臺灣博碩士論文加值系統

本研究以Aspergillus oryzae分生孢子為種麴接種於蒸熟大豆與稻米培養製備豆麴與米麴後作為酵素源，比較豆麴和米麴之β-Glucosidase;活性，顯示豆麴為β-葡萄糖苷酶之良好酵素源。將豆麴粉與黃豆粉(w/w, 1:4)混合，分別加入兩倍體積(v/w)之0、15、30、45及60%酒精溶液，於30℃下培養0、1、3、7天，定期取樣進行萃取及以HPLC分析定量異黃酮素之含量，進而計算其水解率作為評估β-Glucosidase;活性之依據。結果顯示水解率皆隨培養時間之延長而提高，在7天之培養期間，以0%酒精濃度培養者於第1天其水解率即達92.1%、15%酒精濃度培養者則於第3天達92.7%，至於在30、45及60%酒精濃度培養者，經7天之培養，其水解率分別為84.4、58.0及29.7%，顯示在高於30%酒精濃度之條件下，β-葡萄糖苷酶之水解活性明顯受到影響。將上述黃豆粉與豆麴粉混合後添加15%酒精並於30℃之條件培養，分別於5、10、20、30及60分鐘時取樣分析異黃酮成分，並測定β-Glucosidase;之水解活性，其水解率分別為15、41、59、71、75及76%。利用10倍體積水萃取酵素液進行特性探討時，粗酵素液之β-Glucosidase;可以4℃條件保持穩定之活性；在熱安定性試驗中，於37℃之條件其β-Glucosidase;之水解活性亦可維持60分鐘不變化，進而以粗酵素液進行硫酸銨區分、離子交換樹脂及膠體過濾層析分離純化β-Glucosidase;，結果顯示膠體過濾層析時，純化效果受管柱長度、膠體材質(Sephacryl S-200或Sephacryl S-300)及管柱銜接順序之影響，經純化後可分離酵素1與酵素2兩種同功異構酶，酵素1特性：最適反應溫度為40℃；在60℃保持5分鐘時活性會明顯下降；葡萄糖耐受性為2g/l以上時其活性明顯受抑制；儲存於10%酒精以上之條件時其活性隨時間之增加而下降；最適反應pH值為5.5-6.0；Km值為1.31；經native-PAGE電泳分析其分子量為192KDa，SDS-PAGE電泳分析其分子量為95KDa。酵素2之特性：最適反應溫度為30℃；在40℃放置5分鐘其活性會明顯受影響；葡萄糖耐受性於16g/l時其活性仍不受影響；儲存於10%酒精時其活性穩定，但隨酒精濃度增加則有下降趨勢；最適反應pH值6.0；Km值為2.92；經native-PAGE電泳分析其分子量約為60KDa，SDS-PAGE電泳分析其分子量約為50KDa。

Soybeans were soaked with water for 4h, steam-cooked, inoculated with the conidia of Aspergillus oryzae and incubated 3 days for koji preparation and used as an enzyme source. When a series of the pulverized koji were mixed with soybean flour (1:4, w/w) and replenished with 2 volumns (v/w) of aqueous solutions containing 0, 15, 30, 45 and 60% ethanol and incubated at 30℃ for 7 days, hydrolysis of daidzin and genistin increased with an increase of incubation time while decreased with an increase of ethanol concentration. After incubation with 0% ethanol for 1 day, 92.1% of daidzein and genistein were hydrolyzed. After incubation with 15% ethanol for 3 days, 92.7% of daidzin and genistin were hydrolyzed. After incubation with 30, 45 and 60% ethanol for 7 days, the hydrolysis percentages were 84.4, 58.0 and 29.7%, respectively. When the above mixtures were replenished with 15% ethanol and incubated at 30℃ for 0, 5, 10, 20, 30 and 60 min, isoflavone hydrolysis percentages were 15, 41, 59, 71, 75 and 76%, respectively. The pulverized koji was then homogenized, extracted with 10 volumes (v/w) of water and centrifuged to obtain the supernatant as crude enzyme extract. The crude enzyme extract was stable for storage at 4℃ and its β-glucosidase activity did not change after storage at 37℃ for 60min. When the crude enzyme extract was subjected to separation and purification with ammonium sulfate precipitation, DEAE-cellulose ion exchange and gel filtration chromatography, two isoforms of β-glucosidase (1 and 2) were obtained. The chromatographic resolution during gel filtration was affected by column length, packing medium (Sephacryl S-200 or Sephacryl S-300) and sery of column connection. Characteristics of the purified enzyme 1 were : optimal reaction pH was at 5.5-6.0, optimal temperature was at 40℃, Km was 1.31mM, was thermostable at 60℃ stored for 5min, was glucose tolerant up to 2g/l, and was unstable at 10% ethanol for storage. In native- and SDS-PAGE analysis, the estimated molecular weights were 180 KDa and 95 KDa, respectively. Characteristics of the purified enzyme 2 were: optimal reaction pH was at 6.0, optimal reaction temperature was at 30℃, Km was 2.92 mM, was thermostable at 40℃ stored for 5min, was glucose tolerant up to 16.7g/l, and stable for storage at 10% ethanol. In native- and SDS-PAGE analyses, the estimated molecular weights were 60 KDa and 50 KDa, respectively.

