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研究生:黎志宣
研究生(外文):Chih-Hsaun Li
論文名稱:副乾酪乳酸桿菌 NTU 101 所生成降血壓物質之純化與大家優藻褐藻服可醫定膠囊調節血脂之功效評估
論文名稱(外文):Isolation and purification of antihypertensive substances produced by Lactobacillus paracasei subsp. paracasei NTU 101 and study on hypolipidemia effects of fucoidan produced by Cladosiphon okamuranus
指導教授:潘子明潘子明引用關係
指導教授(外文):Tzu-Ming Pan
口試委員:李俊霖林志輝蔣慎思
口試委員(外文):Chun-Lin LeeChih Hui LinShen-Si Jiang
口試日期:2014-04-08
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:112
中文關鍵詞:高血壓乳酸菌血管收縮素轉換抑制劑最適化褐藻醣膠降血脂
外文關鍵詞:hypertensionlactic acid bacteriaangiotensin converting enzymeinhibitorsoptimizationfucoidanhypolipidemia
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Part 1
黃豆是中國人的傳統食物,黃豆蛋白質含有完整的必需胺基酸,為一種相當於動物性蛋白質的植物性蛋白質。許多研究也指出黃豆和黃豆製品對人體健康有許多好處,包括預防心血管疾病、腎臟病、糖尿病、惡性腫瘤、肥胖、骨質疏鬆症、過敏症和便秘等具有改善功能。本研究之目的旨在利用 Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) 發酵尋求 ACEI (angiotensin-converting enzyme inhibitor) 產值最高之基質以及使用反應曲面法 (response surface methodology, RSM) 探討最適發酵條件,分離純化活性成份,並利用核磁共振光譜法 (nuclear magnetic resonance, NMR) 進行純化物鑑定並測定其活性。使用發酵及未發酵的豆漿、牛奶、豆漿加牛奶分別進行水萃和醇萃,並以 MRS (Murashige and Skoo) 培養基發酵之上清液以及菌體,將菌體分成活菌以及以超音波破菌及高壓滅菌釜破菌,共16個組別分別測定 ACEI 活性,結果發現發酵豆漿醇萃組效果最好,最大半抑制濃度為 1.22 mg/mL。RSM 最適化條件培養 (12% 豆粉、起始 pH 9.0和15% 接菌量),其半抑制濃度降為 1.02 mg/mL。接著以不同極性的有機液體分層、Silica gel管柱層析,Sephadex LH20 管柱層析、薄層色層分析以及高效液相色譜法進行純化,最後選用F1-6-2成份進行後續之結構鑑定。
藉由NMR及霍氏轉換紅外光譜儀 (Fourier-Transform Infrared Spectrometer, FTIR)之圖檔數據推論NTU 101發酵豆漿醇萃物中的化合物F1-6-2為daidzein。IUPAC命名為7-hydroxy-3-(4-hydroxyphenyl) chromen-4-one,綜合上述,豆漿經過NTU 101此具有特定生理活性功能之菌株發酵後可提升豆漿中抑制ACE活性的成分,未來或許可將NTU 101發酵豆漿開發為預防及改善高血壓及其衍生疾病之保健食品,以提升傳統豆漿製品之附加價值。

Part 2
血中總膽固醇 (total cholesterol, TC) 越高,冠狀動脈心臟病的死亡率越高,並且飲食中飽和脂肪所提供熱量佔總熱量的百分比與冠狀動脈心臟病的死亡率呈很高的正相關 (相關係數 r = 0.84 ),過量攝取食物中之飽和脂肪酸,會提高血液中 TC 及低密度脂蛋白膽固醇 (low-density lipoprotein cholesterol, LDL-C) 濃度,而促進動脈硬化。另外研究指出,血脂質異常與高脂飲食均為罹患心臟血管疾病相當重要的兩項危險因子。
本研究以餵飼高膽固醇飲食之倉鼠作為評估調節血脂能力之動物模式,給予高膽固醇飲食八週,並以大家優藻生物科技股份有限公司提供之服可醫定膠囊進行體內動物實驗,評估對於血液、糞便與肝臟中的 TC、三酸甘油酯 (triglyceride, TG)、LDL-C 與高密度脂蛋白膽固醇 (high-density lipoprotein cholesterol, HDL-C) 以及體內抗氧化能力之調節效果。結果顯示餵食 fucoidan 一倍劑量可降低血清中 TC 之濃度、肝臟中 TG 濃度、減少血清中脂質過氧化程度與提升超氧歧化&;#37238; (superoxide dismutase, SOD)、過氧化氫&;#37238;及脂肪斑塊堆積之功效 (p < 0.05);餵食 fucoidan 兩倍劑量可降低肝臟中 TC 濃度 (p < 0.05);餵食 fucoidan 五倍劑量可增加糞便中 TC 及 TG 排出量 (p < 0.05)。由本研究可知,餵食褐藻醣膠可改善高膽固醇飲食倉鼠的血脂。

