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論文名稱: | 高粱酒糟抗寒成分之初步鑑定及對吳郭魚之影響
| Preliminary idenyification of the functional compounds of the SDR and the anti-cold stress effect on tilapia
研究生: | 鄭惠玲 Hui-Ling Cheng
指導教授: | 孫寶年 Bonnie Sun Pan
學位類別: | 碩士
學校名稱: | 國立臺灣海洋大學
系所名稱: | 食品科學系
學號: | M95320006
畢業學年度: | 96
語文別: | 中文
關鍵字: | 高粱酒糟 sorghum distillery residue
| 吳郭魚 tilapia
全文影像連結: | | (本論文20110730對校內公開)
| 論文全文(pdf)

論文頁數: | 129
摘要: | 本研究以吳郭魚為模式動物，餵食添加高粱酒糟 (SDR) 95%乙醇萃取物 (SDRE) 及高粱酒糟加單寧結合劑 polyethylene glycol (PEG) 之飼料後，探討SDR對魚耐寒能力之影響，同時分離純化SDRE之有效物質，藉以判定SDRE可能具抗寒之化合物種類。
| 吳郭魚 (133.7 ± 18.6 g) 攝食添加3.6%SDRE之飼料三個月後，進行低溫緊迫試驗，水溫由室溫 (24.5oC)，於10 h內，降溫至13℃，維持5天，再於2天內回溫至24oC。吳郭魚受低溫緊迫後行為變化依序由魚體不平衡、側游、垂直游、翻肚游動、翻肚靜止於缸底、躺平游動、躺平靜止於缸底、終至死亡。降溫後82～130小時，控制組之死亡率為64.3%，明顯高於SDRE組38.4% (p < 0.05)，於回溫2天後，控制組之死亡率提高至92.9%，SDRE組之死亡率為46.2%。吳郭魚 (117.4 ± 27.6 g) 攝食添加20%或30% SDR-PEG之飼料二個月後，以1.1oC / h速率緩慢降溫至13oC，持續8天。於低溫8天，控制組吳郭魚之死亡率為6.8%，30%SDR-PEG組為2.3%，20%SDR-PEG組為0%，三組無統計上之差異 (p > 0.05)。控制組吳郭魚另由行為異常之隻數顯示較SDR-PEG組高。回溫後，控制組吳郭魚之死亡率增加至13.6%，20%SDR-PEG組及30%SDR-PEG組 (2.3%) 皆顯著低於控制組 (p < 0.05)，耐寒能力皆較控制組高。
| 於低溫緊迫試驗中，定時採血檢驗，控制組吳郭魚受低溫緊迫後，血液之起始應力增加，流體行為接近膨脹型 (dilatant fluid)；隨低溫時間延長，紅血球數目顯著上升，血紅素濃度則顯著下降，並產生溶血情形，紅血球膜不飽和脂肪酸與飽和脂肪酸量之比值 (UFA/SFA) 降低或緩慢升高；血漿膽固醇下降；血糖濃度於回溫後，顯著升高；血漿總抗氧化力增加。
| 吳郭魚攝食添加SDRE或SDR-PEG飼料後，於低溫緊迫時，血液起始應力低於控制組，流體行為接近牛頓型；紅血球數目及血色素濃度低於控制組，紅血球形態正常，紅血球膜脂肪酸不飽和度(UFA/SFA) 高於控制組；血糖濃度顯著低於控制組；血小板脂氧合酶活性低於控制組；血漿總抗氧化力與控制組相似。
| 在試管試驗中，高粱酒糟萃取物有延緩人低密度脂蛋白 (LDL) 氧化遲滯期之功效，SDRE之抗氧化功效為Trolox之55.6%，並優於SDR之乙酸乙酯或水萃物。高粱酒糟萃取物亦可清除DPPH自由基，以SDRE之半抑制濃度 (IC50) 最低，清除DPPH自由基之能力最高。SDRE經silica gel 管柱層析區分為正己烷層 (H)、乙酸乙酯層(EA)、丙酮層 (A)、甲醇層 (M) 與水層 (W)，以SDRE-EA產率最高為38.1%，其次為SDRE-M (26.7%)。SDRE-M清除DPPH自由基之能力最高，其半抑制濃度為1.23 ± 0.18 mg/mL，亦可抑制脂氧合酶活性，其半抑制濃度最低為6.12 ± 0.32 μg/mL。SDRE經薄層層析及ninhydrin、vanillin、硫酸呈色後，初步推測含有黃酮、酚類、胜肽及固醇之化合物，與標準品之Rf值比對，推測有酚酸 (ferulic acid) 及固醇 (stigmasterol)。
| 由吳郭魚養殖試驗及試管試驗，SDRE與SDR-PEG之飼料皆具提升調節能量代謝與血液流變性及延緩氧化之功效，可作為魚類抗寒飼料之配方素材。
