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研究生:曾裕漳
研究生(外文):Yu-Chang Tseng
論文名稱:以蛋白質酵素水解大豆豆渣分離蛋白以製備具生理活性之胜肽及性質之研究
論文名稱(外文):Preparation and characterization of biological active peptides obtained by protease hydrolysis of okara protein isolate
指導教授:翁義銘翁義銘引用關係
指導教授(外文):Yih-Ming Weng
學位類別:博士
校院名稱:國立嘉義大學
系所名稱:食品科學系研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:216
中文關鍵詞:豆渣超濾抗氧化血管收縮素轉化酶胺基酸組成3T3-L1前脂肪細胞脂肪分解丙三醇釋放酵素水解
外文關鍵詞:OkaraUltrafiltrationAntioxidationAngiotensin I-converting enzyme3T3-L1 adipocytesLipolysisGlycerol releaseEnzyme hydrolysis
相關次數:
  • 被引用被引用:4
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原料豆渣 (Okara) 先以70℃,20分鐘預熱促進水解後,經脫脂 (Hexane)、加鹼分解 (2N NaOH) 等電點沉澱 (Isoelectric point precipitation) 製備成大豆豆渣分離蛋白 (Okara protein isolate, OPI),並經酵素水解製備大豆豆渣水解物 (Okara protein hydrolysates, OPIH)。酵素水解分成兩種方式,單一酵素水解採用一種酵素進行,使用酵素包括Alcalase, Protamex, Flavourzyme及Papain;兩階段水解 (Two-stage hydrolysis) 採用Protamex及Flavourzyme。在兩階段水解時,第一階段水解條件為:Protamex, 5 % 豆渣溶液、酵素濃度E/S=1 %、50℃、pH 7.0及120分鐘,第二階段水解條件:Flavourzyme, 酵素濃度E/S=0.5%、50℃、pH 7.0及60分鐘,於180分鐘之總水解時間可以得到水解度 (Degree of hydrolysis) 22.3% 之水解液,以二階段雙酵素可得到苦味低及水解度較高之產品。
兩階段水解大豆豆渣水解液經不同分子量限值 (Molecular weight cut- off , MWCO) 之三種超濾膜 (1kDa、3kDa及10kDa) 連續操作,可得不同區分物 (Fractions),區分物可分成濾液 (Permeates, Per) 及保留液 (Retentate, Ret),各區分物分別測試氫氧自由基 (Hydroxyl free radicals) 及ABTS‧+自由基 (ABTS‧+ free radicals) 之清除能力 (Scavenging activity)。結果顯示,水解物 (OPIH) 在濃度10 mg/mL時可顯著清除ABTS‧+及氫氧自由基 49 %以上,超濾後小分子量之濾液如Per 3 (3 kDa> MW > 1kDa) 及Per4 (MW < 1kDa) 為例,在濃度10 mg/mL時對自由基清除率可達85 %,此效果與BHA及Ascobic acid 用量為1.0 mg/mL時之自由基清除率相近。
本研究也探討豆渣分離蛋白及其水解區分物對血管收縮素轉化酶 (Angiotensin-I converting enzyme, ACE) 的抑制能力。豆渣分離蛋白未水解前抑制能力 IC50值為39.1mg/mL,經二階段水解後其IC50值降為0.56 mg/mL,小分子量區分物Per 4之抑制能力 IC50值更降低到0.09 mg/mL,抑制能力約為抗高血壓學名藥Enalapril IC50之25 % 用量之效力。水解物之胺基酸組成分析顯示,經酵素水解後疏水性胺基酸 (HydrOPIHobic amino acid) 佔全體之40.08%,且含高量脯胺酸 (Pro) 及白胺酸 (Leu),這些胺基酸組成與抗氧化及抑制ACE有關。
豆渣兩階段水解液及其區分物加入3T3-L1分化成熟脂肪細胞中,以測試其是否具有增強促進成熟脂肪細胞脂肪分解 (Lipolysis) 之能力。以400ppm之水解液及100 ppm之小分子量水解區分物 (Per 4) 皆可降低脂肪聚集 (Relative lipid accumulation, RLA) 達37%,並使丙三醇 (Glycerol) 之釋放量提高108.3 %。從 Oil Red O細胞染色中明顯觀察到Per 4 (MW<1000Da) 濾液區分物具有使脂肪油滴分解之現象。
由二階段水解及超濾膜所製成之豆渣水解液及區分物其中之小分子量Per 4具有抑制ACE及促進脂肪分解之功效,而小於3 kDa之區分物(Per 3及Per 4)也具有良好的抗氧化功效。
Okara was heated (70℃, 20 min), defatted with hexane, alkaline-treated (2N NaOH) and precipitated at isoelectric point to obtain okara protein isolate (OPI). OPI was further hydrolyzed with proteases either by single enzyme (including Alcalasem, Protamex, Flavourzyme and Papain) or by two-stage hydrolysis consisting of Protamex and Flavourzyme. In the first stage of the two-stage hydrolysis, Protamex was used with the hydrolysis conditions of 5% substrate concentration, E/S=1%, 50℃, pH 7.0 and 120 min; and the hydrolytic product was denoted as P120. In the second stage, Flavourzyme was used with the hydrolysis conditions of E/S=0.5%, 50℃, pH 7.0 and 60 min; and the hydrolytic product was denoted as P120+F60. The results showed that two-stage hydrolysis could achieve the degree of hydrolysis (DH) of OPI to 22.3% with less bitter taste.
