产氢细菌的分离及其对大豆异黄酮转化菌株促转化作用研究
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摘要
大豆异黄酮是大豆在生长过程中形成的次生代谢产物,主要由染料木素(genistein)、黄豆苷原(daidzein)和黄豆黄素(glycitein)组成。它能与雌激素受体结合发挥类似雌激素效应,故称之为植物雌激素。具有防治癌症、降低血脂、预防骨质疏松、改善妇女更年期综合症和抗氧化等多种生理功能。摄入体内的大豆异黄酮,在胃肠道微生物菌群作用下转化为各种不同代谢产物。大量研究结果表明,大豆异黄酮代谢产物具有比大豆异黄酮更高更广的生物学活性。
从保定市动物园饲养的动物和本实验室喂养的鸡粪样中分离出八种兼性厌氧产氢细菌,分别命名为菌株4,7,9,21,26,28,208和G30。通过形态学特征、生理生化特征和16S rDNA序列分析,将菌株4,9,21,26和208鉴定为肺炎克雷伯氏菌(Klebsiella pneumoniae);菌株7为变栖克雷伯氏菌(Klebsiella variicola);菌株28为多粘类芽孢杆菌(Paenibacillus polymyxa);菌株G30为费格森埃希氏菌(Escherichia fergusonii)。这些产氢菌株均能在有氧条件下生长发酵产生氢气。
Sharpea azabuensis Niu-O16(AY263505)是从牛瘤胃胃液分离的能将黄豆苷原(daidzein)还原为二氢黄豆苷原(DHD)的革兰氏阳性细菌菌株。野生型Niu-O16为严格厌氧菌株,经耐氧驯化后能够在有空气氧条件下生长,但转化效率明显降低。本研究尝试,将从不同动物粪样中分离出的兼性厌氧产氢细菌与驯化后的Niu-O16混合培养,以提高Niu-O16的转化效率。研究结果显示,除产氢菌株28与Niu-O16混合培养后转化效率无明显变化外(P>0.05),其它产氢菌株与Niu-O16混合培养后的转化率均显著高于对照(P<0.01),其中以菌株G30与Niu-O16混合培养的转化率最高,高达82.8%,比驯化后单独培养Niu-O16的转化率提高了25.7%。通过对两菌株混合培养后转化动态进行研究发现,混合培养可加快底物黄豆苷原的转化速度,当底物浓度为0.2 mmol/L时,混合培养菌株在12 h内,可将80%以上的黄豆苷原转化为DHD。另外,该混合培养体系能够转化黄豆苷原的最大浓度为1.2 mmol/L。
Isoflavones are the secondary metabolites during the growth of soybeans, mainly composed of daidzein, genistein and glycitein. Isoflavones, one of the plant-produced phytoestrogens have structural and functional similarity to human estrogen by binding with estrogen receptors. It is well known that isoflavones are of various beneficial effects, such as preventing cancer, lowering blood fat, attenuating bone loss, improving women's menopause syndrome, antioxidant activities, and so on. After being absorbed, isoflavones can be degraded into different metabolities. Studies showed that isoflavone metabolites are of higher and wider bioactivities than that of isoflavones.
Eight bacterial strains named strain 4, 7, 9, 21, 26, 28, 208 and G30, which were isolated from Equus quagga, Lama guanicoe, Helarctos malayanus, Cervus albirostris, Grus nigricollis, Crossoptilon mantchuricum, and chicken fecal sample respectively, are facultative bacteria with hydrogen producing activity. Based on morphology feature, physiological and biochemical characteristics as well as analysis of the 16S rDNA sequence, bacterial strains 4, 9, 21, 26 and 208 were identified as Klebsiella pneumoniae 4,Klebsiella pneumoniae 9,Klebsiella pneumoniae 21,Klebsiella pneumoniae 26 and Klebsiella pneumoniae 208. Bacterial strain 7 was identified as Klebsiella variicola 7, strain 28 was identified as Paenibacillus polymyxa 28 and strain G30 was identified as Escherichia fergusonii G30, respectively. All of isolated bacteria can produce hydrogen under aerobic conditions.
Gram positive bacterial strain Sharpea azabuensis Niu-O16 isolated from bovine rumen was an obligate anaerobic strain, which is capable of biotransforming daidzein into DHD under anaerobic conditions. After long time domestication resistant to oxygen, original obligate anaerobic bacterial strain Niu-O16 can grow in BHI liquid medium and produce DHD from daidzein under aerobic conditions. However, the biotransformation capacity of daidzein is significantly decreased. In this study, enhanced the biotansformation activity of domesticated Niu-O16 was investigated by coculture with isolated hydrogen-producing bacterial strains. The rusults showed that all the biotransforming capacity of daidzein was significantly increased by mixed culture (P<0.01) compared with pure culture of strain Niu-O16, except the mixed culture of strain 28 and Niu-O16 (P>0.05). Among all the mixed cultures, the biotransformation efficiency of daidzein by the mixed culture of strain G30 and Niu-O16 was the highest. The biotransforming ratio of daidzein was up to 82.8%, which is increased by 25.7% compared with that of pure culture of strain Niu-O16 under aerobic conditions. According to the biotransformation kinetics, the biotransforming spead of daidzein by the mixed culture of bacterial strain G30 and Niu-O16 became faster than that of pure culture of strain Niu-O16. When the concentration of substrate daidzein was 0.2 mmol / L, more than 80% of daidzein can be transformed into DHD by the mixed culture within 12 hrs, and almost completely biotransformed within 24 hrs incubation under the aerobic conditions. In addition, the highest biotransfoming concentration of substrate daidzein was up to 1.2 mmol / L by the mixed culture of bacterial strain G30 and Niu-O16.
