金頂側耳麥角硫因微波及超音波輔助萃取最適化條件探討及其抗氧化性質與純化

THUIR > College of Agriculture > Department of Food Science > Master's Theses > Item 310901/31400

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Title:	金頂側耳麥角硫因微波及超音波輔助萃取最適化條件探討及其抗氧化性質與純化
Other Titles:	Optimization of microwave and ultrasound assisted extraction of ergothioneine from Pleurotus citrinopileatus and their antioxidant activities and purification
Authors:	呂仲雯 LU, ZHONG-WEN
Contributors:	梁志弘 LIANG,CHIH-HUNG 食品科學系
Keywords:	金頂側耳;麥角硫因;最適化;抗氧化;微波輔助萃取;反應曲面法 Pleurotus citrinopileatus;Ergothioneine;Optimization;Antioxidant activities;Microwave-assisted extraction;Response surface methodology
Date:	2019
Issue Date:	2019-03-21T09:06:41Z (UTC)
Abstract:	微波輔助萃取 (Microwave-assisted extraction, MAE) 及超音波輔助萃取(Ultrasound-assisted extraction, UAE) 是新式之萃取方法，近年來廣泛被運用在食品化學和醫藥方面。與傳統熱水萃取法相比，具有減少萃取溶劑消耗、縮短萃取時間和增加萃取效率等優點。麥角硫因 (Ergothioneine) 是一種含硫的胺基酸，具有抗氧化、抗發炎及抗高血脂之功效，其主要存在於植物及動物組織中，在人體內無法被合成，僅能由食物中攝取。文獻指出金頂側耳 (Pleurotus citrinopileatus) 含高量麥角硫因，因此本研究利用微波及超音波輔助萃取技術，以一次一因子的方式來探討金頂側耳子實體中麥角硫因之最適萃取條件，並以反應曲面法探討各獨立變數之間的交互作用尋求最適萃取條件，最後進行麥角硫因之純化；另以麥角硫因標準品與金頂側耳微波、超音波輔助萃取及熱水萃取液進行抗氧化能力測定。研究結果發現，一次一因子方面，金頂側耳子實體麥角硫因微波輔助萃取之最適條件為功率500 W、時間30分鐘及固液比1：30；超音波輔助萃取之最適條件為時間 40 分鐘、功率75 W及固液比1：30。另本研究亦針對金頂側耳之主要生理活性成分麥角硫因，利用反應曲面法進行探討。由實驗結果得知，麥角硫因微波輔助萃取之最適萃取功率 552 W、時間為 29 分鐘及固液比 1：31，其預測萃取量與利用此條件進行四重複萃取後實際萃取量分別為 1.92 mg/g dw 及 1.92 mg/g dw；超音波輔助萃取之最適萃取條件為時間47分鐘、功率74 W及固液比1：20，其預測萃取量與利用此條件進行四重複萃取後實際萃取量分別為 1.53 mg/g dw 及 1.50 mg/g dw，皆亦無顯著差異，表示實際萃取量符合預測之萃取量。在抗氧化性質方面，金頂側耳子實體粉末微波、超音波輔助萃取及熱水萃取物於 5 mg/mL 時，清除 DPPH 自由基之能力分別為 82.43、83.93及81.39%；螯合亞鐵離子之能力分別為82.80、72.47及72.26%；還原力分別為0.57、0.56 及0.41；清除 ABTS+ 自由基之能力分別為71.16、86.66及84.66%。此外，由EC50值可得知，微波輔助萃取物在清除 DPPH 自由基及還原能力方面具有良好的抗氧化特性；超音波輔助萃取物則在清除 ABTS+ 自由基勝過其他兩者；螯合亞鐵離子能力則為熱水萃取物為優。在麥角硫因純化方面，因微波輔助萃取可得較高量之麥角硫因，因此後續以微波輔助萃取液進行麥角硫因的純化，結果顯示金頂側耳子實體粉末微波輔助萃取液之麥角硫因純度為 24.48%，經切向流過濾後純度為29.03%，最後經 Diol 製備型管柱純化，得到純度為 83.25%，產率為 65.72% 之金頂側耳子實體純化液；最後經由LTQ Orbitrap/MS鑑定，確定純化物為麥角硫因。綜上述結果可得知，以反應曲面法分析進行微波輔助萃取金頂側耳子實體粉末可獲得較高含量之麥角硫因，再搭配新穎技術之切向流過濾後以製備型管柱純化萃取液，可獲得純度高之麥角硫因純化物。因此，新式之萃取方式不僅可減少時間，亦可製備出純度高之機能性純化物，日後可用於菇類機能性成分的開發與應用。 Microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE) are new extraction technology that has been widely used in food chemistry and medicine in recent years. Compared to conventional extraction, MAE and UAE have many advantages, such as less solvent consumption, shorter extraction time and higher extraction rate. Ergothioneine is a sulfur-containing amino acid that has antioxidant, anti-inflammatory and anti-hyperlipidemic effects; it is present throughout the plant and animal tissues, cannot be synthesized by humans and only available from dietary sources. Since some researchers discovered higher content of ergothioneine in Pleurotus citrinopileatus; this study is about using microwave and ultrasound-assisted extraction of ergothioneine by one-factor-at-a-time and response surface methodology (RSM) to investigate the optimum extraction conditions and purified of