臺灣博碩士論文加值系統

目前已知大豆成分之異黃酮類具抗氧化功效，但所含豐富之蛋白質是否也具有抗氧化功效，此點值得深入探討，因此本研究目的在探討不含異黃酮類之大豆蛋白對離乳大白鼠體內抗氧化防禦系統之影響。將16隻雄性離乳SD大白鼠隨機分為兩組，餵予以AIN93G飼料配方為基礎，並以不含異黃酮類之大豆蛋白取代酪蛋白為實驗組。飼養期間紀錄其體重、攝食量及排便量，並分析兩組間蛋白質與脂質消化吸收之差異。飼養至第14天時，經12小時禁食後以斷頭方式犧牲，分析體內抗氧化防禦系統，包括：維生素C、維生素E、還原型及氧化型麩胱甘肽（GSH及GSSG），及抗氧化酵素，包括：超氧歧化酶（SOD）、觸酶（catalase）、麩胱甘肽還原酶（GR）及麩胱甘肽過氧化酶（GPX），並分析脂質過氧化物丙二醛（MDA）、心血管疾病危險因子同半胱胺酸濃度，及體內脂質之變化。結果顯示兩組之體重、攝食量及攝食效應並無差異，隨著飼養天數增加，兩組其累積體重及累積攝食量亦無差異。同時，兩組之蛋白質消化吸收率無差異，且兩組之器官重與相對器官重無差異，而大豆蛋白組脂肪消化吸收率為93.4 ± 1.2％顯著低於酪蛋白組97.0 ± 1.5％（P＜0.05），大豆蛋白組排便量顯著高於酪蛋白組（P＜0.05），可能因大豆蛋白含一種不可消化之高分子量物質可結合脂質排出。大豆蛋白組最後3天平均糞便粗脂肪為78.3 ± 10.0 mg/day顯著高於酪蛋白組62.6 ± 16.6 mg/day（P＜0.05）。在抗氧化防禦系統之結果：兩組血漿中MDA及同半胱胺酸濃度無差異，大豆蛋白組有較高之血漿維生素C濃度（P＜0.05），但兩組血漿中維生素E濃度無差異。分析紅血球之抗氧化酵素結果如下：大豆蛋白組catalase活性顯著低於酪蛋白組（P＜0.05），大豆蛋白組SOD活性為5.48 ± 0.75 U/mg Hb有低於酪蛋白組6.19 ± 0.59 U/mg Hb之趨勢（P＝0.053）。而GSH系統結果如下：兩組血漿GSH、GSSG濃度及GSH：GSSG比值皆無差異，大豆蛋白組GR與GPX活性分別為49.49 ± 13.88與254.6 ± 45.0 U/g Hb皆顯著高於酪蛋白組26.58 ± 2.88與80.3 ± 12.4 U/g Hb（P＜0.05），表示大豆蛋白可能用到GSH系統，清除自由基作用如下：GSH氧化成GSSG，再由GR轉變成GSH，重複使用於對抗自由基。同時GSH也可經由上述的作用模式將氧化的維生素C及維生素E還原，幫助其再生成為具有生理活性的抗氧化物質。大豆蛋白組肝臟中MDA濃度為30.7 ± 6.9 nmol/g liver有低於酪蛋白組35.4 ± 5.2 nmol/g liver之趨勢（P＝0.082），同時，大豆蛋白組有較高之維生素C及維生素E濃度（P＜0.05），因維生素E可清除自由基，且還原型維生素C則幫助維生素E還原，以利重複使用於對抗自由基。而抗氧化酵素catalase、SOD與GSH系統：GSH、GSSG濃度與GSH：GSSG比值及GR與GPX活性，兩組皆無差異。大豆蛋白組腦中MDA濃度顯著低於酪蛋白組（P＜0.05），維生素C濃度顯著高於酪蛋白組（P＜0.05），但兩組腦中維生素E濃度無顯著差異，顯示食用大豆蛋白之大白鼠腦中脂質過氧化物MDA產生較少。在體內脂質濃度方面，兩組血漿之三酸甘油酯、總膽固醇濃度皆無差異。大豆蛋白組肝臟三酸甘油酯濃度為288.0 ± 30.0 mg/total liver顯著低於酪蛋白組355.9 ± 80.0 mg/total liver（P＜0.05），但肝臟總膽固醇濃度無差異。而大豆蛋白組脂肪消化吸收率為93.4 ± 1.2％顯著低於酪蛋白組97.0 ± 1.5％（P＜0.05），此為食用大豆蛋白可結合排出較多糞便粗脂肪所致。
綜合以上結果得知，當給予Sprague-Dawley大白鼠大豆蛋白為蛋白質來源之AIN93G飼料時，體內有較高之維生素C濃度及較高之紅血球GR及GPX活性，因此大豆蛋白可能會對生物體內抗氧化防禦系統造成影響。

The antioxidative activity of soy protein has been attributed to soy isoflavone, while the antioxidative activity of isoflavone-free soy protein isolate has been poorly investigated. This study was to investigate the effect of isoflavone-free soy protein on antioxidant defense system in rats. Sixteen four-week-old male Sprague-Dawley rats were fed AIN-93G diets containing 20％ casein (casein group, n=8) or 20％ isoflavone-free soy protein (soy protein group, n=8) for 14 days. No significant