【作者】 魏欣；

【导师】 袁亚宏；

【作者基本信息】 西北农林科技大学 ， 农产品加工及贮藏工程， 2019， 硕士

【摘要】 硒（Se）作为一种关键的微量元素,在人体及动物体的生长发育、抗氧化等方面发挥重要作用。人体和动物体主要依靠食物来补充硒,但浓度过高的硒会让人产生中毒症状,有机硒相比较于无机硒来说毒性降低并且在机体中的吸收率远高于无机硒。作为一种非聚硒植物,人体使用玉米可避免因为硒盲目摄入过量而引起的中毒症状。因此,本研究以安康地区富硒玉米为原料,分析了硒在玉米中的分布规律、氨基酸构成、富硒玉米硒形态及不同含硒蛋白的二级结构和抗氧化活性;在此基础上对富硒玉米含硒蛋白进行了工艺优化提取、分离纯化和结构表征,以期为开发安康地区富硒玉米及其相关功能添加剂打下基础。主要研究结果如下:（1）安康富硒玉米蛋白含量为8.94%,总硒含量为0.097 mg/kg,其中含硒蛋白占有机硒75.20%,玉米中蛋白主要以碱溶性蛋白和醇溶性蛋白形式存在,但硒结合能力为盐溶性含硒蛋白>碱溶性含硒蛋白>水溶性含硒蛋白>醇溶性含硒蛋白,玉米含硒蛋白主要氨基酸存在形态为硒代胱氨酸,无机硒的存在形态为亚硒酸根。（2）将四种分级提取到的含硒蛋白进行傅里叶红外光谱分析,水溶性含硒蛋白、盐溶性含硒蛋白、醇溶性含硒蛋白、碱溶性含硒蛋白为多种复合蛋白,除碱溶性蛋白外,其余三种蛋白在特征吸收带均有明显特征。进一步通过曲线拟合法分析四种含硒蛋白在1600cm^-1到1700cm^-1的二级结构百分含量,结合红外光谱结果可以看出除碱溶性蛋白外,其余三种是含硒蛋白的含量均有所不同,结构相对稳定。玉米盐溶性含硒蛋白的抗氧化性相对较高,总体来说玉米含硒蛋白抗氧化性（除变性的碱溶性含硒蛋白）与硒结合蛋白的能力的大小存在良好的一致性。（3）采用超声辅助磷酸盐提取法,在单因素试验的基础上,采用BBD响应面分析法对富硒玉米中硒蛋白进行提取优化。优化结果:温度37℃,时间为80min,缓冲液PH选为7.7,玉米含硒蛋白得率可达到41.5345%,通过抗氧化试验可得此方法提取到的含硒蛋白相较上四种含硒蛋白来说有较高的提取率和抗氧化性。因此选定此方法提取到的玉米含硒蛋白作为进一步的研究对象。（4）使用Q Sepharose Fast Flow强阴离子交换柱纯化玉米含硒蛋白,经SDS-PAGE检验后为2个条带,分别命名为Z1和Z2,经过LC-MS/MS检验,对两个个含硒蛋白进行鉴定,Z1鉴定出三种蛋白,一种为谷胱甘肽转移酶,一种为核糖核酸酶,另一种可能为谷胱甘肽过氧化物酶;Z2鉴定出两种蛋白,其中一种为未匹配含硒蛋白,另一种为紫色酸性磷酸酶。更多还原

【Abstract】 As a key trace element,selenium(Se)plays an important role in the growth and development of antioxidants in humans and animals.The human body and the animal mainly rely on food to supplement selenium,but the excessive concentration of selenium can cause poisoning symptoms.The organic selenium is less toxic than inorganic selenium and the absorption rate in the body is much higher than that of inorganic selenium.As a non-selenium-concentrating plant,the use of corn in the human body can avoid the symptoms of poisoning caused by excessive intake of selenium.Therefore,this study used selenium-enriched corn in Ankang as raw material to analyze the distribution of selenium in corn,amino acid composition,selenium form of selenium-enriched corn and secondary structure and antioxidant activity of different selenoproteins.Selenium-enriched corn-containing selenoproteins were optimized,extracted,purified,and characterized,in order to lay the foundation for the development of selenium-enriched corn and related functional additives in Ankang.The main findings are as follows:(1)Ankang selenium-enriched corn protein content is 8.94%,total selenium content is0.097 mg/kg,and selenoprotein accounts for 75.20% of organic selenium.The protein in corn is mainly in the form of alkali-soluble protein and alcohol-soluble protein,but selenium is combined.The ability is salt-soluble selenoprotein> alkali-soluble selenoprotein>water-soluble selenoprotein> alcohol-soluble selenoprotein,the main amino acid of selenoprotein in corn is selenocystine,and the form of inorganic selenium is selenite.(2)The selenoside protein extracted from the four fractions was analyzed by Fourier transform infrared spectroscopy.The water-soluble selenoprotein,salt-soluble selenoprotein,alcohol-soluble selenoprotein and alkali-soluble selenoprotein were various complex proteins,except for alkali solubility.In addition to the protein,the other three proteins have distinct characteristics in the characteristic absorption band.Furthermore,the percentage of secondary structure of four selenoproteins from 1600cm-1 to 1700cm-1 was analyzed by curve fitting method.The results of infrared spectroscopy showed that the contents of sele-containing proteins were all other than alkali-soluble proteins.Different,the structure is relatively stable.The salt-soluble selenoprotein in corn has a relatively high antioxidant activity.In general,the oxidative capacity of selenoprotein in corn(except for denatured alkali-soluble selenoprotein)has a good consistency with the ability of selenium-binding protein.(3)Ultrasound-assisted phosphate extraction method was used to optimize the extraction of selenoprotein from selenium-enriched corn by BBD response surface analysis based on single factor test.Optimization results: temperature 37℃,time 80 min,buffer p H selected as7.7,corn selenoprotein yield can reach 41.5345%,through the antioxidant test can be obtained by this method of selenoprotein extraction compared to the above four The selenoprotein has a higher antioxidant activity.Therefore,selenium-enriched protein extracted by selenium-enriched method was selected as a further research object.(4)The selenoprotein of maize was purified by Q Sepharose Fast Flow strong anion exchange column.After SDS-PAGE,two bands were named,Z1 and Z2,respectively.After LC-MS/MS test,two selenoproteins were used.Identification,Z1 identified three proteins,one is glutathione transferase,one is ribonuclease,and the other may be glutathione peroxidase;Z2 Two proteins were identified,one of which was unmatched selenoprotein and the other was purple acid phosphatase.更多还原