P120+F60 was consecutively separated into four fractions (denoted as Ret 1 , Per 2, Per 3and Per 4) by ultrafiltration membranes with molecular-weight cut-off (MWCO) of 10kDa, 3kDa and 1kDa.The four fractions as well as OPI, P120 and P120+F60 were tested for their hydroxyl and ABTS free radical scavenging activities. The scavenging activities for both free radicals were more than 49% when the concentration of P120+F60 was 10 mg/mL. However, the scavenging activities for both free radicals were 85% when Per 3 and Per 4 were used at the level of 10 mg/mL, which was comparable to BHA and ascorbic acid at the level of 1.0 mg/mL.
The inhibitory activities of angiotensin-I converting enzyme (ACE) of the above okara protein products were also tested. The IC50 values for OPI, P120+F60 and Per 4 were 39.1, 0.56 and 0.09 mg/mL, respectively. The inhibitory activity of Per 4 was about one fourth of the antihypertension drug Enalapril. The amino acid composition analysis revealed that the content of hydrOPIHobic amino acid was 40.08%, and the high level of prolein and leucine might partly explain the antioxidant and anti-AGE activities.
The ability of OPI and hydrolysate fractions for catalyzing lipolysis in differentiated 3T3-L1 adipocytes was tested. Relative lipid accumulation (RLA) was decreased by 37% and the glycerol release was increased by 108.3% when P120+F60 at the level of 400 ppm and Per 4 at the level of 100 ppm were used. Disintegration of fat drops was observed when adipocytes were stained with Oil-Red O.
第一章目錄 i
表目錄 v
圖目錄 vi
中文摘要 10
英文摘要 (Abstract) 12
壹、前言 14
貳、文獻整理 16
一、原料豆渣介紹 16
二、脫脂大豆豆粕 17
三、生物活性肽來源 18
四、生物活性肽之生理功能 20
(一) 延緩血壓 (Blood pressure) 上升 20
(1) 高血壓與血管收縮素轉化酶
(Angiotensin converting enzyme, ACE) 20
(2) 抑制ACE之原理及胜肽結構特性 24
(3) 蛋白水解物對血壓的影響 24
(4) 大豆蛋白水解物與血壓之關係 28
(二) 抗氧化作用 (Antioxidation) 28
(1) 自由基 (Free radicals) 及抗氧化作用 28
(2) 天然抗氧化物質 31
(3) 抗氧化胜肽之結構特性 32
(4) 大豆及豆渣之抗氧化之作用 32
(三) 抗肥胖 (Anti-obecity) 與體重控制 (Weight control) 35
(1) 脂肪組織 (Adipose tissue) 35
(2) 3T3-L1前脂肪細胞株 (3T3-L1 Preadipocytes) 36
(3) 脂肪細胞研究模式 36
(4) 脂肪細胞分化 (Cell differentiation) 過程 37
(5) 大豆蛋白水解物抑制脂肪聚集 (Inhibit lipid accumulation)
及促進脂肪分解 (Lipolysis) 功能 39
五、大豆胜肽的製備方法 42
(一) 酵素水解法 42
(1) 絲胺基酸型蛋白質分解酵素 (Serine proteases) 43
(2) 硫醇型蛋白質分解酵素 (Thiol proteases) 43
(3) 金屬型蛋白質分解酵素 (Metalloproteases) 43
(4) 酸性型蛋白質分解酵素 (Acid protease) 43
(二) 微生物發酵法 45
