【作者】 仲娜；

【导师】 王修林； 王小如；

【作者基本信息】 中国海洋大学 ， 海洋化学， 2008， 博士

【摘要】 硒作为一种重要的微量元素,具有提高GPx活性、SOD酶活性、增强抗氧化及提高免疫力等重要生理功能。机体硒水平下降将直接影响到骨骼、心肌、肝脏等重要脏器的生理功能。目前包括我国在内的世界许多地区都处于缺硒状态,开发富硒产品具有广阔的开发前景和应用价值。由于硒化合物的使用剂量很窄,其毒性及有益性取决于其浓度与存在的化学形态,无机硒具有脂质抗氧化,有机硒的营养水平较高。而硒的化学形态很不稳定,某些复杂的样品前处理易导致硒的损失及形态转变。到目前为止,硒的形态分析在国际上还没有取得重大进展。建立简便有效的硒元素总量分析方法及化学形态定量分析方法,对于有机硒相关产品的开发及应用具有重要的指导意义。本文通过建立胁迫海带富硒培养,实现了硒由无机形态向有机形态的转化。首次采用近年发展起来的ICP-MS元素分析技术,研究了海带对硒的富集能力及规律,ICP-MS技术的高灵敏度、低检出限、宽线性范围、可多元素同时分析等特点,满足了对生物体微量元素总量的准确定量分析。并且,本文通过RP-HPLC高效分离与ICP-MS检测器的联用技术,建立了有效的RP-HPLC-ICP-MS联用技术,并用于富硒海带的硒化学形态的定量分析;比较了四种提取介质辅助超声提取的样品前处理方法,并进行了方法学验证;研究了微量元素硒经海带富集硒多种化学形态的组成及转化分布规律,为富硒海带的开发与应用提供了理论依据。将所建立富硒海带的硒化学形态的HPLC-ICP-MS定量分析方法,进一步应用于富硒蒜苗硒形态的定量分析,并对该方法进一步方法学验证,为生物硒形态的定量分析与产品开发奠定基础。本论文结果主要有以下几方面:（1）采用ICP-MS技术,初步对山东地区23种大型经济海藻的微量元素总量进行了分析研究。实现了23种大型经济海藻多种微量元素多浓度级别的同时在线分析,样品前处理简单、方法快捷、结果准确。实验测得,23种海藻中人体必需微量元素（Na,Mg,Al,K,Ca,Zn,Fe,Cu,As）含量丰富,其中,褐藻门的钾、钙含量较高,绿藻门的磷含量较高,而红藻门的锰、锌含量较高。硒在某些红藻类和褐藻类含量较高,总体来说,硒的含量为:红藻类高于褐藻类,高于绿藻类。海带对微量元素的富集能力较强,富含K,Ca,Na,Mg,Zn,Mn,As等微量元素,Se浓度为0.281μg/g。海带作为大型食用经济藻类,目前在我国养殖规模很大,因此被选作本文富硒培养和研究对象。根据上述测得各微量元素浓度,进行主成分分析与聚类分析,对藻类进行化学识别,大致反映了影响不同门类元素特征的主要因素以及各个门类的微量元素含量特征。分析得出:萱藻和叉开网翼藻、海带和海木耳分别聚为一类,均属于褐藻门类,三叉仙菜、真江蓠、带状蜈蚣藻、条斑紫菜、珊瑚藻相似度较高为一类,属于红藻门,而绿藻门的两种藻,孔石莼和羽藻的元素特征相似度较近。说明,同一门类的元素的组成和分布具有一定程度的相似特征,该研究为不同藻类的识别提供了新的科学依据。（2）建立和比较了三种色谱分离系统的RP-HPLC-ICP-MS联用技术对硒形态混标（亚硒酸钠、硒甲基硒代半胱氨酸、硒代蛋氨酸）的分离。三种色谱系统对硒形态均分离良好,出峰顺序为亚硒酸钠、硒甲基硒代半胱氨酸、硒代蛋氨酸。色谱系统①各峰分离良好,出峰时间较短,其流动相为酸性,对色谱柱性能要求较高,需耐酸性色谱柱;色谱条件②各峰得到良好的分离,硒代蛋氨酸出峰时间相对较长;色谱系统③分离峰形尖锐,出峰时间合适,流动相20mmol/L ammoniun acetic -methanol（95:5）为缓冲盐系统,也满足LC-MS,TOF-MS等仪器进一步结构分析的系统要求,因此该系统被选作为下一步硒形态定量分析的色谱条件。（3）建立了HPLC-ICP-MS联用技术定量分析富硒海带硒形态方法。研究比较了四种提取介质（0.1mol/LHCl,0.1mol/LNaOH,20mmol/L NH₄COOH-CH₃OH（5%）,ProteaseⅩⅣ）辅助超声提取的样品前处理方法对硒形态进行定量分析,并进行方法学验证,同时对富硒海带的硒形态组成及分布规律进行了研究。首先,进行实验室胁迫海带富硒培养。首次采用ICP-MS元素分析技术进行海带硒总量测定,研究了海带对硒的富集转化规律。通过添加一定浓度的亚硒酸盐（硒浓度10mg/l）及氮、磷营养盐,控制水温、光照、加氧等条件,采用挂绳法进行实验室胁迫海带富硒培养,成功实现了元素硒在生物体内的富集转化。采用密闭消解ICP-MS法研究富硒海带硒浓度富集规律,在最初几个小时内,富硒速率较快,硒浓度迅速增加。海带经富硒培养8天,硒浓度可达到900μg/g,是富集前（0.281μg/g）的3000多倍。其次,比较了0.1mol/LHCl,0.1mol/LNaOH,20mmol/LNH₄COOHCH₃OH（5%）,ProteaseⅩⅣ为提取溶剂、辅助超声提取的样品前处理方法,与以往报道的24小时振荡或多种有机溶剂等提取过程相比,样品处理采用超声提取,大大缩短提取时间,步骤简单,提取效果良好,减少了复杂处理过程可能造成的硒浓度损失及形态的转变。经方法学验证,以0.1mol/LHCl为提取介质,辅助超声处理的RP-HPLC-ICPMS定量分析方法,线性（y=2325.9x,r=0.9999）、精密度（RSD=0.5%,n=7）良好,各硒形态浓度和占总硒的回收率可达82.00%,说明该方法用于硒化学形态定量分析简便可行,准确可靠。以0.1mol/LHCl为提取介质的HPLC-ICPMS定量分析,可明显分离出富硒海带四种硒形态,包括Na₂SeO₃,MeSeCys,SeMet以及一种未知硒组分,其中,Na₂SeO₃分布比例较大,为72.26%,据推断大部分Na₂SeO₃在较短富集时间内还未能转化为有机硒形态,通过延长海带富硒时间,可提高有机硒的转化率,同时减少实验误差,提高回收率,该有待于进一步研究。以0.1mol/LNaOH、缓冲盐和蛋白酶为提取介质,主要提取亚硒酸钠和硒代蛋氨酸两种形态,硒回收率较低。不同提取方式的HPLC-ICP-MS联用技术用于生物硒形态的定量分析有待于进一步研究。（4）将上章建立的HPLC-ICP-MS技术进一步用于富硒蒜苗硒化学形态的定量分析研究,并进一步加以方法学验证。