从保定市动物园饲养的动物和本实验室喂养的鸡粪样中分离出八种兼性厌氧产氢细菌,分别命名为菌株4,7,9,21,26,28,208和G30。通过形态学特征、生理生化特征和16S rDNA序列分析,将菌株4,9,21,26和208鉴定为肺炎克雷伯氏菌(Klebsiella pneumoniae);菌株7为变栖克雷伯氏菌(Klebsiella variicola);菌株28为多粘类芽孢杆菌(Paenibacillus polymyxa);菌株G30为费格森埃希氏菌(Escherichia fergusonii)。这些产氢菌株均能在有氧条件下生长发酵产生氢气。
Sharpea azabuensis Niu-O16(AY263505)是从牛瘤胃胃液分离的能将黄豆苷原(daidzein)还原为二氢黄豆苷原(DHD)的革兰氏阳性细菌菌株。野生型Niu-O16为严格厌氧菌株,经耐氧驯化后能够在有空气氧条件下生长,但转化效率明显降低。本研究尝试,将从不同动物粪样中分离出的兼性厌氧产氢细菌与驯化后的Niu-O16混合培养,以提高Niu-O16的转化效率。研究结果显示,除产氢菌株28与Niu-O16混合培养后转化效率无明显变化外(P>0.05),其它产氢菌株与Niu-O16混合培养后的转化率均显著高于对照(P<0.01),其中以菌株G30与Niu-O16混合培养的转化率最高,高达82.8%,比驯化后单独培养Niu-O16的转化率提高了25.7%。通过对两菌株混合培养后转化动态进行研究发现,混合培养可加快底物黄豆苷原的转化速度,当底物浓度为0.2 mmol/L时,混合培养菌株在12 h内,可将80%以上的黄豆苷原转化为DHD。另外,该混合培养体系能够转化黄豆苷原的最大浓度为1.2 mmol/L。
Isoflavones are the secondary metabolites during the growth of soybeans, mainly composed of daidzein, genistein and glycitein. Isoflavones, one of the plant-produced phytoestrogens have structural and functional similarity to human estrogen by binding with estrogen receptors. It is well known that isoflavones are of various beneficial effects, such as preventing cancer, lowering blood fat, attenuating bone loss, improving women's menopause syndrome, antioxidant activities, and so on. After being absorbed, isoflavones can be degraded into different metabolities. Studies showed that isoflavone metabolites are of higher and wider bioactivities than that of isoflavones.
Eight bacterial strains named strain 4, 7, 9, 21, 26, 28, 208 and G30, which were isolated from Equus quagga, Lama guanicoe, Helarctos malayanus, Cervus albirostris, Grus nigricollis, Crossoptilon mantchuricum, and chicken fecal sample respectively, are facultative bacteria with hydrogen producing activity. Based on morphology feature, physiological and biochemical characteristics as well as analysis of the 16S rDNA sequence, bacterial strains 4, 9, 21, 26 and 208 were identified as Klebsiella pneumoniae 4,Klebsiella pneumoniae 9,Klebsiella pneumoniae 21,Klebsiella pneumoniae 26 and Klebsiella pneumoniae 208. Bacterial strain 7 was identified as Klebsiella variicola 7, strain 28 was identified as Paenibacillus polymyxa 28 and strain G30 was identified as Escherichia fergusonii G30, respectively. All of isolated bacteria can produce hydrogen under aerobic conditions.
Gram positive bacterial strain Sharpea azabuensis Niu-O16 isolated from bovine rumen was an obligate anaerobic strain, which is capable of biotransforming daidzein into DHD under anaerobic conditions. After long time domestication resistant to oxygen, original obligate anaerobic bacterial strain Niu-O16 can grow in BHI liquid medium and produce DHD from daidzein under aerobic conditions. However, the biotransformation capacity of daidzein is significantly decreased. In this study, enhanced the biotansformation activity of domesticated Niu-O16 was investigated by coculture with isolated hydrogen-producing bacterial strains. The rusults showed that all the biotransforming capacity of daidzein was significantly increased by mixed culture (P<0.01) compared with pure culture of strain Niu-O16, except the mixed culture of strain 28 and Niu-O16 (P>0.05). Among all the mixed cultures, the biotransformation efficiency of daidzein by the mixed culture of strain G30 and Niu-O16 was the highest. The biotransforming ratio of daidzein was up to 82.8%, which is increased by 25.7% compared with that of pure culture of strain Niu-O16 under aerobic conditions. According to the biotransformation kinetics, the biotransforming spead of daidzein by the mixed culture of bacterial strain G30 and Niu-O16 became faster than that of pure culture of strain Niu-O16. When the concentration of substrate daidzein was 0.2 mmol / L, more than 80% of daidzein can be transformed into DHD by the mixed culture within 12 hrs, and almost completely biotransformed within 24 hrs incubation under the aerobic conditions. In addition, the highest biotransfoming concentration of substrate daidzein was up to 1.2 mmol / L by the mixed culture of bacterial strain G30 and Niu-O16.
引文
[1]毛峻琴,宓鹤鸣.大豆异黄酮的研究进展[J].中草药, 2000, (1): 61-64.
[2]蒋蔡滨.关于大豆异黄酮的研究综述[J].贵阳中医学院学报, 2006, (1): 49-51.
[3]郑德勇,安鑫南.植物抗氧化剂的研究概况与发展趋势[J].林产化学与工业, 2004, 24(3): 113-118.
[4]汪秋安,周冰,单杨.天然黄酮类化合物的抗氧化活性和提取技术研究进展[J].化工生产与技术, 2004, 11(5): 29-32.
[5] Shao Z M, Wu J, Shen Z Z, et al. Genistein exerts multiple suppressive effects on human breast carcinoma cells[J]. Cancer Res, 1998, 58(21): 4851-4857.
[6] Rice S, Whitehead S A. Phytoestrogens and breast cancer-promoters or protectors[J]. Endocr Relat Cancer, 2006, 13: 995-1015.
