富硒大米及富硒酵母中的硒形态分析
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摘要
硒是人体必需的营养元素,在体内发挥着重要的生理、生化功能,如抗氧化、增强免疫力、抗衰老等。同时硒也属于稀散元素,在地球上的含量少且分布不均,全世界有2/3的地区属于缺硒地带,我国缺硒地区占全国总面积的2/3,其中1/3为世界公认的严重缺硒区。当机体硒水平下降时会影响心肌、肝脏、甲状腺等重要的生命器官的正常功能,导致多种疾病的发生。我国科学家通过补硒来治疗和预防克山病、大骨节病等严重危害人们健康的疾病,已取得非常明显的效果。研究表明每天补充一定量的硒还可以使癌症的发生率和死亡率下降50%。植物是自然界硒循环生态链中将无机硒转化为有机硒的关键载体,又是人类硒摄入的重要来源。由于硒化合物存在形式不同,它们在机体的生物利用度,功能有效性,代谢过程都会明显不同,因此对于植物中硒形态分析对于指导人们补硒,保障人们健康有着非常重要的意义。
本文对于富硒大米中的各种蛋白的硒含量进行了测量,根据Osbrone对谷物蛋白质组份分类原则,尝试着采取一种顺序搅拌离心法分离提取大米中存在的四类蛋白,即清蛋白、球蛋白、醇溶蛋白和谷蛋白。采用电感耦合等离子质谱(ICPMS)作为硒的检测方法,测定了四种蛋白提取液中各自的硒含量。运用排阻色谱与电感耦合等离子质谱联用方法对于清蛋白提取液中含硒蛋白组份的分子量进行了测定,初步分析了清蛋白中含硒蛋白组份的分布。通过蛋白酶解和蛋白酸水解法释放富硒大米及富硒酵母中结合在蛋白多肽链中的氨基酸,运用反相离子对色谱和电感耦合等离子质谱联用方法对于酶解液及盐酸水解液中含有的含硒氨基酸成份进行了定性及定量分析,初步探讨了在富硒大米和富硒酵母中与蛋白结合的含硒氨基酸形式。
结果表明:富硒大米中的蛋白提取液的硒含量约占富硒大米中总硒量的73%,由此推测含硒蛋白是富硒大米中硒的主要存在形式,其中谷蛋白提取液中硒含量占总硒量的48%,是主要的含硒蛋白形式。对于清蛋白提取液中含硒蛋白组份的分析,从色谱图中可以看出分子量分别为17.9 KDa、12.6 KDa、8.35 KDa的蛋白中含有硒元素,分子量为12.6 KDa的蛋白含硒量占清蛋白总硒量的50.2%,是清蛋白提取液中主要的含硒蛋白。分析结合到蛋白中的各种含硒氨基酸形式表明:富硒酵母中与蛋白结合的主要含硒氨基酸是硒代蛋氨酸,含量约占总硒量的46%;在富硒大米的酶解液中,只测定出硒代半胱氨酸,未检测到硒代蛋氨酸的存在,另外还检测出一种与硒代蛋氨酸保留时间相近的未知物质,可能是氧化的硒代蛋氨酸形式,但需要做进一步准确鉴定及分析。
本文对于富硒大米中的四类蛋白含硒量测定和清蛋白中含硒蛋白组份分布的分析以及富硒大米中与蛋白结合的含硒氨基酸的检测结果可为今后进一步研究硒在大米蛋白中的结合形态以及评价大米蛋白的生理功能和营养价值提供重要参考。
Selenium is recognized as the necessary nutritional element for human health, which perform many important physiological and biochemical function in the body including anti-oxygenic property、improvement of immunity、prevention of human aging etc. Belonging to trace rare element, selenium is distributed rarely and unevenly on the earth. 2/3 of the area in the world lack selenium, so does in China, of which 1/3 is considered to be extremely lack of selenium. It has been established when the content of selenium is under normal level ,the essential organ such as heart、liver、thyroid grand would be damaged to some extent. Consequently the selenium deficiency will cause many diseases. The Chinese scientists have obtained satisfied results of cureing and preventing the keshan disease and Kashin Beck disease by supplementation of Se. The recently finding show the daily supplement of Se can decrease the occurrence of liver cancer by 50%.Plants is not only the key link through which the inorganic selenium is converted into the organic selenium in the ecological selenium cycle but also the important selenium resource for the human. Selenium in the plants present in many different chemical forms, which determines its bioavailability、efficiency and metabolic pathway. So selenium speciation in plants is much more important in improving effects of selenium supplement and ensuring the human health.