首先进行了胁迫蒜苗富硒培养,蒜苗经富硒培养30d,硒浓度达250μg/g,为富集前（0.121μg/g）的2066倍。采用上章建立的定量分析方法,以0.1mol/LHCl为提取介质、辅助超声的样品处理方法,可明显分离出富硒蒜苗六种硒形态组分:包括Na₂SeO₃,MeSeCys,SeMet以及三种未知硒组分,其中MeSeCys,SeMet的转化率较高,分别占总硒形态的分布比例为64.08%、13.29%,说明Na₂SeO₃经蒜苗吸收可有效转化为有机硒形态,而MeSeCys,据报导为一种抗癌活性前体。经进一步方法学验证,线性及精密度良好,硒浓度回收率达102.5%,方法准确可靠,可用于富硒蒜苗硒形态的定量分析。以20mmol/L ammonium acetic-methanol（5%）,ProteaseⅩⅣ为提取介质,MeSeCys提取率相对也较高,分别占总硒形态的55.94%和59.48%,硒回收率分别为82.87%及82.81%,也可满足富硒蒜苗的硒形态定量分析。该研究进一步为生物硒形态的HPLC-ICPMS定量分析奠定基础,也为富硒蒜苗的开发应用提供理论依据。（5）将上述微波消解-ICP-MS技术对市售富硒产品的硒总量分析,给出科学准确数据。市面上出现越来越多的富硒产品,其硒浓度没有明确的数据标示,而硒的浓度及形态对其生物功能有重要影响,本研究为富硒产品的合理应用和推广提供科学依据。经测定,市售富硒鸡蛋、富硒大葱、富硒茶叶、富硒大米的硒浓度均较普通产品高出数倍。富硒鸡蛋白的硒含量达到15.0μg/g,是普通鸡蛋白（4.07μg/g）的3.69倍,富硒蛋黄硒含量为6.31μg/g,为普通蛋黄（3.09μg/g）的2.04倍。市售富硒大米、富硒茶叶、富硒大葱、富硒元葱的硒浓度均有不同程度增加,其硒浓度依次为0.113μg/g,0.459μg/g,0.214μg/g,12.2μg/g,分别为普通大米（0.054μg/g）、普通茶叶（0.072μg/g）、普通大葱（0.012μg/g）、普通元葱（0.343μg/g）的241、64、20.1、35.6倍。可以看出,植物的富硒倍率显著高于动物,即植物的富集转化能力更强,可以更加有效地将无机硒转化为有机硒。更多还原

【Abstract】 The element Se is known to have enzymatic roles and imuninty.The essentiality of Se was established in the observation of deficiency syptoms which were reduced after Se supplementation.Many regions in the world,including our country are lack of Se.The Se products are greatly expected.The doses of Se is narrow,Besides the total content of selenium,the chemical form is also important due to the differences in bioavailabilty and toxity of the different forms.So the establishment of simple,effective qualative and quantitative analysis technique are of important.As Se is unstabile and trace in amount in biomaterials.The former complex extraction may cause the lose and exchange of Se specitaion.Although there are a few study on the determination of total Se,to the best of our knowledge,the Se sepeciation determination are still of challenge.Laminaria japonica is widely produced in China,by treated with Se enriched nutrients in the laboratory mode for 8 days,different Se species can be got,and the enriched role of Se were firstly studied by ICP-MS.As RP-HPLC-ICP-MS technich has the specility of high seperation,high sensitivity and low detection,we established three chromatagraph systems with ICP-MS detection,three Se specices standards are well seperated.We also compared four solutions（0.1mol/L HCl,0.1mol/L NaOH,20mmol/L NH₄COOH-CH₃OH（5%）, ProteaseⅩⅣ）with ultrasonic extraction,and established a RP-HPLC-ICP-MS method to determine the Se speciation for the first time.with 0.1mol/L HCl,the linearity,precision and recovery are well,as a result,the method is easy and effective and acurate for Se species determination in Laminaria japonica.we continued to have garlic sprout enriched