[7] Taylor, Richard B, Henley E C. Composition for and method of preventing or treating breast cancer [J]. US.6, 300, 367, October9, 2001.
[8] Maximov, O V. Biological active substances from M.a Rupr.et maxim and prospects for using the species in medicine[J]. Rastibelnge Resursy, 1992, 28(3): 157.
[9] Zhou J R, Mukherjee P, Gugger E T, et al. Inhibtion of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis[J]. Cancer Ras, 1998, 58(22): 5231-5238.
[10] Wei H, Cai Q Y, Rahn R O. Inhibition of UV light and Fenton reaction-induced oxidative DNA damage by the soybean isoflavone genistein[J]. Carcinogenesis, 1996, 17(1): 73-77.
[11] Mousavi Y, Adlercreutz H. Genistein is an effective stimulator of sex hormone binding globulin production in hepatocarcinoma human liver cancer cells and suppresses proliferation of these cells in culture[J]. Steroids, 1993, 58(7): 301-304.
[12]曹锋,金泰廙,周袁芬.异黄酮对前列腺癌细胞的激素受体基因表达的影响[J].环境与职业医学, 2005, 22(2): 95-98.
[13]刘颖,张牧,王小雪,等.染料木黄酮对人胃癌细胞生长抑制作用研究[J].营养学报, 2001, 23(1): 62-65.
[14]马吉祥,王勤忠,苏军英,等.大豆异黄酮诱导人食管癌裸鼠移植瘤细胞凋亡[J].中国肿瘤, 2004, 13(1): 34-37.
[15]徐德平,高霞,江汉湖.丹贝异黄酮对肿瘤的抑制效应[J].南京农业大学学报, 2002, 25(1): 97-101.
[16]张晓丹,陈少军,周广红.环境雌激素的生殖毒性作用机制[J].江西医药, 2005, 40(1): 31-33.
[17] Mizunuma H, Kanazawa K, Ogura S, et al. Anticarcinogenic effects of isofavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer[J]. Oncology, 2002, 62(1): 78-84.
[18] Jing Y, Nakaya K, Han R. Differentiation of promyelocytic leukemia cells HL-60 induced bydaidzein in vitro and in vivo[J]. Anticancer Res, 1993, 13(4): 1049.
[19] Potter S M, Baum J M, Teng H, et al. Soy protein and isoflavone:their effeets on blood lipids and density in postmenoposal women[J]. Am J Clin Nutr, 1998, 68: 1375-1379.
[20] Alekel D L, Germain A S, Peterson C T, et al. Isoflavone-rich soy protein isolate attenuates bone in the lumbar spine of perimenopausal women[J]. Am J Clin Nutr, 2000, 72: 844-852.
[21] Harborne J B, Williams C A. Advances in flavonoid research since 1992[J]. Phytochemistry, 2000, 55(6): 481-504.
[22] Magee P J, Rowland I R. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer[J]. The British Journal of Nutrition, 2004, 91(4): 513-531.
[23]张响英,王根林,唐现文,等.大豆黄酮对仔公猪细胞免疫功能的影响[J].黑龙江畜牧兽医, 2005(1): 34-35.
[24]刘皙洁,张桂春,张维生.大豆黄酮对肉仔鸡脂肪代谢的影响[J].东北农业大学学报, 2003, 34(2): 171-175.
[24]程忠刚,林映才,余德谦.大豆黄酮对肥育猪生产性能的影响及其作用机制探讨[J].动物营养学报, 2005, 17(1): 30-34.
[25]孟婷,韩正康,王国杰.大豆黄酮对初产蛋鸡生产性能和血清生理生化指标的影响[J].中国家禽, 2002, 24(13): 13-14.
[26] Payne R L, Bidner T D, Southern L L, et al. Effects of dietary soy isoflavones on growth, carcass traits, and meat quality in growing-fini-shing pigs. J Anim Sci, 2001, 79(5): 1230-1239.
[27] Zarkadas L N, Wiseman J. Influence ofprocessing of full fat soy beans included in diets for piglets. I. Performance[J]. Animal Feed Science and Technology 2005, 118(1-2): 109-119.
[28] Zarkadas L N, Wiseman J. Influence of processing of full fat soya beans included in diets for piglets. II. Digestibility and intestinal morphology[J]. Animal Feed Science and Technology, 2005, 118(3-4): 121-137.
[29] Greiner L L, Stahly T S, Stabel T J. The effect of dietary soy genistein on pig growth and viral replication during a viral challenge[J]. J Anim Sci, 2001, 79(10): 1272-1279.
[30] Greiner L L, Stahly T S, Stabel T J. The effect of dietary soy daidzein on pig growth and viral replication during a viral challenge[J]. J Anim Sci, 2001, 79(3): 3113-3119.
[31] Cassidy A, Brown J E, Hawdon A, et al. Factors affecting the bioavailability of soy isoflavones in humans after ingestion of physiologically relevant levels from different soy foods[J]. J Nutr, 2006, 136: 45-51.
[32] Day A J, Canada F J , Diaz J C, et al. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase[J]. FEBS Lett, 2000, 468: 166-170.
[33] Joannou G E, Kelly G E, Reeder A Y, et al. A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids[J]. Steroid Biochem Mol Biol, 1995, 54: 167-184.
[34] Heinonen S, Wahala K, Adlercreutz H. Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-dma and cis-4-OH-equol in human urine by gas chroma-tography-massspectroscopy using authentic reference compounds[J]. Anal Biochem 1999, 274: 211-219.
[35] Setchell K D, Brown N M, Zimmer-Nechemias L, et al. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability[J]. Am J Clin Nutr, 2002, 76: 447-453.
[36] Day A J, DuPont M S, Ridley S, et al. Eglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity[J]. FEBS Lett 1998,436: 71-75.
[37] Rafii F, Davis C, Park M, et al. Variations in metabolism of the soy isoflavonoid daidzein by human intestinal microfloras from different individuals[J]. Arch Microbiol 2003, 180: 11-16.
