【作者】 周晓玲；

【导师】 崔为正；

【作者基本信息】 山东农业大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 1-脱氧野尻霉素(DNJ)是一种α-葡萄糖苷酶抑制剂,能有效抑制糖吸收,降低血糖值。桑根皮在中国古代就被用作治疗“消渴症”的良药。现代科学已经证明,桑树中降血糖的主要有效成分是DNJ。鉴于此,前人已经对不同桑品种、桑树不同部位、不同叶位、不同季节、不同产地、甚至多种食桑昆虫的DNJ含量进行了研究,但是桑树体内DNJ的来源尚未有明确的研究结果。目前,从中药材中提取以及利用植物内生菌获得具有疗效的活性物质成为药物研究的重要方向。本实验室在前期试验中已经从桑树中分离到两株产DNJ的内生菌:嗜麦芽寡养假单孢杆菌(Stenotrophomonas maltophilia)Y1和藤黄微球菌(Micrococcus luteus)Y2。本文对Y2菌株的生理生化指标和生物学特性进行了测定;研究了Y2菌株产DNJ的最佳发酵条件和发酵配方,并对发酵产物进行了初步分离提纯;建立了体外α-葡萄糖苷酶抑制活性测定模型,测定了Y1和Y2发酵产物粗提物对α-葡萄糖苷酶的抑制活性。同时,为探讨桑树中的DNJ来源,本试验还测定了桑树无菌苗的DNJ含量。主要研究结果如下:1测定了Y2菌株的生理生化指标和生物学特性,结果表明Y2与藤黄微球菌的鉴定特征相符。Y2菌株接种后36 h为对数生长期,36 h~60 h是生长稳定期,60 h后开始衰老;菌株适宜生长的温度是25℃~35℃,可耐受最低生长温度为4℃,最高为40℃,最适生长温度为30℃;该菌株较适宜的pH范围是6.5~8.0,其中最适宜的pH是7.0,适宜在中性偏碱性条件下生长,并有较强的耐盐性。2通过摇瓶培养方法,探讨了环境条件和培养基组分对Y2菌株产DNJ的影响。利用单次单因子试验确定了摇瓶发酵的最佳条件为:接种量2%(v/v)、摇瓶装液量50 mL/250 mL、发酵温度30℃、初始pH 7.0,发酵时间30 h。在此基础上通过单次单因子试验确定了摇瓶发酵配方的最佳碳源、氮源、无机盐组合。运用PB部分因子试验分析了培养基组分:蛋白胨、麦芽糖、MgSO4、KH2PO4、NaCl对Y2菌株产DNJ的影响。结果表明,蛋白胨对Y2菌株发酵产DNJ的影响达到极显著水平,KH2PO4对Y2菌株发酵产DNJ的影响达显著水平,而麦芽糖、MgSO4、NaCl对Y2菌株发酵产DNJ的影响不显著。通过最陡爬坡路径试验逼近最大响应区域,最后用中心组合设计和响应面分析,确定了主要影响因子的最佳浓度。最优化培养基为:麦芽糖20.00 g、蛋白胨36.53 g、KH2PO4 2.22 g、MgSO4 0.5 g、NaCl 4 g、去离子水1000 mL。运用最佳发酵培养基和最佳发酵条件进行摇瓶发酵,利用RP-HPLC-UV法测得发酵液中DNJ的浓度达到了213.18μg/ml,较基础培养基DNJ浓度值提高了184.63%。3对Y2菌株产生的DNJ进行了初步分离纯化。Y2菌株发酵样品进行浓缩后,又经过乙醇沉淀、过NKA-9型大孔吸附树脂和强酸型离子交换树脂,最后的样品经过浓缩、冷冻干燥成样品。提取物衍生化后利用RP-HPLC-UV法对样品进行检测,结果显示,该纯化工艺去除了大量杂质,色谱图中的杂峰大大减少,DNJ含量达到了0.337%,较之前提高了5.3倍。4通过体外α-葡萄糖苷酶抑制模型测定了Y1、Y2菌株发酵液提取物对α-葡萄糖苷酶的抑制活性。结果表明:两种内生菌发酵分离物都具有很强的α-葡萄糖苷酶抑制活性,且最终抑制活性均高于对照药物阿卡波糖,菌株Y2发酵液提取物的抑制中浓度为111.58 mg/L,菌株Y1发酵液提取物的抑制中浓度为2307.56 mg/L,分离物的抑制活性均与浓度呈正相关,其抑制活性具有剂量依赖性。5检测了桑树无菌苗的DNJ含量。运用细菌培养基和真菌培养基对桑树无菌苗内的内生菌检测培养,平板中没有杂菌生长,推断桑树组培苗脱毒成功,已无内生菌在其体内共生。桑树无菌组培苗干物中DNJ含量检测结果为0.197%,与大田中的桑树DNJ含量相当,证明桑树体内的DNJ不只依赖桑树内生菌的产生,桑树本身亦可合成。更多还原

