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研究生:陳蕙筠
研究生(外文):Chen Hui Yun
論文名稱:以生化質譜法分析青黴菌和麴菌孢子
論文名稱(外文):Characterization of Penicillium and Aspergillus Spores by Using Biological Mass Spectrometry
指導教授:陳月枝陳月枝引用關係
指導教授(外文):Chen Yu Chie
學位類別:碩士
校院名稱:國立交通大學
系所名稱:應用化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
畢業學年度:92
語文別:中文
論文頁數:151
中文關鍵詞:基質輔助雷射脫附游離質譜法電噴灑游離質譜法青黴菌麴菌孢子完整的孢子
外文關鍵詞:MALDI-MSESI-MSPenicilliumAspergillusSporesWhole spores
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MALDI-MS及ESI-MS這兩種生化質譜法已被廣泛用於生化分子的分析。而在針對微生物的分析方面,不同的菌株經生化質譜法分析所得到的指紋質譜圖及生物標幟離子可以用以區分不同的菌種,因此如能建立微生物的指紋質譜圖及資料庫,可當作微生物菌種鑑定的參考。本論文以一般常污染穀物、水果或會產生赭麴毒素的七株青黴菌和四株麴菌孢子做為研究物種:包括Penicillium citrinum、 P. chrysogenum、P. digitatum、P. pinophilum、P. italicum、P. brevicompactum、P. expansum、Aspergillus niger、A. awamori、A. sulphureus及A. ochraceus等,利用MALDI-MS及ESI-MS建立可代表青黴菌及麴菌的生物標幟離子峰及指紋質譜圖。
由實驗結果可知,利用已配製在體積比為2:1混合而成之乙�姣P1% 三氟醋酸水溶液的2,5-二羥基苯甲酸或3,5-二甲氧基-4-羥基桂皮酸為基質,混以完整孢子細胞以進行MALDI-MS分析時,可得到再現性極佳的質譜圖。利用MALDI-MS於實際樣品的分析,再將結果比對已建立的孢子生物標幟離子可快速確認菌株種類,例如直接刮取腐敗水果上之真菌的孢子與基質混合後進行分析,所得之質譜圖中可觀察到重覆性極高的離子訊號出現在m/z 2600及m/z 5211,與本論文所得之標準菌株質譜圖相比對之下,可得知導致該水果腐敗之真菌為P. digitatum。
而利用ESI-MS進行分析時,由於無法得到高質量(>1000 Da)的生化分子之離子訊號,因此從實驗結果雖可看出不同屬種之青黴菌及麴菌的ESI質譜圖有些微差異,但仍不足以區分菌株的種類。
中文摘要…………………………………………………………………….. Ⅰ
英文摘要……………………………………………………………………... Ⅱ
謝誌………………………………………………………………………….. Ⅲ
目錄………………………………………………………………………….. Ⅳ
圖表目錄……………………………………………………………………... Ⅷ
壹、緒論…………………………………………………………………….. 1
一、前言………………………………………………………………….. 1
二、質譜法應用在微生物分析之背景介紹…………………………….. 4
2-1質譜法應用在菌種分析之發展…………………………………… 4
2-1-1以氣相層析質譜法進行微生物之研究………………………
…… 4
2-1-2以FAB-MS應用於微生物之研究……………………………
……… 6
2-1-3以MALDI-MS應用於微生物之研究…………………………
…. 7
2-1-4以 ESI-MS應用於微生物的研究……………………………
………………….. 8
三、MALDI -MS的簡介…………………………………………………
9
3-1基質的特性與功能-輔助分析物的脫附游離………………… 9
3-2 MALDI質譜中分析物離子峰的特徵………………………… 11
3-3時間飛行質量分析儀(Time-Of-Flight)的原理………………… 13
3-4 MALDI-TOFMS的優點………………………………………… 15
四、ESI-Ion TrapMS的原理…………………………………………… 16
4-1 ESI游離的機制與原理…………………………………………… 16
4-2 ESI質譜中分析物離子峰的特徵………………………………… 19
4-3 離子阱質量分析儀(Ion Trap)的原理…………………………… 20
五、真菌背景介紹…………………………………………………………
21
5-1真菌的分類法………………………………………………………
21
5-1-1真菌的生長型態………………………………………………
21
5-1-2真菌的生殖方式………………………………………………
22
5-1-3真菌的分類……………………………………………………
24
5-2真菌生物性質與組成成分…………………………………………