六、大豆胜肽之分離 (Separation)、純化 (Purification) 與
鑑定 (Identification) 45
七、超過濾技術 (Ultrafiltration) 46
八、酵素水解物之特性 47
(一) 高溶解度 (Solubility)、低黏度 (Viscosity) 及加工穩定性 47
(二) 高消化性及低過敏性 47
(三) 苦味物質產生 48
參、實驗架構 49
肆、實驗材料 50
一、材料 50
二、儀器設備 50
三、實驗藥品 51
伍、 實驗方法 53
一、大豆豆渣分離蛋白 (Okara protein isolate) 之製備 53
(一) 去油大豆豆渣粉 (Defaterd okara flours) 之製備 53
(二) 大豆豆渣分離蛋白 (Okara protein isolates, OPI) 之製備 53
二、OPI高水解度蛋白酶之篩選 53
(一) 商業混合酵素水解豆渣之條件探討 53
(二) 二階段水解法 54
(三) OPI水解產物分析 56
(1) 水解度 (Degree of hydrolysis, DH) 56
(2) 總氮及產量 (Yield) 測定 57
(3) 回收率 (Recovery) 的計算 57
(4) 胜肽濃度 (Peptide concentration) 測定 57
三、濾膜的系統操作條件之建立 59
(一) 批式超過濾操作流程 59
(二) 連續式酵素膜反應系統 (Enzyme membrane reactor system) 59
四、水解液之分子量測定及區分 60
五、OPI水解物及其濾膜區分物之生理活性測定 60
(一) 抗氧化活性 (Antioxidative activity) 測定 60
(1) DPPH自由基清除能力測定 60
(2) ABTS‧+自由基清除能力 61
(3) 氫氧自由基清除能 (Hydroxyl radicalscavanging activity) 62
(二) 水解物及其濾膜區分物中收縮素I轉化酵素 (Angiotensin-
I converting enzyme, ACEI) 抑制活性測定 69
(1) ACEI抑制活性測定方法 69
(2) ACE抑制活性計算 69
(3) IC50值的測定 70
(三) OPI水解物及其區分物之促脂解 (Lipiolysis) 活性 70
(1) 細胞培養 70
(2) Oil red O染色及相對脂肪累積
(Relative lipid accumulation, RLA) 73
(3) 丙三醇 (Glycerol) 釋放量檢測 73
(4) 三酸甘油酯 (Triglyceride) 含量檢測 73
(5) 蛋白質定量 74
六、胺基酸組成 (Amino acid composition) 分析 75
七、計算水解混合物胜肽之F值 (Fischer ratio) 75
八、統計分析 75
陸、結果與討論 76
一、酵素水解豆渣蛋白試驗 76
(一) 一階段酵素水解 76
(二) 二階段酵素水解 76
二、水解條件對豆渣蛋白水解度之影響 77
三、前處理加熱對水解度之影響 79
四、超濾操作及酵素模反應系統 88
五、大豆豆渣分離蛋白水解物抗氧化能力 90
(一) 清除DPPH自由基能力 90
(二) 水解及超濾區分物對ABTS‧+自由基清除能力之影響 90
(三) 水解及超濾區分物對氫氧自由基 (Hydroxyl radical)
清除能力之影響 91
六、批式酵素膜反應系統水解物分子量分布情形 97
七、連續式酵素模反應系統與批式系統之比較 97
八、大豆豆渣水解產率 (Yield) 102
九、OPIH及超濾區分物對血管收縮轉化酶 (ACE) 抑制能力 105
十、胺基酸分析 109
(一) 水解及超濾區分物胺基酸分析 109
(1) 水解物及區分物中必需胺基酸 109
(2) 水解物及區分物中氨基酸與ACEI之關係 109
(二) 水解及超濾區分物支鏈胺基酸
(Branched chain amino acids, BCAA) 110
(三) 水解物及區分物中氨基酸與抗氧化能力之關係 111
十一、水解條件對相對脂肪堆積量
(Relative lipid accumulation, RLA%) 之影響 113
十二、大豆豆渣水解物對促進脂肪分解 (Lipiolysis) 之影響 116
十三、大豆豆渣蛋白水解液超濾區分物對促進脂肪分解之影響 120
十四、大豆豆渣水解超濾區分物前脂肪細胞油紅染色顯微鏡觀察 120
十五、促進脂肪分解機制探討 122
柒、結論 127
捌、參考文獻 128
第二章目錄

目錄 i
表目錄 v
圖目錄 vi
中文摘要 151
英文摘要 (Abstract) 152
壹、前言 153
貳、文獻整理 154
一、蛋白質的功能特性 (Fumctional properties) 154
(一) 水合性質 (Hydration properties) 154
(二) 沉澱與凝膠 (Gelling) 作用 154
(三) 表面性質 (Surface properties) 154
二、大豆蛋白質的功能特性 (Functional properties) 154
(一) 蛋白質溶解度 (Protein solubility, PS) 154
(二) 表面疏水性 (Surface hydrOPIHbicity, Ho) 155
(三) 乳化能力 (Emulsifying capacity) 與乳化安定性
(Emulsifying stability) 155
