臺灣博碩士論文加值系統

酵素輔助萃取(Enzyme assisted extraction)做為新穎的綠色萃取技術，應用於各種工業或是取代傳統溶劑萃取以獲得機能性成分。圓筒絲瓜(Luffa cylindriac)在台灣被廣泛耕種，並成為國民日常飲食中常見的食用蔬菜，許多文獻指出絲瓜富含多種機能性成分，包含酚酸、類黃酮、齊墩果酸及葫蘆素等，並具有良好的抗氧化活性，然而鮮少有研究探討酵素輔助萃取對絲瓜中機能性成分含量及抗氧化活性之影響。因此本研究旨在探討是使用不同商業酵素進行酵素輔助萃取能否提升絲瓜中機能性成分並評估其最佳劑量，在純化後探討酵素輔助萃取對酚酸含量的影響及其抗氧化活性。結果顯示，三種商業酵素在工作劑量5% (w/w)下進行酵素輔助萃取時能獲得最高的總酚及總類黃酮含量且顯著高於水萃取組，又以使用FoodProⓇ 30L的組別顯著高於其他酵素，分別得到總酚3.39±0.26 mg GAE/g以及總類黃酮0.36±0.00 mg QE/g。掃描式電子顯微鏡的結果顯示經FoodProⓇ CBL處理後，絲瓜纖維結構被破壞。傅立葉轉換紅外線吸收光譜儀的結果顯示，纖維素酶透過破壞纖維素中的C=O及C-O-C等官能基達到水解的效果。純化後以使用5% (w/w) FoodProⓇ CBL的組別能獲得最高的對-香豆酸(p-coumaric acid)及阿魏酸(ferulic acid)，分別為11.85±0.42 mg/g purified extract及11.96±0.38 mg/g purified extract，然而與純化水萃取物相比沒有檢測到咖啡酸。在抗氧化活性的部分，純化液中以使用5% (w/w) FoodProⓇ CBL的組別具有最佳的抗氧化活性，分別為67.4% DPPH自由基清除能力、125.47 µM TE/mL TECA值以及215.74 µM TE/mL FRAP還原能力。上述結果表示酵素輔助萃取能有效提升絲瓜果肉萃取物中的機能性成分含量並做為具有潛力的抗氧化劑來源。

Enzyme assisted extraction (EAE) is a novel green extraction technology that is used in various industries or to replace traditional solvent extraction to obtain functional components. Luffa cylindrica is widely cultivated in Taiwan and has become a common edible vegetable in the daily diet of the people. Previous studies reported that luffa is rich in various functional components, including phenolic acids, flavonoids, oleanolic acid, and cucurbitacin, and has significant antioxidant activity. However, few studies have been investigated on the effect of EAE on Luffa cylindrica. Therefore, this study aimed to investigate EAE with different commercial enzymes on the functional components in luffa and evaluate the optimal dose, and investigate the effect of EAE on the phenolic acid content and the antioxidant activity of luffa after purification. The results showed that the highest total phenolics and total flavonoids content were obtained when the three commercial enzymes were subjected to EAE at a working dose of 5% (w/w), which was significantly higher than the water extraction group, and the group using FoodProⓇ 30L was significantly higher than the others enzyme, with total phenolics 3.39±0.26 mg GAE/g and total flavonoids of 0.36±0.00 mg QE/g, respectively. SEM results showed that the structure of luffa fibers was damaged after treatment with FoodProⓇ CBL, and FTIR results showed that cellulase hydrolyzed cellulose by destroying its functional groups such as C=O and C-O-C. After purification, the highest p-coumaric acid and ferulic acid were obtained in the group using 5% (w/w) FoodProⓇ CBL, 11.85±0.42 mg/g purified extract and 11.96±0.38 g/g mg/g purified extract, respectively. However, there is no caffeic acid was detected compared to the purified water extract. In the result of antioxidant activity, the best antioxidant activity was observed in the purified extract which used 5% (w/w) FoodProⓇ CBL with 67.4% DPPH free radical scavenging effect, 125.47 µM TE/mL TECA value and 215.74 µM TE/mL FRAP reducing power. These results indicate that enzyme assisted extraction was shown to be effective in increasing the functional contents in luffa pulp extract and as a potential source of antioxidants.

