circinalis and Cylindrospermopsis raciborskii under different growth phases. Intracellular BMAA was detected in both cyanobacterial laboratory culture and environmental samples under different growth phases in the range concentration of 3.76-16.64 µg/g and 9.66-14.45 µg/g, respectively. While extracellular BMAA was detected in the range concentration of 0.0103-0.0446 µg/L. DAB was confirmed to be present in some of the cyanobacterial samples.
論文目次 Abstract I
摘要 II
Acknowledgement III
Table of Contents V
List of Figures VIII
List of Tables XI
Chapter I–Introduction 1
1.1 Background 1
1.2 Research Objectives 2
Chapter II–Literature Review 3
2.1 Cyanobacteria and Cyanotoxins 3
2.2 Growth Phases of Cyanobacteria 6
2.3 β-methylamino-L-alanine (BMAA) 8
2.3.1 Structure and Properties 8
2.3.2 BMAA Fractions 11
2.3.3 BMAA and Its Isomers 12
2.3.4 Occurrence of BMAA in Cyanobacteria 13
2.3.5 Occurrence of BMAA in Aquatic Environments 16
2.4 Extraction Methods for BMAA 17
2.4.1 Protein Precipitation 17
2.4.2 Solid Phase Extraction (SPE) 18
2.5 Analytical Methods for the Determination of BMAA in Cyanobacteria 19
2.5.1 Hydrophilic Interaction Chromatography (HILIC) 21
2.5.2 Liquid Chromatography Tandem Mass Spectrometer (LC-MS/MS) 23
Chapter III–Materials and Methods 27
3.1 Experimental Scope 27
3.2 Cyanobacteria Culturing 28
3.2.1 Preparation of Artificial Sputum Medium (ASM) and Jaworski’s Medium (JM) 28
3.3.1 Sedgewick-Rafter counting cell 31
3.3.2 Microcystis aeruginosa and Anabaena circinalis cell enumeration 32
3.3.3 Cylindrospermopsis raciborskii cell enumeration 32
3.4 Sample Preparation 33
3.5. Extraction Methods 34
3.5.1 Protein Precipitation 34
3.5.2 Solid Phase Extraction (SPE) 39
3.6 Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) 42
3.6.1 Liquid Chromatography Condition 42
3.6.2 Mass Spectrometric Condition 43
3.6.3 Reagents and Equipments 45
3.7 BMAA and DAB Quantification 45
Chapter IV – Results and Discussion 47
4.1 Extraction Protocol Adjustment 47
4.1.1 Initial Protein Precipitation Protocol 47
4.1.2 Initial Solid Phase Extraction (SPE) Protocol 51
4.2 Preliminary Study 56
4.2.1 Cell Enumeration 56
4.2.2 Trial of Extraction 59
4.2.3 Hydrolysis Process Confirmation on the Recovery of d3-BMAA and BMAA 62
4.2.4 Effect of Different Volume of HCl on the Recovery of d3-BMAA and BMAA 63
4.3 BMAA Extraction 65
4.3.1 Lag Phase 66
4.3.2 Exponential Phase 70
4.3.3 Stationary Phase 74
4.5 Environmental Sample 83
4.6 Comparison Between Cyanobacterial Laboratory Culture and Environmental Sample 88
Chapter V–Conclusions 91
5.1 Conclusions 91
5.2 Suggestions 92
References 93
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