Study on the fatty acid composition of jellyfish Rhopilema esculentum

The fatty acids composition in different parts of full-grown Rhopilema esculentum jellyfish from Yellow Sea was investigated. The lipids, extracted from the umbrella and oral arms and gonads of R. eculentum jellyfish, respectively were analysed by combined capillary gas chromatography/mass spectrometry. The results show that there are more than thirty kinds of fatty acids in jellyfish, and the fatty acid compositions of three parts of R. esculentum are almost the same. In the three parts, percentages of polyunsaturated fatty acids (PUFA) are high, and range from 36.23% to 38.74%. Docosahexaenoic acid (DHA), eicosatetraenoic acid (AA) and eicosapentaenoic acid (EPA) are three major PUFA.

Effects of nitrogen source and concentration on growth rate and fatty acid composition of Ellipsoidion sp (Eustigmatophyta)

In order to study the effects of different nitrogen source and concentration on the growth rate and fatty acid composition, a marine microalga Ellipsoidion sp. with a high content of eicosapentaenoic acid (EPA) was cultured in media with different nitrogen sources and concentrations. During the pre-logarithmic phase, the alga grew faster with ammonium as N source than with nitrate, but the reverse applied during the post-logarithmic phase. The alga grew poorly in N-free medium or medium with urea as the sole N source. In the same growth phase, ammonium medium resulted in higher yield of total lipid, but the EPA yield did not differ significantly different from that using nitrate medium. The maximum growth rate occurred in medium containing 1.28 mmol L-1 sodium nitrate, while maximum EPA and total lipid contents were reached at 1.92 mmol L-1, when EPA accounted for 27.9% total fatty acids. The growth rate kept stable when NH4Cl ranged from 0.64 to 2.56 mmol L-1, and the maximum content of total lipid and EPA occurred in the medium with 2.56 mmol L-1 NH4Cl. The EPA content was higher in the pre- than post-logarithmic phase, though the total lipid content was lower. The highest EPA content expressed as percent total fatty acid was 27.9% in nitrate medium and and 39.0% in ammonium medium.