difference in body weight, food intake, feed efficiency, cumulative food intake and cumulative body weight. There was no difference in apparent protein digestibility between two groups. There was no difference in organs weight and relative organs weight between two groups. The soy protein group had lower apparent fat digestibility. Significantly greater amounts of feces and fecal fat were excreted by rats fed the soy protein group compared with rats fed the casein group. There was no difference in MDA and homocysteine concentration of plasma between two groups, and the soy protein group had higher vitamin C concentration of plasma (P＜0.05) , while there was no difference in vitamin E concentration of plasma. The SOD activities of RBC was tended to be reduced by soy protein (P=0.053), and the soy protein group had lower catalase activities. The soy protein group had both higher GR and GPX activities in RBC. There was no difference in GSH and GSSG concentration and GSH: GSSG ratio of plasma between two groups. The soy protein may regulate the GSH system to maintain the antioxidant defense system in blood. The liver MDA concentration was tended to be reduced by soy protein (P=0.082) , and the soy protein group had both higher vitamin C and vitamin E concentration of liver (P＜0.05). The concentration of GSH, GSSG and GSH: GSSG ratio in plasma and liver were no difference between two groups. The MDA concentration in brain was lower than casein group (P＜0.05), and the soy protein group had higher vitamin C concentration in brain (P＜0.05), while there was no difference in vitamin E concentration in brain. There was no difference in triglyceride and total cholesterol of plasma between two groups. Significantly greater concentration of liver triglyceride by rats fed the soy protein group compared with rats fed the casein group. There was no difference in total cholesterol of liver between two groups.
In conclusion, when Sprague-Dawley rats were fed AIN-93G diets containing 20% isoflavones-free soy protein, there were higher concentrations in vitamin C, and higher activities in GR and GPX of RBC. Soy protein may affect on antioxidant defense system in Sprague-Dawley rats.