第一章、緒言 1
第二章、文獻回顧 3
一、圓筒絲瓜簡介 3
(一) 生長分佈 3
(二) 基本成分 3
(三) 植化素 4
(四) 生理活性及應用 5
1 抗氧化活性 5
2 抗發炎活性 5
3 抗高血糖活性 6
4 創傷癒合活性 7
5 固定化載體 7
二、酚類簡介 7
(一) 基本結構 8
(二) 酚酸 8
1 基本結構與分佈 8
2 生理活性 9
(三) 類黃酮 11
1 基本結構及分佈 11
2 生理活性 11
(四) 抗氧化機制 12
1 酚酸 13
2 類黃酮 13
三、皂苷簡介 14
(一) 基本結構 14
(二) 生物合成途徑 15
(三) 齊墩果酸 15
1 基本結構與分佈 15
2 生理活性 15
(四) 葫蘆素 17
1 基本結構與分佈 17
2 生物合成途徑 17
3 生理活性 17
(五) 抗氧化機制 18
四、水解酵素簡介 19
(一) 酵素的分類 19
(二) 纖維素酶 20
1 纖維素酶的種類 20
2 纖維素酶的應用 21
(三) 蛋白酶 21
1 蛋白酶的種類 21
2 蛋白酶的應用 22
五、抗氧化系統簡介 23
(一) 自由基與活性氧的危害 23
(二) 酵素性系統 23
(三) 非酵素性系統 24
(四) 抗氧化活性分析 24
第三章、材料與方法 25
一、研究架構 25
二、材料與儀器 26
(一) 原料 26
(二) 化學試藥 26
(三) 商業酵素 27
(四) 儀器設備 27
三、實驗方法 30
(一) 酵素輔助萃取最適條件之評估 30
1 不同萃取條件之絲瓜萃取液製備 30
2 絲瓜萃取液機能性成分分析 31
3 酵素處理前後絲瓜之物化性質變化 33
(二) 絲瓜萃取液之純化及機能性成分分析 34
1 萃取液製備 34
2 皂苷之純化與分析 34
3 酚酸之純化與分析 36
(三) 絲瓜純化液之抗氧化活性分析 39
1 DPPH 自由基清除能力 39
2 TEAC 值 40
3 FRAP 還原能力 41
4 亞鐵離子螯合能力 42
(四) 統計分析 42
第四章、結果與討論 43
一、酵素輔助萃取最適條件之評估 43
(一) 不同酵素劑量對絲瓜萃取液中機能性成分之影響 43
1 以CBL 評估酵素使用劑量 43
2 酵素種類與劑量對萃取液中總酚及總類黃酮含量之影響 44
(二) 不同酵素處理後絲瓜果肉之物化性質變化 45
1 表面結構檢測 45
2 表面官能基檢測 46
(三) 酵素輔助萃取之最適酵素種類及工作劑量歸納 47
二、絲瓜萃取液之純化及機能性成分分析 47
(一) 皂苷之檢測 47
1 HPLC 等度沖提之皂苷分析條件評估 48
2 等度沖提條件下之皂苷鑑定結果 48
3 HPLC 梯度沖提之葫蘆素B 分析條件評估 49
4 梯度沖提條件下之葫蘆素B 鑑定結果 50
5 討論 50
(二) 酚酸之檢測 51
1 酸化對純化結果之影響 51
2 分配次數對純化結果之影響 52
3 酚酸之UPLC-DAD 鑑定結果 52
4 酚酸成分之定量 54
5 討論 54
(三) 總酚及總類黃酮之檢測 55
(四) 純化後機能性成分含量與物化性質變化之歸納 56
三、絲瓜純化液之抗氧化活性分析 56
(一) DPPH 自由基清除能力 57
(二) TECA 值 58
(三) FRAP 鐵離子還原抗氧化能力 60
(四) 亞鐵離子螯合能力 61
第五章、結論 63
第六章、參考文獻 102

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