麻疯树种子油体以及种子萌发的蛋白质组学研究

麻疯树（Jatropha curcas L.）属大戟科麻疯树属多年生亚乔木，耐干旱、高温和贫瘠等，具很强的抗逆性，在干热河谷等边际土地上生长良好。其种子富含油脂，是制备生物柴油的理想材料，为重要的能源植物之一。油体（oil body）是种子细胞中重要的细胞器, 脂肪酸以三脂酰甘油（triacylglyeerols，TAG）的形式储存其内，是种子萌发和幼苗生长时所需碳骨架和能量的主要来源。种子萌发为生命萌动并构建成自养个体的过程，是高等植物生长发育中的重要事件。 本论文运用高通量的蛋白质组学研究手段，结合电镜技术和生理学分析，对麻疯树种子油体以及种子萌发过程中蛋白质表达、生理学响应和细胞结构变化进行了研究。 从麻疯树种子胚乳中分离油体，再从油体中提取蛋白，经双向凝胶电泳后，得到油体蛋白质组的二维表达谱，这些蛋白质主要分布在等电点5 ~ 10、分子量12 ~ 66 kDa的范围内;图像分析表明，油体蛋白质组至少有141个蛋白点，其中酸性蛋白74个，碱性蛋白67个，表达丰度较高的多为低分子量碱性蛋白。对其中36个重要蛋白点进行LC-MS/MS质谱分析，得到鉴定的蛋白分别为30个基因的表达产物，主要包括油体重要的结构蛋白油质蛋白（oleosin）和caleosin，麻疯树种子毒蛋白curcin，以及新鉴定得到的另一种可能的麻疯树种子毒蛋白，人体过敏反应蛋白橡胶延伸因子（REF）。还有四个与脂肪酸代谢相关的酶，其中3-羟酰-酰基载体蛋白（ACP）脱水/催化酶和醇酰基转移酶与脂肪酸合成有关，而脂氧合酶和磷脂酶D在脂肪酸降解中发挥作用，显示部分脂肪酸代谢相关的酶在油体储存状态就已附着在油体上，为种子萌发时动员油脂做好了准备。 麻疯树种子胚乳发达，在32℃湿润土壤中很快就会萌动，胚轴伸长露出胚根，长出新根，约4天后形成出土子叶幼苗。种子萌发过程中胚乳主要成分含量测定表明，含水量在前24小时迅速上升，至48小时增加缓慢，此后开始较快上升，可分为三个阶段，呈现“S”型的变化;粗脂肪和粗蛋白在前两个阶段变化不大，进入第三阶段后其含量迅速下降，前者先于后者，分别在萌发后72小时和96小时后开始明显减少，说明被大量降解、转化，供萌发生长利用，其中主要组分亚油酸最为明显。细胞超微结构观察发现，排列整齐充满整个胚乳细胞的油体和嵌合在油体中的蛋白储存泡在种子萌发过程中，随着线粒体、乙醛酸循环体和液泡的出现增多或增大而被逐渐解体、减少或消失;同时，发现脂肪酸主要在乙醛酸循环体、蛋白颗粒主要在液泡中被降解或转化。 蛋白质组学分析表明，麻疯树种子在萌发72小时过程中变化量在两倍以上的差异蛋白点共有141个，所有的差异蛋白均通过LC-MS/MS分析和NCBI蛋白数据库搜索得到鉴定。其中包括多个参与降解储藏油脂的酶，如乙醛酸循环途径中的顺乌头酸酶，异柠檬酸裂解酶和苹果酸脱氢酶等，均从种子萌发48小时开始表达量明显上升;葡糖异生途径中的酶在种子萌发中的积累略晚于乙醛酸循环途径，如烯醇酶，磷酸甘油酸变位酶，磷酸甘油酸激酶，磷酸丙糖异构酶和醛缩酶大多在萌发约60小时后表达量开始上调。分析结果表明，乙醛酸循环途径在种子萌发48小时后被激活，与电镜观察胚乳细胞发现油脂在萌发48小时时开始被动员相一致，因而大规模的油脂动员开始于种子萌发的第三阶段。 同时，蛋白质组学的分析结果也得到了种子胚乳组分变化分析及电镜观察结果的印证。超微细胞结构观察显示种子储藏蛋白降解在萌发第二阶段启动，主要在液泡中进行降解。粗脂肪的含量在72小时时显著降低，而电镜观察显示此时胚乳细胞中出现中央大液泡，出现大量的线粒体和乙醛酸循环体，细胞结构发生重大变化，萌发96小时后仅有少量油体残留于胚乳细胞中，这些都为储藏油脂在麻疯树种子萌发过程中的降解方式提供了重要证据。许多其他的功能蛋白在种子萌发过程中也发生了变化，表明种子萌发过程中不仅发生储藏物质的动员，也发生抗逆反应以及植物形态的构建等众多其他生理生化反应。 本研究首次对麻疯树种子油体进行了蛋白组成分析，并结合电镜技术及生理分析深入探讨了种子储藏物质在萌发中的降解方式，为更好的理解油体结构、木本油料种子的萌发机制和对麻疯树进行品种的改良提供了参考。

Lipase-catalyzed synthesis of biodiesel from acid oil in fixed bed reactor

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols and is a feedstock for production of biodiesel fuel now. The transesterification of acid oil and methanol to biodiesel was catalyzed by immobilized Candida lipase in fixed bed reactors. The reactant solution was a mixture of acid oil, water, methanol and solvent (hexane) and the main product was biodiesel composed of fatty acid methyl ester (FAME) of which the main component was methyl oleate. The effects of lipase content, solvent content, water content temperature and flow velocity of the reactant on the reaction were analyzed. The experimental results indicate that a maximum FAME content of 90.18% can be obtained in the end product under optimum conditions. Most of the chemical and physical properties of the biodiesel were superior to the standards for 0(#) diesel (GB/T 19147) and biodiesel (DIN V51606 and ASTM D6751).

Optimization of biodiesel production from trap grease via acid catalysis

As a kind of waste collected from restaurants, trap grease is a chemically challenging feedstock for biodiesel production for its high free fatty acid (FFA) content. A central composite design was used to evaluate the effect of methanol quantity, acid concentration and reaction time on the synthesis of biodiesel from the trap grease with 50% free fatty acid, while the reaction temperature was selected at 95 degrees C. Using response surface methodology, a quadratic polynomial equation was obtained for ester content by multiple regression analysis. Verification experiments confirmed the validity of the predicted model. To achieve the highest ester content of crude biodiesel (89.67%), the critical values of the three variables were 35.00 (methanol-to-oil molar ratio), 11.27 wt% (catalyst concentration based on trap grease) and 4.59 h (reaction time). The crude biodiesel could be purified by a second distillation to meet the requirement of biodiesel specification of Korea.