目 次
中文摘要------------------------------------------------------Ⅰ
英文摘要------------------------------------------------------Ⅲ
表次--------------------------------------------------------- XI圖次---------------------------------------------------------XII
第一章前言-----------------------------------------------------1
第二章文獻回顧
2.1大豆之介紹--------------------------------------------------3
2.2大豆之營養成分----------------------------------------------4
2.3大豆異黃酮素之種類------------------------------------------7
2.4大豆之異黃酮素----------------------------------------------9
2.5大豆主要異黃酮素的物化特性---------------------------------12
2.6大豆異黃酮素之吸收代謝-------------------------------------14
2.7大豆異黃酮素之機能特性-------------------------------------17
2.8β-葡萄糖苷酶之介紹----------------------------------------25
第三章材料與方法
3.1材料
3.1.1原料-----------------------------------------------------29
3.1.2層析管---------------------------------------------------29
3.1.3標準蛋白-------------------------------------------------29
3.1.4重要化學藥劑---------------------------------------------30
3.1.5儀器-----------------------------------------------------31
3.2實驗方法
3.2.1豆麴之製備及特性探討-------------------------------------32
3.2.2異黃酮分析與β-葡萄糖苷酶粗酵素活性測定------------------33
3.2.2.1異黃酮之萃取-------------------------------------------33
3.2.2.2 HPLC分析條件------------------------------------------33
3.2.2.3β-葡萄糖苷酶活性分析----------------------------------34
3.2.3豆麴β-葡萄糖苷酶粗酵素液之生化特性探討------------------34
3.2.4豆麴β-葡萄糖苷酶之純化分離------------------------------36
3.2.5膠體過濾層析純化酵素之生化特性探討-----------------------38
3.2.6豆麴純化β-葡萄糖苷酶電泳檢定----------------------------39
3.3統計分析---------------------------------------------------43
第四章結果與討論
4.1豆麴之製備及特性探討
4.1.1豆麴、米麴之β-葡萄糖苷酶活性之比較----------------------44
4.1.2酒精濃度對β-葡萄糖苷酶水解異黃酮糖苷活性之影響----------46
4.1.3大豆粉與豆麴粉於15%酒精濃度下之異黃酮糖苷水解活性-------48
4.2豆麴β-葡萄糖苷酶粗酵素液之生化特性探討
4.2.1豆麴β-葡萄糖苷酶粗酵素液之反應曲線----------------------50
4.2.2豆麴β-葡萄糖苷酶粗酵素液之最適反應溫度------------------50
4.2.3豆麴β-葡萄糖苷酶粗酵素液之熱安定性----------------------50
4.2.4豆麴β-葡萄糖苷酶粗酵素液之最適pH值----------------------54
4.2.5豆麴β-葡萄糖苷酶粗酵素液之葡萄糖耐受性試驗--------------54
4.2.6豆麴β-葡萄糖苷酶粗酵素液於不同酒精濃度及溫度條件下之
安定性--------------------------------------------------------54
4.3豆麴β-葡萄糖苷酶之純化分離
4.3.1陰離子交換與膠體過濾管柱層析-----------------------------58
4.3.2不同膠體過濾層析管柱之純化結果比較-----------------------60
4.4膠體過濾層析純化酵素之生化特性探討
4.4.1純化β-葡萄糖苷酶1和2之最適反應溫度------------------62
4.4.2純化β-葡萄糖苷酶 1和2之熱安定性-------------------------62
4.4.3純化β-葡萄糖苷酶 1和2之最適pH值-------------------------67
4.4.4純化β-葡萄糖苷酶 1和2之葡萄糖耐受性試驗-----------------67
4.4.5酒精濃度對純化β-葡萄糖苷酶 1和2活性之影響---------------72
4.4.6純化β-葡萄糖苷酶1和2之Km值------------------------------72
4.4.7純化β-葡萄糖苷酶1和2之水解異黃酮糖苷活性探討------------77
4.4.8純化β-葡萄糖苷酶1和2水解異黃酮糖苷之HPLC圖譜分析--------77
4.4.9水解異黃酮素後葡萄糖含量測定----------------------------81
4.5豆麴β-葡萄糖苷酶電泳分析
4.5.1 SDS-PAGE------------------------------------------------83
4.5.2 Native-PAGE---------------------------------------------83
第五章結論----------------------------------------------------88第六章參考文獻------------------------------------------------90
表 次
Table 1. Isoflavone contents in soybean products and fermented
soybean products---------------------------------------5
Table 2. Formula of the separation and stacking gels used for
SDS- and Native-PAGE analyses-------------------------42
Table 3. Purification and specific activity of β-glucosidase 1
and 2 extracted from soybean koji---------------------85
圖 次
Figure 1. Coronary heart disease death rates in six countries
in 1950-92 of men aged 25-64-------------------------6
Figure 2. Proposed scheme for the formation of 8-OHD and 8-OHG-8