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論文目次: | 目錄
| 中文摘要    i
| 英文摘要    iv
| 目錄    vii
| 圖目錄    xii
| 壹、前言    1
| 貳、文獻回顧    4
| 一、低溫緊迫對魚類成長及生理影響    4
| 1. 對行為及成長之影響    5
| 2. 內分泌之變化    7
| 3. 代謝作用之影響    9
| 4. 細胞膜上脂肪酸組成之變化    10
| 5. 血液參數之變化    11
| 二、飼料中添加抗緊迫物質之影響    12
| 1. 一條根95%乙醇萃取物    12
| 2. 維生素E及維生素C    13
| 3. 卵磷脂    13
| 4. 抗凍蛋白    14
| 三、高粱與高粱酒糟    14
| 1. 構造與一般組成    14
| 2. 高粱之有效成分及其生理活性    15
| 2.1 酚類化合物    15
| 2.2 植物固醇    19
| 2.3 Policosanol    20
| 3. 高粱酒糟之成分與生理活性    21
| 參、材料與方法    23
| 一、材料    23
| 1. 吳郭魚    23
| 2. 高粱酒糟    23
| 2.1 高粱酒糟帶殼與去殼之粉末    23
| 2.2 去油高粱酒糟    24
| 2.3 高粱酒糟水、95%乙醇及乙酸乙酯萃取物    24
| 3. 飼料製備    25
| 3.1 實驗一飼料: 高粱酒糟95%乙醇萃取物    25
| 3.2 實驗二飼料: 20%或30%SDR加單寧結合劑PEG    25
| 3.3 實驗三飼料: 3.6%或5.4% SDRE    26
| 4. 吳郭魚之血液及血漿    26
| 5. 人的血漿    27
| 二、方法    28
| 1. 飼料一般成分分析    28
| 2. 一般生理指標    28
| 2.1 比成長率    28
| 2.2 死亡率    28
| 3. 低溫緊迫試驗    28
| 3.1 實驗一: 高粱酒糟95%乙醇萃取物 (SDRE)    28
| 3.2 實驗二: 高粱酒糟加單寧結合劑 (SDR-PEG)    29
| 3.3 實驗三: 3.6%及5.4% SDRE (急速低溫緊迫)    30
| 4. 血液生化分析    30
| 4.1紅血球之形態    30
| 4.2 血液流變性之測定    31
| 4.3 血液參數之測定    31
| 4.3.1 血比容積    31
| 4.3.2 紅血球計數    32
| 4.3.3 血色素濃度    32
| 4.4紅血球之形態    32
| 4.5 紅血球膜之脂肪酸分析    32
| 4.5.1 紅血球膜之萃取    32
| 4.5.2 微量甲酯化    33
| 4.6 血漿生化分析    34
| 4.6.1 血糖定量    34
| 4.6.2 膽固醇定量    34
| 4.6.3 血漿總抗氧化力    35
| 4.7 血漿脂氧合酶之測定    36
| 4.7.1 粗酵素液之製備    36
| 4.7.2 蛋白質定量    36
| 4.7.3 脂氧合酶異構酶活性之測定    36
| 5. 去油高粱酒糟95%乙醇萃取物之分離    37
| 5.1 silica gel 管柱層析    37
| 5.2 薄層液相層析    38
| 5.3 呈色反應    38
| 5.3.1 Sulfuric acid    38
| 5.3.2 Vanillin-sulfuric acid    39
| 5.3.3 Ninhydrin    39
| 6. 抗氧化力之測定    39
| 6.1 延緩低密度脂蛋白氧化之能力    39
| 6.1.1 脂蛋白分離    39
| 6.1.2 LDL之透析    40
| 6.1.3 Cu2+誘導LDL之氧化反應    41
| 6.1.4 LDL氧化遲滯期之計算    41
| 6.2 清除DPPH自由基之能力    41
| 6.3 總多酚定量    42
| 6.4抑制脂氧合酶活性之能力    43
| 7. 統計分析    44
| 肆、結果    45
| 一、高粱酒糟對吳郭魚成長、血液性質及耐寒之影響    45
| (一) 高粱酒糟95%乙醇萃取物之飼料    45
| 1. 吳郭魚攝食後之比成長率    45
| 2. 吳郭魚攝食後血液之變化    46
| 3. 吳郭魚攝食後受低溫緊迫之生理變化    47
