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研究生:董靜馨
研究生(外文):Ching-Hsin Tung
論文名稱:金針菇冷水萃取物中金針菇溶血蛋白質與真菌免疫調節蛋白的熱安定性差異
論文名稱(外文):Study of thermal stability difference between flammutoxin (FTX) and fungal immunomodulatory protein-fve (FIP-fve) in cold-water extracts of Flammulina velutipes
指導教授:呂廷璋
指導教授(外文):Ting-Jang Lu
口試委員:陳頌方許輔何國榮陳宏彰
口試委員(外文):Sung-Fang ChenFuu SheuHer, Guor-RongHong-Jhang Chen
口試日期:2018-06-19
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:食品科技研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:141
中文關鍵詞:金針菇子實體真菌免疫調節蛋白金針菇溶血蛋白質HPLC-UV-ESI-MS蛋白質的穩定性
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真菌免疫調節蛋白 (FIP-fve) 是一個具有潛在發展功能性食品的成分。然而,在 FIP-fve 的萃取物中不希望出現金針菇溶血蛋白質 (flammutoxin FTX),本研究應用加熱處理代替強化分離過程來分離 FIP-fve,同時排除了FTX。本實驗開發以 on-line desalting HPLC-UV-ESI-MS 方法,監測金針菇子實體中這兩種蛋白質熱穩定性的情形。以 molecular weight cut-off centrifugal filtration 和 on-line desalting HPLC-UV-ESI-MS 的方法簡化前處理過程並有效的移除鹽類,在移動相中添加 0.1% 三氟醋酸配合孔徑300 Å 逆相層析管柱可達到好的分離效果。在 HPLC-UV-ESI-MS 系統,使用 bovine serum albumin 作為標準蛋白質在 UV 280 nm 下進行檢測,100g 新鮮子實體可獲得 11 mg FIP-fve,質譜儀結果得到 FIP-fve 分子量為 12.74 kDa.,flammutoxin 分子量為 21.91 kDa.。在熱安定實驗結果顯示金針菇子實體加熱後,真菌免疫調節蛋白可能與其他成分作用,所以無法萃取出來。金針菇子實體以低溫萃取的蛋白質溶液進行耐熱試驗,顯示在較低濃度的溶液中,FIP-fve 和 FTX 都具有較高的熱穩定性,加入 0.1 M 海藻糖不會顯著改變兩種蛋白質的穩定性,加入 20% 乙醇結果顯示,兩種蛋白質對乙醇的耐受性是差的。含有 580μg/mL FIP-fve 和 452 μg/mL FTX 的冷水萃取物在 60℃ 下加熱 5 分鐘,可以有效地排除 FTX,並保持 75% 的 FIP-fve。
Fungal immunomodulatory protein (FIP-fve) is a potential functional food ingredient. However, undesirable component flammutoxin (FTX) would occur in the extracted fraction of FIP-fve. In this paper, an application of heating processing instead of the intensive separation process was employed in fractionation of FIP-fve, meanwhile, exclusion of FTX was reached. A rapid analytical approach, on-line desalting HPLC-UV-ESI-MS method, for the analysis of FIP-fve and flammutoxin (FTX), two important bioactive proteins in the fruiting bodies of Flammulina velutipes, was developed. In this study, a highly efficient desalting method is provided using molecular weight cut-off centrifugal filtration and on-line desalting. Results indicated that using trifluoroacetic acid as a modifier on a 300 Å reversed-phase column renders effective separation. ESI-MS revealed that the apparent molecular masses of FIP-fve and FTX were 12.74 kDa and 21.91 kDa, respectively. Eleven milligrams of FIP-fve was obtained from 100 g of fresh fruiting bodies, and UV detection was performed at 280 nm using bovine serum albumin as the standard protein. The results of the heat stability test show that fungal immunomodulatory proteins cannot be extracted after the mushroom body is heated. Both FIP-fve and FTX had higher thermal stability in a lower concentration solution using a low-temperature extraction. Adding 0.1 M trehalose or 20% ethanol did not significantly alter the stability of both proteins. Heating cold water extract contained 580 μg/mL FIP-fve and 452 μg/mL FTX at 60℃ for 5 minutes could effectively exclude FTX and remain 75% of FIP-fve.