with Selinite for one months,and determined the tatol Se and Se species with the established method,and do further methodology validation to establish a theoretic fundation for the application of Se enriched Laminaria japonica.Besides,19 elements of 23 large economical algae in ShanDong Province were determined by ICP-MS with closed digestion,and then identified by clustering analysis.Furthermore,some Se enriched products at present time were also determind for scientific and accurate data with microwave-ICP-MS.The main results were summarized as follows:（1）Determination of 19 elements in 23 large economical algae by ICP-MS with closed digestion.in the result,the algea are rich of the body necessary elements,such as Na,Mg,Al,K,Ca,Zn,Fe,and so on.13 algea are all of K、Ca、P macroelements and Fe、Mn、Cu、Zn microelements.Laminaria has the high ability of enriching elements,itt was rich of K,Ca,Na,Mg,Zn,Mn,As etal,and selenium is about 0.281μg/g,As one of the economic and edible algea,it was prouduced widely in our country,so was choosed as the study object.The elements concentration in those samples were calculated and the algae sort were identified according to the elements components and concentration by the PCA and CA methods.such as,Scytosiphon lomentarius（Lyngb.）J.Ag and Dictyopteris divaricata（Okamura）Okamura,Laminaria japonica Aresch and Sarcodia montagneana（Hooker et Harvey）J.Agardh clustered as one group seperately,which all belongs to phaeophyta.The Ceramium kondoi Yendo,Gracilaria vermiculophylla, Grateloupia filicina（Lamx.）C.Ag,Porphyra yezoensis Ueda and Corallina sp.are all Rhodophyta.so the same phylum are of similar elements symptom the research provides new theory for the identification of algea.（2）We established three chromatographic system in conjunction with ICP-MS to separate Se species standards（Na₂SeO₃,MeSeCys,SeMet）.The chromatographic systems③were:Alltima C18 column（250mm×4.6mm,5μm）,with pre-column,mobile phase:20mmol/L ammonium acetic-methanol（95:5）,flow 1.0ml/min,injection volumn 20μL.The system①has a low pH,so it was of a higher requirement of the column.the standards’ peaks were relatively sharp and seperated well in short time by system③,and the system was also suitable for further LC-MS,TOF-MS analysis,and so were choosed as the system for the next quantative analysis of Se.