[38] Rafii F, Hotchkiss C, Heinze T M, et al. Meta-bolism of daidzein by intestinal bacteria from rhesus monkeys(Macaca mulatta) [J]. Comp Med 2004, 54: 165-169.
[39] Barnes S, Coward L, Kirk M, et al. HPLC mass spectrometry analysis of isoflavones[J]. Proc Soc Exp Biol Med, 1998, 217: 254-262.
[40] Coldham N G, Howells L C, Santi A, et al. Biotransformation of genistein in the rat: Elucidation of metabolite structure by product ion mass fragmentology[J]. J Steroid Biochem Mol Biol, 1999, 70: 168-184.
[41] Bowey E, Adlercreutz H, Rowland I. Metabolismof isoflavones and lignans by the gut microflora:a study in germ-free and human flora associated rats[J]. Food Chem Toxicol 2003, 41: 631-636.
[42] Rowland I, Wiseman H, Sanders T, et al. Metabolism of oestrogens and phytoes-trogens:role of the gut microflora[J]. Biochem SocTrans 1999,27: 304-308.
[43] Kostelac D, Rechkemmer G, Briviba K. Phytoes-trogens modulate binding response of estrogen receptors alpha and beta to the estrogen responseelement[J]. J Agric Food Chem 2003,51: 7632-7635.
[44] Muthyala R S, Ju Y H, Sheng S, et al. Equol, a natural estrogenic metabolite from soy isoflavones:convenient prepa-ration and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta[J]. Bioorg Med Chem 2004, 12: 1559-1567.
[45] Mitchell J H, Gardner P T, McPhail D B, et al. Antioxidant efficacy of phytoestrogens in chemical and biological model systems[J]. Arch Biochem Biophys 1998, 360: 142-148.
[46] Brown N M, Setchell K D. Animal models impac-ted by phytoestrogens in commercial chow: implications for pathways influenced by hormones[J]. Lab Invest 2001,81: 735-747.
[47] Morito K, Hirose T, Kinjo J, et al. Interaction of phytoestrogens with estrogen receptors alpha and beta[J]. Biol Pharm Bull 2001,24: 351-356.
[48] Morito K, Aomori T, Hirose T, et al. Interaction of phytoestrogens with estrogenreceptors alpha and beta(II)[J]. Biol Pharm Bull 2002,25: 48-52.
[49] Hodgson J M, Croft K D, Puddey I B, et al. Soybean isoflavonoids and their metabolic products inhibit in vitro lipo-protein oxidation in serum.[J]. Nutr. Biochem, 1996, 7: 664-669.
[50] Arora A, Nair M G, Strasburg G M. Antioxidant activities of isoflavones and their biological meta-bolites in a liposomal system[J]. Arch Biochem Biophys 1998, 356: 133-141.
[51] Schneider H, Schwiertz A, Collins M D, et al. Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract. Arch Microbiol, 1999, 171: 81-91.
[52] Krishnamurty H G, Cheng K J, Jones G A, et al. Identification of products produced by anaerobic degradation of rutin and related flavonoids by Butyrivibrio sp. C3[J]. Can J Microbiol, 1970, 16: 759-767.
[53] Decroos K, Vanhemmens S, Cattoir S, et al. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions[J]. Arch Microbiol 2005, 183: 45-55.
[54] Schoefer L, Mohan R, Schwiertz A, et al. Anaerobic degradation of flavonoids by Clostridium orbiscindens[J]. Appl Environ Microbiol, 2003, 69: 5849-5854.
[55] Hur H G, Lay J, Rafii F, et al. Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin[J]. Arch Microbiol, 2000, 174: 422-428.
[56] Wang X L, Shin K H, Hur H G, et al. Enhanced biosynthesis of dihydrodaizein and dihydrogenistein by a newly isolated bovine rumen anaerobic bacterium[J]. Journal of Biotechnology, 2005, 115(3): 261-269.
[57] Hur H G, Beger R D, Rafii F, et al. Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein[J]. Archives of Microbiology, 2002, 178: 8-12.
[58] Wang X L, Hur H G, Kim Su IL. C-ring cleavage of isoflavones daidzein and genistein by a newly-isolated human intestinal bacterium Eubacterium ramulus julong601[J]. Journal of Microbiology and Biotechnology, 2004, 14(4): 766-771.
[59] Wang X L, Hur H G, Kim Su IL, et al. Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium[J]. Applied and Environmental Microbiology, 2005, 71: 214-219.
[60] Minamida K, Tanaka M, Abe A. Production of equol from daidzein by gram-positive rod-shaped bacterium isolated from rat intestine[J]. Journal of Bioscience and Bioengineering, 2006, 102(3): 247-250.
[61] Ueno T, Uchiyama S, Kikuchi N. The role of intestinal bacteria on biological effects of soy isoflavones in humans[J]. J Nutr, 2002, 594S, 132.
[62] Beck V, Rohr U, Jungbauer A. Phytoestrogens derived from red clover:an alternative to estrogen replacement therapy[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2005, 94(5): 499-518.
[63] Chin-Dusting J P, Fisher L J, Lewis T V, et al. The vascular activity of some isoflavone metabolites: implications for a cardioprotective role[J]. British Journal of Pharmacology, 2001, 133(4): 595-605.
[64] Jiang F, Jones G T, Husband A J, et al. Cardiovascular protective effects of synthetic isoflavones derivatives in apolipoprotein edeficient mice[J]. Journal of Vascular Research, 2003, 40(3): 276-284.
[65]李燕红,林钰,等.农作物秸秆废弃物厌氧发酵生物制氢的研究[J].环境科学与技术, 2006,29(11): 8-9.
[66]卢怡,尹德升,等.牛粪、鸡粪发酵产氢潜力的研究[J].可再生能源, 2004, (2): 37-39.
