【作者】 汪兴平；

【导师】 谢笔钧；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2006， 博士

【摘要】 葛仙米以其资源稀少、营养价值丰富、独特的生物活性和广泛的应用价值而引起人们地关注。葛仙米作为一种食药同源的传统食品已有几千年的历史，但对其主要活性成分的研究很少。为探讨葛仙米中的生物活性成分，本文以野生葛仙米为研究对象，充分利用生物大分子研究的最新技术和现代分离、分析手段，首次系统地研究了葛仙米蛋白质的提取、分离和优化，并对藻蓝蛋白的纯化和藻蓝蛋白的一级结构、空间结构进行了研究，且分析了藻蓝蛋白抗氧化活性。其主要结果如下：1葛仙米的生物学特性与生化组成研究以湖北省鹤峰县走马镇野生葛仙米为研究对象，分析了生态条件及分布规律。葛仙米主要分布于亚热带二高山地区，属亚热带大陆性季风湿润气候；土壤含Fe、Mn相对较高；年最高温度36℃，最低温度0.7℃，年均降雨量1836.4mm，年均日照1342h。葛仙米营养丰富，蛋白质、多糖含量高，达54％；富含维生素，V_B1 1.2mg／100g，V_B2 11.8mg／100g，Vc 550mg／100g，V_E 7.0mg／100g，β-胡萝卜素114mg／100g，高于一般食物；脂肪含量为5.50％，以中碳链为主；含大量矿质元素和微量元素。2葛仙米蛋白的提取、分离和优化及藻蓝蛋白的纯化和鉴定试验选取pH值、温度和提取时间这三个影响葛仙米蛋白得率的最重要的因素，采用三因素二次旋转正交回归组合实验，优化出葛仙米蛋白提取的最佳工艺条件为，缓冲液pH为7.3、浸提时间4.3h、盐浓度0.21mol／L，葛仙米蛋白得率达7.13％。采用反相分段梯度盐析法，即先用20％（NH₄）₂SO₄饱和度沉淀除去杂质，60％（NH₄）₂SO₄沉淀PC，沉淀溶解以后第2次和第3次盐析时逐渐变窄沉淀范围，提高了藻蓝蛋白的纯度，保证了较高的得率。葛仙米蛋白提取工艺为：原料—复水—反复冻融—超声波破壁—磷酸缓冲液浸提（4℃，4h／次，3次，pH值为7.0的0.05M磷酸钾缓冲液+0.2M NaCl）—高速冷冻离心（14000r／min，4℃，45min）—反相硫酸铵分步盐析法—透析—冷冻干燥得粗葛仙米蛋白。利用DEAE-Cellulose 52离子交换层析，采用步进式洗脱模式成功地将葛仙米蛋白分离出葛仙米藻蓝蛋白。采用羟基磷灰石（HA）柱层析、DEAE-Sepharose FF，进一步纯化后得到均一的级分。经柱层析法、分光光度法、荧光光谱法和电泳法鉴定其为具有色谱纯和电泳纯的样品。SDS-PAGE凝胶电泳法测定了葛仙米藻蓝蛋白相对分子质量，即为38.245K，是为两条不同分子质量（即19.852K和18.383K）的肽链通过二硫键连接而成。3葛仙米藻蓝蛋白一级结构的表征采用MALDI-TOF-MS测定NSPC的分子质量，获得了NSPC精确分子质量为37715。根据氨基酸自动分析仪测定的氨基酸组成和精确分子质量计算出NSPC分子总共有331个氨基酸残基，分别是由40个Asp、22个Thr、18个Ser、22个Glu、1个Gly、31个Ala、27个Cys、17个Val、13个Ile、22个Leu、5个Tyr、7个Phe、7个Lys、24个His、51个Arg、14个Pro、1个Met和9个Trp组成。胰蛋白水解酶分别酶解NSPC-1和NSPC-2后，分别采用MALDI-TOF-MS分析，其肽指纹谱图显示胰蛋白酶可将NSPC-1和NSPC-2水解成大小不等的多条肽段，质量数在800以上的NSPC-2有11段，即水解的肽质量分别为899.54、1454.77、1544.9、1683.87、1803.93、1811.91、1987.92、2003.90、2536.26、2915.42和3492.72。NSPC-2的氨基酸序列为：1 MKTPLTEAVS IADSQGRFLS STEIQVAFGR FRQAKAGLEAAKALTSKADS51 LISGAAQAVY NKFPYTTQMQ GPNYAADQRG KDKCARDIGY YLRMVTYCLI101 AGGTGPMDEY LIAGIDEINR TFELSPSWYI EALKYIKANH GLSGDAAVEA151 NSYLDYAINA LS肽质量指纹图谱和肽序列标签结合用于蛋白质数据库搜索，证明NSPC-2为极太螺旋藻α链，而NSPC-1未从数据库中检索到与此匹配的蛋白。4葛仙米藻蓝蛋白分子构象的表征采用FTIR、CD、AFM、SEM、X-衍射、DSC和FR等分析技术，测定了NSPC的空间结构及变性温度。FTIR和CD分析结果显示NSPC主要以α-螺旋结构为主，β-折叠或卷曲结构含量较少。荧光光谱显示，在水溶液中NSPC分子中的Trp残基两种类型都存在，既有暴露于分子外部的Trp残基，也有处于疏水内部的Trp残基。其溶液构象在pH7时最稳定，但酸碱度对其构象影响较小，在pH＜7时NSPC构象相对变化略大于在碱性环境。有机溶剂对其构象变化影响较大。通过AFM观察，在中性环境下，NSPC分子呈链状形式存在：DSC结果表明，NSPC变性温度为41.6℃。通过扫描电镜观察，NSPC聚集念主要以片层、针状和蜂窝状形式。X-射线衍射分析结果表明经冻干得到的NSPC粉术结晶度高，且结晶区分子的规整性较高。5葛仙米蛋白稳定性研究研究了温度、pH、光强、乙醇浓度、中性盐浓度、饱和度的硫酸铵溶液和不同蔗糖浓度等因子对其稳定性的影响。结果表明，葛仙米蛋白在自然光照下，温度低于40℃，pH5～8，乙醇浓度小于40°，低浓度中性盐和低浓度蔗糖溶液中能保持较好地稳定性，60％饱和度的硫酸铵溶液有利于其稳定性。6葛仙米藻蓝蛋白的抗氧化活性非脂质体系抗氧化实验、体外抗氧化实验等结果表明葛仙米蛋白粗提物具有良好的抗氧化性能作用。清除氧自由基的能力为：粗提物＞藻红蛋白＞藻蓝蛋白；这可能是由于粗提物中还含有多糖和酚类物质等共同作用的结果，葛仙米藻蓝蛋白对清除O₂^-、·OH自由基和H₂O₂具有较好的量效关系。在体外反应体系中，NSPC能显著地降低MDA生成和血和肝中过氧化物的含量，起到保护细胞膜和红血球的作用。更多还原

