臺灣博碩士論文加值系統

液化澱粉芽孢桿菌(Bacillus amyloliquefaciens)已有許多文獻指出其可產抗生物質伊枯草菌素 (iturin)、表面活性素(surfactin)與豐原素 (fengycin)的能力，用以抑制植物病原真菌或細菌的增長，進而發展成生物製劑以取代化學農藥。本研究利用高效能液相層析連接螢光偵測器法(HPLC-FLD)取代傳統使用之高效能液相層析連接紫外光偵測器法(HPLC-UVD) ，以進行液化澱粉芽孢桿菌之二次代謝物iturin A與surfactin濃度分析。因其HPLC-FLD之選檡性與靈敏度較HPLC-UVD為佳，且分析樣品的極性範圍廣，故將二次代謝物衍生化，再以HPLC-FLD分析之，將可提高化學分析法之靈敏度。
高純級鄰苯二甲醛(o-phthaldialdehyde, OPA)為一HPLC螢光標記試劑，本研究以OPA/2-ME為衍生試劑與iturin A、surfactin進行衍生化反應，以螢光偵測器(激發波長: 334 nm，散射波長: 440 nm)進行分析之。研究結果顯示，硼酸鹽緩衝液以pH為10.5時顯示較佳之偵測訊號，較能促使目標物與OPA/2-ME衍生試劑進行反應。而目標物與試劑反應3分鐘內即刻上機分析，所得之偵測訊號最佳，隨著與試劑衍生時間(10 min與30 min)愈長，所得之偵測訊號則愈弱。Bacillus amyloliquefaciens經M-1、M-2與M-3不同配方培養基培養3、5、7天，所產之二次代謝物與OPA/2ME試劑衍生反應後可產螢光訊號，surfactin與iturin A個別於4.9與8.7分鐘時先沖堤出，其中以M-1 medium培養5天之抗生物質濃度最佳，iturin A為407.62 mg/L，surfactin則為8678.42 mg/L。其螢光檢知器之偵測感度約為紫外光檢知器之2~4.5倍。理論板數N值則約為1,296~14,299，理論板數越高，表示其具較高分離能力。而選擇係數α值約為1.046~1.475，且某些波峰分離度小於1.5，其代表此分析法存在著訊號干擾，因此測試其硫酸銨，作為配方之無機氮源，因結構之立體障礙小，且可使蛋白質沉澱，進而能降低螢光偵測器之訊號干擾。本研究雖可提高化學分析法之靈敏度，但尚須克服衍生物螢光強度不夠，以及干擾過多等現象。

目錄
中文摘要 III
英文摘要 V
壹、前言………………………………………………………………….1
貳、文獻回顧 3
2-1 液化澱粉芽孢桿菌( Bacillus amyloliquefaciens )簡介 3
2-1-1液化澱粉芽孢桿菌( Bacillus amyloliquefaciens )之生理特性 3
2-2 二次代謝產物－細菌性環脂胜肽 4
2-2-1 伊枯草菌素(Iturin) 5
2-2-2 表面素(Surfactin) 5
2-3 螢光衍生 6
2-3-1 衍生試劑 7
2-3-1-1鄰苯二甲醛(o-phthalaldehyde, OPA) 9
2-3-2 衍生試劑最適波長探討 9
2-3-3 螢光衍生化方法 10
2-4 影響樣品衍生反應之因素 15
2-4-1 樣品衍生反應時間探討 15
2-4-2樣品衍生反應溫度探討 16
2-4-3試劑緩衝溶液及其酸鹼值 16
2-4-4流動相溶劑之選擇 16
2-5偵測器之干擾 17
2-5-1添加硫酸銨對偵測器干擾之影響 17
2-6 檢知器之選擇性 18
參、材料與方法 19
3-1 實驗材料與器材 19
3-1-1 實驗儀器設備 19
3-1-2 實驗藥品 20
3-1-3 實驗菌株來源 21
3-2 實驗方法 21
3-2-1 菌體保存與培養方法 21
3-2-2 實驗中使用之培養基 22
3-3 液態發酵 24
3-3-1 發酵液之搖瓶培養 25
3-3-2 樣品取樣 25
3-4 二次代謝物萃取 26
3-4-1發酵液之二次代謝物萃取 26
3-4-2 代謝萃取物之HPLC分析 26
3-5衍生試劑之配製 27
3-5-1 硼酸鹽緩衝液(Borate buffer)之配製 27
3-5-2 OPA/2-ME 螢光衍生試劑之配製 27
3-6 添加硫酸銨對偵測器干擾之影響 27
3-7 波峰理論板數(N值)之計算 27
3-8 選擇係數(a值)之計算 28
3-9 波峰分離度之計算 28
3-10 HPLC-FLD之層析條件 29
肆、結果 30
4-1 OPA/2-ME衍生試劑對Iturin A與surfactin標準品分析之影響
…………………………………………………………………30
4-2硼酸鹽緩衝液之pH值對OPA/2-ME衍生試劑之影響 30
4-3不同反應時間對OPA/2-ME衍生試劑之影響 33
4-4不同反應溫度對OPA/2-ME衍生試劑之影響 33
4-5不同配方培養基對HPLC-FLD偵測訊號之影響 34
4-6不同菌種於不同配方培養基中所產之二次代謝產物對其HPLC偵測訊號之影響 34
4-6-1 液化澱粉芽孢桿菌A1 (Bacillus amyloliquefaciens A1) 35
4-6-1-1於M-1 培養基中經不同天數培養對其HPLC偵測訊號之影響 35
4-6-1-2於M-2 培養基中經不同天數培養對其HPLC偵測訊號之影響 35
4-6-1-3於M-3 培養基中經不同天數培養對其HPLC偵測訊號之影響 36
4-6-2 液化澱粉芽孢桿菌F1 (Bacillus amyloliquefaciens F1) 37
4-6-2-1於M-1 培養基中經不同天數培養對其HPLC偵測訊號之影響 37
4-6-2-2於M-2 培養基中經不同天數培養對其HPLC偵測訊號之影響 37
4-6-2-3於M-3 培養基中經不同天數培養對其HPLC偵測訊號之影響 38
4-7不同檢知器對Iturin A與Surfactin之偵測感度(peak area)之影響 38
4-7-1液化澱粉芽孢桿菌A1 (Bacillus amyloliquefaciens A1) 38
4-7-2液化澱粉芽孢桿菌F1 (Bacillus amyloliquefaciens F1) 39
4-8不同檢知器對Iturin A與Surfactin濃度之影響 40
4-8-1液化澱粉芽孢桿菌A1 (Bacillus amyloliquefaciens A1) 40
4-8-2液化澱粉芽孢桿菌F1 (Bacillus amyloliquefaciens F1) 41
4-9螢光檢知器之選擇性、理論板數與波峰分離度 42
伍、討論 45
5-1 OPA/2-ME衍生試劑對標準品分析之影響…………………..45
5-2影響樣品衍生反應之因素 45
5-2-1硼酸鹽緩衝液之pH值對OPA/2-ME衍生試劑之影響 46
5-2-2樣品衍生反應時間之探討 47
5-2-3樣品衍生反應溫度探討 47
5-3不同氮源培養基對HPLC-FLD偵測訊號之影響 48
5-4不同檢知器對二次代謝物之偵測感度(peak area)之影響 49
5-5不同檢知器對二次代謝物濃度之影響 50
5-6螢光檢知器之選擇性、理論板數與波峰分離度之探討 50
陸、結論 52
柒、參考文獻 54
捌、圖表 60
玖、附錄 98

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