【作者】 陈德文；

【导师】 潘思轶； 汪兴平；

【作者基本信息】 华中农业大学 ， 产品加工及贮藏工程， 2005， 硕士

【摘要】 葛仙米以其资源稀少、营养价值丰富,独特的生物活性和药理作用而引起人们广泛地关注。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。近年来,仅少数人对其生理和基本的生化组成进行了研究。为探讨葛仙米中的生物活性成分,本文以野生葛仙米藻红蛋白为研究对象,在系统研究葛仙米藻红蛋白的提取、分离和纯化的基础上,利用生物大分子研究的最新技术和现代分离、分析手段,尝试揭示葛仙米藻红蛋白的结构与生物活性及二者间的关系。其主要结果如下: 1 葛仙米的生态条件及分布规律与生化组成研究 以湖北省鹤峰县走马镇野生葛仙米为研究对象,对其生态条件及分布规律与生化组成作了较系统的分析。测定了其水分、蛋白质、脂肪、多糖、类胡萝卜素、叶绿素、矿物元素的含量。 2 葛仙米藻红蛋白的提取、分离、纯化和鉴定 以脱脂葛仙米为原料,采用盐溶和盐析的方法,首次从葛仙米中制备出清蛋白、球蛋白和盐溶蛋白,并确定了适合葛仙米藻胆蛋白提取制备的最佳工艺,其最佳工艺条件为,缓冲液pH值为7.3、浸提时间4.3h、盐浓度0.21mol/L,葛仙米藻胆蛋白最大得率为7.13%。 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盐G-25凝胶色谱,进一步纯化葛仙米藻红蛋白,得到均一的藻红蛋白。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。 3 葛仙米藻胆蛋白稳定性研究 研究了温度、pH、光强、酒精浓度、中性盐浓度和蔗糖浓度等因子对其稳定性的影响。结果表明,葛仙米藻胆蛋白在自然光照下,温度为40℃以下,pH为5～8,酒精浓度小于40°,低浓度中性盐和低浓度蔗糖溶液能保持较好地稳定性。 4 葛仙米藻红蛋白部分分子构象的表征 采用SDS-PAGE和常规PAGE电泳,FFIR、CD、AFM和DSC等分析技术,更多还原

【Abstract】 The significant biological and pharmacological activity 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 phycobiliproteins was used as material. Relationships between its bioactivity and its conformation were attempted to discovery, on the basis of systemically studies on preparation and purification of NSPE by using modern separation technology and modern chemical analysis method. The main results were shown as follows:1 The ecological conditions and distribution for NSK and its biochemisty componentOn the basis of the ecological conditions for NSK and their analysis having been investigated, in order to determine its biochemistry components, such as 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 NSPEDefatted 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 wassuccessful to obtain five fractions, by the step gradient. PE was further purified by DEAE-Separose FF, HA and Sephadex G-25 chromatography. Some methods such as spectrophotometer and chromatography and electrophoresis were applied to identify the purity of PE, and it was identified to be chromatography and electrophoresis grade pure.3 The stability of NSPPThe effect of temperature, pH, light intensity, alcohol concentration, neutral salt concentration and sucrose concentration on the stability of NSPP was investigated. The results showed that, NSPP kept still stability under the appropriate circumstance: under natural sunlight, below 40°C, pH 5-8, alcohol concentration < 40°.4 The structure characterization of NSPPThe molecular weight (MW) of PE was measured by non-denaturing polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the results demonstrated that the MW of PE is 51.808kd; As the result of circular dichroism (CD) spectrum and Fourier transformation infared (FTIR) spectrum shown, the secondary structure of PE was mainlya-helix t and the content of B-shee or coil was very small; PE’s agglutination form was observed by force atom microscope (AFM), there were mainly different size globular form. Meanwhile, its denatured temperature was also studied by differential scan calorimeter (DSC), the results showed that the temperatute was 56.1°C.5 Antioxidant activity of NSPEScavenge capability of oxygen free radical was studied by chemiluminescence, the results showed that scavenge capability was orderly, H2O2 radical, -OH radical, O2~’ radical, respectively.更多还原