【Abstract】 The significant biological and apply value of Nostoc Sphaeroids kutz with abound nutrition and infrequent distribution had been paid people’s much attention. Although NSK as a kind of food and medicine function had been used for several thousand years, the main active component of activity and function is little investigated. In recent years, it was by a few people that its partial physiology and primary biochemistry composition was reported. In order to ascertain the active component of NSK, the phycocyanin was used as material, taking advantage of modern separation technology and biomacromolecules analysis method, first systemically studies on preparation and purification of NSPP, and its primary structure and high structure were also investigated. The main results were shown as follows:1 The biological trait of NSK and its biochemisty componentOn the basis of the biological trait of NSK having been investigated, in order to determine its biochemistry components, such as ecological condition and its contribution, Reproduction mechanism, water, protein, fat, polysaccharide, carotene and chlorophyll as well as mineral element, wild NSK, Zouma town, Hefeng county, Hubei province, was used as crude material.2 Extraction, separation, purification and identification of phycocyaninDefatted NSK as material, albumin, globin and salt-soluble protein were separated from NSK by salting-in and salting-out methods for the first time and the the optimal extraction technology were studied. The results were as follows: pH 7.0, salt concentration 0.2 mol/L and extracted time was 4 h, and the output of phycobiliproteins was 7.13 percent. DEAE-Cellulose ion-exchange chromatography was used to separation NSPP. It was successful to obtain five fractions, by the step gradient. NSPP was further purified by DEAE-Separose FF and HA chromatography. Some methods such as spectrophotometer and chromatography and electrophoresis were applied to identify the purity of phycocyanin, and it was identified to be chromatography and electrophoresis grade pure.3 Primary structure characterization of phycocyaninThe primary structure of phycocyanin was studied with matrix-assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and sodium dodecyl sulfate ployacrylamide gel electrophoresis （SDS-PAGE）, its molecular weight and their peptide mass fingerprinting were analysed. The results demonstrated that the measured molecular weight of phycocyanin was 37.715Kd, and consisted of two peptides which were similar with their molecular weight and amino acid sequences.4 The comformation of phycocyaninPhycocyanin high structure was investigated, as the result of circular dichroism （CD） spectrum and Fourier transformation infared （FTIR） spectrum shown, the secondary structure of NSPC was mainlyα-helix structure and the content ofβ-shee or coil was very small. While NSPC’s agglutination form was observed by force atom microscope （AFM） and scan electron microscope （SEM） there existed mainly catenulate distribution. Meanwhile, they denatured temperature was also studied by differential scan calorimeter （DSC）, the results showed that the temperatute of NSPC 41.6℃.It was shown by fluorescence spectrum that there were both the Trp residues which lie outside the molecular and the Trp residues which lie in the inside hydrophilic structure in NSPC.5 The stability of protions in NSPThe effect of temperature, pH, light intensity, alcohol concentration, difffrent concentration of neutral salt, different saturation ammonium sulfate solution and sucrose concentration on the stability of protions in NSP was investigated. The results showed that, protions in NSP kept still stability under the appropriate circumstance: under natural sunlight, below 40℃, pH 5-8, alcohol concentration＜40°, and 60% aturation ammonium sulfate solution was beneficial to stability. 6 Antioxidation activity of phycocyaninExperiments about the anti-oxidation effect in non-lipidosome and in vitro mice all indicated anti-oxidation activity of NSPP was significantly. The results showed that scavenge capability was orderly, H₂O₂ radical,·OH radical. O₂^- radical with chemiluminescence, respectively. Through the experiment in vitro, Phycocyanin could reduce the content of MAD and LOP in blood and liver.更多还原