豬血紅蛋白之酵素水解及抗氧化活性研究 吳桂菁、張基郁

豬血紅蛋白之酵素水解及抗氧化活性研究 吳桂菁、張基郁

E-mail: [email protected]

摘 要

本研究以豬血紅蛋白為材料，探討以Alcalase 與Flavourzyme水解前後之抗氧化活性。抗氧化活性測定項目，包含還原力、

亞鐵離子螯合能力及DPPH自由基清除能力等三種，並與BHA、α-生育醇或ETDA之抗氧化性做比較。 結果發現未經酵素 水解處理之豬血紅蛋白不具還原力，但在亞鐵離子螯合能力方面，則隨濃度之增大而有增加的趨勢，於濃度5.0 mg/mL 時

，具有74.46 %之螯合亞鐵離子能力；在DPPH自由基清除能力方面，於濃度2.0 mg/mL時，其清除自由基能力為21.53 %。

使用Alcalase和Flavourzyme兩種蛋白質水解酵素對豬血紅蛋白進行水解之水解度變化方面，使用Alcalase水解豬血紅蛋白10 小時後，得到水解度為9.84 %；使用Flavourzyme者，可得到18.75 %之較高水解度，約為使用Alcalase者之1.91倍。在使 用Alcalase和Flavourzyme進行兩階段水解之水解度中，以單一2.0 % Alcalase進行10小時水解作用，所得到水解度遠比使 用2.0 % Alcalase進行水解4小時後，再加入0.5、1.0與2.0 % Flavourzyme進行2、4、6小時水解得到之水解度要來的低。 在 豬血紅蛋白水解物之抗氧化活性中，其還原力並不顯著，但其亞鐵離子螯合能力則隨濃度之增大而有增加的趨勢，以2.0

% Alcalase水解4小時後再經1.0 % Flavourzyme水解6小時所得豬血紅蛋白水解物具有最強之亞鐵離子螯合能力，於樣品重對 溶劑體積比值濃度為5.0 mg/mL時，具有63.54 %之螯合亞鐵離子能力；在DPPH自由基清除能力方面，以2.0 % Alcalase水 解4小時所得豬血紅蛋白水解物，其對DPPH自由基清除效果較佳，於樣品重對溶劑體積比值濃度為5.0 mg/mL時，具 有41.94 %的清除自由基能力。將豬血紅蛋白水解物，進行膠體過濾層析，並將層析圖所有尖峰分成三個不同蛋白區分，

然後與抗氧化活性進行相關性分析，結果顯示水解物之抗氧化活性與FractionⅠ含量呈現顯著之正相關，而與FractionⅢ含 量呈顯著之負相關。 綜合本研究之結果，可知豬血紅蛋白與其水解物具有亞鐵離子螯合能力，部分豬血紅蛋白水解物具有 清除DPPH自由基能力，故可做為開發天然抗氧化劑之參考。

關鍵詞 : 豬血紅蛋白 ; 抗氧化性 ; 還原力 ; 亞鐵離子螯合能力 ; DPPH自由基清除能力 ; 酵素水解 目錄

目錄 封面內頁 簽名頁 授權書………iii 中文摘要………

………iv 英文摘要………vi 誌謝………

………viii 目錄………ix 圖目錄

………xiii 表目錄………

……xiv 第一章　緒論………1 第二章　文獻回顧………

………3 2.1 蛋白質之功能與構造………3 2.2 豬血液之一般性狀與組成………

………4 2.2.1 全血量及放血量………4 2.2.2 血液的組成………4 2.2.3 物理性 狀………4 2.2.4 血液的營養組成………6 2.3 血紅蛋白之構造性質…………

………9 2.4 血液的利用………9 2.5 蛋白質之水解………

………14 2.6 影響蛋白質酵素水解之因子………15 2.6.1 酵素的種類與濃度………16 2.6.2 受質的選擇………18 2.6.3 酵素與受質比例………18 2.6.4 pH值與濃度

………19 2.7 蛋白質水解物之特性與利用………19 2.7.1 促進礦物質吸收…………

………20 2.7.2 抗氧化性………20 2.7.3 抗高血壓………

…24 2.7.4 類鴉片作用………25 2.7.5 調節免疫力………26 2.8 自由基、

活性氧與人體健康………27 2.8.1 自由基與活性氧………27 2.8.2 自由基與活性氧的生 成………28 2.9 脂質的氧化………29 2.9.1 防止食品氧化之方法………