[67] Chen W M, Tseng Z J, Lee K S, et al. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge[J]. Int J Hydrogen Energy, 2005,30(10): 1063-1070.
[68] Collet C, Adler N, Schwitzguébel J P, et al. Hydrogen production by Clostridium thermolacticum during continuous fermentation of lactose[J]. Hydrogen Energy, 2004,29: 1479-1485.
[69] Liu G, Shen J. Effects of culture medium and medium conditions on hydrogen production from starch using anaerobic bacteria[J]. Biosci Bioeng, 2004,98: 251-256.
[70] Evvyernie D, Morimoto K, Karita S, et al. Conversion of chitinouswaste to hydrogen gas by Clostridium paraputrificum M-21[J]. Biosci Bioeng, 2001,91: 339-343.
[71] Wang C C, Chang C W, Chu C P, et al. Producing hydrogen from wastewater sludge by Clostridum bifermentans[J]. Biotechnol, 2003,102: 83-92.
[72] Nakashimada Y, Rachman M A, Kakizono T, et al. Hydrogen production of Enterobacter aerogene saltered by extracellular and intracellular redox states[J]. Hydrogen Energy, 2002, 27: 1399-1405.
[73] Kumar N, Das D. Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08[J]. Process Biochem, 2000, 35: 589-593.
[74] Mandal B, Nath K, Das D. Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae. Biotechnol Lett, 2006, 28(11): 831-835.
[75] PodestáJ J, Navarro A M, Estrella C N, et al. Electrochemical measurements of trace concentrations of biological hydrogen produced by Enterobacteriaceae[J]. Inst Pasteur, 1997, 148: 87-93.
[76]李永峰,任南琪,杨传平,等.一株高效产氢产酸细菌的鉴定与产氢特性[J].中国环境科学, 2005, 25(2): 210-213.
[77] Oh Y K, Park M S, Seol E H, et al. Isolation of hydrogen producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor[J]. Biotechnol Bioprocess Eng, 2003, 8: 54-57.
[78] Shin H S, Youn J H, Kim S H. Hydrogen production from foodwaste in anaerobic mesophilic and thermophilic acidogenesis[J]. Hydrogen Energy, 2004, 29: 1355-1363.
[79]Tanisho S, Kuromoto M, Kadokura N. Effect of CO2 removal of hydrogen production by fermentation[J]. Hydrogen Energy, 1998, 23(7): 559-563.
[80]王继华,赵爱萍.生物制氢技术的研究进展与应用前景[J].环境科学研究, 2005, 18(4): 129-135。
[81]冯树,张忠泽.混合菌——一类值得重视的微生物资源[J].世界科技研究与发展, 2000, 22(3): 44-47.
[82]韩梅,李春龙,韩晓日,等.大豆根瘤菌和胶质芽孢杆菌混合培养研究[J].沈阳农业大学学报, 2009, 40(2): 188-192.
[83] Weimer, P J. Mixed cultures in Biotechnology[M]. Mc Graw Hill, New York, 1991.
[84]齐云,袁月祥,陈飞,等.一组纤维素分解菌的分离、筛选及其产酶条件的研究[J].天然产物研究与开发, 2003, 15(6): 510-512.
[85]李霞,王向东,林松,等.混合型微生物絮凝剂产生菌的最佳培养条件[J].应用与环境生物学报, 2005, 11(3): 377-380.
[86]林红雨,陈策实,尹光林.欧文氏菌和棒杆菌的属间融合研究[J].微生物学通报, 1999, 26(1), 3-6.
[87]马辉文,王疆元.利用微生物混合培养物生产甜菜渣单细胞蛋白[J].武汉大学学报, 1994, (4), 107-114.
[88]谢忠,陈荣.利用微生物混合培养技术生产玉米废渣单细胞蛋白[J].粮食与饲料工业, 1994, (7), 28-31.
[89]代小江,王礼德,贺锡勤.利用微生物混合培养物生产沙棘果渣单细胞蛋白[J].微生物学通报, 1995, 22(5): 267-270.
[90]冯树,周樱桥,张忠泽.微生物混合培养极其应用[J].微生物学通报, 2001, 28(3): 92-95.
[91]东秀珠,蔡妙英.常见细菌系统鉴定手册(第一版)[M].北京:科学出版社, 2001.
[92]杜连祥,等.工业微生物学试验技术[M].天津科学技术出版社, 1992.
[93] Lane, D J. 16S/23S rRNA sequencing. In E. Stackebrandt and M. Goodfellow (eds.), Nucleic Acid techniques in Bacterial Systematics[M]. New York: John Wiley and Sons, 1991.
[94] Karel D, Steffi V, Sofie C, et al. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions[J]. Arch Microbiol, 2005, 183: 45–55.
[95]李朝东.大豆异黄酮还原菌株的耐氧驯化与诱变选育[D].河北农业大学, 2009.
[2]蒋蔡滨.关于大豆异黄酮的研究综述[J].贵阳中医学院学报, 2006, (1): 49-51.
[3]郑德勇,安鑫南.植物抗氧化剂的研究概况与发展趋势[J].林产化学与工业, 2004, 24(3): 113-118.
[4]汪秋安,周冰,单杨.天然黄酮类化合物的抗氧化活性和提取技术研究进展[J].化工生产与技术, 2004, 11(5): 29-32.
[5] Shao Z M, Wu J, Shen Z Z, et al. Genistein exerts multiple suppressive effects on human breast carcinoma cells[J]. Cancer Res, 1998, 58(21): 4851-4857.
[6] Rice S, Whitehead S A. Phytoestrogens and breast cancer-promoters or protectors[J]. Endocr Relat Cancer, 2006, 13: 995-1015.
[7] Taylor, Richard B, Henley E C. Composition for and method of preventing or treating breast cancer [J]. US.6, 300, 367, October9, 2001.
