【作者】 陈填烽；

【导师】 郑文杰；

【作者基本信息】 暨南大学 ， 无机化学， 2005， 硕士

【摘要】 硒（Selenium）是唯一受基因调控的人体必需微量元素,螺旋藻（Spirulina platensis）是富集和转化硒的理想载体。本实验比较了不同加硒方法对螺旋藻富硒的影响,并在优化的加硒条件下对富硒螺旋藻进行混养研究;对藻体中的硒及藻代谢后排出藻细胞外的气态烷基硒进行形态分析;并对自养、异养和混养条件下螺旋藻超微结构的差异进行表征,取得了一些有意义的结果。 1.硒对螺旋藻具有刺激促进与致毒抑制的双重作用。对于一次性加硒法,在适当的硒浓度（≤150mg/L）下,钝顶螺旋藻能有效的富集、转化培养基中的无机硒为有机硒,并可同时观察到藻体中多种活性物质如类叶黄素、类胡萝卜素及叶绿素a、藻胆蛋白等含量的提高。但是,更高的硒浓度（≥200mg/L）胁迫处理在获得较高的硒富集量的同时,明显的抑制藻的生长,导致螺旋藻生物量及多种活性物质（包括光合色素及藻胆蛋白等）含量的降低。大部分硒的富集转化都是在大约3天内达到平衡,并且大部分（约90%）以有机硒的形式存在于藻体中。 2.在螺旋藻生长周期的第7、8、9三天分次加硒,既可提高富硒量又可降低高硒对螺旋藻的抑制作用。结果表明此加硒方法可提高其最终生物量（1.34g/L,对照组为1.18g/L）,硒产量高达1743.06μg/L,且藻体中各种主要活性物质均有不同程度的增加,证明这是一种理想的富硒处理方法。 3.建立了Dansyl柱前衍生化-高效液相色谱法及Waters AccQ·Tag氨基酸HPLC分析法用于分离测定18种通用氨基酸及硒代蛋氨酸,效果满意。 4.按优化方法培养的富硒螺旋藻干粉中总硒含量为1.176mg/g,其中有机硒为1.105mg/g,有机化率为94%。Dansyl柱前衍生化-高效液相色谱法测定结果显示:藻体中含硒氨基酸主要形态为Se-Met,含量为0.261mg/g,按硒计则为0.105mg/g,占有机硒的9.5%,说明藻体内尚存在较多的其他形式的有机硒。 5.甲基化是硒在生物体中的重要代谢途径。本实验采用吹扫捕集-GC-MS方法测定气态硒化合物,共检测出二甲基硒、二甲基二硒两种,效果满意。并对其MS谱图的裂解机理进行探讨。基于硒的6种同位素在自然界中的分布规律,含硒有机化合物的MS裂解图谱一般都易于指认。一般80_Se、⁸¹Se的丰度较高,可以它们作为含硒片断的指认依据。更多还原

【Abstract】 The trace mineral selenium （Se） is an essential nutrient of fundamental importance to human biology. Spirulina platensis （S. platensis ） was found to possess a good tolerance to high levels of Se, indicating that it was a good carrier for Se biotransformation. The effects of different Se adding methods on S. platensis were investigated. The speciation of Se existing in the microalgae cells and volatile Se metabolized to the environment, the morphological changes of S. platensis cultured under autotrophic, heterotrophic and mixotrophic conditions were also carried out in this study. Some meaningful results were obtained.1. Se has either stimulating or toxic effects on the culture of S. platensis depending on the Se levels in media. S. platensis could bioaccumulate Se efficiently during the cultivation time and the accumulation amount increased with Se stress （≤ 150 mg/L）. The higher initial Se concentrations （≥200 mg/L） led to high Se enrichment, but inducing much lower biomass concentrations and some active substances. The results revealed that the majority of the total Se was accumulated in a short time （about 3 days） and most of them were biotransformed into organic Se （about 90 %） by integrating with the algal bioligands, such as proteins, lipids, polysaccharides and other algal components.2. Effects of different Se adding methods on the growth, Se accumulation and transformation of S. platensis were investigated. The results indicated that the high Se-enriched S. platensis with a total Se content of 1301.17 μg/g and an organic ratio of 86.85 % can be obtained while the Se was added in the 7th, 8th, 9th day respectively with an accumulative concentration of 1000 mg/L. A stimulative effect in biomass of Se-enriched cultures was observed in the 7 th experimental set, comparing with the control. Furthermore, the contents of phycobiliproteins, and photosynthetic pigments （Lutein and β -carotene） increased to some extend. About 67.12% of the organic Se existed in the part of soluble proteins.3. A reversed phase high performance liquid chromatographic （RP-HPLC）method with precolumn derivazation of dansyl （Dns-Cl）, and a Waters AccQ-Tag method for simultaneous determination of 18 common amino acids and selenomethionine （Se-Met） was established.4. The amino acids in Se-enriched S. platensis were analyzed with the Dns-Cl HPLC method. The peaks area of the 19 amino acids linearly correlated with their concentrations. Detection limit was 3.5×10^-8 mol/L. The quantitative analysis showed that Se-enriched treatment didn’t change the content of 18 common amino acids in Se-SP. The content of Se-Met was 0.261 mg/g in the Se-SP. The detection linearity, precision and recovery associated with this assay were evaluated.5. Methylation is an effective detoxification mechanism. A new method was developed for the fast determination of trace amounts of volatile Se in culture media of S. platensis. The results indicated that the chief methylation productions of Se biotransformation by S. platensis were dimethylselenide （DmSe） and dimethyldiselenide （DMDSe）.6. The morphological changes of S. platensis cultured under autotrophic, heterotrophic and mixotrophic conditions were also studied. The difference among the ultrastructure of S. platensis cells may be due to the photosynthesis and rapid intake of organic carbon resource.更多还原