Positional distribution of fatty acids on the glycerol backbone during the biosynthesis of glycerolipids in Ectocarpus fasciculatus

The biosynthesis of glycolipids in E. fasciculatus was studied by C-14 label and chase. The fatty acids in sulphoquinovosyl diacylglycerol (SQDG) were almost 16-carbon and 18-carbon ones. In addition to the two fatty acids, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) contained 8.5 mol% and 31.0 mol% of eicosapentaenoic acid (20 : 5), respectively, and this fatty acid was usually distributed in the sn-1 position of the glycerol backbone. When plants were incubated with [2-C-14] acetate, differences existed in the positional distribution of the labeled fatty acids in sn-1 and sn-2 among the three glycerolipids. In SQDG C-14-labeled fatty acids were distributed uniformly in the sn-1 and sn-2 positions. In DGDG, C-14-labeled fatty acids were mainly distributed in the sn-2 position. In MGDG, the radioactivity of fatty acids in sn-1 position was far greater than that in sn-2 position after a 30 min pulse label, and the difference in radioactivity between the two positions decreased rapidly. The above results indicated that differences in the positional distribution of C-14-labeled fatty acids between sn-1 and sn-2 positions might be related to 20 : 5 and the biosynthesis of DGDG. Our results also suggested that E. fasciculatus had the same DGDG biosynthetic pathway as that in higher plants and galactosyl transferase was selective for MGDC.

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Laminaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as Alterornonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that: (1) The blades of L. japonica exhibited symptoms of lesion, bleaching and deterioration when infected by the bacterium, and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species (ROS) generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde (MDA) and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. (2) The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase (SOD) and catalase (CAT) increased greatly when infected by the bacterium, but glutathione peroxidase (Gpx) and ascorbate peroxidase (APX) did not exhibit active responses to the bacterium throughout the experiment. (3) The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

Fatty acids of some algae from the Bohai Sea

The fatty acid compositions of 22 species of marine macrophytes, belonging to the Ceramiales, Cryptonemiales, Nemalionales, Laminariales, Chordariales, Scytosiphonales, Desmarestiales, Dictyosiphonales, Fucales, Dictyotales and Ulvales and collected from the Bohai Sea, were determined by capillary gas chromatography. The contents of polyunsaturated fatty acids (FAs) in the Bohai Sea algae, in comparison with the same species from the Yellow Sea were found to be lower. Red algae had relatively high levels of the acids 16:0, 18:1(n-7), 18:1(n-9), 20:5(n-3) and 20:4(n-6), and those examined were rich in C-20 PUFAs, these chiefly being arachidonic and eicosapentaenoic acids. The major FAs encountered in the Phaeophyta were 14:0, 16:0, 18:1(n-9), 18:2(n-6), 18:3(n-3), 18:4(n-3), 20:4(n-6) and 20:5(n-3). C18PUFAs are of greater abundance in the brown algae than in the red algae examined. All three green algae from the Ulvales had similar fatty acid patterns with major components, 16:0, 16:4(n-3), 18:1(n-7), 18:2(n-6), 18:3(n-3), and 18:4(n-3). They contained 16:3(n-3) and more 16:4(n-3), were rich in C18PUFAs, chiefly 18:3(n-3) and 18:4(n-3) and had 18:1(n-7)/18:1 (n-9) ratios higher than 1. (C) 2002 Elsevier Science Ltd. All rights reserved.

Necessity of dietary lecithin and eicosapentaenoic acid for growth, survival, stress resistance and lipoprotein formation in gilthead sea bream Sparus aurata