[8] Maximov, O V. Biological active substances from M.a Rupr.et maxim and prospects for using the species in medicine[J]. Rastibelnge Resursy, 1992, 28(3): 157.
[9] Zhou J R, Mukherjee P, Gugger E T, et al. Inhibtion of murine bladder tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and angiogenesis[J]. Cancer Ras, 1998, 58(22): 5231-5238.
[10] Wei H, Cai Q Y, Rahn R O. Inhibition of UV light and Fenton reaction-induced oxidative DNA damage by the soybean isoflavone genistein[J]. Carcinogenesis, 1996, 17(1): 73-77.
[11] Mousavi Y, Adlercreutz H. Genistein is an effective stimulator of sex hormone binding globulin production in hepatocarcinoma human liver cancer cells and suppresses proliferation of these cells in culture[J]. Steroids, 1993, 58(7): 301-304.
[12]曹锋,金泰廙,周袁芬.异黄酮对前列腺癌细胞的激素受体基因表达的影响[J].环境与职业医学, 2005, 22(2): 95-98.
[13]刘颖,张牧,王小雪,等.染料木黄酮对人胃癌细胞生长抑制作用研究[J].营养学报, 2001, 23(1): 62-65.
[14]马吉祥,王勤忠,苏军英,等.大豆异黄酮诱导人食管癌裸鼠移植瘤细胞凋亡[J].中国肿瘤, 2004, 13(1): 34-37.
[15]徐德平,高霞,江汉湖.丹贝异黄酮对肿瘤的抑制效应[J].南京农业大学学报, 2002, 25(1): 97-101.
[16]张晓丹,陈少军,周广红.环境雌激素的生殖毒性作用机制[J].江西医药, 2005, 40(1): 31-33.
[17] Mizunuma H, Kanazawa K, Ogura S, et al. Anticarcinogenic effects of isofavones may be mediated by genistein in mouse mammary tumor virus-induced breast cancer[J]. Oncology, 2002, 62(1): 78-84.
[18] Jing Y, Nakaya K, Han R. Differentiation of promyelocytic leukemia cells HL-60 induced bydaidzein in vitro and in vivo[J]. Anticancer Res, 1993, 13(4): 1049.
[19] Potter S M, Baum J M, Teng H, et al. Soy protein and isoflavone:their effeets on blood lipids and density in postmenoposal women[J]. Am J Clin Nutr, 1998, 68: 1375-1379.
[20] Alekel D L, Germain A S, Peterson C T, et al. Isoflavone-rich soy protein isolate attenuates bone in the lumbar spine of perimenopausal women[J]. Am J Clin Nutr, 2000, 72: 844-852.
[21] Harborne J B, Williams C A. Advances in flavonoid research since 1992[J]. Phytochemistry, 2000, 55(6): 481-504.
[22] Magee P J, Rowland I R. Phyto-oestrogens, their mechanism of action: current evidence for a role in breast and prostate cancer[J]. The British Journal of Nutrition, 2004, 91(4): 513-531.
[23]张响英,王根林,唐现文,等.大豆黄酮对仔公猪细胞免疫功能的影响[J].黑龙江畜牧兽医, 2005(1): 34-35.
[24]刘皙洁,张桂春,张维生.大豆黄酮对肉仔鸡脂肪代谢的影响[J].东北农业大学学报, 2003, 34(2): 171-175.
[24]程忠刚,林映才,余德谦.大豆黄酮对肥育猪生产性能的影响及其作用机制探讨[J].动物营养学报, 2005, 17(1): 30-34.
[25]孟婷,韩正康,王国杰.大豆黄酮对初产蛋鸡生产性能和血清生理生化指标的影响[J].中国家禽, 2002, 24(13): 13-14.
[26] Payne R L, Bidner T D, Southern L L, et al. Effects of dietary soy isoflavones on growth, carcass traits, and meat quality in growing-fini-shing pigs. J Anim Sci, 2001, 79(5): 1230-1239.
[27] Zarkadas L N, Wiseman J. Influence ofprocessing of full fat soy beans included in diets for piglets. I. Performance[J]. Animal Feed Science and Technology 2005, 118(1-2): 109-119.
[28] Zarkadas L N, Wiseman J. Influence of processing of full fat soya beans included in diets for piglets. II. Digestibility and intestinal morphology[J]. Animal Feed Science and Technology, 2005, 118(3-4): 121-137.
[29] Greiner L L, Stahly T S, Stabel T J. The effect of dietary soy genistein on pig growth and viral replication during a viral challenge[J]. J Anim Sci, 2001, 79(10): 1272-1279.
[30] Greiner L L, Stahly T S, Stabel T J. The effect of dietary soy daidzein on pig growth and viral replication during a viral challenge[J]. J Anim Sci, 2001, 79(3): 3113-3119.
[31] Cassidy A, Brown J E, Hawdon A, et al. Factors affecting the bioavailability of soy isoflavones in humans after ingestion of physiologically relevant levels from different soy foods[J]. J Nutr, 2006, 136: 45-51.
[32] Day A J, Canada F J , Diaz J C, et al. Dietary flavonoid and isoflavone glycosides are hydrolysed by the lactase site of lactase phlorizin hydrolase[J]. FEBS Lett, 2000, 468: 166-170.
[33] Joannou G E, Kelly G E, Reeder A Y, et al. A urinary profile study of dietary phytoestrogens. The identification and mode of metabolism of new isoflavonoids[J]. Steroid Biochem Mol Biol, 1995, 54: 167-184.
[34] Heinonen S, Wahala K, Adlercreutz H. Identification of isoflavone metabolites dihydrodaidzein, dihydrogenistein, 6'-OH-O-dma and cis-4-OH-equol in human urine by gas chroma-tography-massspectroscopy using authentic reference compounds[J]. Anal Biochem 1999, 274: 211-219.