On the basis of Osborne fractionation , employing the sequence milled and centrifugal procedure separate and extract protein from the selenium-rich rice including albumin、globulin、prolamin as well as gluletin. Inductively Coupled Plasms Mass (ICPMS) serve as selenium-selected detection method to determine the selenium concentration of protein extraction. Then the using of hyphenated technology of the Size exclusion chromatography coupled to ICPMS (SEC-ICPMS) identifies the molecular mass and character the distribution of selenium contained protein in the albumin extraction. Another hyphenated technology of Reversed-phase ion pairing chromatography combined with ICPMS is used for qualitative and quantitative analysis of selenoaminoacid after extracted through enzymatic digestion and acid hydrolysis from selenium-rich rice and selenized yeast.
The result obtained show: the selenium content of protein extraction account for 73% of the total selenium content in selenium-rich rice, so it could be presumed that selenium contained protein is the primary form present in selenium-rich rice. Gluletin is dominating the selenium contained protein which its content is 48% of the total selenium content in selenium-rich rice. It is observed from chromatography of SEC-ICPMS that selenium is identified in the protein with molecular mass 17.9 KDa、12.6KDa、8.35KDa respectively from the albumin extraction, the protein with molecular weight 12.6 KDa is the main selenium contained protein which it take up 50.2% of the total selenium content of the albumin extraction. With respect to the study on selenoaminoacid incorporated into protein, the result is that selenomethionine (SeMet) is the major selenoaminoacid in the selenized yeast which the content is 46% of the overall selenium content of the yeast. In the selenium-rich rice, however, selenomethionine could not be found, the selenocystine (SeCys) and another selenium contained substance with a similar retention time to standard selenomethionine are detected in the chromatography. It needs to further identify and analysis to ensuring the new substance is oxidation of selenomethionine.
The results obtained including the selenium concentration of four type of protein extracted from selenium-rich rice、the distribution of selenium contained protein in albumin extraction and the form of selenoaminoacid incorporated into protein can provide the valuable reference about further studying selenium species of associated with protein in selenium-rich rice and assessing the function and nutritional value of rice protein.
本文对于富硒大米中的各种蛋白的硒含量进行了测量,根据Osbrone对谷物蛋白质组份分类原则,尝试着采取一种顺序搅拌离心法分离提取大米中存在的四类蛋白,即清蛋白、球蛋白、醇溶蛋白和谷蛋白。采用电感耦合等离子质谱(ICPMS)作为硒的检测方法,测定了四种蛋白提取液中各自的硒含量。运用排阻色谱与电感耦合等离子质谱联用方法对于清蛋白提取液中含硒蛋白组份的分子量进行了测定,初步分析了清蛋白中含硒蛋白组份的分布。通过蛋白酶解和蛋白酸水解法释放富硒大米及富硒酵母中结合在蛋白多肽链中的氨基酸,运用反相离子对色谱和电感耦合等离子质谱联用方法对于酶解液及盐酸水解液中含有的含硒氨基酸成份进行了定性及定量分析,初步探讨了在富硒大米和富硒酵母中与蛋白结合的含硒氨基酸形式。
结果表明:富硒大米中的蛋白提取液的硒含量约占富硒大米中总硒量的73%,由此推测含硒蛋白是富硒大米中硒的主要存在形式,其中谷蛋白提取液中硒含量占总硒量的48%,是主要的含硒蛋白形式。对于清蛋白提取液中含硒蛋白组份的分析,从色谱图中可以看出分子量分别为17.9 KDa、12.6 KDa、8.35 KDa的蛋白中含有硒元素,分子量为12.6 KDa的蛋白含硒量占清蛋白总硒量的50.2%,是清蛋白提取液中主要的含硒蛋白。分析结合到蛋白中的各种含硒氨基酸形式表明:富硒酵母中与蛋白结合的主要含硒氨基酸是硒代蛋氨酸,含量约占总硒量的46%;在富硒大米的酶解液中,只测定出硒代半胱氨酸,未检测到硒代蛋氨酸的存在,另外还检测出一种与硒代蛋氨酸保留时间相近的未知物质,可能是氧化的硒代蛋氨酸形式,但需要做进一步准确鉴定及分析。
本文对于富硒大米中的四类蛋白含硒量测定和清蛋白中含硒蛋白组份分布的分析以及富硒大米中与蛋白结合的含硒氨基酸的检测结果可为今后进一步研究硒在大米蛋白中的结合形态以及评价大米蛋白的生理功能和营养价值提供重要参考。