目 錄
致謝 2
中文摘要 3
Abstract 4
目錄 5
圖目錄 8
表目錄 12
前言 13
壹、文獻整理 14
1. 金針菇生理活性成分: 14
1.1 蛋白質 14
1.1.1 真菌免疫調節蛋白 14
1.1.2 真菌免疫調節蛋白的分子生物學研究 25
1.1.3 溶血蛋白(hemolysin) 27
1.2 多醣 32
1.2.1 β-D-葡聚糖 32
1.2.2 金針菇多醣類 34
1.3 Ergothioneine 35
2. 蛋白質分析技術: 36
2.1 蛋白質純化 36
2.2 蛋白質定量分析 37
2.2.1 Biuret Method 37
2.2.2 Lowry Method 38
2.2.3 UV Absorbance 39
2.2.4 Bradford Method(Comassie Brilliant Blue G-250) 40
2.2.5 其他方法 40
2.3 利用質譜來鑑定及定序蛋白質 41
2.3.1 一般質譜掃描(MS scan) 41
2.3.2 串聯式質譜掃描(MS/MS scan 或 MS2 scan) 46
2.3.2.1 Bottom-up 48
2.3.2.2 Top-down 51
2.4 On-line LC–MS 去塩系統 58
3. 蛋白質的加工安定性之探討 60
3.1 熱處理應用 62
3.2 有機溶劑應用--乙醇 63
3.3 保護劑-海藻糖應用 65
3.3.1冷凍保護劑(cryoprotectants) 66
3.3.2生物保護(bioprotection) 66
貳、材料與方法 69
1. 實驗材料: 69
2. 化學試藥、器材及實驗方法 69
2.1.檢體前處理 69
2.1.1試藥: 69
2.1.2 實驗操作: 69
2.1.2. 子實體中蛋白質的耐熱試驗 69
2.1.2.2金針菇蛋白質區分物的耐熱試驗: 69
2.1.2.3金針菇蛋白質冷水萃取法: 69
2.1.2.4金針菇冷水粗萃液熱安定性試驗: 70
2.2. SDS膠體電泳分析 74
2.2.1試藥: 74
2.2.2 實驗操作: 74
2.3. 一般質譜掃描(MS scan)LC-UV-ESI/MS 分析 79
2.3.1試藥: 79
2.3.2 儀器及設備: 79
2.3.3 高效液相層析儀條件: 79
2.3.4質譜儀條件: 80
2.3.5 On-line desalting LC–MS系統: 81
2.3.6標準曲線的製作: 82
2.3.7鑑別試驗及含量測定: 82
2.4、Bottom-up LC- ESI/MS 分析 83
2.4.1 儀器及設備: 83
2.4.2 Digestion 83
2.4.3 In-gel digestion: 84
2.4.4高效液相層析儀條件: 84
2.4.5 質譜儀條件: 84
參、結果 86
1. 金針菇樣品 FIP-fve 與 FTX 分析平台的優化 86
1.1 HPLC 檢驗條件的優化 86
1.2. LC-UV vs. LC/MS Data 86
1.2.1 FIP-fve 86
1.2.1.1. LC-UV-ESI/MS 86
1.2.1.2. Bottom-up LC- ESI/MS 87
1.2.1.3 FIP-fve 的定量 87
1.2.2 Flammutoxin 87
1.2.2.1. LC-UV-ESI/MS 87
1.2.2.2. Bottom-up LC- ESI/MS 88
1.2.2.3. Flammutoxin 的定量 89
2. 子實體受熱後 FIP-fve 與 FTX 之萃取率差異 89
3. 萃取液中 FIP-fve 與 FTX 熱穩定性試驗 90
4. FIP-fve 和 FTX 在水萃液中耐熱試驗 91
肆、討論 93
1. 金針菇樣品 FIP-fve 與 FTX 分析平台的優化 93
2. FIP-fve 與 FTX 熱安定性差異的應用 95
伍、結論 97
文獻參考 99
縮寫 115
圖次 116
表次 132
附錄 133
文獻參考
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