The substitution of dietary docosahexaenoic acid (DHA) with eicosapentaenoic acid (EPA) reduces larval growth in gilthead sea bream. However, the value of EPA when dietary DHA is able to meet the requirements of the larvae has not been sufficiently studied. Dietary phosphoacylgliceride levels also affect fish growth and it has been suggested that they enhance lipid transport in developing larvae. The present experiment was carried out to further study the effect of dietary lecithin and eicosapentaenoic acid on growth, survival, stress resistance,. larval fatty acid composition and lipid transport, when DHA is present in the microdiets of gilthead:sea bream. Eighteen thousand gilt-head sea bream larvae of 4.99+/-0.53 mm total length were fed three microdiets tested by triplicate: a control diet [2% soybean lecithin (SBL) and 2.89% EPA], a low EPA diet,(2% SBL and 1.63% EPA) and a no SBL diet (0% SBL and 2.71% EPA). Handling, temperature and salinity tests determined larval resistance to stress. The results show that when dietary DHA levels are high, but dietary arachidonic acid (ARA) levels are about 0.2%, EPA is necessary to improve larval growth, and survival. Larval EPA content, but not DHA or ARA, was affected by dietary EPA levels. Increased dietary EPA improved larval stress resistance to handling and temperature tests, which could be related to its possible role as a regulator of cortisol production whereas it did not affect stress resistance after salinity shock. Larvae fed the no SBL diet showed a lower lipid content characterized by a low proportion of saturated and monounsaturated fatty acids, together with a significant reduction in the appearance of lipoprotein particles in the lamina propria and in the size of such particles, denoting a critical reduction in dietary lipid transport and utilization, and lower larval growth and survival rates.

Extraction of Nitraria tangutorum seed oil by supercritical carbon dioxide and determination of free fatty acids by HPLC/APC/IMS with fluorescence detection

The seed oil from Nitraria tangutorum samples was obtained by supercritical carbon dioxide extraction methods. The extraction parameters for this methodology, including pressure, temperature, particle size and extraction time, were optimized. The free fatty acids in the seed oil were separated with a pre-column derivation method and 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as a labeling regent, followed by high-performance liquid chromatography (HPLC) with fluorescence detection. The target compounds were identified by mass spectrometry with atmospheric pressure chemical ionization (APCI in positive-ion mode). HPLC analysis shows that the main compositions of the seed oil samples were free fatty acids (FFAs) in high to low concentrations as follows: linoleic acid, oleic acid, hexadecanoic acid and octadecanoic acid. The assay detection limits (at signal-to-noise of 3:1) were 3.378-6.572 nmol/L. Excellent linear responses were observed, with correlation coefficients greater than 0.999. The facile BDETS derivatization coupled with mass spectrometry detection allowed the development of a highly sensitive method for analyzing free fatty acids in seed oil by supercritical CO2 extraction. The established method is highly efficient for seed oil extraction and extremely sensitive for fatty acid profile determination. (C) 2007 Elsevier B.V. All rights reserved.

Determination of free fatty acids in soil and bryophyte plants by precolumn derivatization via HPLC with fluorescence detection and tandem mass spectrometry (HPLC-MS/MS)

A sensitive method for the determination of 30 kinds of free fatty acids (FFAs, C-1-C-30) with 1-[2-(p-toluenesulfonate)-ethyl]-2-phenylimidazole-[4,5-f] 9,10-phenan- threne (TSPP) as labeling reagent and using high performance liquid chromatography with fluorescence detection and identification by online postcolumn mass spectrometry with atmospheric pressure chemical ionization (APCI) source in positive-ion mode (HPLC/MS/APCI) has been developed. TSPP could easily and quickly label FFAs in the presence of K2CO3 catalyst at 90 degrees C for 30 min in N,N-dimethylformamide (DMF) solvent, and maximal labeling yields close to 100% were observed with a 5-fold excess of molar reagent. Derivatives were stable enough to be efficiently analyzed by high performance liquid chromatography. TSPP was introduced into fatty acid molecules and effectively augmented MS ionization of fatty acid derivatives and led to regular MS and MS/MS information. The collision induced cleavage of protonated molecular ions formed specific fragment ions at m/z [MH](+)(molecular ion), m/z [M'+CH2CH2](+)(M' was molecular mass of the corresponding FFA) and m/z 295.0 (the, mass of protonated molecular core structure of TSPP). Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C-8 column (4.6 x 150 mm, 5 mu m, Agilent) with a good baseline resolution in combination with a gradient elution. Linear ranges of 30 FFAs are 2.441 x 10(-3) to 20 mu mol/L, detection limits are 3.24 similar to 36.97 fmol (injection volume 10 mu L, at a signal-to-noise ratio of 3, S/N 3:1). The mean interday precision ranged from 93.4 to 106.2% with the largest mean coefficients of variation (R.S.D.) < 7,5%. The mean intraday precision for all standards was < 6.4% of the expected concentration. Excellent linear responses were observed with correlation coefficients of > 0.9991. Good compositional data could be obtained from the analysis of extracted fatty acids from as little as 200 mg of bryophyte plant samples.Therefore, the facile TSPP derivatization coupled with HPLC/MS/APCI analysis allowed the development of a highly sensitive method for the quantitation of trace levels of short and long chain fatty acids from biological and natural environmental samples.

