【作者】 樊进茹；

【导师】 王秀伶；

【作者基本信息】 河北农业大学 ， 微生物与生化药学， 2010， 硕士

【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。更多还原

【Abstract】 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.更多还原