ergothioneine. In addition, test the antioxidant capacity by using ergothioneine standard and microwave, ultrasound assisted extraction and hot water extract. The result in one-factor-at-a-time showed that microwave-assisted optimum extraction conditions of ergothioneine microwave power was 500 W, extraction time was 30 min and solid to liquid was 1:30; ultrasound-assisted optimum extraction conditions of ergothioneine extraction time was 40 min, ultrasound power was 75 W and solid to liquid was 1:30. Another result in RSM was focus on the main active components of Pleurotus citrinopileatus, which was ergothioneine, the optimum extraction conditions of MAE microwave power was 552 W, extraction time was 29 min and solid to liquid was 1:31; UAE extraction time was 47 min, ultrasound power was 74 W and solid to liquid was 1:20, which the predict value and the experimental value (n=4) respectively were 1.92 mg/g dw, 1.92 mg/g dw and 1.53 mg/g dw, 1.50 mg/g dw, no significant differences, the ergothioneine equation was valid. From these results, it was found that the MAE of ergothioneine extraction was superior to UAE. On the antioxidant acticity of microwave-assisted extraction extracts from Pleurotus citrinopileatus fruiting body powder (MEP), ultrasound-assisted extraction extracts from Pleurotus citrinopileatus fruiting body powder (UEP) and hot extracts from Pleurotus citrinopileatus fruiting body powder (HWEP). As the concentration was 5 mg/mL, the scavengling ability of DPPH radicals were 82.43, 83.93 and 81.39%; the chelating ability on ferrous ions were 82.80, 72.47 and 72.26%; the reducing power were 0.57, 0.56 and 0.41, and the scavenging ability on ABTS+ radicals were 71.16, 86.66 and 84.66%. According to EC50 value, MEP has good antioxidant properties on scavegling DPPH radicals and reducing power ability; UEP was more efficient than MEP and HWEP on scavenging ability of ABTS+ radicals. Moreover, the chelating ability on ferrous ions, HWEP was the best. MEP, which had most ergothioneine content, was further separated successively by tangential flow filtration (TFF) and IntersilTM Diol column to obtain higher purify ergothioneine. The purity of ergothioneine in MEP was 24.48%, after tangential flow filtration, the purity was 29.03%. In IntersilTM Diol column step, the purify of ergothioneine was 83.25%, recovery was 65.72%. The results show that using LTQ Orbitrap/MS examination purified compound in MEP is ergothioneine.In conclusion, high content of ergothioneine can obtained by MAE of Pleurotus citrinopileatus dried fruiting body powder. Next, with TFF and IntersilTM Diol column, we can get high purity ergothioneine purified product. Therefore, the new extraction technology can not only reduce time but also can prepare functional purified substances with high purity, which used for the development and application of functional components of mushrooms in the future.
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