中文摘要..................................................i
英文摘要..................................................iii
謝誌......................................................v
目錄......................................................vi
表目錄....................................................viii
圖目錄....................................................ix
縮寫表....................................................x
第一章 前言...............................................1
第二章 文獻回顧...........................................3
第一節 自由基與氧化壓力...................................3
一、自由基及活性氧物質.................................3
二、自由基及活性氧物質對生物體之傷害...................3
三、同半胱胺酸.........................................4
第二節 生物體內抗氧化防禦系統之簡介.......................6
第三節 大豆與大豆蛋白簡介.................................10
第四節 大豆蛋白對抗氧化防禦系統之影響.....................11
第五節 大豆蛋白對脂質之影響...............................14
第六節 實驗目的...........................................16
第三章 材料與方法.........................................24
第一節 實驗設計...........................................24
第二節 抗氧化防禦系統之分析...............................24
一、維生素C...............................................24
二、維生素E...............................................26
三、還原型麩胱甘肽及氧化型麩胱甘肽........................27
四、抗氧化酵素............................................28
（一）超氧歧化酶（Superoxide dismutase；SOD）.............29
（二）觸酶（catalase）....................................31
（三）麩胱甘肽還原酶（Glutathione reductase；GR）.........31
（四）麩胱甘肽過氧化酶（Glutathione peroxidase；GPX）.....32
五、脂質過氧化物..........................................33
六、含硫化合物............................................34
第三節 脂質之分析.........................................35
一、血脂質................................................35
（一）總膽固醇（Total cholesterol；TC）...................36
（二）三酸甘油酯（Triglycerides；TG）.....................36
二、肝臟脂質..............................................36
三、糞便與飼料之脂質與蛋白質..............................37
第四節 統計分析...........................................39
第四章 結果...............................................45
第一節 生長狀況之變化.....................................45
第二節 抗氧化防禦系統之變化...............................45
第三節 脂質之變化.........................................48
第五章 討論...............................................61
第一節 生長狀況之變化.....................................61
第二節 抗氧化防禦系統之變化...............................61
第三節 脂質之變化.........................................64
參考文獻..................................................66

表目錄
表一、分離大豆蛋白之組成..................................41
表二、酪蛋白與大豆蛋白之胺基酸組成........................42
表三、AIN-93G之飼料配方...................................43
表四、AIN-93-VX 維生素飼料配方............................44
表五、餵予SD大白鼠不同蛋白質14天對其體重、攝食量及攝食效
應之影響............................................49
表六、餵予SD大白鼠大豆蛋白14天對其蛋白質與脂肪消化吸收率
之影響..............................................52
表七、餵予SD大白鼠大豆蛋白14天對其器官重與相對器官重之影
響..................................................54
表八、餵予SD大白鼠大豆蛋白14天對其體內維生素C、維生素E
及丙二醛之含量......................................55
表九、餵予SD大白鼠大豆蛋白14天對其還原型及氧化型麩胱甘肽
之含量..............................................56
表十、餵予SD大白鼠大豆蛋白14天對其抗氧化酵素活性之影響....57
表十一、餵予SD大白鼠大豆蛋白14天對其血漿含硫化合物之影響..58
表十二、餵予SD大白鼠大豆蛋白14天對其血漿及肝臟中脂質含量..59

圖目錄
圖一、脂質過氧化作用......................................18
圖二、生物體內之抗氧化防禦系..............................19
圖三、生物膜上維生素C、E、麩胱甘肽、麩胱甘肽過氧化酶及麩胱
甘肽還原酶之抗氧化機制..............................20
圖四、大豆加工食品及利用..................................21
圖五、分離大豆蛋白之一般製法..............................22
圖六、大豆蛋白影響動物體內膽固醇濃度之可能機制............23
圖七、實驗設計............................................40
圖八、餵予SD大白鼠大豆蛋白14天對其累積體重之變化..........50
圖九、餵予SD大白鼠大豆蛋白14天對其累積攝食量之變化........51
圖十、餵予SD大白鼠大豆蛋白14天對其排便量之影響............53
圖十一、餵予SD大白鼠大豆蛋白14天對其糞便脂質之影響........60

1. Roberts, W. C. (1995) Preventing and arresting coronary atherosclerosis. Am. Heart J. 130: 580-600.
2. Potter, S. M. (1995) Overview of proposed mechanisms for the hypocholesterolemic effect of soy. J. Nutr. 125: 606S-611S.
