响应面法优化红胞藻中藻红蛋白提取工艺及体外抗氧化性研究

利用响应面分析法优化藻红蛋白的提取工艺。在单因素实验基础上,使用硫酸铵分级沉淀提取纯化藻红蛋白,选超声时间、温度、功率为影响因素,以藻红蛋白得率为响应值,根据Box-Behnken实验设计原理对藻红蛋白提取工艺进行响应面分析,优化藻红蛋白提取工艺。结果表明:在超声时间为16 min、温度为33℃、功率为750 W时藻红蛋白得率取得最大值。在此优化条件下进行验证性实验,测得蛋白得率为15.93%,与预测值相对误差为2.50%,表明该优化工艺具有良好的可行性。藻红蛋白对O-2·和ABTS+·具有一定的清除作用,其IC_(50)分别为0.025 mg/m L和0.023 mg/m L。

红胞藻藻红蛋白的分离纯化及稳定性研究

文章旨在从红胞藻中分离纯化得到试剂级的藻红蛋白,并对其相关性质进行研究。以红胞藻为原料,采用反复冻融、硫酸铵盐析和疏水层析的方法分离纯化藻红蛋白,并对其稳定性进行研究。结果表明,红胞藻经过4次反复冻融后藻红蛋白溶出率最高,两步硫酸铵分级沉淀可使藻红蛋白纯度(A_(545)/A_(280))提高到2.45,进一步采用Butyl-S-QZT 6FF疏水层析纯化后藻红蛋白纯度(A_(545)/A_(280))达到试剂级,为6.63,回收率为48.9%。纯化的藻红蛋白在545 nm处有最大特征吸收峰,荧光激发峰位于541 nm和553 nm处,发射峰位于587.5 nm处,二级结构主要以α-螺旋的形式存在。藻红蛋白含有3个亚基,分子质量分别为9、10 ku和20 ku,在30~50℃、pH6~8条件下能保持相对稳定。Cu^(2+)对藻红蛋白的光谱学性质有一定的影响,在Ca^(2+)、Mn^(2+)、Zn^(2+)、Fe^(2+)、Fe^(3+)等离子存在时,藻红蛋白光谱学性质没有发生明显变化。结论:采用硫酸铵分级沉淀结合一步疏水层析获得了试剂级的藻红蛋白,得率高,工艺简便。研究结果为红胞藻藻红蛋白的大规模生产提供了参考。

甲醛对中华绒鳌蟹各期溞状幼体及隐藻的急性毒性

通过静水试验法，用甲醛对中华绒螯蟹各期状幼体及隐藻进行急性毒性试验，结果分别为：Z1：24hLC50为41．214mg／L，48hLC50为33．364mg／L，SC为3．34mg／L。Z2：24hLC50为125．497mg／L，48hLC50为54．258mg／L，SC为5．43mg／L。Z3：24hLC50为143．617mg／L，48hLC50为93．817mg／L，SC为9．38mg／L。Z4：24hLC50为200．679mg／L，48hLC50为98．414mg／L，SC为9．84mg／L。甲醛对Z5的毒性状况比较特殊，虽经10次试验仍无规律性。试验表明，30mg／L以下的甲醛，24小时内波海红胞藻全部成活，甲醛对波海红胞藻的6hLC50为：90mg／L，12hLC50为：60．77mg／L，24hLC50为：57．5mg／L。一定浓度的甲醛可杀死苗池水中的赤潮生物，蓄水池中的赤潮生物可用含氯化合物杀灭，以降低赤潮生物对中华绒螯蟹幼体的毒性，提高幼体的变态成活率和育苗产量。

