本研究以7.5 % ( w/w of protein ) 之Alcalase 、Flavourzyme 及二種混合 (1:1) 之酵素 (Mix) 分別對8 % 鵝蛋黃蛋白質 (goose egg yolk protein; GEYP) 進行水解，並依水解時間製備0、0.5、4、8、12h水解物以探討水解時間 (hydrolysis time; HT) 與酵素種類對GEYP水解物之水解率 (degree of hydrolysis; DH)、產率及抗氧化特性之影響。結果顯示，三組酵素水解物之DH和產率皆隨HT增加而上升，其中以混合酵素水解GEYP12h所得之水解物 (MH12h) 其DH最高可達56.7 %，其次為Flavourzyme 的36.7 %。就抗氧化性而言，MH12h具最佳的α, α-diphenyl-β-picrylhydrazyl (DPPH)自由基清除能力、氫氧自由基清除能力、還原力及亞鐵離子螯合力。將MH12h依序經30、10、1及0.15 kDa分子量限值濾膜進行區分，分別可得30、10、1與0.15 kDa濃縮液與0.15 kDa濾液，30 kDa 濃縮液具最佳的亞鐵離子螯合力與DPPH自由基清除能力，此區分物之Tyr與Ser含量顯著增加，這可能與其抗氧化力之提升有關。1 kDa濃縮液具有最佳的還原力與清除氫氧自由基的能力，可能與胺基酸Pro含量增加有關。上述兩區分物之分子量主要小於1,000 Da，推測此分子量之胜肽提供較佳的抗氧化力。由此可知MH12h有良好的抗氧化力，未來具開發為天然抗氧化物質應用於食品系統中之潛力。

The purpose of this study was to investigate the effects of hydrolysis time (HT) (0, 0.5, 4, 8, 12 h) and enzyme types (Alcalase, Flavourzyme, and Mixed enzyme Alcalase + Flavourzyme) on degree of hydrolysis (DH), yield, and antioxidant activities during hydrolysis of 8 % goose egg yolk protein (GEYP). Each enzymes concentration was 7.5% (w/w of protein). Both DH and yield of the hydrolysates increased with increase of HT. The hydrolysate obtained from hydrolysis of GEYP with mixed enzyme for 12h (MH12h) had the highest DH of 56.7 % than Flavourzyme’s 36.7 %. For antioxidative activities of the hydrolysates, MH12h had the strongest α, α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radical scavenging activity, reducing power, and ferrous ion chelating activity. MH12h were further factionated by ultrafiltration membranes with 30, 10, 1, and 0.15 kDa molecular weight cut-off (MWCO) sequentially to obtained 30, 10, 1, 0.15 kDa concentrate and 0.15 kDa permeate, respectively. The result showed 30 kDa concentrate had higher ferrous ion chelating activity and DPPH radical scavenging activity than other fractions. For the amino acid composition, 30 kDa concentrate had higher Tyr and Ser. 1 kDa concentrate had higher reducing power and hydroxyl radical scavenging activity than others. It might be due to its higher Pro content. The molecular weight profile of 30 kDa and 1 kDa concentrate had mainly 1,000 Da. Therefore, MH12h had stronger antioxidative activity , and may serve as potential nature antioxidative materials for food system in the future.