（3）A new RP-HPLC-ICPMS method to determin different Se species in Laminaria japonica with Comparing four solution of 0.1mol/L HCl,0.1mol/L NaOH, 20mmol/L ammonium acetic-methanol（5%）,nonspecific ProteaseⅩⅣ,with ultrasonic extraction were studied,and the methodology validation were also done.With 0.1mol/L HCl,three species（Na₂Se₂O₃,MeSeCys,SeMet）and one unknown species were identified and determined,Na₂SeO₃ was 72.26%,and MeSeCys 5.73%of the sum.the linearity（y=2325.9x,r=0.9999） and the precision（RSD=0.5%, n=7） are good by methodology validation,the recovery reached 82.00%.As a result,the method is easy,usible and reliable.First,A laboratory method was established for Cultivating Laminaria japonica with the addition of selenite（10mg/L） and N（300μg/L）,P（30μg/L） nutrient solution for 8 days,and the tatol Se and the enriched role were also studied by the new technich ICP-MS for the first time.the enriched rate was fastest in the first few hours and the Se concentration of the enriched Laminaria japonica reached 900μg/g,about 3000 times of the unriched（0.281μg/g）.Second,with 0.1mol/L NaOH,20mmol/L ammonium acetic-methanol（5%） and nonspecific ProteaseⅩⅣas the extract solution,the main Se species identified were Na₂Se₂O₃ and SeMet,the SeMeCys was not detected.and the recoverity was low relatively,so different extract solution result in different Se specices,more study are needed for the quantitive and qualitive analysis of HPLC-ICP-MS on bio-Se.（4）We continued to determine the Se species in Se enriched garlic sprouts by the established RP-HPLC-ICPMS methods for further methodological validation.Se concentration reached 250μg/g by cultivating in Se enriched solution for one monthes,about 2066 times of the unenriched（0.121μg/g）.six species,including selenite,MeSeCys,SeMet and three unknown species were well separated and determined,selenite were greatly transformed to MeSeCys and SeMet,Se recovery was 102.5%by 0.1mol/L HCl extract solution with ultrasonic processing.with 20mmol/L ammonium acetic-methanol（5%） and proteaseⅩⅣsolution extraction,the recovery were 82.87%and 82.81%.the results provided further methodological validation for the established RP-HPLC-ICPMS technich for Se species determination in biomaterials.（5）The Se enriched eggs,onion,tea and rice sold in the market were studied by ICP-MS with microwave digestion.The Se concentration was all improved to some extend.such as,the Se of enriched egg white was 1.50*10⁴μg/g,3.69 times of the unenriched（4.07*10³μg/g）;the enriched egg yellow was 6.31*10³μg/g,2.04 times of the unenriched（3.09*10³μg/g）;the enriched rice（113.5μg/g）,tea（459.8μg/g）,green chinese onion（214μg/g）,onion（1.22*10⁴μg/g）were 241,72,64,20.1 times of that of the unenriched rice（54μg/g）,tea（72μg/g）,green chinese onion（12μg/g）,onion（342.8μg/g）. from the results,we can also find that the plants have the higher ability of enriching microelements.the Se species of the Se enriched product are also expected for further study.更多还原