与纳豆激酶有亲和吸附特性的大豆蛋白性质研究

采用偶联纳豆激酶的Sepharose-4B作为吸附介质,用亲和层析技术从大豆蛋白中得到了与纳豆激酶具有亲和吸附作用的目的蛋白,采用变性电泳技术测得其分子量分别约为30.0和26.0 kDa;采用表面等离子共振技术测定了目的蛋白与纳豆激酶的亲和吸附,检测出此蛋白中含有与纳豆激酶偶联的结构,并且推测得出2种目的蛋白是11S(大豆球蛋白)的2个亚基对。     

纳豆激酶的发酵和纯化方法

对纳豆激酶在菌种选育、发酵条件及分离纯化工艺方面的研究进展作了综述,介绍了提高纳豆激酶产量的多种固、液体发酵条件和菌种选育结果,并归纳了膨胀床吸附、金属螯合双水相亲和纳豆分配、反胶束萃取、磁性微球分离等分离技术在纳豆激酶分离纯化中的应用.     

纳豆激酶的亲和吸附工艺研究

采用静态吸附方法验证了大豆颗粒对纳豆激酶的亲和吸附特性,测定了其静态吸附动力学特性和吸附等温线以及一些吸附条件。结果表明:该亲和吸附等温线符合Langmiur方程,吸附动力学符合扩散方程;吸附的最佳缓冲液选择pH6.0、0.01 mol.L-1的PBS,在静态时选用大豆颗粒的最大吸附量为6351.58 IU.g-1,洗脱后收率达到81.30%,纯化倍数约30.23倍。初步推断大豆蛋白中含有与纳豆激酶特异性吸附的的配体结构。     

豆渣固体发酵产纳豆激酶的工艺优化及其部分酶学性质研究

研究纳豆菌固体发酵产纳豆激酶的工艺及其部分酶学性质.采用单因素和正交试验,对以豆渣为原料纳豆菌固体发酵生产纳豆激酶的工艺条件进行优化,并利用最佳发酵工艺制备纳豆激酶粗品,对纳豆激酶的部分性质进行研究.结果表明固体发酵培养基最佳配比:豆渣:麸皮=5:2,初始含水量65%,接种量为10%,初始pH为8.0,培养温度30℃.采用最适培养基和优化工艺,在250 m1三角瓶中进行验证实验,纳豆激酶的酶的产率可达到1577U·g-1.酶学性质研究表明,最适反应温度为60℃,37℃以下稳定,最适反应pH为8.0,在pH7-9溶液中基本稳定.体外溶栓作用表明,纳豆激酶溶解纤维蛋白的方式是直接溶解,而不是通过激活纤溶酶原.     

益康纳豆的研制

益康纳豆是以大豆为原料经纳豆芽孢杆菌发酵制得的健康食品。本文从多种发酵豆制品中分离筛选到一株纳豆激酶活性好的菌株，并对该菌株的形态、生理生化特性、影响固体制备的因素、固体培养物保藏试验以及该菌株纳豆激酶的酶学性质等内容进行了研究。研究表明，经发酵后的纳豆可溶性总氮及多种氨基酸含量明显增高，富含多种生物活性酶，同时也是理想的微生态调节剂。     

高产纳豆激酶突变菌株产酶条件的研究

为了获得最佳产酶条件,通过研究不同碳源(葡萄糖、木糖、麦芽糖、蔗糖、淀粉),氮源(牛肉膏、蛋白胨、大豆蛋白胨、酵母膏、硫酸铵),碳氮浓度比和无机离子组成的培养基发酵,利用纤维蛋白平板法测定纳豆激酶的活性.得到纳豆激酶的最佳产酶条件为:1.5%蛋白胨,1.5%麦芽糖,0.05%硫酸镁,0.2%氯化钙,0.2%磷酸二氢钠和0.1%磷酸氢二钠;碳氮浓度比为1:1.     

聚丙烯酰胺固相微球与黄曲霉毒素B1抗体偶联条件的研究

以表面带羧基的聚丙烯酰胺固相微球为载体,通过碳二亚胺(EDC)活化,共价结合兔黄曲霉毒素B1抗体.研究结果表明最适偶联pH值为6.0～6.5,最适反应时间为17～20h,最佳EDC用量为5～15mg/10mg微球.抗体的最大偶联效率达60.40%,为利用聚丙烯酰胺微球建立黄曲霉毒素快速检测技术奠定了基础.     