3. Tsai, P. J. & Huang, P. C. (1999) Effects of isoflavones containing soy protein isolate compared with fish protein on serum lipids and susceptibility of low density lipoprotein and liver lipids to in vitro oxidation in hamsters. J. Nutr. Biochem. 10: 631-637.
4. Fukui, K., Tachibana, N., Wanezaki, S., Tsuzaki, S., Takamatsu, K., Yamamoto, T., Hashimoto, Y. & Shimoda, T. (2002) Isoflavone-free soy protein prepared by column chromatography reduces plasma cholesterol in rats. J. Agric. Food Chem. 50: 5717-5721.
5. Damasceno, N. R. T., Goto, H., Rodrigues, F. M. D., Dias, C. T. S., Okawabata, F. S., Abdalla, D. S. P. & Gidlund, M. (2000) Soy protein isolate reduces the oxidizability of LDL and the generation of oxidized LDL autoantibodies in rabbits with diet-induced atherosclerosis. J. Nutr. 130: 2641-2647.
6. Aoki, H., Otaka, Y., Igarashi, K. & Takenaka, A. (2002) Soy protein reduces paraquat-induced oxidative stress in rats. J. Nutr. 132: 2258-2262.
7. Takenaka, A., Annaka, H., Kimura, Y., Aoki, H. & Igarashi, K. (2003) Reduction of paraquat-induced oxidative stress in rats by dietary soy peptide. Biosci. Biotechnol. Biochem. 67: 278-283.
8. Chen, H. M., Muramoto, K., Yamauchi, F. & Nokihara, K. (1996) Antioxidant activity of designed peptides based on the antioxidative peptide isolated from digests of a soybean protein. J. Agric. Food Chem. 44: 2619-2623.s
9. Thomas, M. J. (1995) The role of free radicals and antioxidants: how do we know that they are working? Crit. Rev. Food Sci. Nutr. 35: 21-39.
10. McLaveu, D. S.（1996）Dietary antioxidants and disease. Med. Digest. 14: 5-10.
11. Halliwell, B., Evans, P., Kaur, H. & Aruoma, O. Free radicals, tissue injury, and human disease: a potential for therapeutic use of antioxidants? In: Kinny, J. M. and Tuck, H. M., eds. Organ Metablism and Nutrition: Ideals for future critical care. New York: Raven Press, 1994: 425-445.
12. Stadtman, E. R. (1992) Protein oxidation and aging. Science 257: 1220-1224.
13. Gutteridge, J. M. C. & Halliwell, B. (1990) The measurement and mechanism of lipid peroxidation in biological systems. Trends. Biochem. Sci. 15: 129-135.
14. Kang, S. S., Wong, P. W. & Malinow, M. R. (1992) Hyperhomocyst(e)inemia as a risk factor for occlusive vascular disease. Annu. Rev. Nutr. 12: 279-98.
15. Perna, A. F., Ingrosso, D. & De Santo, N. G. (2003) Homocysteine and oxidative stress. Amino Acids. 25: 409-417.
16. Berman, R. S. & Martin, W. (1993) Arterial endothelial barrier dysfunction: actions of homocysteine and the hypoxantine-xanthine oxidase free radical generating system. Br. J. Pharmacol. 108: 920–926.
17. El-Khairy, L., Ueland, P. M., Refsum, H., Graham, I. M. & Vollset, S. E. European Concerted Action Project (2001) Plasma total cysteine as a risk factor for vascular disease: The European Concerted Action Project. Circulation 103: 2544-2549.
18. Blom, H. J. (2000) Consequences of homocysteine export and oxidation in the vascular system. Semin. Thromb. Hemost. 26: 227-232.
19. Chambers, J. C., McGregor, A., Jean-Marie, J. & Kooner, J. S. (1998) Acute hyperhomocysteinaemia and endothelial dysfunction. Lancet 351: 36-37.
