臺灣博碩士論文加值系統

　　肥胖為 21 世紀的一種代謝性疾病，肥胖者長期藉由飲食控制或運動來改善體重，其效果有限，需要其它輔助治療，如利用藥物或機能性食品。本研究室已篩選出具有活性物質之酵母菌，並經由細胞及動物試驗證實酵母菌之降血糖功效，因此本研究更進一步以細胞及動物模式探討該酵母菌對血脂之影響以及抗肥胖潛力。以 3T3-L1 脂肪細胞進行活性分析，在未添加胰島素時破碎酵母菌組 (Broken yeast, BY)、酵母菌殘留物組 (Yeast residue, YR) 及酵母菌萃取物組 (Yeast extract, YE) 葡萄糖攝取量顯著高於控制組，其中以 YE 組效果為最佳，在濃度 250 μg/mL 時顯著高於控制組 7.2%，另外添加 10 nM 胰島素，各組在濃度 250 μg/mL 時即有顯著差異。油紅 O 染色結果顯示 BY 組、YR 組及 YE 組脂滴堆積情形顯著低於控制組，以 YE 組效果為最佳，於 250 μg/mL 的濃度下顯著低於控制組 17.5%。BY 組及 YE 組能顯著降低脂肪細胞中三酸甘油酯含量，YE 組於濃度大於 375 μg/mL 時，顯著低於控制組 10.9%。以 48 隻五週齡雄性 Sprague-Dawley 大鼠進行九週試驗，隨機分為 6 組：(1) 正常飲食組 (Normal control diet, NC) (n = 8)，其餘 5 組以高脂飲食誘導肥胖三週，分為 (2) 高脂飲食組 (High fat diet, HF)、(3) 破碎酵母菌組 (High fat diet + 200 mg/kg Broken yeast, BY)、(4) 酵母菌殘留物組 (High fat diet + 200 mg/kg Yeast residue, YR)、(5) 兩倍酵母菌殘留物組 (High fat diet + 400 mg/kg Yeast residue, 2YR) 及 (6) 酵母菌萃取物組 (High fat diet + 200 mg/kg Yeast extract, YE)，結果顯示，各實驗組均能降低大鼠之體重增加量及腎周圍脂肪組織，BY 組、2YR 組及 YE 組能減少血漿中 Total cholesterol (TC)、Low density lipoprotein-Cholesterol (LDL-C) 及 Alanine aminotransferase (ALT) 且能減少肝臟中 Triglyceride (TG) 的含量，BY 組及 YE 組能減少血漿中脂質過氧化物含量，YR 組及 2YR 組能減少肝臟中 TC 的含量，BY 組及 2YR 組能降低肝臟中脂質過氧化物含量，2YR 能改善葡萄糖耐受性不良及增加 High density lipoprotein-Cholesterol (HDL-C)。綜合上述結果，酵母菌可能具有抗肥胖之潛力。

Obesity is a metabolic disease in the 21st century. Obese people weight management based on diet or physical activity is often unsuccessful in the long term, most studies show weight regain. Therefore, it need adjuvant therapy, such as the use of medical drugs or natural products. The laboratory has isolated yeast with active substances and confirmed the hypoglycemic effect of yeasts through cell and animal experiments. Therefore, this study further explored the effects of yeast on blood lipids and anti-obesity potential through cell and animal models.The activity analysis by 3T3-L1 adipocytes, the results demonstrate that the glucose uptake (without insulin added) of broken yeast (BY), yeast residue (YR) and yeast extract (YE) was significantly higher than the control group, and the effect of the YE group was the best, at the concentration of 250 μg/mL, it was significantly higher than the control group 7.2%. In addition, 10 nM insulin was added, and each group had significantly higher than the control group 250 μg/mL. Oil red-O staining results showed that the BY, YR and YE were significantly lower than the control group, and the YE was the best, which was significantly lower than the control group 17.5% at 250 μg/mL. The BY and the YE significantly reduced the triglyceride content in the adipocytes, and the YE was significantly lower than the control group 10.9% at 375 μg/mL. Five-week-old male Sprague-Dawley rats (n = 48) were divided into six groups as follows: (1) Normal control diet (NC) (n = 8), and another five groups were induced with high-fat diet, divided into (2) High fat diet (HF), (3) High fat diet + 200 mg/kg Broken yeast (BY), (4) High fat diet + 200 mg/kg Yeast residue (YR), (5) High fat diet + 400 mg/kg Yeast residue (2YR) and (6) High fat diet + 200 mg/kg Yeast extract (YE), the results showed that each experimental group can reduce the weight gain and perirenal adipose tissue of rats. BY, 2YR and YE can reduce total cholesterol (TC), Low density lipoprotein-Cholesterol (LDL-C) and Alanine aminotransferase (ALT) in plasma, also can reduce the content of triglyceride (TG) in the liver. BY and YE can reduce malondialdehyde (MDA) in plasma. YR and 2YR can reduce the TC content in the liver. BY and 2YR group can reduce MDA in the liver. 2YR can improve glucose tolerance and increase High density lipoprotein-Cholesterol (HDL-C). In conclusion, the novel yeast may have the potential to antiobesity.

