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

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

黎志宣(Chih-Hsaun Li)

指導教授 ： 潘子明

共同指導教授 ： 蔡宗佑(Tsung-Yu Tsai)

國立臺灣大學/生命科學院/生化科技學系/碩士(2014年)

https://doi.org/10.6342/NTU.2014.01253

摘要

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酶 (superoxide dismutase, SOD)、過氧化氫酶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。

關鍵字

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

並列摘要

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.

並列關鍵字

hypertension ； lactic acid bacteria ； angiotensin converting enzyme ； inhibitors ； optimization ； fucoidan ； hypolipidemia

參考文獻

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