…………30 2.9.2 抗氧化劑之抗氧化機制………31 2.10天然抗氧化劑種類………34 第三章　實驗材料與方法………37 3.1 實驗材料………

…37 3.2 蛋白?選擇………37 3.3 化學試藥………37 3.4 豬血紅蛋白之製備………39 3.5 實驗方法………39 3.5.1 豬血紅 蛋白之色澤與基本成分分析…………41 3.5.2 抗氧化活性之分析………44 3.5.3 蛋白質組成分析…………

………46 3.5.4 酵素水解物之製備………47 3.5.5 豬血紅蛋白及其水解物之組成探討………

…50 3.5.6 統計分析………50 第四章　結果與討論………

…51 4.1 豬血紅蛋白之色澤、基本成分及電泳分析…………51 4.1.1 豬血紅蛋白之色澤與基本成分分析…………51 4.1.2 豬

血紅蛋白之電泳分析………51 4.2 豬血紅蛋白之抗氧化性………54 4.2.1 還原力…………

………54 4.2.2 亞鐵離子螯合能力………56 4.2.3 清除α, α-diphenyl-

β-picrylhydrazyl (DPPH)自由基能力………58 4.3 豬血紅蛋白之酵素水解………60 4.3.1 以Alcalase和Flavourzyme水解豬血紅蛋白 之水解度變化………60 4.3.2 以Alcalase和Flavourzyme 進行兩階段水解 之水解度變化………62 4.4 豬血紅蛋白水解物之抗氧化活性………64 4.4.1 不同酵素水解處理豬血紅蛋白之還原力……64 4.4.2 不同酵素水解處理豬血紅蛋白之亞鐵離子螯 合能力………

………67 4.4.3 不同酵素水解處理豬血紅蛋白之清除α, α- diphenyl-β-picrylhydrazyl(DPPH)自由基 能力…

………69 4.5 豬血紅蛋白經酵素水解處理前後之抗氧化活性比較 ………

………71 4.6 豬血紅蛋白及其水解物之Sephadex G-50膠體過濾 層析………

………74 4.7 豬血紅蛋白及其水解物之組成………74 4.8 豬血紅蛋白水解物之組成與其水解物抗氧化性之相 關性………79 第五章　結論與展望………82 參考文獻………84 圖目錄 圖2.1 豬隻血液之一般組成………

………5 圖2.2 (Ⅰ)血色質(Ⅱ)血色質與血球蛋白結合區位之結構………10 圖2.3 血液之利用………

………12 圖3.1 本研究之實驗流程………40 圖4.1 豬血紅蛋白

之SDS-PAGE電泳圖………53 圖4.2 豬血紅蛋白與BHA、α-生育醇之還原力比較……….55 圖4.3 豬血紅蛋白與BHA、α-生育醇和EDTA之亞鐵離子螯合能力 比較………

…57 圖4.4 豬血紅蛋白及BHA、α-生育醇之DPPH自由基清除能力比較 ………

………59 圖4.5 使用Alcalase和Flavourzyme水解豬血紅蛋白之水解度….61 圖4.6 使用Alcalase和Flavourzyme進行豬血紅蛋白 兩階段水 解之水解度………63 圖4.7 豬血紅蛋白水解物與BHA、α-生育醇之還原 力比較……….66 圖4.8 豬血紅蛋白水解物與EDTA之亞鐵離子螯合能力比較………68 圖4.9 豬血紅蛋白水解物與BHA、α- 生育醇之DPPH自由基清 除能力比較………70 圖4.10 Sephadex G-50膠體過濾層析 下豬血紅蛋白水解前後之分 子量分佈………76 表目錄 表2.1 豬血液的化學組成…

………7 表2.2 動物血液的胺基酸組成………8 表2.3 血液的利用

………13 表2.4 蛋白質水解物之理化及功能特性………21 表4.1 豬血紅蛋白之色澤與基本組成分分析………52 表4.2 豬血紅蛋白經酵素水解處理前後之抗氧化活性比較……

…72 表4.3 豬血紅蛋白及其水解物中不同蛋白質區分之含量百分比…78 表4.4 豬血紅蛋白水解物之不同蛋白質區分含量與 其抗氧化活性 之相關係數………81

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