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

Abstract

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.