目錄 ……………………………………………………………………Ｉ
圖次 ……………………………………………………………………Ⅴ
表次 ……………………………………………………………………Ⅶ
附錄次…………………………………………………………………..Ⅷ
中文摘要………………………………………………………………..Ⅸ
英文摘要……………………………………………………………….ⅩⅠ
壹、 前言……………..……………………………………………...…1
貳、 文獻回顧……………………………………………………...…..3
一、 國內養鵝業之生產情形…………………………………....…..3
(一) 白羅曼鵝之簡介………………………………….….……........4
(二) 鵝蛋之特性與組成……………………..…………….……...…5
(三) 蛋黃之組成及應用……………………..…………………...….5
二、 脂質氧化作用……………………….…………………....……11
(一) 脂質自氧化作用……..………….………………….……...…..11
(二) 脂質氧化與疾病……………...…...………………………..…15
三、 自由基與活性氧..…..…….………………..……….…………17
(一) 自由基與活性氧的種類…………...……………...…….…….17
(二) 自由基與活性氧的來源………………….…………......…….18
(三) 自由基與活性氧對生物體的影響……...………..…..…….....20
四、 抗氧化機制與抗氧化物質……………………………………20
五、 影響胜肽抗氧化活性之因子…………………………………24
六、 蛋白質之水解作用……………………………………………31
參、 材料與方法………………………………….…………....……..41
一、 試驗材料………………………………………………………41
二、 實驗設計……………………..………………………………..41
三、 鵝蛋黃蛋白質水解物之製備…………………………………43
(一) 粗鵝蛋黃蛋白質之製備………………..……..........….........…43
(二) 實驗酵素……………………………………..….……....…......45
(三) 鵝蛋黃蛋白質酵素水解之條件篩選………………...…..…....45
(四) 鵝蛋黃蛋白質水解物之製備…………………….…………....46
(五) 基本成分分析……………………….….…..……………..…...46
(六) GEYP水解物之水解率測定……………….……..........…...50
(七) 產率之測定…………………...………….………….….…..….53
四、 抗氧化能力之測定………………………………..…………..53
五、 磷含量之測定…………………………………………………58
六、 膜反應系統之區分作用…………………………........……....59
七、 分子量分佈分析……………………………………..…….….60
八、 胺基酸組成分析………………………………….….……......63
九、 統計分析…………………………………..…………...……...67
肆、 結果與討論……….…………………………………...………...68
一、 GEYP基本成分分析………………………………………….68
二、 GEYP水解條件探討………………………………………….70
(一) 較適基質濃度之探討……………………………….............…70
(二) 較適酵素濃度之探討………………………….….........…...…72
(三) 在相同水解條件下不同酵素對於水解率之影響及其pH值之變化………………75
三、 GEYP抗氧化性水解物的製備條件及其特性之比較…….....78
(一) GEYP水解物產率之比較…………………....……...…...……79
(二) GEYP水解物抗氧化能力之比較………...….…..……………82
1. α, α-diphenyl-β-picrylhydrazyl (DPPH) 自由基清除能力之比較……82
2. 亞鐵離子螯合能力之比較…………………………..…...…...85
3. 還原力之比較…………………………………….…………...89
4. 清除氫氧自由基能力之比較……………………..…...……...94
四、 探討MH12h經不同分子量限值區分所得之區分物對抗氧化力的影響………………………………………………..…………97
(一) GEYP、MH12h及其區分物之分子量分佈…………………...97
(二) MH12h區分物之還原力、金屬螯合力、氫氧自由基與DPPH自由基清除能力……99
(三) GEYP最具抗氧化力區分物之胺基酸組成……...……..…....102
伍、 結論…………………………………………………….…….....106
陸、 參考文獻………………………………………………...….…..107
柒、 附錄……………………………………………………..............125
圖次
圖一、脂質自氧化的反應階段………………………………………...12
圖二、不飽和脂肪酸自氧化反應中最初的氫過氧化物的形成………14
圖三、脂質氧化反應之最終產物………………………………………16
圖四、氧分子之氧化還原及激發狀態…………………………………19
圖五、電子捕捉劑所捕捉到的蛋白質之側鏈自由基之結構…………28
圖六、甲硫胺酸側鏈還原脂質氫過氧化物的機制….………………...30
圖七、試驗設計………………………………………………………....42
圖八、粗鵝蛋黃蛋白質之製備流程…………………………………... 44
圖九、濾膜區分流程圖…………………………………………………61
圖十、不同酵素與GEYP濃度對其水解物水解率的影響…………..71
圖十一、Alcalase (a)與Flavourzyme (b) 濃度與水解時間對GEYP水解物之水解率之影響………………………………………..73
圖十二、不同酵素與水解時間對GEYP水解物水解率之影響 (a) 及其pH值之變化 (b)…………………………………..…..…76
圖十三、酵素種類與水解時間對GEYP水解物產率之影響......…...80
圖十四、水解時間及酵素種類對GEYP水解物之DPPH自由基清除能力與BHT當量之影響…………………………………...83
圖十五、水解時間及酵素種類對GEYP水解物螯合亞鐵離子能力與 EDTA當量之影響……86
圖十六、GEYP水解物之水解率對亞鐵離子螯合能力的關係……...87
圖十七、水解時間及酵素種類對GEYP水解物還原力和相對BHT當量之影響……………..92
圖十八、GEYP水解物之水解率對還原力的關係…………………...93
圖十九、水解時間及酵素種類對GEYP水解物氫氧自由基清除能力之影響．．．．．．．95
表次
表一、各種禽蛋之重量、蛋白、蛋黃及蛋殼所占百分比………….6
表二、禽類蛋黃蛋白質之組成………………………………...….….8
表三、鵝蛋黃及GEYP之基本組成分…………....….……….…….69
表四、GEYP水解物中總磷的含量……………...………………….90
表五、GEYP、MH12h 及其膜區分物之分子量分佈………….….98
表六、MH12h 經不同分子量限制濾膜區分對其還原力、螯合力、氫氧自由基和DPPH自由基清除能力之影響……….……100
表七、GEYP、MH12h、30kDa 與1kDa濃縮液的胺基酸組成...103
附錄次
附錄一、L-Leucine 標準檢量線…………..…………………….….125
附錄二、BHT清除DPPH自由基清除能力之標準檢量線…..…...125
附錄三、EDTA螯合亞鐵離子之標準檢量線……………….…..…126
附錄四、KH2PO4之標準檢量線…………………………………....126
附錄五、還原力之標準檢量線…………………………………..…127
附錄六、MH12h經不同分子量限值濾膜區分所得區分物之分子量
分佈…………………………………………………..……128
附錄七、標準品分子量與滯留時間檢量線……………...…...……129
附錄八、分子量標準品之高效液相層析圖………………………..130
附錄九、胺基酸標準品之高效液相層析圖…………………..……131

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