【Abstract】 1-deoxynojirimycin(DNJ) is anα-glucosidase inhibitor and could lower blood glucose efficiently. In ancient China, the root of mulberry tree was thought to be an excellent treatment for Diabetes. The modern science now proved that DNJ is the main active constituent of the mulberry tree lowering the blood glucose. View of that, former studies has determined the DNJ concentration from these materials as follows: different mulberry varieties, different mulberry positions, different leaf positions, different seasons, different metropolis as well as insects feeding on mulberry. However, there is no conclusion of the exact origin of DNJ in mulberry.Now it becomes an important direction of researche works that obtained to active compound was extracted frome Chinese crude drug and plant endophyte. In our former studies endophytes producing DNJ stably, Y1 Stenotrophomonas maltophilia and Y2 Micrococcus luteus, have been isolated. Further researches were as follows: the determination Physiology and biochemistry indicator and bionomics of Y2, the optimization of Y2 fermenting conditions, the initial identification and characterization of DNJ produced by the endophytes, theα-glucosidase inhibition activity of fermentation crude extract. At the same time, We determined the DNJ content of germ free mulberry. The main results are as follows:1 The determination Physiology and biochemistry indicator and bionomics of Y2. Contrast with Micrococcus luteus, the characteristic of Y2 is similar to it. Strain Y2, inoculating it, enter logarithmic growth phase in 36 h, stationary phase is 36 h~60 h, enter senescence phase after 60 h. The suitable temperature is 25℃~35℃, and minimum 4℃. maximum 40℃. The suitable pH is 6.5~8.0, and the most suitable one is 7.0. At neutral and alkaline conditions, it could grow well, and strong salt tolerance.2 The effects of environmental conditions and nutrition of strain Y2 producing DNJ were studied through the cultivation in shake-flask fermentation.The optimal shaking flask fermentation conditions were confirmed by one-factor-at-a-time experiment.The optimized fermentation conditions for DNJ production were: inoculum volume 2%(v/v), solution amount 50 mL/250 mL, fermentation temperature 30℃, initial pH 7.0, fermentation time 30 h.Based on the results above, the effects of nutrition on production of DNJ were studied involving the optimal carbon, the optimal nitrogen, inorganic salt combination by one-factor-at-a-time experiment. In the optimization of medium, the influences of peptone, maltose, MgSO4, KH2PO4 and NaCl on DNJ production were first evaluated using Plackett Burman design. Among of the test components, the effect of peptone reach extremely significant level and the KH2PO4 shows significant effect on the DNJ production. However, maltose, MgSO4 and NaCl have no significant effects on DNJ production. The path of steepest ascent was used to approach the optimal region of the medium composition. In the last step, the optimal concentrations of test components were determined by central composite design and response surface analysis.The components of optimum medium for production of DNJ were: maltose 20.0g/L, peptone 36.53 g/L, KH2PO4 2.22 g, MgSO4 0.5 g/L, NaCl 4 g/L, demineralized water 1000 mL.Based on the optimal fermentation condition and medium, the DNJ concentration of fermentation broth after RP-HPLC-UV reached 213.18μg/ml, 184.63% higher than that of basal medium.3 The initial extraction, purification of DNJ produced by strain Y2 was studied by Rep-HPLC-UV analysis. Strain Y2 fermentation broth was concentrated, precipitated by ethanol, separated by NKA-9 and duolite, concentrated again, and freezed to dehydration. Determined by RP-HPLC-UV analysis after derivatization, the DNJ concentration was up to 0.337%, enhanced by 5.3 times, the sample product was purified effective.4 Determination of theα-glucosidase inhibition activity of fermentation crude extract byα-glucosidase inhibition model in vitro. The results are as follows: The sample products both better than acarbose have effectα-glucosidase inhibition activity. The IC50 of strain Y2 is 111.58 mg/L, and Y1 is 2307.56 mg/L. Its activity is positively relative with concentration in certain range, and exhibits dose-dependent inhibition. 5 Determination of the germ free mulberry DNJ concentration. The germ free mulberry were detected with bacteria medium and fungus medium, and were proved no parachorium endophyte. Compared with mulberry growing in field, the DNJ concentration of germ free mulberry are equivalent, 0.197%. The DNJ production depended on not only endophyte but also mulberry.更多还原