20. Bellamy, M. F., McDowell, I. F., Ramsey, M. W., Brownlee, M., Bones, C., Newcombe, R. G. & Lewis, M. J. (1998) Hyperhomocysteinemia after an oral methionine load acutely impairs endothelial function in healthy adults. Circulation 98: 1848–1852.
21. Upchurch, G. R., Welch, G. N., Fabian, A. J., Freedman, J. E., Johnson, J. L., Keaney, J. F. & Loscaloz, J. (1997) Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. 272: 17012-17017.
22. Fang, Y. Z., Yang, S. & Wu, G. (2002) Free radicals, antioxidants, and nutrition. Nutrition 18: 872-879.
23. Feri, B. (1991) Ascorbic acid protects lipids in human plasma and low density lipoprotein against oxidative damage. Am. J. Clin. Nutr. 54: 1113S-1118S.
24. Burton, G. W. & Ingold, K. U. (1990) Mechanisms of antioxidant action: preventive and chain-breaking antioxidants. In: Miguel, A., Quintanilha, A. T. and Weber, H. (ed). Handbook of free radicals and antioxidants in biomedicine. CRC. Press. Boca. Raton, F. L. pp. 29-43.
25. Shan, X. Q., Aw, T. Y. & Jones, D. P. (1990) Glutathione-dependent protection against oxidative injury. Pharmacol. Ther. 47: 61-71.
26. Packer, L. (1991) Protective role of vitamin E in biological systems. 53: 1050S-1055S.
27. Fishwe, A. B. (1998) Intracellular production of oxygen derived free radicals. In: Halliwell, B (ed). Free radicals and tissue injury. Proc. Upjohn. Sympo. F. A. S. E. B. Press, Rockville, Bethesda, M. D. pp. 34-42.
28. Fridovich, I. (1975) Superoxide dismutase. Annu. Rev. Biochem. 44: 147-159.
29. Cohen, G. & Hochstein, P. (1963) Glutathione peroxidase: the primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 2: 1420-1428.
30. 賴滋漢、賴業超 （1994）食品科技辭典，p1372-1374，富林出版社，台中市。
31. Bishov, S. J. & Henick, A. S. (1972) Antioxidant effect of protein hydrolysates in a freeze-dried model system. J. Food Sci. 37: 873-875.
32. Bishov, S. J., Henick, A. S. (1975) Antioxidant effect of protein hydrolysates in freeze-dried model system. Synergistic action with a series of phenolic antioxidants. J. Food Sci. 40: 345-348.
33. Yamaguchi, N., Yokoo, Y. & Fujimaki, M. (1975) Studues on antioxidative activities of amino compounds on fat and oils. Part III. Antioxidative activities of soybean protein hydrolysate and synergistic effect of hydrolyzate on tocopherol. Nippon Shokuhin Kogyo Gakkaishi 22: 431-435.
34. Yamaguchi, N., Yokoo, Y. & Fujimaki, M. (1979) Antioxidative activities of protein hydrolyzates. Nippon Shokuhin Kogyo Gakkaishi 26: 65-70.
35. Yamaguchi, N., Naito, S., Yokoo, Y. & Fujimaki, M. (1980) Oxidative stability of dried model food consisted of soybean protein hydrolyzate and lard. Nippon Shokuhin Kogyo Gakkaishi 27: 51-55.
36. Yee, J. J., Shipe, W. F. & Kinsella, J. E. (1980) Antioxidant effects of soy protein hydrolysates on copper-catalyzed methyl linoleate oxidation. J. Food Sci. 45: 1082-1083.
37. Tsuge, N., Eikawa, Y., Yamamoto, M. & Sugisawa, K. (1991) Antioxidative activity of peptides prepared by emzymatic hydrolysis of egg-white albumin. Nippon Nogei Kagaku Kaishi 65: 1635-1641.