25
5-3實驗室鑑定真菌的方法……………………………………………
26
5-4青黴菌與麴菌的特性介紹…………………………………………
26
5-4-1青黴菌對環境及人體的影響…………………………………
26
5-4-2麴菌對環境及人體的影響……………………………………
30
六、論文目標………………………………………………………………
33
貳、實驗………………………………………………………………………
35
一、實驗材料………………………………………………………………
35
1-1實驗藥品與儀器……………………………………………………
35
1-1-1實驗藥品………………………………………………………
35
1-1-2實驗儀器………………………………………………………
36
1-2菌種培養……………………………………………………………
36
1-2-1菌種來源………………………………………………………
36
1-2-2培養菌種所需之材料…………………………………………
38
1-2-3培養基之製備………………………………………………… 39
1-2-4菌種培養………………………………………………………
39
二、以MALDI-MS進行真菌之分析……………………………………
40
2-1樣品的製備方法……………………………………………………
40
2-1-1基質的配製. …………………………………………………
40
2-1-2樣品的處理…………………………………………………… 41
2-2實際樣品的鑑定分析…………………………………………… 41
2-2-1水果與果汁的樣品……………………………………………
41
2-3利用PSD為分析標準菌株與污染源菌株的方法…………………
41
2-4偵測極限之探討……………………………………………………
42
2-4-1孢子在去離子水中之偵測極限………………………………
42
2-4-2孢子在果汁中之偵測極限……………………………………
43
2-4-3孢子在咖啡豆中之偵測極限…………………………………
44
三、以ESI-Ion Trap進行真菌之分析……………………………………
45
3-1青黴菌孢子的前處理方法…………………………………………
45
3-2樣品在ESI分析之條件……………………………………………
45
參、結果與討論………………………………………………………………
46
一、建立青黴菌及麴菌孢子在MALDI質譜圖中之生物標幟離子……
46
1-1 MALDI基質的選擇………………………………………………
46
1-2 溶解MALDI基質溶劑選擇………………………………………
47
1-3培養基的選擇………………………………………………………
48
1-4 探討真菌培養天數對分析結果的影響…………………………
54
1-5探討青黴菌及麴菌亞種孢子在MALDI質譜圖中之生物標幟離
子之差異……………………………………………………………
差異


55
1-6應用於實際樣品的分析……………………………………………
99
1-7利用PSD(Post Source Decay)鑑定菌種……………………………
102
1-8偵測極限的探討……………………………………………………
104
1-8-1孢子在去離子水中之偵測極限………………………………
104
1-8-2孢子在果汁中之偵測極限……………………………………
117
1-8-3孢子在咖啡豆中之偵測極限…………………………………
123
二、以ESI-MS建立青黴菌孢子及麴菌之生物標幟離子………………
126
2-1以不同萃取溶液處理孢子對分析結果的影響……………………
126
2-2 ESI溶劑對分析結果的影響………………………………………
128
2-3不同屬種之青黴菌與麴菌在ESI質譜圖中的差異………………
128
肆、結論………………………………………………………………………
135
一、以MALDI-MS分析青黴菌及麴菌孢子……………………………
135
1-1最佳分析條件………………………………………………………
135
1-2代表青黴菌及麴菌孢子的生物標幟峰離子………………………
135
1-3分析實際樣品的結果………………………………………………
136
1-4 PSD的分析結果……………………………………………………
136
1-5偵測極限……………………………………………………………
137
二、以ESI-Ion Trap分析青黴菌及麴菌孢子……………………………
138
2-1最佳分析條件………………………………………………………
138
2-2不同種之青黴菌及麴菌孢子的ESI質譜圖………………………
138
伍、參考文獻…………………………………………………………………
139
陸、附錄……………………………………………………………………… 151
一、菌種保存的方法………………………………………………………
151
1-1保存菌種所需材料…………………………………………………
151
1-2保存步驟……………………………………………………………
151
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