| 3.1 行為變化與死亡率    47
| 3.2 血液參數    49
| 3.2.1 血比容積    49
| 3.2.2 紅血球數目    50
| 3.2.3 血色素濃度    51
| 3.3. 紅血球膜脂肪酸組成    53
| 3.4. 血漿生化指標    57
| 3.4.1 血糖    57
| 3.4.2 膽固醇    58
| (二) 高粱酒糟加單寧結合劑PEG之飼料    59
| 1. 吳郭魚攝食後之比成長率    59
| 2. 吳郭魚攝食後血液之變化    60
| 3. 吳郭魚攝食後受低溫緊迫之生理變化    62
| 3.1 行為變化與死亡率    62
| 3.2. 血液流變性    67
| 3.2.1 起始應力    67
| 3.2.2 流體行為指標    69
| 3.2.3 表黏度    70
| 3.2.4 血液黏度指標    70
| 3.3. 血液參數    72
| 3.3.2 紅血球數目    73
| 3.3.3 血色素濃度    75
| 3.4. 紅血球形態    76
| 3.5 紅血球膜脂肪酸組成    79
| 3.6 血漿生化指標    84
| 3.6.1 血糖    84
| 3.6.2 膽固醇    86
| 3.6.3 血漿總抗氧化力    87
| 3.7 血小板脂氧合酶活性    88
| 二、攝食高粱酒糟乙醇萃取物對吳郭魚受急速低溫緊迫之影響    93
| 三、高粱酒糟有效成分之分離鑑定    95
| (一) 高粱酒糟之製備條件及其抗氧化力    95
| 1. 乾燥條件    95
| 2. 萃取溶劑    96
| 3. 抗氧化力    97
| 3.1 清除DPPH自由基之能力    97
| 3.2 延緩LDL氧化遲滯期之功效    98
| (二) 高粱酒糟95%乙醇萃取物之抗氧化區分    102
| 1. 油脂對SDRE總多酚含量與抗氧化力之影響    102
| 2. SDRE經silica gel 管柱區分之產率與抗氧化性    103
| (三) 黃酮、胜肽、固醇類之薄層層析鑑定    106
| 1. SDRE    106
| 2. SDRE之silica gel 管柱區分物    107
| 伍、討論    110
| 一、攝食高粱酒糟之飼料對吳郭魚成長之影響    110
| 二、攝食高粱酒糟之飼料對吳郭魚受低溫緊迫後生理之影響    110
| (一) 血液攜氧能力    110
| (二) 能量代謝    113
| (三) 高粱酒糟成分對溶血抑制與血糖濃度降低之影響    114
| 1. 抑制溶血之成分    114
| 2. 減少血糖濃度之可能成分    115
| 陸、結論    118
| 柒、參考文獻    120
|
| 表目錄
| 表一、吳郭魚飼料配方及一般成分    26
| 表二、吳郭魚攝食添加高粱酒糟95%乙醇萃取物飼料2個月後之體重與比成長率    45
| 表三、吳郭魚攝食含高粱酒糟乙醇萃取物之飼料2個月後血液參數之變化    46
| 表四、吳郭魚攝食一般飼料3個月經低溫緊迫後紅血球膜脂肪酸組成之變化    54
| 表五、吳郭魚攝食高粱酒糟95%乙醇萃取物飼料3個月經低溫緊迫後紅血球膜脂肪酸組成之變化    55
| 表六、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後之體重與比成長率    59
| 表七、吳郭魚攝食含20%或30%高粱酒糟加PEG飼料2個月後血液參數之變化    61
| 表八、吳郭魚攝食一般飼料2個月經低溫緊迫後紅血球膜脂肪酸組成之變化    80
| 表九、吳郭魚攝食20%SDR-PEG經低溫緊迫後紅血球膜脂肪酸組成之變化    81
| 表十、吳郭魚攝食30%SDR-PEG經低溫緊迫後紅血球膜脂肪酸組成之變化    82
| 表十一、高粱酒糟乾燥過程中多酚含量之差異    95
| 表十二、高粱酒糟以二次水、95%乙醇、乙酸乙酯萃取之產率及清除DPPH自由基之半抑制濃度    96
| 表十三、高粱酒糟萃取物對人LDL氧化遲滯期之影響    101
| 表十四、去油高粱酒糟之95%乙醇萃取物與SDRE之產率、總多酚類含量與清除DPPH自由基之半抑制濃度    102
| 表十五、高粱酒糟95%乙醇萃取物經silica gel管柱層析之各層乾重、產率及清除DPPH自由基之半抑制濃度    104
| 表十六、高粱酒糟95%乙醇萃取物及其silica gel管柱層析區分物對脂氧合酶之抑制效果    105