Cell cycles and proliferation patterns in Haematococcus pluvialis

Most studies on Haematococcus pluvialis have been focused on cell growth and astaxanthin accumulation;far less attention has been paid to cell cycles and proliferation patterns.The purpose of this study was to clarify cell cycles and proliferation patterns in H.pluvialis microscopically using a camera and video recorder system.The complicated life history of H.pluvial i s can be divided into two stages:the motile stage and the non-motile stage.All the cells can be classifi ed into forms as follows:motile cell,nonmotile cell,zoospore and aplanospore.The main cell proliferation,both in the motile phase and non-motile phase in H.pluvialis,is by asexual reproduction.Under normal growth conditions,a motile cell usually produces two,sometimes four,and exceptionally eight zoospores.Under unfavorable conditions,the motile cell loses its fl agella and transforms into a non-motile cell,and the non-motile cell usually produces 2,4 or 8 aplanospores,and occasionally 20–32 aplanospores,which further develop into non-motile cells.Under suitable conditions,the non-motile cell is also able to release zoospores.The larger non-motile cells produce more than 16 zoospores,and the smaller ones produce 4 or 8 zoospores.Vegetative reproduction is by direct cell division in the motile phase and by occasional cell budding in the non-motile phase.There is,as yet,no convincing direct evidence for sexual reproduction.

Effects of iron electrovalence and species on growth and astaxanthin production of Haematococcus pluvialis

To increase the cell concentration and the accumulation of astaxanthin in the cultivation of Haematococcus pluvialis, effects of different iron electrovalencies （Fe^2＋-EDTA and Fe^3＋-EDTA） and species （Fe-EDTA, Fe（OH）x^32x and FeC6H5O7） addition on cell growth and accumulation of astaxanthin were studied. Results show that different iron electrovalencies have various effects on cell growth and astaxanthin accumulation of H. pluvialis. Compared with Fe^3＋-EDTA, Fe^2＋-EDTA stimulate more effectively the formation of astaxanthin. The maximum astaxanthin content （30.70 mg/g biomass cell） was obtained under conditions of 18 μmol/L Fe^2＋-EDTA, despite the lower cell density （2.3×10^5 cell/ml） in such condition. Fe^3＋-EDTA is more effective than Fe^2＋-EDTA in improving the cell growth. Especially, the maximal steady-state cell density, 2.9×10^5 cell/ml was obtained at 18 μmol/L Fe^3＋-EDTA addition. On the other hand, all the various species of iron （EDTA-Fe, Fe（OH）x^32x, FeC6H5O7） are capable to improve the growth of the algae and astaxanthin production. Among the three iron species, FeC6H5O7 performed the best. Under the condition of a higher concentration （36 μmol/L） of FeC6H5O7, the cell density and astaxanthin production is 2 and 7 times higher than those of iron-limited group, respectively. The present study demonstrates that the effects of the stimulation with different iron species increased in the order of FeC6H5O7, Fe（OH）x^32x and EDTA-Fe.

DNA barcode assessment of Ceramiales(Rhodophyta) in the intertidal zone of the northwestern Yellow Sea

A total of 142 specimens of Ceramiales （Rhodophyta） were collected each month from October 2011 to November 2012 in the intertidal zone of the northwestern Yellow Sea. These specimens covered 21 species, 14 genera, and four families. Cluster analyses show that the specimens had a high diversity for the three DNA markers, namely, partial large subunit rRNA gene （LSU）, universal plastid amplicon （UPA）, and partial mitochondrial cytochrome c oxidase subunit I gene （COl）. No intraspecific divergence was found in our collection for these markers, except for a 1-3 bp divergence in the COI of Ceramium kondoi, Syrnphyocladia latiuscula, and Neosiphoniajaponica. Because short DNA markers were used, the phylogenetic relationships of higher taxonomic levels were hard to evaluate with poor branch support. More than half species of our collection failed to find their matched sequences owing to shortage information of DNA barcodes for macroalgae in GenBank or BOLD （Barcode of Life Data） Systems. Three specimens were presumed as Heterosiphonia crispella by cluster analyses on DNA barcodes assisted by morphological identification, which was the first record in the investigated area, implying that it might he a cryptic or invasive species in the coastal area of northwestern Yellow Sea. In the neighbor-joining trees of all three DNA markers, Heterosiphonia japonica converged with Dasya spp. and was distant from the other Heterosiphonia spp., implying that H.japonica had affinities to the genus Dasya. The LSU and UPA markers amplified and sequenced easier than the COI marker across the Ceramiales species, but the COI had a higher ability to discriminate between species.