Figure 3. Structures of the isoflavone family of phytoestroges-1-------------------------------------------------------------10
Figure 4. The structures of each isoflavones------------------11
Figure 5. Metabolism of isoflavone in humans------------------16
Figure 6. Chemical structures of estrone, estradiol, genistein,
genistin,daidzein, and daidzin-----------------------19
Figure 7. Scheme for the liberation of daidzein and genistein
from daidzin and genistin----------------------------28
Figure 8. The β-glucosidase activity of steamed soybean and
rice inoculated with the conidia of Aspergillus oryzae
and subsequent incubation----------------------------45
Figure 9. Effect of ethanol on the hydrolytic activity of
isoflavones in the soybean flour mixed with koji flour
for incubation---------------------------------------47
Figure 10. The hydrolytic activity of β-glucosidases extracted
from soybean and koji with 15% ethanol---------------49
Figure 11. Time curve of the crudeβ-glucosidases extracted
from soybean koji------------------------------------51
Figure 12. Effect of temperature on the crude β-glucosidases
extracted from soybean koji--------------------------52
Figure 13. Termostability of the crudeβ-glucosidases extracted
from soybean koji------------------------------------53
Figure 14. The optimal reaction pH of the crude β-glucosidases
extracted from soybean koji--------------------------55
Figure 15. Glucose tolerance of the crudeβ-glucosidases
extracted from soybean koji--------------------------56
Figure 16. The stability of the β-glucosidases in 10% or 20%
ethanol stored at room temperature and 4℃-----------57
Figure 17. Fractionation of the purified β-glucosidase 1 and 2
by DEAE-Cellulose and Sephacryl S-300 + Sephacryl S-
200 HR column----------------------------------------59
Figure 18. Chromatograms of various gel filtration systems----61
Figure 19. Activity of the purified β-glucosidase 1 as
affected by temperature------------------------------63
Figure 20. Activity of the purified β-glucosidase 2 as
affected by temperature------------------------------64
Figure 21. Termostability of the purified β-glucosidase 1----65
Figure 22. Termostability of the purified β-glucosidase 2----66
Figure 23. Activity of the purified β-glucosidase 1 as
affected by pH---------------------------------------68
Figure 24. Activity of the purified β-glucosidase 2 as
affected by pH---------------------------------------69
Figure 25. Glucose tolerance of the purified β-glucosidase 1-70
Figure 26. Glucose tolerance of the purified β-glucosidase 2-71
Figure 27. Activity of the purified β-glucosidase 1 as
affected by ethanol---------------------------------73
Figure 28. Activity of the purified β-glucosidase 2 as
affected by ethanol --------------------------------74
Figure 29. The Km for the purified β-glucosidase 1-----------75
Figure 30. The Km for the purified β-glucosidase 2-----------76
Figure 31. Hydrolysis of isoflavones in the soybean flour by
the purified β-glucosidase 1 and 2-----------------78
Figure 32. Hydrolysis of isoflavones in the soybean flour by
the purified β-glucosidase 1----------------------79
Figure 33. Hydrolysis of isoflavones in the soybean flour by
the purified β-glucosidase 2----------------------80
Figure 34. Glucose contents in the soybean flour suspension
after incubation with soybean koji for isoflavone
hydrolysis--82
Figure 35. SDS-PAGE protein patterns--------------------------86
Figure 36. Native-PAGE protein patterns-----------------------87

第六章 參考文獻
江文德. 1998. 簡介大豆中異黃酮素. 食品工業. 30(9): 6-12.