| 表十七、SDRE經silica gel管柱區分物之呈色反應    109
|
| 圖目錄
| 圖一、高粱中已確定之酚酸單體    16
| 圖二、常見花青素之化學結構 (A) 與高粱之3-去氧花青素及其衍生物 (B) 之比較    17
| 圖三、高粱中縮合單寧polyflavan-3-ols之結構    18
| 圖四、吳郭魚攝食添加高粱酒糟95%乙醇萃取物飼料3個月後受低溫緊迫之死亡率    47
| 圖五、吳郭魚攝食添加高粱酒糟乙醇萃取物飼料3個月後受低溫緊迫5天及回溫2天，吳郭魚之活動狀態    48
| 圖六、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後受低溫緊迫血比容積之變化    49
| 圖七、吳郭魚攝食添加高粱酒糟95%乙醇萃取物飼料3個月後，經低溫緊迫5天紅血球數目之變化    51
| 圖八、吳郭魚攝食添加高粱酒糟95%乙醇萃取物飼料3個月後經低溫緊迫，血紅素濃度之變化    52
| 圖九、吳郭魚攝食高粱酒糟95%乙醇萃取物3個月後經低溫緊迫，紅血球膜脂肪酸組成之百分比    56
| 圖十、吳郭魚攝食高粱酒糟95%乙醇萃取物飼料3個月後受低溫緊迫，血糖 (A) 與血漿膽固醇 (B) 濃度之變化    57
| 圖十一、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫之行為變化    63
| 圖十二、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後，受低溫緊迫6.5天之行為變化    64
| 圖十三、吳郭魚受低溫緊迫及回溫後行為異常數目    65
| 圖十四、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫之死亡率    66
| 圖十五、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月，受低溫緊迫後血液起始應力、流體行為指標、表黏度及黏稠指標之變化    68
| 圖十六、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後，受低溫緊迫血比容積之變化    73
| 圖十七、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後受低溫緊迫紅血球數量之變化    75
| 圖十八、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後受低溫緊迫血紅素濃度之變化    76
| 圖十九、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫後，紅血球型態之變化    78
| 圖二十、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫後，紅血球膜脂肪酸組成之百分比    83
| 圖二十一、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫後，紅血球膜脂肪酸不飽和度之變化    83
| 圖二十二、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月後受低溫緊迫，血糖含量之變化    85
| 圖二十三、吳郭魚攝食添加高粱酒糟95%乙醇萃取物飼料3個月後，受低溫緊迫，血漿膽固醇之變化    87
| 圖二十四、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫後，血漿總抗氧化力之變化    88
| 圖二十五、吳郭魚攝食添加20%或30%高粱酒糟加PEG飼料2個月受低溫緊迫後，血小板脂氧合酶異構物活性之變化    89
| 圖二十六、吳郭魚攝食添加3.6%或5.4%高粱酒糟乙醇萃取物飼料1個月受急速低溫緊迫之死亡率    93
| 圖二十七、濕高粱酒糟、乾燥高粱酒糟與Trolox、對人LDL氧化之影響    100
| 圖二十八、沒食子酸之標準檢量曲線    102
| 圖二十九、SDRE之薄層層析圖譜    106
| 圖三十、SDRE管柱區分物之薄層層析圖譜    107
|
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