摘要 i
目錄 iii
圖目錄 vi
表目錄 viii
附錄 ix
壹、 前言 1
貳、 文獻整理 2
一、 肥胖 (Obesity) 2
1. 現況 2
2. 定義 2
二、 脂肪組織與肥胖之相關性 2
1. 脂肪組織 2
2. 脂肪組織與肥胖 3
三、 脂質代謝相關酵素 3
1. 荷爾蒙敏感性脂解酶 (Hormone-sensitive lipase, HSL) 3
2. 脂蛋白脂解酶 (Lipoprotein lipase, LPL) 3
3. 脂肪酸合成酶 (Fatty acid synthase, FAS) 及乙醯輔酶 A 羧化酶 (Acetyl-CoA carboxylase, ACC) 4
四、 血脂 4
1. 簡介 4
2. 脂質代謝 5
五、 酵母菌 5
1. 簡介 5
2. 細胞壁組成 5
3. 酵母菌活性物質 5
(1) 酵母菌水解物 5
(2) β-葡聚醣 6
參、 實驗設計 7
肆、 實驗材料與方法 9
一、 實驗材料 9
1. 菌株 9
2. 細胞株 9
3. 細菌培養基 9
4. 細胞培養基 9
5. 動物飼料 9
6. 實驗動物 9
7. 分析套組 10
8. 化學藥品 10
二、 儀器設備 11
三、 實驗方法 12
1. 菌株保存與活化 12
2. 菌體培養與萃取 12
3. β-葡聚醣含量分析 12
(1) 總葡聚醣測定方法 12
(2) α-葡聚醣含量分析 13
3. 3T3-L1 細胞分析模式 13
(1) 3T3-L1 細胞分化培養 13
(2) 細胞存活率 (Cell viability assay) 13
(3) 活性分析 13
(4) Oil Red-O 染色 14
(5) 細胞內 TG 含量分析 14
4. 動物試驗 14
(1) 動物分組 14
(2) 動物飼養及樣品收集 14
(3) 葡萄糖耐受性試驗 15
(4) 血漿中葡萄糖含量分析 15
(5) 血漿中 TC 含量測定 15
(6) 血漿中 TG 含量測定 16
(7) 血漿中 Lipoprotein 分離與測定 17
(8) 血漿中 AST、ALT 活性測定 17
(9) 血漿中 Creatinine 含量測定 17
(10) 血漿中 Uric acid 含量測定 18
(11) 血漿中 TBARS 含量測定 18
(12) 肝臟 TC 及 TG 含量測定 18
(13) 肝臟 TBARS 含量測定 19
(14) 肝臟酵素含量測定 19
A. ACC 含量測定 19
B. FAS 含量測定 19
(15) 脂肪組織酵素含量測定 19
A. LPL 含量測定 19
B. HSL 含量測定 20
(16) 糞便 TC 及 TG 含量測定 20
5. 統計分析 20
伍、 結果與討論 21
陸、 結論 26
柒、 參考文獻 27
實驗圖表 32
附錄 60

王勝弘。2003。酵母菌所產葡萄糖耐量因子活性及其最佳生產條件之探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
吳建輝。2011。酵母菌降血糖活性物質之生產、分離純化與作用機制探討。國立臺灣海洋大學食品科學系博士學位論文。基隆，臺灣。
陳艷琳。2014。幾丁聚醣對改善高脂飲食誘發大白鼠肥胖及脂肪肝之機制探討。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
蔡國珍，蕭連豐，劉益善。1984。啤酒酵母中葡萄糖耐量因子生物活性之強化。酒類試驗所年報 73:95-102。
衛生福利部。2016。成人健康體位標準。台北，臺灣。
鍾明娟。2010。酵母菌降血糖活性物質之萃取及其降血糖活性評估。國立臺灣海洋大學食品科學系碩士學位論文。基隆，臺灣。
Aguilar‐Uscanga B, Francois JM. (2003). A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Letters in Applied Microbiology, 37(3), 268–274.