[35] Setchell K D, Brown N M, Zimmer-Nechemias L, et al. Evidence for lack of absorption of soy isoflavone glycosides in humans, supporting the crucial role of intestinal metabolism for bioavailability[J]. Am J Clin Nutr, 2002, 76: 447-453.
[36] Day A J, DuPont M S, Ridley S, et al. Eglycosylation of flavonoid and isoflavonoid glycosides by human small intestine and liver beta-glucosidase activity[J]. FEBS Lett 1998,436: 71-75.
[37] Rafii F, Davis C, Park M, et al. Variations in metabolism of the soy isoflavonoid daidzein by human intestinal microfloras from different individuals[J]. Arch Microbiol 2003, 180: 11-16.
[38] Rafii F, Hotchkiss C, Heinze T M, et al. Meta-bolism of daidzein by intestinal bacteria from rhesus monkeys(Macaca mulatta) [J]. Comp Med 2004, 54: 165-169.
[39] Barnes S, Coward L, Kirk M, et al. HPLC mass spectrometry analysis of isoflavones[J]. Proc Soc Exp Biol Med, 1998, 217: 254-262.
[40] Coldham N G, Howells L C, Santi A, et al. Biotransformation of genistein in the rat: Elucidation of metabolite structure by product ion mass fragmentology[J]. J Steroid Biochem Mol Biol, 1999, 70: 168-184.
[41] Bowey E, Adlercreutz H, Rowland I. Metabolismof isoflavones and lignans by the gut microflora:a study in germ-free and human flora associated rats[J]. Food Chem Toxicol 2003, 41: 631-636.
[42] Rowland I, Wiseman H, Sanders T, et al. Metabolism of oestrogens and phytoes-trogens:role of the gut microflora[J]. Biochem SocTrans 1999,27: 304-308.
[43] Kostelac D, Rechkemmer G, Briviba K. Phytoes-trogens modulate binding response of estrogen receptors alpha and beta to the estrogen responseelement[J]. J Agric Food Chem 2003,51: 7632-7635.
[44] Muthyala R S, Ju Y H, Sheng S, et al. Equol, a natural estrogenic metabolite from soy isoflavones:convenient prepa-ration and resolution of R- and S-equols and their differing binding and biological activity through estrogen receptors alpha and beta[J]. Bioorg Med Chem 2004, 12: 1559-1567.
[45] Mitchell J H, Gardner P T, McPhail D B, et al. Antioxidant efficacy of phytoestrogens in chemical and biological model systems[J]. Arch Biochem Biophys 1998, 360: 142-148.
[46] Brown N M, Setchell K D. Animal models impac-ted by phytoestrogens in commercial chow: implications for pathways influenced by hormones[J]. Lab Invest 2001,81: 735-747.
[47] Morito K, Hirose T, Kinjo J, et al. Interaction of phytoestrogens with estrogen receptors alpha and beta[J]. Biol Pharm Bull 2001,24: 351-356.
[48] Morito K, Aomori T, Hirose T, et al. Interaction of phytoestrogens with estrogenreceptors alpha and beta(II)[J]. Biol Pharm Bull 2002,25: 48-52.
[49] Hodgson J M, Croft K D, Puddey I B, et al. Soybean isoflavonoids and their metabolic products inhibit in vitro lipo-protein oxidation in serum.[J]. Nutr. Biochem, 1996, 7: 664-669.
[50] Arora A, Nair M G, Strasburg G M. Antioxidant activities of isoflavones and their biological meta-bolites in a liposomal system[J]. Arch Biochem Biophys 1998, 356: 133-141.
[51] Schneider H, Schwiertz A, Collins M D, et al. Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract. Arch Microbiol, 1999, 171: 81-91.
[52] Krishnamurty H G, Cheng K J, Jones G A, et al. Identification of products produced by anaerobic degradation of rutin and related flavonoids by Butyrivibrio sp. C3[J]. Can J Microbiol, 1970, 16: 759-767.
[53] Decroos K, Vanhemmens S, Cattoir S, et al. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions[J]. Arch Microbiol 2005, 183: 45-55.
[54] Schoefer L, Mohan R, Schwiertz A, et al. Anaerobic degradation of flavonoids by Clostridium orbiscindens[J]. Appl Environ Microbiol, 2003, 69: 5849-5854.
[55] Hur H G, Lay J, Rafii F, et al. Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin[J]. Arch Microbiol, 2000, 174: 422-428.
[56] Wang X L, Shin K H, Hur H G, et al. Enhanced biosynthesis of dihydrodaizein and dihydrogenistein by a newly isolated bovine rumen anaerobic bacterium[J]. Journal of Biotechnology, 2005, 115(3): 261-269.
[57] Hur H G, Beger R D, Rafii F, et al. Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein[J]. Archives of Microbiology, 2002, 178: 8-12.
[58] Wang X L, Hur H G, Kim Su IL. C-ring cleavage of isoflavones daidzein and genistein by a newly-isolated human intestinal bacterium Eubacterium ramulus julong601[J]. Journal of Microbiology and Biotechnology, 2004, 14(4): 766-771.
[59] Wang X L, Hur H G, Kim Su IL, et al. Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated anaerobic human intestinal bacterium[J]. Applied and Environmental Microbiology, 2005, 71: 214-219.
[60] Minamida K, Tanaka M, Abe A. Production of equol from daidzein by gram-positive rod-shaped bacterium isolated from rat intestine[J]. Journal of Bioscience and Bioengineering, 2006, 102(3): 247-250.
[61] Ueno T, Uchiyama S, Kikuchi N. The role of intestinal bacteria on biological effects of soy isoflavones in humans[J]. J Nutr, 2002, 594S, 132.
[62] Beck V, Rohr U, Jungbauer A. Phytoestrogens derived from red clover:an alternative to estrogen replacement therapy[J]. The Journal of Steroid Biochemistry and Molecular Biology, 2005, 94(5): 499-518.