纳豆激酶的分离纯化及体外溶栓特性研究

通过硫酸铵分步沉淀和Sephadex G-100凝胶过滤层析,分离纯化纳豆激酶,并用SDS-PAGE和PAGE电泳检测纯化效果,体外溶栓试验检测纳豆激酶的溶栓特性。SDS-PAGE显示纳豆激酶为单一条带,分子量为33kDa,而PAGE至少有3个具有纤溶活性的区域,表明纳豆激酶可能由几种同工酶组成,体外溶栓结果表明:与蚓激酶相比,纳豆激酶具有较强的的体外溶栓和抗凝作用,并有一定的溶血作用。     

纳豆菌固定化材料优化的研究

为获得固定化纳豆菌材料,以海藻酸钠(SA)和聚乙烯醇(PVA)为包埋材料,采用固定化细胞技术对纳豆菌生产纳豆激酶进行了研究.结果表明:在PVA中加入SA进行细胞包埋可获得渗透性能好强度高的固定化细胞.通过正交试验进一步确定,当PVA的浓度为9%、SA的浓度为1%、硼酸的浓度为5%、CaCl2的浓度为6%时,固定化细胞的强度最好,采用摇床培养可连续发酵使用6次,活性也很高,产生的纳豆激酶酶活溶纤圈直径积达87.69 mm2·15μL-1.     

纳豆芽孢杆菌发酵产品研究进展

纳豆是日本的传统食品,由纳豆芽孢杆菌发酵制作而成,其分泌的纳豆激酶具有安全性高、溶栓性能力强等特点。近些年来,纳豆芽孢杆菌(Bacillus natto)在食品及农副产物深加工领域应用越来越广泛,生产了多种活性产物并有效地提高了食品的营养特性。纳豆激酶除溶栓外更多最新的功能活性也逐渐被研究者发现,在稳定性及气味改良等方面的改善也有着很大的进步。本文综述了纳豆芽孢杆菌及纳豆激酶的开发及应用现状及存在的瓶颈问题和解决对策等研究进展,以期为纳豆相关食品的开发提供支持。     

球状壳聚糖树脂对茶多酚的吸附热力学和动力学研究

采用反相悬浮交联法制备了球状壳聚糖树脂(RCM),通过静态吸附实验,研究了RCM对茶多酚的吸附热力学和动力学特性。结果表明:吸附等温线符合Laugmiur等温曲线,且平衡常数Kb随着温度升高而升高。吸附是非自发的、吸热的、熵增加的过程。吸附过程符合二级动力学吸附模型,吸附过程主要受粒子内扩散模型控制。     

Preparation and BSA Adsorption Behavior of Chitosan-arginine Based Nanofiber Membranes

In this work, an affinity nanofiber membrane was successfully prepared by solution blowing of arginine-modified chitosan (CS-Arg) for bovine serum albumin (BSA) adsorption. CS-Arg was firstly synthesized by coupling L-arginine onto chitosan backbone. Then, CS-Arg nanofiber membranes (CANFs) were fabricated using solution blowing process with Polylactide (PLA) as assistant polymer. The results showed that CANFs effectively adsorbed BSA, and the adsorption capacities were influenced by the degrees of substitution (DS) of arginine in CS, pH value, contact time, and initial protein concentration. The highest adsorption capacity of 445.19 mg/g was achieved uvnder the following conditions: DS of 43.7 %, pH of 7.14, and initial concentration of 3.0 mg/ml. BSA adsorbed on the CANFs membrane conformed to Langmuir model, and the adsorption rate was consistent with the second-order kinetics model. This work implies that an arginine-modified chitosan nanofiber-based novel biomaterial has a potential application in adsorption of BSA.     

Novel Spray Dried Glycerol 2-Phosphate Cross-Linked Chitosan Microparticulate Vaginal Delivery System—Development,Characterization and Cytotoxicity Studies

Chitosan microparticulate delivery systems containing clotrimazole were prepared by a spray drying technique using glycerol 2-phosphate as an ion cross-linker. The impact of a cross-linking ratio on microparticle characteristics was evaluated. Drug-free and drug-loaded unmodified or ion cross-linked chitosan microparticles were examined for the in vitro cytotoxicity in VK2/E6E7 human vaginal epithelial cells. The presence of glycerol 2-phosphate influenced drug loading and encapsulation efficacy in chitosan microparticles. By increasing the cross-linking ratio, the microparticles with lower diameter, moisture content and smoother surface were observed. Mucoadhesive studies displayed that all formulations possessed mucoadhesive properties. The in vitro release profile of clotrimazole was found to alter considerably by changing the glycerol 2-phosphate/chitosan ratio. Results from cytotoxicity studies showed occurrence of apoptotic cells in the presence of chitosan and ion cross-linked chitosan microparticles, followed by a loss of membrane potential suggesting that cell death might go through the mitochondrial apoptotic pathway.     