Aoki S, Iwai A, Kawata K, Muramatsu D, Uchiyama H, Okabe M, avd Uede T. (2015). Oral administration of the Aureobasidium pullulans-derived β-glucan effectively prevents the development of high fat diet-induced fatty liver in mice. Scientific Reports, 5, 10457.
Battilana P, Ornstein K, Minehira K, Schwarz JM, Acheson K, Schneiter P, Burri J, Jéquier E, Tappy L. (2001). Mechanisms of action of β-glucan in postprandial glucose metabolism in healthy men. European Journal of Clinical Nutrition, 55(5), 327.
Carlson E, Goldford S. (1979). A sensitive enzymatic method for determination of free and esterified tissue cholesterol. Clinica Chimica Acta, 79(3), 575-582.
Caro JF, Dohm LG, Pories WJ, Sinha MK. (1989). Cellular alterations in liver, skeletal muscle, and adipose tissue responsible for insulin resistance in obesity and type II diabetes. Diabetes/Metabolism Research and Reviews, 5(8), 665-689.
Carruther M, Trinick TR, Jankowaka E, Traish AM. (2008). Are the adverse effects of glitazones linked to induced testosterone deficiency? Cardiovascular Diabetology, 7(1), 30.
Carver JD. (1994). Dietary nucleotides: cellular immune, intestinal and hepatic system effects. The Journal of Nutrition, 124(suppl_1), 144S-148S.
Casey GP, Ingledew WM. (1983). High gravity brewing: influence of pitching rate and wort gravity on early yeast viability. Journal of the American Society of Brewing Chemists, 41(4), 148-152.
Chell M. (1997). New developments in breadmaking. Food Manufacture, 72(2), 21-22.
Cohen JC, Horton JD, Hobbs HH. (2011). Human fatty liver disease: old questions and new insights. Science, 332(6037), 1519-1523.
Darlington GJ, Ross SE, MacDougald OA. (1998). The role of C/EBP genes in adipocyte differentiation. Journal of Biological Chemistry, 273(46), 30057-30060.
De Araújo TV, Andrade EF, Lobato RV, Orlando DR, Gomes NF, de Sousa RV, Pereira LJ. (2017). Effects of ꞵ‐glucans ingestion (Saccharomyces cerevisiae) on metabolism of rats receiving high‐fat diet. Journal of Animal Physiology and Animal Nutrition, 101(2), 349-358.
Donnelly KL, Smith CI, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ. (2005). Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. The Journal of Clinical Investigation, 115(5), 1343-1351.
Faust IM, Miller WH, Sclafani A, Aravich PF, Triscari J, Sullivan A. (1984). Diet-dependent hyperplastic growth of adipose tissue in hypothalamic obese rats. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 247(6), R1038-R1046.
Formiguera X, Cantόn A. (2004). Obesity: epidemiologyand clinical aspects. Best Practice and Research: Clinical Gastroenterology, 18(6), 1125-1146.
Fungwe TV, Cagen L, Wilcox HG, Heimberg M. (1992). Regulation of hepatic secretion of very low density lipoprotein by dietary cholesterol. The Journal of Lipid Research, 33(2), 179-191.
Green PHR, Tall AR, Glickman RM. (1978). Rat intestine secretes discoid HDL. Journal of Clinical Investigation, 61(2), 528-534.
