臺灣博碩士論文加值系統

赭麴毒素A (Ochratoxin A，OTA)由真菌中麴黴菌屬(Aspergillus sp.) 和青黴菌屬(Penicillium sp.) 菌株產生的次級代謝產物，廣泛污染糧食和飼料。赭麴毒素A具有很強的腎臟和肝臟毒性，並有致畸、致突變、免疫抑制和致癌等作用，對動物和人體健康有很大的潛在危害。本研究利用免疫親合管柱結合液相層析串聯式質譜儀，針對自2012年7月至2014年9月間亞洲八個地區229件之穀物及飼料樣品中之赭麴毒素A進行定量檢測，結果顯示平均含量為5 ng/mL，最高含量96 ng/mL，總檢出率達31 %。根據此文進一步分析區域污染分佈及赭麴毒素A與其他黴菌毒素共發性。本研究利用蒙托土及羅勒籽和奇亞籽作為赭麴毒素A吸附除污之資材，體外模擬禽畜動物腸胃酸性環境下比較二者吸附赭麴毒素A之吸附效能。結果顯示，當蒙托土與赭麴毒素A重量比超過3105其吸附成效可達90%，羅勒籽和奇亞籽重量比必需超過6106吸附率方能達90%。據此結果將討論二項天然資材進行毒素吸附之機制。

Ochratoxin A (OTA) is a mycotoxin mainly produced as a secondary metabolite by fungi of the Aspergillus (Aspergillus sp.) and Penicillium (Penicillium sp.) species. It contaminates food and feed and has strong kidney and liver toxicity. It's teratogenic, mutagenic, and immunosuppressive and carcinogenic effects are potential hazards to animal and human health. In this study, we applied by immunoaffinity column integrated with high performance liquid chromatography tandem mass (LC/MS/MS) to analyse. The OTA quantitatively in 229 cereal and feed samples, which are collected from eight regions of Asia during July 2012 to September 2014. The results showed that the average level of OTA in cereal samples was 5 ng/mL, the maximum value was 96 ng/mL, and the occurrence is 31%. The difference of OTA contamination level among these regions and the co-occurrence of OTA with other toxins further discussed. Meanwhile we used montmorillonite and chia seed and basil seed as decontamination materials. The efficiency of OTA adsorption by montmorillonite and chia seed and basil seed was compared under the environment, simulate the livestock animal stomach for acidity in vitro. The results showed that when the weight ratio of montmorillonite to OTA over 3x105, decontamination efficiency achieved 90%; however, the weight ration of basil seed and chia seed to OTA needs to be over 6x106 to approach 90% adsorption of OTA. Based on the results, the mechanism of toxin adsorption by these two decontamination materials is discussed.

摘要 I
Abstract II
誌謝 III
目錄 V
表目錄 VI
圖目錄 VIII
第壹章 緒論 1
1.1 前言 1
1.2 研究動機 3
第貳章 文獻探討 4
2.1 黴菌毒素 4
2.1.1 赭麴毒素A介紹 5
2.1.2 赭麴毒素A毒性及毒理 6
2.1.3 赭麴毒素A 對禽畜動物的影響 8
2.1.4 赭麴毒素A在各國最大殘留量與分佈情形及相關規範 9
2.2 黴菌毒素除污方式介紹 11
2.2.1 物理方法 12
2.2.2 化學方法 13
2.2.3 生物方法 14
2.3 黴菌毒素吸附劑-黏土介紹 16
2.3.1 蒙托土介紹 17
2.3.2 奇亞籽與羅勒籽介紹 18
2.4 赭麴毒素A 檢驗方法 19
2.4.1高效能液相層析儀(HPLC) 20
2.4.2 液相層析串聯式質譜儀 (LC/MS/MS) 23
2.5 免疫親合管柱 (Immunoaffinity column) 25
第參章 材料與實驗方法 43
3.1 實驗材料 43
3.1.1 實驗藥品 43
3.1.2 實驗器材 44
3.1.3 實驗設備 44
3.1.4 實驗樣品來源 45
3.2 儀器條件 46
3.2.1 液相層析串聯式質譜儀 (LC/MS/MS) 46
3.2.2 高效能液相層析法-螢光偵測器(HPLC-FLD) 47
3.3 試劑配製 48
3.3.1 液相層析串聯式質譜儀 (LC/MS/MS) 48
3.3.2 高效能液相層析法-螢光偵測器(HPLC-FLD) 49
3.4 實驗步驟 51
3.4.1 赭麴毒素A-萃取前處理 51
3.4.2 吸附劑-吸附赭麴毒素A實驗 52
第肆章 結果與討論 62
4.1 儀器確效 62
4.1.1 液相層析串聯式質譜儀 (LC/MS/MS)確效 63
4.1.1.1 線性(Linearity) 63
4.1.1.2 基質效應(Matrix effect) 63
4.1.1.3 偵測極限(LOD)與定量極限(LOQ) 65
4.1.1.4 準確度(Accuracy)度與精密度(Precision) 65
4.1.1.5 量測不確定度(Uncertainty)分析 66
4.1.1.6 亞洲地區污染分佈情形及程度 71
4.1.1.7 赭麴毒素 A 與其他黴菌毒素共發之相關性 72
4.1.2 高效能液相層析法-螢光偵測器HPLC-FLD確效 74
4.1.2.1 線性(Linearity) 74
4.1.2.2 偵測極限(LOD)與定量極限(LOQ) 74
4.1.2.3 準確度(Accuracy)度與精密度(Precision) 76
4.1.2.4 量測不確定度(Uncertainty)分析 76
4.1.2.5 吸附實驗 81
4.1.2.6 吸附實驗優化 81
4.1.2.7 吸附實驗成效 82
第伍章 結論 116
參考文獻 118