Preparation and acid dye adsorption behavior of polyurethane/chitosan composite foams

We prepared a series of polyurethane(PU)/chitosan composite foams with different chitosan content of 5∼20 wt% and investigated their adsorption performance of acid dye (Acid Violet 48) in aqueous solutions with various dye concentrations and pH values. It was observed that PU/chitosan composite foams exhibited well-developed open cell structures. Dye adsorption capacities of the composite foams increased with the increment of chitosan content in composite foams, because amine groups of chitosan serve as the binding sites for sulfonic ions of acid dyes in aqueous solutions. In addition, dye adsorption capacities of composite foams were found to increase with decreasing the pH value, which stems from the fact that the enhanced chemisorption between protonated amine groups of chitosan and sulfonic ions of acid dye is available in acidic solutions. The dye adsoption kinetics and equilibrium isotherm of the composite foams were well described with the pseudo-second order kinetic model and Langmuir isotherm model, respectively. The maximum adsorption capacity (q _max) for the PU/chitosan composite foams with 20 wt% chitosan content is evaluated to be ca. 30 mg/g.     

Sulfur-modified chitosan hydrogel as an adsorbent for removal of Hg(II) from effluents

Sulfur-modified chitosan hydrogel (SMCH) was successfully synthesized by grafting dimethyl 3,3′-dithiodipropionate onto chitosan and then crosslinking with N,N′-methylene diacrylamide (MBA). The structure and properties of chitosan and sulfur-modified chitosan (SMC) were characterized and analyzed by Fourier transform infrared spectroscopy (FT-IR), Nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and Thermogravimetric analysis (TGA). Meanwhile, chitosan hydrogel and SMCH were characterized by Scanning electron microscope (SEM). In addition, static adsorption Hg(II) ions properties of chitosan hydrogel and SMCH were also investigated. The FT-IR and ¹H NMR manifested that SMC was synthesized successfully. The XRD and TGA showed that the crystallinity and thermal stability of SMC decreased. SEM showed that the SMCH had much more pores and bigger specific surface area than chitosan hydrogel. The result of adsorption experiment indicated that the SMCH showed noticeable improvements in the adsorption capacity of Hg(II), and had the highest adsorption capacity (187.5 mg/g) at pH 5.0. The equilibrium was achieved at 40 min. And the maximum adsorption capacities were 186.9 mg/g of SMCH.     

Chitosan-Genipin Microspheres for the Controlled Release of Drugs: Clarithromycin,Tramadol and Heparin

The aim of this study was to first evaluate whether the chitosan hydrochloride-genipin crosslinking reaction is influenced by factors such as time, and polymer/genipin concentration, and second, to develop crosslinked drug loaded microspheres to improve the control over drug release. Once the crosslinking process was characterized as a function of the factors mentioned above, drug loaded hydrochloride chitosan microspheres with different degrees of crosslinking were obtained. Microspheres were characterized in terms of size, morphology, drug content, surface charge and capacity to control in vitro drug release. Clarithromycin, tramadol hydrochloride, and low molecular weight heparin (LMWH) were used as model drugs. The obtained particles were spherical, positively charged, with a diameter of 1–10 μm. X-Ray diffraction showed that there was an interaction of genipin and each drug with chitosan in the microspheres. In relation to the release profiles, a higher degree of crosslinking led to more control of drug release in the case of clarithromycin and tramadol. For these drugs, optimal release profiles were obtained for microspheres crosslinked with 1 mM genipin at 50 ºC for 5 h and with 5 mM genipin at 50 ºC for 5 h, respectively. In LMWH microspheres, the best release profile corresponded to 0.5 mM genipin, 50 ºC, 5 h. In conclusion, genipin showed to be eligible as a chemical-crosslinking agent delaying the outflow of drugs from the microspheres. However, more studies in vitro and in vivo must be carried out to determine adequate crosslinking conditions for different drugs.     

Synthesis,Characterization, and Antibacterial Activity of Cross-Linked Chitosan-Glutaraldehyde

This present study deals with synthesis, characterization and antibacterial activity of cross-linked chitosan-glutaraldehyde. Results from this study indicated that cross-linked chitosan-glutaraldehyde markedly inhibited the growth of antibiotic-resistant Burkholderia cepacia complex regardless of bacterial species and incubation time while bacterial growth was unaffected by solid chitosan. Furthermore, high temperature treated cross-linked chitosan-glutaraldehyde showed strong antibacterial activity against the selected strain 0901 although the inhibitory effects varied with different temperatures. In addition, physical-chemical and structural characterization revealed that the cross-linking of chitosan with glutaraldehyde resulted in a rougher surface morphology, a characteristic Fourier transform infrared (FTIR) band at 1559 cm⁻¹, a specific X-ray diffraction peak centered at 2θ = 15°, a lower contents of carbon, hydrogen and nitrogen, and a higher stability of glucose units compared to chitosan based on scanning electron microscopic observation, FTIR spectra, X-ray diffraction pattern, as well as elemental and thermo gravimetric analysis. Overall, this study indicated that cross-linked chitosan-glutaraldehyde is promising to be developed as a new antibacterial drug.     

大豆主要过敏原Gly m Bd30K蛋白单克隆抗体的制备与应用

利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合.采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化.采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体...