[63] Chin-Dusting J P, Fisher L J, Lewis T V, et al. The vascular activity of some isoflavone metabolites: implications for a cardioprotective role[J]. British Journal of Pharmacology, 2001, 133(4): 595-605.
[64] Jiang F, Jones G T, Husband A J, et al. Cardiovascular protective effects of synthetic isoflavones derivatives in apolipoprotein edeficient mice[J]. Journal of Vascular Research, 2003, 40(3): 276-284.
[65]李燕红,林钰,等.农作物秸秆废弃物厌氧发酵生物制氢的研究[J].环境科学与技术, 2006,29(11): 8-9.
[66]卢怡,尹德升,等.牛粪、鸡粪发酵产氢潜力的研究[J].可再生能源, 2004, (2): 37-39.
[67] Chen W M, Tseng Z J, Lee K S, et al. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge[J]. Int J Hydrogen Energy, 2005,30(10): 1063-1070.
[68] Collet C, Adler N, Schwitzguébel J P, et al. Hydrogen production by Clostridium thermolacticum during continuous fermentation of lactose[J]. Hydrogen Energy, 2004,29: 1479-1485.
[69] Liu G, Shen J. Effects of culture medium and medium conditions on hydrogen production from starch using anaerobic bacteria[J]. Biosci Bioeng, 2004,98: 251-256.
[70] Evvyernie D, Morimoto K, Karita S, et al. Conversion of chitinouswaste to hydrogen gas by Clostridium paraputrificum M-21[J]. Biosci Bioeng, 2001,91: 339-343.
[71] Wang C C, Chang C W, Chu C P, et al. Producing hydrogen from wastewater sludge by Clostridum bifermentans[J]. Biotechnol, 2003,102: 83-92.
[72] Nakashimada Y, Rachman M A, Kakizono T, et al. Hydrogen production of Enterobacter aerogene saltered by extracellular and intracellular redox states[J]. Hydrogen Energy, 2002, 27: 1399-1405.
[73] Kumar N, Das D. Enhancement of hydrogen production by Enterobacter cloacae IIT-BT 08[J]. Process Biochem, 2000, 35: 589-593.
[74] Mandal B, Nath K, Das D. Improvement of biohydrogen production under decreased partial pressure of H2 by Enterobacter cloacae. Biotechnol Lett, 2006, 28(11): 831-835.
[75] PodestáJ J, Navarro A M, Estrella C N, et al. Electrochemical measurements of trace concentrations of biological hydrogen produced by Enterobacteriaceae[J]. Inst Pasteur, 1997, 148: 87-93.
[76]李永峰,任南琪,杨传平,等.一株高效产氢产酸细菌的鉴定与产氢特性[J].中国环境科学, 2005, 25(2): 210-213.
[77] Oh Y K, Park M S, Seol E H, et al. Isolation of hydrogen producing bacteria from granular sludge of an upflow anaerobic sludge blanket reactor[J]. Biotechnol Bioprocess Eng, 2003, 8: 54-57.
[78] Shin H S, Youn J H, Kim S H. Hydrogen production from foodwaste in anaerobic mesophilic and thermophilic acidogenesis[J]. Hydrogen Energy, 2004, 29: 1355-1363.
[79]Tanisho S, Kuromoto M, Kadokura N. Effect of CO2 removal of hydrogen production by fermentation[J]. Hydrogen Energy, 1998, 23(7): 559-563.
[80]王继华,赵爱萍.生物制氢技术的研究进展与应用前景[J].环境科学研究, 2005, 18(4): 129-135。
[81]冯树,张忠泽.混合菌——一类值得重视的微生物资源[J].世界科技研究与发展, 2000, 22(3): 44-47.
[82]韩梅,李春龙,韩晓日,等.大豆根瘤菌和胶质芽孢杆菌混合培养研究[J].沈阳农业大学学报, 2009, 40(2): 188-192.
[83] Weimer, P J. Mixed cultures in Biotechnology[M]. Mc Graw Hill, New York, 1991.
[84]齐云,袁月祥,陈飞,等.一组纤维素分解菌的分离、筛选及其产酶条件的研究[J].天然产物研究与开发, 2003, 15(6): 510-512.
[85]李霞,王向东,林松,等.混合型微生物絮凝剂产生菌的最佳培养条件[J].应用与环境生物学报, 2005, 11(3): 377-380.
[86]林红雨,陈策实,尹光林.欧文氏菌和棒杆菌的属间融合研究[J].微生物学通报, 1999, 26(1), 3-6.
[87]马辉文,王疆元.利用微生物混合培养物生产甜菜渣单细胞蛋白[J].武汉大学学报, 1994, (4), 107-114.
[88]谢忠,陈荣.利用微生物混合培养技术生产玉米废渣单细胞蛋白[J].粮食与饲料工业, 1994, (7), 28-31.
[89]代小江,王礼德,贺锡勤.利用微生物混合培养物生产沙棘果渣单细胞蛋白[J].微生物学通报, 1995, 22(5): 267-270.
[90]冯树,周樱桥,张忠泽.微生物混合培养极其应用[J].微生物学通报, 2001, 28(3): 92-95.
[91]东秀珠,蔡妙英.常见细菌系统鉴定手册(第一版)[M].北京:科学出版社, 2001.
[92]杜连祥,等.工业微生物学试验技术[M].天津科学技术出版社, 1992.
[93] Lane, D J. 16S/23S rRNA sequencing. In E. Stackebrandt and M. Goodfellow (eds.), Nucleic Acid techniques in Bacterial Systematics[M]. New York: John Wiley and Sons, 1991.
[94] Karel D, Steffi V, Sofie C, et al. Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions[J]. Arch Microbiol, 2005, 183: 45–55.
[95]李朝东.大豆异黄酮还原菌株的耐氧驯化与诱变选育[D].河北农业大学, 2009.