Selenium is recognized as the necessary nutritional element for human health, which perform many important physiological and biochemical function in the body including anti-oxygenic property、improvement of immunity、prevention of human aging etc. Belonging to trace rare element, selenium is distributed rarely and unevenly on the earth. 2/3 of the area in the world lack selenium, so does in China, of which 1/3 is considered to be extremely lack of selenium. It has been established when the content of selenium is under normal level ,the essential organ such as heart、liver、thyroid grand would be damaged to some extent. Consequently the selenium deficiency will cause many diseases. The Chinese scientists have obtained satisfied results of cureing and preventing the keshan disease and Kashin Beck disease by supplementation of Se. The recently finding show the daily supplement of Se can decrease the occurrence of liver cancer by 50%.Plants is not only the key link through which the inorganic selenium is converted into the organic selenium in the ecological selenium cycle but also the important selenium resource for the human. Selenium in the plants present in many different chemical forms, which determines its bioavailability、efficiency and metabolic pathway. So selenium speciation in plants is much more important in improving effects of selenium supplement and ensuring the human health.
On the basis of Osborne fractionation , employing the sequence milled and centrifugal procedure separate and extract protein from the selenium-rich rice including albumin、globulin、prolamin as well as gluletin. Inductively Coupled Plasms Mass (ICPMS) serve as selenium-selected detection method to determine the selenium concentration of protein extraction. Then the using of hyphenated technology of the Size exclusion chromatography coupled to ICPMS (SEC-ICPMS) identifies the molecular mass and character the distribution of selenium contained protein in the albumin extraction. Another hyphenated technology of Reversed-phase ion pairing chromatography combined with ICPMS is used for qualitative and quantitative analysis of selenoaminoacid after extracted through enzymatic digestion and acid hydrolysis from selenium-rich rice and selenized yeast.
The result obtained show: the selenium content of protein extraction account for 73% of the total selenium content in selenium-rich rice, so it could be presumed that selenium contained protein is the primary form present in selenium-rich rice. Gluletin is dominating the selenium contained protein which its content is 48% of the total selenium content in selenium-rich rice. It is observed from chromatography of SEC-ICPMS that selenium is identified in the protein with molecular mass 17.9 KDa、12.6KDa、8.35KDa respectively from the albumin extraction, the protein with molecular weight 12.6 KDa is the main selenium contained protein which it take up 50.2% of the total selenium content of the albumin extraction. With respect to the study on selenoaminoacid incorporated into protein, the result is that selenomethionine (SeMet) is the major selenoaminoacid in the selenized yeast which the content is 46% of the overall selenium content of the yeast. In the selenium-rich rice, however, selenomethionine could not be found, the selenocystine (SeCys) and another selenium contained substance with a similar retention time to standard selenomethionine are detected in the chromatography. It needs to further identify and analysis to ensuring the new substance is oxidation of selenomethionine.
The results obtained including the selenium concentration of four type of protein extracted from selenium-rich rice、the distribution of selenium contained protein in albumin extraction and the form of selenoaminoacid incorporated into protein can provide the valuable reference about further studying selenium species of associated with protein in selenium-rich rice and assessing the function and nutritional value of rice protein.
引文
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[63] Thompson J J, Houk R S. Inductively coupled plasma mass spectrometric detection for multielement flow injection analysis and elemental speciation by reversed-phase liquid chromatography[J].Aanl Chem, 1986, 58(12): 2541- 2548.