吳鳴鈴，發酵大豆食品及其健康機能性，財團法人食品工業發產研究所
菌種保存及研究簡訊，第41期，第13卷第3期，1-5。
余碧. 曾浩洋. 1996. 不同生長階段家兔小腸β-Galactosidase;及β-
Glucosidase;之膠體過濾層析比較. 中國畜牧學會會誌 25(1): 67-74.
余碧，曾浩洋. 1995. 家兔腸道β-Galactosidase;及β-Glucosidase;之
純化與特性探討. 中國農業化學會會誌 33(6): 724-730.
邱義源. 2001. 迎合保健時代的傳統發酵食品. 台北國際食品展專刊.
75-78.
高嘉鴻. 1998. 加工條件對大豆製品異黃酮含量之影響及以單細胞電泳法
探討其哺乳類細胞DNA損傷影響之研究. 國立中興大學食品科學碩士論
文.
陳昶宇. 2000. 富含異黃酮素大豆食品之製造方法. 國立台灣大學食品科
技所碩士論文.
許玉妹. 曾夢蛟. 林金和. 2002. 淹水逆境下蓮霧根部酒精脫氫酵素同功
異構酵素之純化與生化特性. 台灣農業化學與食品科學 40(1): 19-27.
張惠琴. 2000. 不同計量之大豆異黃酮素對大白鼠乳腺腫瘤之影響. 靜宜
大學食品營養系碩士論文.
戴蔭方. 劉成軍. 張超良. 曹慶榮. 李保真. 1995. 藥用蔬果. 度假出版
社 86-90.
葉娟美. 楊明哲. 蔡英傑. 2002. 簡化重組subtilisin YaB純化方法之探
討. 台灣農業化學與食品科學 40(1): 45-54.
楊開聰. 2000. 黑豆發芽期間抗氧化效力及異黃酮含量變化之探討. 中國
文化大學生活應用科學系碩士論文.
Adlercreutz, H., Honjo, H., Higashi, A., Fotsis, T.,
Hämäläinen, E., Hasegawa, T., and Okada, H. 1991. Urinary
excretion of lignans and isoflavonid phytoestrogens in
Japanese men and women consuming a traditional Japanese diet.
Am. J. Clin. Nutr. 54: 1093-1100.
Adlercreutz, H., Bannwart, C., Wahala, K., Makela, T., Brunow,
G., Hase, T., Arosemena, P. J., Kellis, J. T., and Vickery,
L. E. 1993. Inhibition of human aromatase by mammalian
lignans and isoflavonoid phytoestrogens. J. Steroid Biochem.
Mol. Biol. 44: 147-153.
Adlercreutz, H., Mousavi, Y., Clark, J., Hockerstedt, K., and
Hase, T. 1992. Dietary phytoestrogens and cancer: in vivo and
in vitro studies. J. Steroid Biochem. Mol. Biol. 41: 331-337.
Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe,
S. I., Itoh, N., Shibuya, M., and Fukami, Y. 1987. Genistein,
a specific inhibitor of tyrosine-specific protein kinase. J.
Biol. Chem. 262: 5592-5595.
Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe,
S. I., and Itoh, N. 1987. Genistein, a specific inhibitor of
tyrosine specific protein kinase. J. Biol. Chem. 262: 5592-
5595.
Axelson, M., and Stechell, K. D. R. 1981. The excretion of
lignans in rats-evidence for an intestinal bacterial source
for this new group of compounds. FEBS Letters 123: 337-342.
Axelson, M., Sjovall, J., Gustafsson, B. E., and Setchell, K.
D. R. 1984. Soya - a dietary source of the non-steroidal
estrogen equol in man and animals. J. Endocrinol. 102: 49-56.
Baggott, J. E., Ha, T., Vaughn, W. H., Juliana, M. M., Hardin,
J. M., and Grubbs, C, J. 1990. Effect of miso (Japanese
soybean paste) and NaCl and DMBA-induced rat mammary tumors.
Nutr. Cancer 14: 103-109.
Barnes, S., Peterson, T. G., and Coward, L. 1995. Rationale for
the use of genistein-containing soy matrixes in
chemoprevention trials for breast and prostate cancer. J.
Cell Biochem. Suppl. 22: 181-187.
Bertrand, R., Kerrigan, D., Sarang, M., and Pommier, Y. 1991.