Ha SK, Chae C. (2010). Inducible nitric oxide distribution in the fatty liver of a mouse with high fat diet-induced obesity. Experimental Animals, 59(5), 595-604.
Horton JD, Goldstein JL, Brown MS. (2002). SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver. The Journal of Clinical Investigation, 109(9), 1125-1131.
Jensen B, Farach-Carson MC, Erin K, Kamil AA. (2004). High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes. Experimental Cell Research, 301(2), 280-292.
Jensen-Urstad AP, Semenkovich CF. (2012). Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger? Biochimica et Biophysica Acta, 1821(5), 747-753.
Jeon T, Hwang SG, Hirai S, Matsui T, Yano H, Kawada T, Park DK. (2004). Red yeast rice extracts suppress adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 cells. Life Sciences, 75(26), 3195-3203.
Jung EY, Cho MK, Hong YH, Kim JH, Park Y, Chang UJ, Suh HJ. (2014). Yeast hydrolysate can reduce body weight and abdominal fat accumulation in obese adults. Nutrition, 30(1), 25-32.
Jung EY, Kim SY, Bae SH, Chang UJ, Choi JW, Suh HJ. (2011). Weight reduction effects of yeast hydrolysate below 10 kDa on obese young women. Journal of Food Biochemistry, 35(2), 337-350.
Kanagasabapathy G, Chua KH, Malek SNA, Vikineswary S, Kuppusamy UR. (2014). AMP-activated protein kinase mediates insulin-like and lipo-mobilising effects of β-glucan-rich polysaccharides isolated from Pleurotus sajor-caju (Fr.), Singer mushroom, in 3T3-L1 cells. Food Chemistry, 145, 198-204.
Kawano Y, Cohen DE. (2013). Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. Journal of Gastroenterology, 48(4), 434-441.
Kim KM, Chang UJ, Kang DH, Kim JM, Choi YM, Suh HJ. (2004). Yeast hydrolysate reduces body fat of dietary obese rats. Phytotherapy Research, 18(11), 950-953.
Kim MJ, Kim OH, Cheong C, Jang KH, Kim CH, Kang SA. (2015). β-Glucan from Aureobasidum species inhibits fat accumulation in 3T3-L1 adipocyte differentiation. Food Science and Biotechnology, 24(3), 1147-1150.
Klis FM, Mol P, Hellingwerf K, Brul S. (2002). Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiology Reviews, 26(3), 239-256.
Kogani G, Pajtinka M, Babincova M, Miadokova E, Rauko P, Slamenova D, Korolenko TA. (2008). Yeast cell wall polysaccharides as antioxidants and antimutagens: Can they fight cancer? Minireview. Neoplasma, 55(5), 387.
Kraus D, Yang Q, Kahn BB. (2015). Lipid Extraction from Mouse Feces. Bio-Protocol, 5, e1375.
Kroeger CM, Hoddy KK, Varady KA. (2014). Impact of weight regain on metabolic disease risk: a review of human trials. Journal of Obesity, 2014.
Lanaspa MA, Sanchez-Lozada LG, Choi YJ, Cicerchi C, Kanbay M, Roncal-Jimenez CA, Schreiner G. (2012). Uric Acid induces hepatic steatosis by generation of mitochondrial oxidative stress potential role in fructose-dependent and independent fatty liver. Journal of Biological Chemistry, 287(48), 40732-40744.
Lee I, Kim J, Ryoo I, Kim Y, Choo S, Yoo I, Bae K. (2010). Lanostane triterpenes from Ganoderma lucidum suppress the adipogenesis in 3T3-L1 cells through down-regulation of SREBP-1c. Bioorganic and Medicinal Chemistry Letters, 20(18), 5577-5581.
Li T, Owsley E, Matozel M, Hsu P, Novak CM, Chiang JY. (2010). Transgenic expression of cholesterol 7α‐hydroxylase in the liver prevents high‐fat diet–induced obesity and insulin resistance in mice. Hepatology, 52(2), 678-690.
Loeb WF, Quimby FW. (1999). The Clinical Chemistry of Laboratory Animals (Pergamon, New York).