[64] Caruso J A, Klaue B, Michalke B, et al. Group assessment : elemental speciation[J].Ecotoxicolohy and Environmental Safety, 2003,56 (1): 32-44.
[65] Michalke B. Element speciation definitions, analytical methodology,and some examples[J].Ecotoxicology and Environmental Safety, 2003,56 (1):122-139.
[66] Sutton K L, Caruso J A. Liquid chromatography-inductively coupled plasma mass spectrometry[J].Journal of Chromatography A,1999,856(1-2):243-258.
[67] Uden P C. Modern trends in the speciation of selenium by hyphenated techniques[J].Anal Bioanal Chem, 2002,373(6):422-431.
[68] Wallschiager D, London J. Determination of inorganic selenium species in rain and sea waters by anion exchange chromatography-hydride generation-inductively-coupled plasma-dynamic reaction cell-massspectrometry (AEC-HG-ICP-DRC-MS)[J].Journal of Analytical Atomic Spectrometry,2004,19(9):1119-1127.
[69] Mazan S, Gilon N, Cretier G, et al. Inorganic selenium speciation using HPLC-ICP-hexapole colllision/reaction cell-MS[J].Journal of Analytical Atomic Spectrometry,2002,17(4):366-370.
[70] Cole R B. Electrospray Ionization Mass Spectrometry—Fundamentals, Instrumentation & Applications[M]. Wiley, New York, 1997:25
[71] Larsen E H, Hansen M, Fan T, et al. Speciation of selenoamino acids, selenonium ions and inorganic selenium by ion exchange HPLC with mass spectrometric detection and its application to yeast and algae[J].Journal of Analytical Atomic Spectrometry,2001,16(12):1403-1408.
[72] Huerta V D, Reyes L H, Marchante-Gayon J M, et al. Total determination and quantitative speciation analysis of selenium in yeast and whea flour by isotope dilution analysis ICP-MS[J]. J Anal At Spectrom, 2003,(18):1243-1247
[73] Capelo J L, Fernandez C, Pedras B, et al. Trends in selenium determination/speciation by hyphenated techniques based on AAS or AFS[J].Talanta,2006,68(5):1442-1447.
[74] Moreno M E, Perez-Conde C, Camara C. The effect of the presence of volatile organoselenium compounds on the determination of inorganic selenium by hydride generation [J].Anal Bioanal Chem,2003,375 (5): 666-672.
[75] Tsopelas F N,Ochsenkuhn-Petropoulou M T,Mergias I G, et al. Comparison of ultra-violet and inductively coupled plasma-atomic emission spectrometry for the on-line quantification of selenium species after their separation by reversed-phase liquid chromatography[J].Analytica Chimica Acta,2005,539(1-2):327-333.
[1] Morita T, Kiriyama S. Mass production method for rice protein isolate and nutritional evaluation[J]. Journal of food science, 1993,58(6):1393-1396
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[3] 简少文,梅承恩,梁永能,等.富硒米提取液对 EB 病毒转化脐积血 B 淋巴细胞及EBV 早期抗原表达的影响[J],癌症,2003,22(1):26-29
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[66] Sutton K L, Caruso J A. Liquid chromatography-inductively coupled plasma mass spectrometry[J].Journal of Chromatography A,1999,856(1-2):243-258.
[67] Uden P C. Modern trends in the speciation of selenium by hyphenated techniques[J].Anal Bioanal Chem, 2002,373(6):422-431.
[68] Wallschiager D, London J. Determination of inorganic selenium species in rain and sea waters by anion exchange chromatography-hydride generation-inductively-coupled plasma-dynamic reaction cell-massspectrometry (AEC-HG-ICP-DRC-MS)[J].Journal of Analytical Atomic Spectrometry,2004,19(9):1119-1127.
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[73] Capelo J L, Fernandez C, Pedras B, et al. Trends in selenium determination/speciation by hyphenated techniques based on AAS or AFS[J].Talanta,2006,68(5):1442-1447.
[74] Moreno M E, Perez-Conde C, Camara C. The effect of the presence of volatile organoselenium compounds on the determination of inorganic selenium by hydride generation [J].Anal Bioanal Chem,2003,375 (5): 666-672.