Cell death induced by topoisomerase inhibitors: role of
calcium in mammalian cells. Biochem. Pharmacol. 42: 77-85.
Booth, C., Hargreaves, D. F., O’Shea, J. A., and Potten, C. S.
1999. In vivo adiministration of genistein has no effect on
small intestinal epithelial proliferation and apoptosis, but
a modest effect on clonogen survival. Cancer Letters 144: 169-
175.
Brown, J. P. 1988. Hydrolysis of glycosides and esters. In:
Role of the Gut Flora in toxicity and cancer (Rowland, I. R.,
ed.), pp. 109-144. Academic Pree, San Diego, CA.
Cai, Q., and Wei, H. 1996. Effect of dietary genistein on
antioxidant enzyme activities in SENCAR mice. Nutr. Cancer
25: 1-7.
Chen, Y., Liu, X., Pisha, E., Constantinou, A. I., Hua, Y.,
Shen, L., Breemen, R. B. V., Elguindi, E. C., Blond, S. Y.,
Zhang, F., and Bolton J. L. 2000. A metabolite of equine
estrogens, 4-hydroxyequilenin, induces DNA damage and
apoptosis in breast cancer cell lines. Chem. Res. Toxicol.
13: 342-350.
Chevion, M. 1988. A site-specific mechanism for free radical
induced biological damage: The essential role of redox-active
transition metals. Free Radic. Biol. Med. 5: 27-37.
Chiou, R. Y. -Y. and Cheng, S. L. 2001. Isoflavone
transformation during soybean koji preparation and subsequent
miso fermentation supplemented with ethanol and NaCl. J.
Agri. Food Chem. 49(8): 3656-3660.
Chiou, R. Y. -Y. 1999. Salt-free fermentation of miso using
ethanol, sugars and polyols. J. Food Sci. 64: 918-920.
Chiou, R. Y. -Y., Ku, K. -L., Ferng, S., and Beuchat, L. R.
1999. Fermentation of low-salt miso as affected by
supplementation with ethanol. Int. J. Food Microbiol. 48: 11-
20.
Chiou, R. Y. -Y., Ku, K. -L., Lai, Y. -S., and Chang, L. -G.
2001. Antioxidative characteristics of oils in ground pork-
fat patties cooked with soy sauce. JAOCS 78: 7-11.
Coward, L., Barnes, N. C., Setchell, K. D. R., and Barnes, S.
1993. Genistein, daidzein, and β-glycoside conjugates:
antitumor isoflavones in soybean foods from American and
Asian diets. J. Agric. Food Chem. 41: 1961-1967.
Devary, Y., Gottlieb, R. A., Smeal, T., and Karin, M. 1992. The
mammalian ultraviolet respone is triggered by activation of
src tyrosine kinase. Cell 71: 1081-1091.
Duthie, S. J., Johnson, W., and Dobson, V. L. 1997. The effect
of dietary flavonoids on DNA damage (strand breaks and
oxidized pyrimdines) and growth in human cells. Mutat. Res.
390: 141-151.
Donn, J. -E. Jr. 1975. Cancer epidemiology in populations of
the United States-with emphasis on Hawaii and California-and
Japan. Cancer res. 35: 3240-3245.
Dizdaroglu, M. 1992. Oxidative damage to DNA in mammalian
chromatin. Mutat. Res. 275: 331-324.
Esaki, H.; Watanabe, R.; Onozaki, H.; Kawakishi, S., and Osawa,
T. 1999. Formation mechanism for potent antioxidative o-
dihydroxyisoflavones in soybeans fermented with Aspergillus
saitoi. Biosci. Biotechnol. Biochem. 63 (5): 851-858.
Esaki, H.; Watanabe, R.; Onozaki, H.; Kawakishi, S., and Osawa,
T. 1998. Potent antioxidative isoflavones isolated from
soybeans fermented with Aspergillus saitoi. Biosci.
Biotechnol. Biochem. 62 (4): 740-746.
Fairbairn, D. W., Olive, P. L., and O’Neill, K. L. 1995. The
comet assay: a comprehensive review. Mutat. Res. 339: 37-59.
Farmakalidis, E., and Murphy, P. A. 1985. Isolation of 6’-O-
acetyldaidzein and 6’-O-acetylgenistein from toasted
defatted soy flakes. J. Agric. Food Chem. 33: 385-389.
Fotsis, T., Pepper, M., Adlercreutz, H., Fleischmann, G., Hase,
T., Montesano, R., and Schweigerer, L. 1993. Genistein, a
dietary-derived inhibitor of in vitro angiogenesis. Proc.
