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微生物纤溶酶和纳米银抗栓抗菌复合系统的构建
Alternative TitleConstruction of the antithrombotic antibacterial composite system of microbial fibrinolytic enzyme and nanosilver
魏雪团
Subtype博士
Thesis Advisor刘会洲
2013-05-01
Degree Grantor中国科学院研究生院
Degree Discipline生物化工
Keyword纤溶酶   鹰嘴豆   固体发酵   纳米银   抗栓抗菌复合系统
Abstract本文从微生物纤溶酶和纳米银的制备,以及其复合抗栓抗菌应用系统的构建等方面开展应用基础研究。首先,通过纤溶活性筛选法获得一株纤溶酶高产菌株-解淀粉芽胞杆菌LSSE-62。在34°C和50%初始含水量条件下发酵鹰嘴豆,纤溶活性达39.28 FU/g干重,经硫酸铵沉淀获得高活性冻干粉(3569.68 FU/g)。发酵后,鹰嘴豆的总酚、总黄酮含量和抗氧化活性得到提高,并具有强大的抗凝活性和丰富的营养成分。 其次,通过基因扩增法筛选到一株高产纳豆激酶纤溶酶(NK)的枯草芽胞杆菌,发酵鹰嘴豆的NK活性达356.25 FU/g,副产物聚谷氨酸含量较低。发酵的鹰嘴豆经50%乙醇萃取和75%乙醇沉淀,NK活性回收率达89%,聚谷氨酸回收率降至21%,获得高活性冻干粉(4000.58 FU/g)。通过甲基丙烯酸-丙烯酸乙酯聚合物包衣技术制备了NK肠溶胶囊,实现了NK的抗酸保护和肠道控制释放。 为开发NK在抗栓抗菌复合系统中的应用,首次通过阳光诱导不同生物介质高效制备了4种纳米银。通过阳光诱导解淀粉芽胞杆菌细胞抽提物合成纳米银(E-Ag)。在光强度70,000 lx、抽提物3 mg/mL、NaCl 2 mM的条件下,80 min可完成1 mM Ag+的还原形成E-Ag。E-Ag主要是平均粒径为14.6 nm的球形银单质纳米晶,Zeta电位达-70.84 mV,抗菌性能良好。根据红外分析推测其反应机理为:蛋白肽类分子螯合Ag+,光驱动下发生还原反应形成稳定的纳米分散体系。相比E-Ag,解淀粉芽胞杆菌发酵上清液合成的纳米银(S-Ag)平均粒径增大,稳定性降低,且反应速率降低。另外,通过胰蛋白胨和酵母抽提物合成两种纳米银(T-Ag和Y-Ag),胰蛋白胨可在60 min内还原1 mM Ag+形成T-Ag。T-Ag和Y-Ag均为螯合蛋白肽类分子的球形单质银纳米晶,抗菌性能良好,酸性pH和长期阳光照射可显著降低其稳定性。 最后,通过T-Ag吸附NK制备了纳豆激酶-纳米银抗栓抗菌复合颗粒(NK-Ag),T-Ag对NK的饱和吸附量高达24.35 FU/mg,NK-Ag还增强了NK的热稳定性和抗凝活性。在静电作用力驱动下,NK-Ag与聚乙烯亚胺(PEI)层层自组装成规则、均匀的NK-Ag-PEI复合膜,具有良好的抗栓活性和抗菌活性。
Other AbstractIn this study, microbial fibrinolytic enzymes and nanosilver were prepared, and their antithrombotic and antibacterial composite system was constructed for further application. Firstly, a fibrinolytic enzyme high productive strain of Bacillus amyloliquefaciens LSSE-62 was isolated by fibrinolytic activity based isolation method. By fermentation of chickpeas under the conditions of 34°C and 50% initial moisture content, the fibrinolytic activity reached 39.28 FU/g, and the high activity lyophilized powder (3569.68 FU/g) were obtained by ammonium sulfate precipitation. After fermentation, the total phenolic, total flavonoid contents and the antioxidant activities of chickpeas were improved significantly. Moreover, the fermented chickpeas possessed potent anticoagulant activity and various nutritional ingredients. Secondly, a nattokinase high productive strain of Bacillus subtilis was isolated by gene amplification screening method. By fermentation of chickpea with B. subtilis, the NK activity reached 356.25 FU/g, with a low concentration of poly-γ-glutamic acid (PGA), a main by-product of nattokinase. By extraction with 50% and precipitation with 75% ethanol solution, the NK activity recovery reached 89%, with a reduced PGA recovery (21%), and 4000.58 FU/g of NK powders were obtained. By enteric coating of nattokinase capsule with methacrylic acid-ethyl acrylate copolymer, the nattokinase was protected from being denatured under various acid conditions, and controlled release at simulated intestinal fluid was realized. To apply the nattokinase in antithrombotic and antibacterial composite system, 4 kinds of nanosilver (AgNPs) were prepared by sunlight-induced biosynthesis methods. AgNPs (E-Ag) were obtained by solar irradiation of cell-free extracts of B. amyloliquefaciens and AgNO3. Under optimized conditions (solar intensity 70000 lx, extract concentration 3 mg/mL, and NaCl content 2 mM), 1 mM of the Ag+ was reduced to AgNPs within 80 min. E-Ag was in the form of spherical pure Ag nanocrystalline with a mean diameter of 14.6 nm. The Zeta potential of E-Ag reached -70.84 mV, and it showed significant antimicrobial activity. For a possible mechanism from the infrared analysis, Ag+ was capped with proteins (peptides), and then reduction reaction were drived by light energy to form stable nanosilver dispersion system. Compared with cell extracts, the supernatant-prepared AgNPs showed a larger average diameter and a reduced stability, as well as a lower reaction rate. Additionally, T-Ag and Y-Ag were synthesized with tryptone and yeast extract, respectively. Reduction of 1 mM Ag+ could be completed within 60 min with tryptone to form T-Ag. T-Ag and Y-Ag were in the form of peptides-capped spherical pure Ag nanocrystals, showing obvious antimicrobial activity, and the colloidal stability of AgNPs was obviously reduced by acidic pH and long-time sunlight radiation. Finally, the antithrombotic and antibacterial complex (NK-Ag) was prepared by adsorption of NK with T-Ag, and a high sturation adsorption capacity of 24.35 FU/mg AgNPs was obtained. The NK-Ag also enhanced the thermal stability and anticoagulant activity of NK. Based on the electrostatic force between NK-Ag and polyethylenimine (PEI), uniform and regular NK-Ag-PEI composite film was prepared using layer by layer self-assembly technology, and the composite film showed potent antithrombotic activity and antibacterial activity.
Pages127
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/8254
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
魏雪团. 微生物纤溶酶和纳米银抗栓抗菌复合系统的构建[D]. 中国科学院研究生院,2013.
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