Part 1
Soy is a traditional Chinese food, its protein contains a complete essential amino acids as an equivalent of animal protein. Many studies have also pointed out that soy and soy products have many benefits on human health, including the prevention of cardiovascular disease, kidney disease, diabetes, malignancy, obesity, osteoporosis, allergies, constipation, etc and improved health condition. The purpose of this study is using Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) to optimize the production conditions of angiotensin-converting enzyme (ACE) inhibition value with Response Surface Methodology (RSM). After separating and purifing active ingredients, nuclear magnetic resonance (NMR) was applied to identify isolated bioactive compounds. Among 16 groups measured, fermented soil milk extraction had the highest ACEI activity, with half-maximal inhibitory concentration (IC50) reduced from 1.22 mg/mL to 1.02 mg/mL under the follow condition: 12% soy milk, starting from pH 9.0 and 15% inoculum. NTU 101 soil milk alcohol extraction were separated and purified by different polarity organic liquids, Silica gel column chromatography, Sephadex LH20 column chromatography, TLC and HPLC. After that, purified compounds F1-6-2 had the most inhibitory activity. Purified compounds F1-6-2 was identified as Daidzein by NMR and FTIR. IUPAC name is 7-Hydroxy-3- (4-hydroxyphenyl) chromen-4-one. In conclusion, soy milk fermented by NTU 101 can produce bioactivity substances that inhibited ACE activity. In the future, to enhance the value of traditional soy milk products, this multifunction component can be applied to the development of functional foods to prevent the development of hypertension and associated diseases.

Part 2
Many studies have pointed out a higher total cholesterol (TC) value in serum contributed to a higher death rate from coronary heart disease, and the percentage of saturated fat accounted for total calories in dietary showed a strong positive correlation with coronary heart disease (correlation coefficient r = 0.84). Excessive intake of saturated fatty acids in food will increase blood TC and low-density lipoprotein cholesterol (LDL-C), and promote atherosclerosis. And there are many studies indicating that abnormal blood lipids and high cholesterol diet are both important risk factors of cardiovascular diseases.
The objective of this study was to determine the effect of fucoidan on lipid modulation of hypercholesterolemic hamsters fed with high cholesterol diet, evaluation for TC, triglycerides (TG), LDL-C, high-density lipoprotein cholesterol (HDL-C) and the antioxidant ability of blood, feces and liver. The results indicated that TC ,TG, the ratio of LDL-C to HDL-C of serum, and total liver TC and TG were significant decreased after feeding with fucoidan, compared with high cholesterol group. In addition, the level of the activities of superoxide dismutase and catalase were significantly increased in the fucoidan groups (p < 0.05), as compared to those in the high cholesterol group. In conclusion, fucoidan can regulate the blood lipid of hamsters fed with high cholesterol diet.