Natl. Acad. Sci. 90: 2690-2694.
Fotsis, T., Pepper, M., Adlercreutz, H., Hase, T., Montesano,
R., and Schweigerer, L. 1995. Genistein, a dietary ingested
isoflavonoid, inhibits cell proliferation and in vitro
angiogenesis. J. Nutr. 125: 790S-797S.
Frey, R.S., Li J., and Singletary, K.W. 2001. Effects of
genistein on cell proliferation and cell cycle arrest in
nonneoplastic human mammarymepithelial cells: involvement of
Cdc2, p21waf/cip1, p27kip1, and Cdc25C expression. Biochem.
Pharmacol. 61: 979-989.
Fukutake, M., Takahashi, M., Ishida, K., Kawamura, H.,
Sugimura, T., and Wakabayashi, K. 1996. Quantification of
genistein and genistin in soybeans and soybean products. Food
Chem. Toxic. 34: 457-461.
Giri, A. K., and Lu, L. —J. W. 1995. Genetic damage and the
inhibition of 7, 12-dimethylbenz[a]anthraceneinduced genetic
damage by the phytoestrogen, genistein and daidzein, in
female ICR mice. Cancer Letters 95: 125-133.
Goldin, B. R., Adlercreutz, H., Gorbach, S. L., Woods, M. N.,
Dwyer, J. T., Conlon, T., Bohn, E., and Gershoff, S. N. 1986.
The relationship between estrogen and diets of Caucasian
American and Oriental immigrant women. Am. J. Clin. Nutr. 44:
945-953.
Gorbach, S. L., Plaut, A. G., Nahas, L., Weinstein, L.,
Spanknebel, G., and Levitan, R. 1967. Studies of intestinal
microflora Ⅱ-microorganisms of the small intestine and their
relations to oral and faecal flora. Gastroenterology 53: 856-
867.
Günata, Z., and Vallier, M. J. 1999. Production of a highly
glucose-tolerant extracellularβ-glucosidase by Aspergillus
strains. Biotech. Letters 21: 219-223.
Gyorgy, P., Maurata, K., and Ikehara, H. 1964. Antioxidants
isolated from fermented soybeans (Tempeh). Nature 203: 870.
Hawksworth, G., Drasar, B. S., and Hill, M. J. 1971. Intestinal
bacteria and the hydrolysis of glycosidic bonds. J. Med.
Microbiol. 4: 451-459.
Hendrich, S., Lee, K. W., Xu, X., Wang, H. J., and Murphy, P.
A. 1994. Defining food components as new nutrients. J. Nutr.
124: 1789s-1792s.
Heyworth, R., and Walkwe, P.G. 1962. Almond-emulsion β-D-
glucosidase and β-D-galactosidase. Biochem. J. 83: 331-336.
Hsu, W. C, Cho, P. J., Wu, M. J., and Chiou, R. Y. Y. 2002. A
rapid and small-scale method for estimating antioxidative
potency of peanut sprouts. (in press)
Ikeda, R., Ohta, N., and Watanabe, T. 1995. Changes of
isoflavones at various stages of fermentation in defatted
soybeans. Nippon Shokuhin Kagaku Kogaku Kaishi 42(5): 322-327.
Kapiotis, S., Hermann, I., Held, C. S., Ehringer, H., and
Gmeiner, B. M. K. 1997. Genistein, the dietary-derived
angiogenesis inhibitor, prevents LDL oxidation and protects
endothelial cells from damage by atherogenic LDL.
Arterioscler Thromb Vasc Biol. 17: 2868-2874.
Karr, S. C., Lampe, J. W., Hutchins, A. M., and Slavin, J.
1997. Urinary isoflavonoid excretion in humans is dose
dependent at low to moderate levels of soy-protein
consumption. Am. J. Clin. Nutr. 66: 46-51.
Kennedy, A. R. 1995. The evidence for soybean products as
cancer preventive agents. J. Nutr. 125: 733S-743S.
Kennedy, A. R. 1998. The Bowman-Birk inhibitor from soybeans as
an anticarcinogenic agent. Am. J. Clin. Nutr. 68(S): 1406S-
1412S.
Kim, J. —W., Liou, B. B., Lai, M. —Y. Ponce, E., and
Grabowski, G. A. 1996. Gaucher disease: Identification of
three new mutations in the Korean and Chinese (Taiwanese)
populations. Early Human Development. 46: 203.
Kuo, S. M. 1996. Antiproliferative potency of structurally
distinct dietary flavonoids on human colon cancer cells.