[75] Tsopelas F N,Ochsenkuhn-Petropoulou M T,Mergias I G, et al. Comparison of ultra-violet and inductively coupled plasma-atomic emission spectrometry for the on-line quantification of selenium species after their separation by reversed-phase liquid chromatography[J].Analytica Chimica Acta,2005,539(1-2):327-333.
[1] Morita T, Kiriyama S. Mass production method for rice protein isolate and nutritional evaluation[J]. Journal of food science, 1993,58(6):1393-1396
[2] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72: 248-254
[3] Shih F F, Daigle K W. Preparation and Characterization of rice protein isolates[J]. Journal of the American Oil Chemists` Society, 2000,77(8):885-889
[4] Cagampang G B, Cruz L J, Espiritu S G et al. Studies on the extraction and composition of rice proteins[J]. Cereal Chem, 1966,43:145-155
[5] Shih F F, Champagne K D, Zarins Z. Use of enzymes in the processing of protein products from rice bran and rice flour[J]. Nahrung, 1999,43(1):14-18
[6] 吴永尧, 罗泽民, 陈建英 等. 水稻硒蛋白及其硒结合形态研究[J]. 华中师范大学学报(自然科学版), 2000,34(2):223-225
[7] Krishnan H B, White J A. Morphometric analysis of rice seed protein bodies[J]. Plant Physiol, 1965,109:1491-1495
[8] 牛洪斌, 覃怀德, 王益华. 水稻谷蛋白的一个新基因克隆及表达分析[J].作物学报. 2007,33(3):349-355
[9] 曾广文, 蒋德安. 植物生理学[M]. 成都科技大学出版社, 1998,58-150
[1] Brown KM, Arthur JR. Selenium,selenoproteins and human health: a review[J]. Public health nutrition, 2001,4:593-599
[2] 向天勇,吴永尧,陈建英。天然大豆硒蛋白抗肿瘤作用研究[J],氨基酸和生物资源,2005,27(1):10-12,21
[3] 简少文,梅承恩,梁永能,等.富硒米提取液对 EB 病毒转化脐积血 B 淋巴细胞及EBV 早期抗原表达的影响[J],癌症,2003,22(1):26-29
[4] Szpunar J, Lobinski R. Species-selective analysis for metal biomacoromolecular complexes using hyphenated techniques [J], Pure Appl Chem,1999, 71: 899-918
[1] Lobinski R, Edmonds J S, Suzuki K T. Species-selective determination of selenium compounds in biological materials.[J] Pure Appl Chem, 2000, 72(3): 447-461
[2] Zheng Jian, Masaki Ohata, Naoki Furuta et al. Speciation of selenium compounds with ion-pair reversed-phase liquid chromatography using inductively coupled plasma mass spectrometry as element-specific detection.[J] Journal of Chromatography A, 2000, 874: 55-64
[3] 王玉琪, 彭新湘. 适用于水稻叶片蛋白质组分析的双向电泳技术.[J] 植物生理与分子生物学学报,2006,32(2):252-256
[4] 谢丽琪,欧阳政.一种改良的硒蛋白的水解方法.[J] 色谱,1990,8(4):250-251
[5] 王俊,黄明,徐幸莲 等. 硒及富硒功能食品研究进展[J]. 江苏农业科学, 2003, (2): 53-56
[6] Aleksandra Polatajko, Magdalena Sliwka-Kaszynska, Mihaly Dernovics, et al. A systematic approach to selenium speciation in selenized yeast.[J] Journal of Analytical Atomic Spectrometry,2004,19:114-120
[7] Clark L C, Combs G F, Turnbull B W, et al . Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin.[J] Am Med Association, 1996,276:1957-1985
[8] Uden P C, Boakye H T, Kahakachchi Chethaka, et al. Element selective characterization of stability and reactivity of selenium species inselenized yeast.[J] Journal of Analytical Atomic Spectrometry, 2004,19:65-73
[9] Lobinski R, Edmonds J S, Suzuki K T, et al. Species-selective determination of selenium compounds in biological materials.[J] Pure Appl. Chem., 2000, 72 (3) : 447-461
[10] Korhola M, Vainio A, Edelmann K. Selenium yeast.[J] Ann Clin Res, 1986 , 18 (1) : 65-68
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