Misra A, Khurana L. (2008). Obesity and the metabolic syndrome in developing countries. The Journal of Clinical Endocrinology and Metabolism, 93(11_supplement_1), s9-s30.
Nicolosi R, Bell SJ, Bistrian BR, Greenberg I, Forse RA, Blackburn GL. (1999). Plasma lipid changes after supplementation with β-glucan fiber from yeast. The American Journal of Clinical Nutrition, 70(2), 208-212.
Reyna NY, Cano C, Bermúdez VJ, Medina MT, Souki AJ, Ambard M, Nuñez M, Ferrer MA, Inglett GE. (2003). Sweeteners and β-glucans improve metabolic and anthropometrics variables in well controlled type 2 diabetic patients. American Journal of Therapeutics, 10(6), 438-443.
Santas J, Lázaro E, Cuñé J. (2017). Effect of a polysaccharide‐rich hydrolysate from Saccharomyces cerevisiae (LipiGo®) in body weight loss: randomised, double‐blind, placebo‐controlled clinical trial in overweight and obese adults. Journal of the Science of Food and Agriculture, 97(12), 4250-4257.
Schreuder MP, Mooren AT, Toschka HY, Verrips CT, Klis FM. (1996). Immobilizing proteins on the surface of yeast cells. Trends in Biotechnology, 14(4), 115-120.
Sell H, Berger JP, Samson P, Castriota G, Lalonde J, Deshaies Y, Richard D. (2004). Peroxisome proliferator-activated receptor γ agonism increases the capacity for sympathetically mediated thermogenesis in lean and ob/ob mice. Endocrinology, 145(8), 3925-3934.
Shepherd J, Packard CJ. (1987). Lipid transport through the plasma: the metabolic basis of hyperlipidaemia. Baillière's Clinical Endocrinology and Metabolism, 1(3), 495-514.
Shepherd PR, Kahn BB. (1999). Glucose transporters and insulin action-implications for insulin resistance and diabetes mellitus. New England Journal of Medicine, 341(4), 248-257.
Steiner G, Poapst ME, Shumak SL, Foster DM. (1986). Metabolism of the apolipoprotein B-containing lipoproteins. Methods in Enzymology, 129, 395-420.
Student AK, Hsu RY, Lane MD. (1980). Induction of fatty acid synthetase synthesis in differentiating 3T3-L1 preadipocytes. Journal of Biological Chemistry, 255(10), 4745-4750.
Silva AZ, Costa FP, Souza IL, Ribeiro MC, Giordani MA, Queiroz DA, Dekker RF. (2018). Botryosphaeran reduces obesity, hepatic steatosis, dyslipidaemia, insulin resistance and glucose intolerance in diet-induced obese rats. Life Sciences, 211, 147-156.
Silva-Sena GG, Malini M, Delarmelina JM, Dutra JCV, Gervásio SV, Leal MAS, Batitucci MDCP. (2018). In-vivo antimutagenic and antiatherogenic effects of the (1→3)(1→6)-β-D-glucan botryosphaeran. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 826, 6-14.
Tapia E, Cristóbal M, García-Arroyo FE, Soto V, Monroy-Sánchez F, Pacheco U, Madero M. (2012). Synergistic effect of uricase blockade plus physiological amounts of fructose-glucose on glomerular hypertension and oxidative stress in rats. American Journal of Physiology-Renal Physiology, 304(6), F727-F736.
Trayhurn P, Beattie JH. (2001). Physiological role of adipose tissue: white adipose tissue as an endocrine and secretory organ. Proceedings of the Nutrition Society, 60(3), 329-339.
Wang L, Behr SR, Newman RK, Newman CW. (1997). Comparative cholesterol-lowering effects of barley β-glucan and barley oil in golden syrian hamsters. Nutrition Research, 17(1), 77-88.
World Health Organization. (2018). Obesity and overweight. Retrieves February 16, 2018 from http://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight.
Wu CH, Lin HT, Wu GJ, Wang SH, Tsai GJ. (2011). Effects of cultural medium and conditions on the proliferation and hypoglycemic activity of Saccharomyces pastorianus no. 54. Journal of bioscience and bioengineering, 112(2), 159-165.