【作者】 张帆；

【导师】 许美玉；

【作者基本信息】 北京林业大学 ， 农产品加工与贮藏， 2011， 硕士

【摘要】 本项研究以苦杏仁蛋白为研究对象,用碱溶酸沉法、分离蛋白法、盐析法、反胶束法及盐碱两步法等五种方法从苦杏仁中提取蛋白,通过测定蛋白含量、蛋白提取率以及蛋白组成成分,分别在蛋白含量、提取率及组成蛋白种类等各层面,确定较理想的苦杏仁蛋白提取方法,并分析苦杏仁蛋白组成成分。将用不同方法提取的苦杏仁蛋白应用于细胞增殖分析试验,研究苦杏仁蛋白对人结肠腺癌细胞生长的影响,并初步分析具有抑制该细胞生长的苦杏仁蛋白结构。主要结果如下：1)以苦杏仁为原料,用碱溶酸沉法、分离蛋白法、盐析法、反胶束法及盐碱两步法等五种不同方法分别提取苦杏仁蛋白,通过用凯氏定氮法测定蛋白含量,分析蛋白提取率,结果表明用盐析法提取的苦杏仁蛋白含量最高,用盐碱两步法提取的苦杏仁蛋白提取率最高。2)用凝胶过滤层析、SDS-聚丙烯酰胺凝胶电泳及提取具有不同溶解性蛋白组分等方法,分析用上述五种不同方法提取的苦杏仁蛋白的组成成分,结果显示用盐析法提取的苦杏仁蛋白中所含蛋白种类最多。3)用凝胶过滤层析和SDS-聚丙烯酰胺凝胶电泳法研究用上述五种不同方法提取的苦杏仁蛋白成分,结果表明苦杏仁蛋白是由相对分子量为22 KDa到63 KDa之间的不同亚基以不同方式组成,用不同提取方法提取的苦杏仁蛋白中所含蛋白种类有所不同。4)按溶解性不同从苦杏仁中提取清蛋白,球蛋白,醇溶蛋白,谷蛋白,结果表明苦杏仁蛋白中包含清蛋白、球蛋白、醇溶蛋白和谷蛋白,其所占百分比分别约为75%、14%、6%和5%。5)利用细胞增殖分析试验,研究用上述五种方法提取的苦杏仁蛋白对人结肠腺癌细胞生长的影响,结果发现用反胶束法提取苦杏仁蛋白具有显著抑制人结肠腺癌细胞生长作用,该作用具有浓度依赖性。6)研究按不同溶解性提取的苦杏仁清蛋白、球蛋白、醇溶蛋白和谷蛋白对人结肠腺癌细胞生长的影响,结果表明苦杏仁中醇溶蛋白和谷蛋白分别具有显著抑制人结肠腺癌细胞生长作用,该作用也同样具有浓度依赖性。7)SDS-聚丙烯酰胺凝胶电泳法分析苦杏仁中醇溶蛋白和谷蛋白结构,结果表明醇溶蛋白显示相对分子量为43 KDa、40 KDa、31 KDa、23 KDa、15 KDa、14 KDa这6条泳带,谷蛋白为40 KDa、31 KDa、23 KDa、15 KDa、14 KDa这5条泳带。经过测定蛋白含量,结果表明两者均为结合了其它非蛋白分子的蛋白复合物。更多还原

【Abstract】 To study apricot kernel Protein, protein was extracted from apricot kernel via five kinds of methods as follow:alkali-solution and acid-isolation, protein isolation, salt fractionation, reverse micelles and salt/alkali two-step method. The appropriate extraction methods were determined by measuring the protein content, protein extraction rate and protein composition, and analyze the composition of the apricot kernel protein. Protein extracted by different methods was used in cell proliferation assay test to study effect on human colon adenocarcinoma cell growth of the protein, and analyze preliminarily structures of the protein inhibited human colon adenocarcinoma cells growth. The main results were as follows:(1) Protein was extracted from apricot kernel by five kinds of methods about alkali-solution and acid-isolation, protein isolates, salt fractionation, reverse micelles, and salt and alkali two-step method. Protein content and extraction rate were analyzed though Kjeldahl determination. The results showed that protein extracted by salt fractionation and salt and alkali two-step method has the highest content and extraction rate, respectively.(2) Composition of protein extracted in the above five ways was analyzed via gel filtration chromatography, SDS-polyacrylamide gel electrophoresis and extracting protein fractions with the different solubility. The results indicated apricot kernelprotein extracted by salt fractionation contains most types of protein.(3) The protein components extracted via the above-mentioned five different methods were studied by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. The results showed that apricot kernelprotein consist of different monomer with the diverse molecular weight (from 22 KDa to 63 KDa) in different ways. The protein extracted by different methods contains different types of protein. (4) Albumin, globulin, gliadin and glutenin were extracted from apricot kernelas different solubility. The results indicated that their contents were about 75%,14%,6% and 5%, respectively.(5) Effect on human colon adenocarcinoma cell growth of apricot kernelprotein extracted by the above-mentioned five methods was tested via cell proliferation assay. The results showed that protein extracted via reverse micelles method significantly inhibits human colon cancer cell growth and the effect was concentration dependent.(6) Effect on human colon adenocarcinoma cell growth of albumin, globulin, gliadin and glutenin extracted as different solubility was analyzed. The results showed that gliadin and glutenin have respectively a significant role in inhibiting the growth of human colon cancer cells, likewise, the role also depends on concentration.(7) The structures of gliadin and glutenin were analyzed via SDS-polyacrylamide gel electrophoresis. The results showed that the relative molecular weight of gliadin with six transects were respectively 43 KDa,40 KDa,31 KDa,23 KDa,15 KDa and 14 KDa and glutenin with five were 40 KDa,31 KDa,23 KDa,15 KDa and 14 KDa. The results of determination of protein content indicated that both of them were protein complex combined with other non-protein molecule.更多还原