38. Reeves, P. G., Nielsen, F. H. & Fahey, G. C. Jr. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123: 1939–1951.
39. Mitton, K. P. & Trevithick, J. R. (1994) High-performance liquid chromatography-electrochemical detection of antioxidants in vertebrate lens: glutathione, tocopherol, and ascorbate. Methods Enzymol. 233: 523-539.
40. Nierenberg, D. W. & Nann, S. L. (1992) A method for determining concentrations of retionol, tocopherol, and five carotenoids in human plasma and tissue samples. Am. J. Clin. Nutr. 56: 417-426.
41. Hissin, P. J. & Hilf, R. (1976) A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74: 214-226.
42. Aebi, H. (1984) Catalase in vitro. Methods in Enzymology. 105: 121-126.
43. Goldberg, D. M. & Spooner, R. J. (1983) In methods of enzymatic analysis: In: Bergmeten, H. V. (ed). pp: 258-265.
44. Paglia, D. E. & Valentine, W. N. (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70: 158-169.
45. Tatum, V. L., Changchit, C. & Chow, C. K. (1990) Measurement of malondialdehyde by high performance liquid chromatography with fluorescence detection. Lipids 25: 226-229.
46. Durand, P., Fortin, L. J., Lussier-Cacan, S., Davignon, J. and Blache, D. (1996) Hyperhomocysteinemia induced by folic acid deficiency and methionine load-applications of a modified HPLC method. Clinica. Chimica. Acta. 252: 83-96.
47. Richmond, N. (1973) Preparation and properties of a cholesterol oxidase from nocardia sp. and its application to the enzymatic assay of total cholesterol in serum. Clin. Chem. 19: 1350-1356.
48. Roeschlau, P., Bernt, E. & Gruber, W. J. (1974) Clin. Chem. Clin. Biochem. 12: 403.
49. Trinder, P. (1969). Determination of glucose in blood using glucose oxidase with an alternative oxygen acceptor. Ann. Clin. Biochem. 6: 24.
47. Folch, J., Lees, M. & Stanel, G. S. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226: 497-509.
48. AOAC. (1995) Meat and meat products. In: CunniffP, editor. Official methods of analysis of AOAC International. 16th ed. Washington, D.C. AOAC International. pp. 1-23.
49. Kanauchi, O., Deuchi, K., Imasato, Y. & Kobayashi, E. (1994) Increasing effect of a chitosan and ascorbic acid mixture on fecal dietary fat excretion. Biotech. Biochem. 58: 1617-1620.
50. Deuchi, K., Kanauchi, O., Imasato, Y. & Kobayashi, E. (1995) Effect of the viscosity or deacetylation degree of chitosan on fecal fat excreted. Biosci. Biotech. Biochem. 59: 781-785.
51. Sugano, M. & Goto, S. (1990) Steroid-binding peptides from dietary proteins. J. Nutr. Sci. Vitaminol. 36: 147S-150S.
52. Thorburne, S.K. & Juurlink, B.H. (1996) Low glutathione and high iron govern the susceptibility of oligodendroglial precursors to oxidative stress. J. Neurochem. 67: 1014-1022.
53. Madani, S., Lopez, S., Blond, J. P., Prost, J. & Belleville, J. (1998) Highly purified soybean protein is not hypocholesterolemic in rats but stimulates cholesterol synthesis and excretion and reduces polyunsaturated fatty acid biosynthesis. J. Nutr. 128: 1084-1091.
54. Iritani, N., Suga, A., Fukuda, H., Katsurada, A. & Tanaka, T. (1988) Effects of dietary casein and soybean protein on triglyceride turnover in rat liver. J. Nutr. Sci. Vitaminol (Tokyo) 34: 309-315.
55. Wang, Y., Jone, P. J. H., Ausman, L. M. & Lichtenstein, A. H. (2004) Soy protein reduces triglyceride levels and triglyceride fatty acid fractional synthesis rate in hypercholesterolemic subjects. Atherosclerosis 173: 269-275.