Determination of 30 free fatty acids in two famous Tibetan medicines by HPLC with fluorescence detection and mass spectrometric identification

A simple and sensitive high-performance liquid chromatographic (HPLC) method with fluorescence detection and mass spectrometric identification has been developed for analysis of 30 long-chain and short-chain free Fatty acids (FFAs). The fatty acids were derivatized to their esters with 1-[2-(p-toluenesulfonate)ethyl]-2-phenylimidazole-[4,5-f]-9,10-phenanthrene (TSPP) in N,N-dimethylformamide (DMF) at 90 degrees C with anhydrous K2CO3 as catalyst. A mixture Of C-1-C-30 fatty acids was completely separated within 60 min by gradient elution on a reversed-phase C-8 column. Qualitative identification of the acids was performed by atmospheric-pressure chemical ionization mass spectrometry (APCI-MS) in positive-ion mode. The fluorescence excitation and emission wavelengths were 260 and 380 nm, respectively. Quantitative determination of the 30 acids in two Tibetan medicines Gentiana straminea and G. dahurica was performed. The results indicated that the medicines contained many FFAs. Linear correlation coefficients for the FFA derivatives were > 0.9991. Relative standard deviations (RSDs, n = 6) for the fatty acid derivatives were < 3%. Detection limits (at a signal-to-noise ratio of 3:1) were 3.1-38 fmol. When the fatty acid derivatives were determined in the two real samples results were satisfactory and the sensitivity and reproducibility of the method were good.

Analysis of saturated free fatty acids from pollen by HPLC with fluorescence detection

A sensitive method for the determination of free fatty acids using 2-(2-(anthracen-10-yl)-1H-naphtho[2,3-dimidazol-1-yl) ethyl-p-toluenesuIfonate (ANITS) as tagging reagent with fluorescence detection has been developed. ANITS could easily and quickly label fatty acids in the presence of the K2CO3 catalyst at 90 degrees C for 40 min in N,N-dimethylformamide solvent. From the extracts of rape bee pollen samples, 20 free fatty acids were sensitively determined. Fatty acid derivatives were separated on a reversed-phase Eclipse XDB-C8 column by HPLC in conjunction with gradient elution. The corresponding derivatives were identified by post-column APCI/MS in positive-ion detection mode. ANITS-fatty acid derivatives gave an intense molecular ion peak at mlz [M+H](+); with MS/MS analysis, the collision-induced dissociation spectra of m/z [M+H](+) produced the specific fragment ions at mlz [M-345](+) and mlz 345.0 (here, m/z 345 is the core structural moiety of the ANITS molecule). The fluorescence excitation and emission wavelengths of the derivatives were lambda(ex) = 250 nm and lambda(em) = 512 nm, respectively. Linear correlation coefficients for all fatty acid derivatives are > 0.9999. Detection limits, at a signal-to-noise ratio of 3 : 1, are 24.76-98.79 fmol for the labeled fatty acids.

HPLC-APCI-MS determination of free fatty acids in Tibet folk medicine Lomatogonium rotatum with fluorescence detection and mass spectrometric identification

A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone-9-ethyl-p-toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB-C-8 column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at lambda(ex) 404 and lambda(em) 440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post-column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive-ion detection mode. Nineteen FFAs from the extract of Lomatogonium rotatum are sensitively determined. The results indicate that the plant Lomatogonium rotatum is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C-14, C-16, and C-18. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are > 0.9989, and detection limits (at signal-to-noise of 3: 1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are < 2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of Lomatogonium rotatum with satisfactory results.

Simple derivatization method for sensitive determination of fatty acids with fluorescence detection by high-performance liquid chromatography using 9-(2-hydroxyethyl)-carbazole as derivatization reagent

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60 degreesC with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C-1-C-20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C-18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (lambda (ex) 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n = 6) for each fatty acid derivative are <4%. The detection limits are at 45-68 fmol levels for C-14-C-20 fatty acids and even lower levels for fatty acids. (C) 2001 Elsevier Science B.V. All rights reserved.