(四) 起泡能力 (Foaming capacity) 與起泡安定性
(Foaming stability) 156
(五) 影響泡沫生成與安定性的因子 157
(1) 蛋白質的結構 157
(2) 蛋白質的濃度 157
(3) 溫度 157
(4) pH和離子強度 (Ionic strength, I) 157
三、酵素水解對蛋白質功能特性之影響 157
(一) 蛋白質溶解度與表面疏水性 157
(二) 乳化能力與乳化安定性 158
(三) 起泡能力與起泡安定性 159
四、實驗目的 160
参、實驗材料 161
一、材料 161
二、儀器設備 161
三、實驗藥品 161
肆、實驗方法 163
一、豆渣分離蛋白水解物之胜肽製備 163
(一) 去油大豆豆渣粉 (Defatted okara flour) 之製備 163
(二) 大豆豆渣分離蛋白 (Okara protein isolate, OPI) 之製備 163
(三) 大豆豆渣蛋白水解物 (Okara protein hydrolysate, OPIH)
之製備 163
(1) 單階段水解 163
(2) 二階段水解 163
二、大豆豆渣蛋白水解物之超濾 (Ultrafiltration) 164
三、大豆豆渣水解酵素及超濾區分物對功能性質之影響 164
四、實驗分析方法 164
(一) 蛋白質定量 (Protein assay) 164
(二) 蛋白質溶解度 (Protein solubility, PS%) 164
(三) 表面疏水性 (Surface hydrophobicity) 165
(四) 乳化能力指數 (Emulsifying activity index, EAI) 及乳化
安定指數(Emulsifing stability index, ESI) 166
(五) 起泡能力 (Foamung capcity, FC %) 及泡沫穩定度
(Foaming stability, FS %) 166
五、計算分析 167
伍、結果與討論 168
一、酵素水解及超濾對蛋白質溶解度(Protein solubility) 之影響 168
二、酵素水解及超濾對表面疏水性 (Surface hydrophobicity, Ho)
之影響 171
三、水解及超濾之分子量分布圖 (Molecular weight distribution) 172
四、酵素水解及超濾對乳化性質 (Emulsifying properties) 之影響 132
五、泡沫性質 179
六、功能性質之利用之相關性 183
陸、結論 184
柒、參考文獻 185
第三章目錄

目錄 i
表目錄 v
圖目錄 vi
中文摘要 199
英文摘要 (Abstract) 200
壹、前言 201
貳、文獻整理 202
一、豆渣 (Okara) 之利用 202
二、低脂漢堡肉 (Low fat burger) 與脂肪取代物 (Fat replacer) 203
(一) 蛋白質基礎 (Protein base) 之取代物 203
(1) 乳清 (Whey) 203
(2) 膠原蛋白 (Collagen) 204
(3) 大豆蛋白 (Soy protein) 204
(二) 脂肪 (Fat-base) 為主體之取代物 205
(三) 碳水化合物 (Carbohydrate) 為主體之取代物 205
(1) 纖維 (Fiber) 205
(2) 膠類 (Gums) 206
(3) 多種混合物 206
三、漢堡肉之風味感官品評 206
四、可溶性膳食纖維 (Soluble dietary fiber) 207
五、大豆膳食纖維 (Dietary fibers) 之生理功能與製程技術 208
參、實驗材料 210
一、材料 210
二、儀器設備 210
三、實驗藥品 211
肆、實驗方法 212
一、濕豆渣一般成分分析 212
(一) 水分含量 (Moisturr content) 212
(二) 粗蛋白分析 212
(三) 粗脂肪 (Crude fat) 212
(四) 灰份 (Ash) 212
(五) 膳食纖維 (Fotal dietary fiber) 分析 213
二、濕豆渣前處理 214
三、濕豆渣之酵素水解 217
四、豬肉漢堡肉之製備 217
五、添加濕豆渣豬肉漢堡品質變化 217
(一) 色澤 (Color) 217
(二) 烹煮損耗 (Cook loss) 219
(三) 肉品收縮度 (Dimensional shrinkage) 219
(四) 保水能力 (Water-holding capacity, WHC) 測定 219
(五) 膽固醇 (Cholesterol) 測定 220
(六) 感官品評 220
六、統計分析 220
伍、結果與討論 223
一、濕豆渣原料及豬肉漢堡一般成份分析 223
二、豆渣可溶性膳食纖維之提升 223
(一) 加熱前處理對可溶性膳食纖維之影響 223
(二) 纖維分解酵素處理對可溶性膳食纖維之影響 224
三、添加濕豆渣對豬肉漢堡成份之影響 225
四、添加濕豆渣對豬肉漢堡之影響 232
(一) pH值 232
(二) 烹煮流失 (Cook loss) 232
(三) 色澤 (Color) 232
(四) 膽固醇 (Cholesterol) 235
(五) 感官品評 (Sensory evaluation) 235
陸、結論 238
柒、參考文獻 239
第一章、參考文獻

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