Cancer Letters 110: 41-48.
Lee, HP., Gourlery, L., Duffy, SW., Esteve, J., and Day, NE.
1991. Dietary effects on breast-cancer risk in Singapore.
Lancet 337: 1197-1200.
Matsuura, M., and Obata, A. 1993. β-Glucosidase from soybeans
hydrolyze daidzein and genistein. J. Food Sci. 58(1): 144-147.
Matsuura, M., Obata, A., and Fukushima, D. 1989. Objectionable
flavor of soy milk developed during the soaking of soybeans
and its control. J. Food Sci. 54(3): 602.
Matsuura, M., Sasaki, J., and Murao, S. 1995. Studies on β-
Glucosidase from soybean that hydrolyze daidzin and genistin:
isolation and characterization of an isozyme. Biosci.
Biotech. Biochem. 59(9): 1623-1627.
Matskawa, Y., Marui, N., Sakai, T., Satome, Y., Yoshida, M.,
and Matsumoto, M. 1993. Genisetin arrests cell cycle
progression at G2-M. Cancer Res. 53: 1328-1331.
Messina, M. J., and Barnes, S. 1991. The role of soy products
in reducing cancer risk. J. Natl. Cancer Inst. 83: 541-546.
Messina, M. J., Persky, V., Setchell, K. D. R., and Barnes, S.
1994. Soy intake and cancer risk: a review of the in vitro
and in vivo data. Nutr. Cancer 21: 113-131.
Meneghini, R., and Martins, E. L. In: Halliwell, B. and Aruoma,
O. L. eds. Hydrogen peroxide and DNA damage. DNA and free
radicals. New York: Eillis Horwood; 1993: 83-93.
Mitsuoka, T. 1982. Recent trends in research on intestinal
flora: Bifidobacteria Microflora 3: 3-24.
Miyazawa, M., Sakano, K., Nakamura, S. -I., and Kosaka, H.
2001. Antimutagenic activity of isoflavone from Pueraria
lobata. J. Agric. Food Chem. 49: 336-341.
Murphy, C. T., Kellie, S., and Westwick, J. 1993. Tyrosine-
kinase activity in rabbit platelts stiumulated with platelet-
activating factor. Eur. J. Biochem. 216: 639-651.
Nagayami, F. and Saito, Y. 1968. Distribution of amylase, α-
and β-glucosidase, and β-galactosidase in fish. Bull. Jpn.
Soc. Sci. 34: 944-948.
Pandjaitan, N., Hettiarachchy, N., Ju, Z. Y., Crandall, P.,
Sneller, C., and Dombek, D. 2000a. Evaluation of genistin and
genistein contents in soybean varieties and soy protein
concentrate prepared with 3 basic methods. J. Food Sci. 65
(3): 399-402.
Pandjaitan, N., Hettiarachchy, N., Ju, Z. Y., Crandall, P.,
Sneller, C., and Dombek, D. 2000b. Enrichment of genistein in
soy protein concentrate with hydrocolloids and β-
glucosidase. J. Food Sci. 65(4): 591-595.
Peterson, G. 1995. Evaluation of the biochemical targets of
genistein in tumor cells. J. Nutr. 125(suppl.): 784S-789S.
Peterson, G., and Barnes, S. 1993.Genistein and biochanin A
inhibit the growth of human prostate cancer cells but not
epidermal growth factor receptr tyrosine autophosphorylation.
The Prostate 22: 335-345.
Pratt, D. E., and Birac, P. M. 1979. Source of antioxidant
activity of soybeans and soy products. J. Food Sci. 44: 1720-
1723.
Record, I. R., Dreosti, I. E., and McInerney, J. K. 1995. The
antioxidant activity of genistein in vitro. J. Nur. Biochem.
6: 481-485.
Richard R. Love, M. D. 1991. Antiestrogen chemoprevention of
breast cancer: critical issues and research. Preventive
Medicine 20: 64-78.
Riou, C., Salmon, J. -M., Vallier, M..-J., Günata, Z., and
Barre, P. 1998. Purification, characterization, and substrate
specificity of a novel highly glucose-tolerant β-glucosidase
from Aspergillus oryzae. Appl. and Environ. microbiol. 64
(10): 3607-3614.
Ruiz-Larrea, M. B., Mohan, A. R., Paganga, G., Miller, N. J.,
and Bolwell, G. P. 1997. Antioxidant activity of
phytoestrogenic isoflavones. Free Rad. Res. 26(1): 63-70.