縮寫表 i
中文摘要 iii
英文摘要 v
目錄 vii
表目錄 xii
圖目錄 xiii
第一部份 副乾酪乳酸桿菌 NTU 101 所生成降血壓物質之分離純化 1
第一章 序言 2
第二章 文獻回顧 3
ㄧ、益生菌及其應用 3
(一) 益生菌 3
(二) 乳酸菌 3
二、發酵大豆製品 7
(一) 大豆 7
(二) 大豆的營養素 7
(三) 大豆的營養醫療功效 8
三、高血壓回顧與研究 9
(ㄧ) 高血壓的定義與分類 9
(二) 降高血壓藥物治療 10
四、調節血壓的機制 10
(一) 血管收縮素I 轉換酵素 (Angiotensin I-converting enzyme, ACE) 與血壓的 調控 10
(二) 血管收縮素I 轉換酵素與血管收縮素I 轉換酵素抑制劑(Angiotensin I-converting enzyme inhibitor, ACEI)的關係 11
第三章 實驗目的及大綱 15
第四章 材料與方法 17
ㄧ、儀器 17
二、藥品 18
三、試驗方法 19
(一) 最適基質探討 19
1. 菌株來源及培養方法 19
2. 發酵豆奶與牛奶製備 19
3. 發酵豆奶水萃取之製備 19
4. 發酵豆奶醇萃取之製備 19
(二) 以反應曲面法探討最適培養條件方法 20
(三) 萃取物之純化分離 20
1. 矽膠管柱層析 20
2. Sephadex LH20層析 21
3. HPLC分析 21
(四) 體外ACE 抑制活性測定方法的改良 21
1. 藥品配置 22
2. 步驟 23
第五章 結果與討論 24
ㄧ、NTU 101 發酵乳發酵條件之最適化 24
(一) 最適基質探討 24
1. 水萃與醇萃 24
2. 發酵與未發酵 24
3. 豆漿、牛奶、75%豆漿加25%牛奶 24
4. MRS 發酵之上清液以及菌體 26
(二) 不同發酵條件對 NTU 101 發酵乳之影響 26
1. 不同豆漿濃度對 NTU 101 發酵乳中 ACEI 活性之影響 26
2. 不同起始 pH 值對 NTU 101 發酵乳中 ACEI 活性之影響 26
3. 不同接菌量對 NTU 101 發酵乳中 ACEI 活性之影響 28
4. 不同轉速對 NTU 101 發酵乳中 ACEI 活性之影響 28
5. 不同溫度對 NTU 101 發酵乳中 ACEI 活性之影響 28
6. 不同發酵時間對 NTU 101 發酵乳中 ACEI 活性之影響 28
(三) NTU 101 發酵乳於三因子三階層反應曲面法 (response surface ethodology, RSM) 之試驗 29
二、活性物質分離純化 29
三、發酵豆奶醇萃純化物之鑑定 34
四、探討發酵前後發酵豆奶有效成分的改變 45
五、Daidzein 與其他黃酮類 ACEI 功效比較 46
第六章 結論 49
第七章 參考文獻 50
第二部份 大家優藻褐藻服可醫定膠囊調節血脂之功效評估 58
第一章 序言 59
第二章 文獻回顧 60
ㄧ、代謝症候群 60
(一) 代謝症候群定義 61
(二) 代謝症候群與自由基及血脂之相關 61
二、高血脂症 62
(一) 高血脂症的定義 62
(二) 膽固醇對心血管疾病的影響 64
三、海藻 64
(ㄧ) 海藻的應用 65
(二) 岡村枝管藻 65
(三) 褐藻醣膠 66
第三章 實驗目的及大綱 70
第四章 材料與方法 72
ㄧ、儀器 72
二、藥品 72
三、試驗方法 73
(一) 高膽固醇飲食動物試驗模式系統 73
1. 試驗動物來源與飼養 73
2. 餵食劑量之計算 78
3. 飼料配方配製與投予途徑 79
(二) 動物犧牲 79
1. 動物犧牲法 79
2. 抽血法 79
3. 臟器與糞便之採集 82
(三) 調節血脂之功效評估 82
1. 血液中脂質分析 82
2. 肝組織或糞便脂質分析 83
(四) 血液中各種氧化指標測定 85
1. 脂質過氧化程度分析 85
2. 超氧歧化&;#37238; (superoxide dismutase, SOD) 含量分析 86
3. 過氧化氫&;#37238; (catalase) 含量分析 86
(五) 肝腎組織病理學檢查與判讀 88
(六) 胸腔主動脈之脂質斑塊染色與分析 88
(七) 生物統計分析方法 88
第五章 結果與討論 89
ㄧ、以褐藻醣膠對動物體內血脂調節之影響 89
(一) 血清中總膽固醇、三酸甘油酯、低密度脂蛋白膽固醇及高密度脂蛋白膽固醇之濃度 89
(二) 肝臟組織及糞便中總膽固醇、三酸甘油酯之濃度 91
二、血液中各項氧化指標之分 93
(一) 血液中脂質過氧化程度 93
(二) 血液中抗氧化酵素之活性 94
1. 超氧歧化&;#37238; (superoxide dismutase, SOD) 94
2. 過氧化氫&;#37238; (catalase) 94
(三) 心臟主動脈脂質堆積程度 98
三、肝腎組織病理學檢查與判讀 98
四、褐藻醣膠調節膽固醇動物試驗中各試驗結果討論 103
第六章 結論 105
第七章 參考文獻 106

part1
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