表目錄
表1、常見主要黴菌屬及黴菌毒素對照表 26
表2、赭麴毒素(Ochratoxin A)對不同動物之LD50值 27
表3、各國對於各類食品中赭麴毒素A(OchratoxinA)之最大殘留量 28
表4、以HPLC-FLD分析赭麴毒素A(OchratoxinA)之使用條件一覽表 29
表5、VICAM不同黴菌毒素之免疫親合管柱載體容量(Capacity) 30
表6、液相層析串聯式質譜儀-液相層析條件設定 54
表7、液相層析串聯式質譜儀-液相層析梯度沖提設定 54
表8、液相層析串聯式質譜儀-離子源條件設定 54
表9、赭麴毒素A之質譜多重反應偵測模式參數 54
表10、液相層析串聯式質譜儀-相對離子強度 55
表11、高效能液相層析法-螢光偵測器條件設定 56
表12、基質效應之影響情形 84
表13、LC/MS/MS分析赭麴毒素A之偵測極限與定量極限評估資料 85
表14、LC/MS/MS檢測-赭麴毒素A之精密度、準確度評估 86
表15、LC/MS/MS檢測-赭麴毒素A之計算其平均濃度之標準偏差SD值 87
表16、LC/MS/MS檢測-赭麴毒素A之查核樣品回收率之不確定度評估 88
表17、LC/MS/MS檢測-赭麴毒素A之添加樣品之不確定度評估 89
表18、赭麴毒素A檢量線之不確定度評估 90
表19、赭麴毒素A檢量線之不確定度評估計算公式 91
表20、黴菌毒素相關性分析 92
表21、HPLC-FLD分析赭麴毒素A之偵測極限與定量極限評估資料 94
表22、HPLC-FLD檢測-赭麴毒素A之精密、準確度評估 95
表23、HPLC-FLD檢測-赭麴毒素A之計算其平均濃度之標準偏差SD值 96
表24、HPLC-FLD檢測-赭麴毒素A之查核樣品回收率之不確定度評估 97
表25、HPLC-FLD檢測-赭麴毒素A之添加樣品之不確定度評估 98
表26、赭麴毒素A檢量線之不確定度評估 99
表27、赭麴毒素A檢量線之不確定度評估計算公式 100


圖目錄

圖1、赭麴毒素之化學結構異構物。 31
圖2、赭麴毒素α(Ochratoxinα，OTα)之化學結構異構物。 32
圖3、赭麴毒素A致突變性頻率與人體酵素P450的關係 33
圖4、蒙托土結構模型示意圖 34
圖5、蒙托土在各種工業上的運用 35
圖6、高效液相層析儀基本結構示意圖 36
圖7、正相層析法和逆相層析法分與移動相極性強弱之分離時間之關係圖 37
圖8、HPLC-FLD檢測紅葡萄酒中OTA 光譜圖 38
圖9、5 ng /mL OTA之HPLC-FLD光譜圖 39
圖10、電噴灑游離法(Electrospray Ionization, ESI+)示意圖 40
圖11、串聯式三段四極桿(Triple quadruple, QQQ)示意圖 41
圖12、VICAM免疫親合管柱作用機制 42
圖13、50 ng/mg赭麴毒素A之LC/MS/MS質譜圖 57
圖14、基質不需過親合管柱純化之流程圖 58
圖15、基質需過親合管柱純化之流程圖 59
圖16、赭麴毒素A-萃取前處理流程圖 60
圖17、吸附劑-吸附赭麴毒素A實驗流程圖 61
圖18、LC/MS/MS分析赭麴毒素A之標準檢量線 101
圖19、標準品之檢量線與基質批配檢量線 102
圖20、量測不確定度來源支鏈圖 103
圖21、赭麴毒素 A在亞洲地區污染情形 104
圖22、各國污染分佈情形及程度 105
圖23、赭麴毒素A吸收波長測試 106
圖24、HPLC-UV與HPLC-FLD背景訊號(Background)光譜圖 107
圖25、赭麴毒素 A 100 ng/mL標準品之HPLC-UV與HPLC-FLD光譜圖 108
圖26、HPLC-FLD分析赭麴毒素A之標準檢量線標準檢量線 109
圖27、四種吸附劑資材吸附成效比較 110
圖28、吸附劑製備過程優化 111
圖29、反應時間過程優化 112
圖30、離心優化 113
圖31、蒙托土Calibrin-Z、Calibrin-A吸附劑資材之吸附成效 114
圖32、羅勒籽和奇亞籽之吸附劑資材之吸附成效 115

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