Salti, G.I., Grewel, S., Mehta, R.R., Das Gupta, T.K., Boddie
Jr, A.W., and Constantinou, A.I. 2000. Genistein induces
apoptosis and topoⅡ-mediated DNA breakage in colon cancer
cells. European J. Cancer 36: 796-802.
Setchell, K. D. R., Borriello, S. P., Hulme, P., Kirk, D. N.,
and Axelson, M. 1984. Nonsteroidal estrogens of dietary
origin: possible role in hormone-dependent disease. Am. J.
Clin. Nutr. 40: 569-578.
Sharma, O. P., Adlercreutz, H., Strandberg, J. D., Zirkin, B.
R., Coffey, D. S., and Ewing, L. L. 1992. Soy of dietary
source plays in rats. J. Steroid Bichem. Mol. Biol. 43: 557-
564.
Shiihara, T., Oka, A., Suzaki, I., Ida, H., and Takeshita, K.
2000. Communicating hydrocephalus in a with Gaucher’s
disease type 3. Pediatric neurol. 22:234-236.
Su, C. and Zhu, X. 1979. The metabolic fate of the effective
components of radix puerariae. 103. The absorption,
distribution and elimination of 14C-daidzein. Yao Hsueh Hsueh
Pao 14(3): 129-134. 1988. Influence of soybean processing,
habitual diet, and soy dose on urinary isoflavonoid
excretion. Am. J. Clin. Nutr. 68: 1492S-1495S.
Sue, M., Ishihara, A., and Iwamura H. 2000. Purification and
characterization of a β-glucosidase from rye (Secale cereale
L.) seedlings. Plant Sci. 155: 67-74.
Takashima S., Nakamura, A., Hidaka, M., Masaki, H., and Uozumi,
T. 1999. Moleclar cloning and expression of the novel fungal
β-glucosidase genes from Humicola grisea and Trichoderma
reesei. J. Biochem. 125: 728-736.
Tew, B. Y., Xu, X., Wang, H. J., Murphy, P. A., and Hendrich,
S. 1996. A diet high in wheat fiber decreases the
bioavailability of soybean isoflavones in a single meal fed
to women. J. Nutr. 126: 871-877.
Waltzl, B. 1996. Health-promoting effects of phytochemicals.
In: Proceeding of IUFoST ’96’ regional symposium.
Walz, E. 1931. Isoflavone- and saponin-glucosidase in soja
hispida. Ann. Chem. 489: 118-122.
Wang, H. —J., and Murphy, P. A. 1994. Isoflavone composition
of American and Japanese soybeans in Iowa: effects of
variety, crop year, and location. J. Agric. Food Chem. 42:
1674-1677.
Welling, P. G. 1986. First-pass metabolism, enterohepatic
circulation, and physicochemical factors affecting
absorption. In: Pharmacokinetics-Processes and Mathematics
(Welling, P. G. ed). Am. Chem. Society, Washington, DC. 35-44.
Wei, H., Cai, Q., Rahn, R., and Zhang, X. 1997. Singlet oxygen
involvement in ultraviolet (254 nm) radiation-induced
formation of 8-hydroxy-
deoxyguanosine in DNA. Free Radic. Biol. Med. 23(1): 148-154.
Wei, H., Wei, L., Frankel, K., Bowen, R., and Barnes, S.1993.
Inhibition of tumor promoter-induced hydrogen peroxide
formation in vitro and in vivo by genistein. Nutr. Cancer 20:
1-12.
Winter, J., Moore, L. H., Dowell, V. R., Jr., and Bokkenheuser,
V. D. 1989. C-ring cleavage of flavonoids by human intestinal
bacteria. Appl. Environ. Microbiol. 55: 1203-1208.
Wolf, W. J., and Cowan, J. C. 1975. Soybean as a food source.
Boca Ration, FL: CCRC press.
World Health Organization, World Health Statistics 1992, 1993.
Womg, E. 1975. The isoflavonoids. In The flavonoids. Harborne,
J. B., Mabry, T. J., and Murphy, H. pp.743-799. New York.
Xu, X., Harris, K., Wang, H. J., Murphy, P. A., and Hendrich,
S. 1995. Bioavailability of soybean isoflavones depends upon
gut microflora in women. J. Nutr. 125: 2307-2315.
Zhang, X., Rosenstein, B. S., Wang, Y., Lebwohl, M., and Wei,
H. 1997. Identification of possible reactive oxygen species
involved in ultraviolet radiation-induced oxidative DNA
damage. Free Radic. Biol. Med. 23(7): 980-985