Formation and early development of tetraspores of Polysiphonia urceolata (Rhodomelaceae,Rhodophyta)

Polysiphonia urceolata is one type of potential commercial red seaweeds used for breeding and cultivation, because of its significant biochemical and biomedical application. However, the information of breeding and seedling incubation for cultivation is limited, especially the early development. In this study, tetrasporohyte and gametophyte of P. urceolata were taken as the study materials in Huiquan Bay, Qingdao, China. The cleaned and sterilized tetrasporophytes and gametophytes were pre-cultured in sterilized seawater, then nurtured at 18℃, 25 μmol photons m^-2 s^-1 in 12：12 h （light：dark） photoperiod. Continuous observation under microscope showed that the early development consists of bipolar division stage and seedling stage. In the division stage, tetraspores germinate into bipolar sporelings that further differentiate into a colorless rhizoidal portion and a lightly pigmented upright shoot. The lightly pigmented rhizoidal cell develops to a rhizoid and the larger pigmented cell transforms to an erect axis. In the seedling stage, several quasi-protuberances appear on the erect axis and form juvenile seedlings. The results demonstrate the culture of P. urceolata from tetraspores under laboratory conditions.

Isolation and optimization of production of Astaxanthin from Antarctic yeast Rhodotorula sp. NJ298

Rhodotorula sp. NJ298 which could produce carotenoids was isolated from Antarctic sea ice. The major carotenoid was identified as astaxanthin by Liquid Chromatography/Mass Spectrometry （LC/MS）, and its content accounted for 87.62% of total carotenoids （1,786 μg/g）. High Performance Liquid Chromatogrephy （HPLC） analysis showed that the purity of the astaxanthin reached about 96. 16% through a simple purification. Maximum astaxanthin production （1,908μg/g） was obtained when the yeast was grown at 10 ℃ in seawater medium containing 5 g/L sodium acetate, 5 g/L peptone, 0.5 g/L NaC1, 0.01 g/L KH2PO4; 0.01 g/L MgSO4·7H20 and 0.001 g/L FeSO4·7H20 at pH 7.5.

Characterization of calcium deposition induced by Synechocystis sp. PCC6803 in BG11 culture medium

Calcium carbonate （CaCO3） crystals in their preferred orientation were obtained in BG11 culture media inoculated with Synechocystis sp. PCC6803 （inoculated BG11）. In this study, the features of calcium carbonate deposition were investigated. Inoculated BGll in different calcium ion concentrations was used for the experimental group, while the BGll culture medium was used for the control group. The surface morphologies of the calcium carbonate deposits in the experimental and control groups were determined by scanning and transmission electron microscopy. The deposits were analyzed by electronic probe micro-analysis, Fourier transform infrared spectrum, X-ray diffraction, thermal gravimetric analysis and differential scanning calorimetry. The results show that the surfaces of the crystals in the experimental group were hexahedral in a scaly pattern. The particle sizes were micrometer-sized and larger than those in the control group. The deposits of the control group contained calcium （Ca）, carbon （C）, oxygen （O）, phosphorus （P）, iron （Fe）, copper （Cu）, zinc （Zn）, and other elements. The deposits in the experimental group contained Ca, C, and O only. The deposits of both groups contained calcite. The thermal decomposition temperature of the deposits in the control group was lower than those in the experimental group. It showed that the CaCO3 deposits of the experimental group had higher thermal stability than those of the control group. This may be due to the secondary metabolites produced by the algae cells, which affect the carbonate crystal structure and result in a close-packed structure. The algae cells that remained after thermal weight loss were heavier in higher calcium concentrations in BGll culture media. There may be more calcium- containing crystals inside and outside of these cells. These results shall be beneficial for understanding the formation mechanism of carbonate minerals.

A new ketosteroid from red alga Acanthophora spicifera

A new ketosteroid, along with six known steroids, was isolated from the ethanolic extracts of red alga Acanthophora spicifera （Vahl.） Boergesen. The structures, identified using chemical and spectroscopic methods including 2D NMR, were： （1） 22-hydroxy-5α-eholest-3,6-dione, （2） 6-hydroxycholest-4-ene-3-one, （3） cholest-4-ene-3,6-dione, （4） cholest-5-ene-3β-ol, （5） 5u-cholestane-3,6-dione, （6） β-Sitosterol and （7） Saringosterol. The MTT method was used to test the cytotoxicity of the compounds against the human cancer cell lines, HCT-8, Bel-7402, BGC-823, A549 and HELA. Compounds 1, 2, 3 and 5 showed moderate cytotoxic activity against human cancer cell lines.