微孔草籽油的超临界CO2流体萃取及HPLC—MS分析

利用超临界CO2萃取微孔草籽油,并对籽油进行了HPLC／MS分析。实验确定的最佳超临界CO2流体萃取条件是：萃取温度45℃,萃取压力20MPa,CO2流量为35-40kg／h,萃取时间120min,在此条件下白刺籽油的萃取率为16．12％。利用HPLC／MS对微孔草籽油分析,发现其不饱和脂肪酸的相对含量高达73．19％。比较了超临界CO2萃取微孔草籽油油样和石油醚萃取微孔草籽油油样的理化性质,发现超临界CO2流体萃取的籽油质量优于传统溶剂萃取的籽油。     

An ecofriendly approach to process rice bran for high quality rice bran oil using supercritical carbon dioxide for nutraceutical applications

An integrated approach to extraction and refining of RBO using supercritical carbon dioxide (SC-CO2) in order to preserve the nutritionally important phytochemicals is reported here. Process variables such as pressure, temperature, time, solvent flow rate and packing material on extraction yield and quality of RBO were investigated using a pilot model SC-CO2 extraction system. Three isobaric (350, 425 and 500 bar), three isothermal temperatures (50, 60 and 70 degrees C), three extraction times (0.5, 1 and 1.5h), at 40/min CO2 flow rate and three packing materials (pebbles, glass beads and structured SS rings) were employed. The RBO yield with SC-CO2 extraction increased with temperature and time under isobaric conditions. At the 60 degrees C isotherm, an increase in the RBO yield was obtained with an increase in the pressure and time. The RBO yield increased significantly with structured SS rings used as packing material. The RBO extracted with SC-CO2 had negligible phosphatides, wax and prooxidant metals (Fe and Cu) and was far superior in color quality when compared with RBO extracted with hexane. At the optimum condition of extraction at 500 bar, 60 degrees C for 1.5h, with structured SS rings used as packing material, the yield of RBO was comparable with that of hexane extraction (22.5%). The phytochemical contents of the RBO under the optimum conditions were in the range of tocols, 1500-1800 ppm; sterols, 15,350-19,120 ppm and oryzanol 5800-11,110 ppm.     

Influence of Seizures on Lipids of Homogenate and Neuronal and Glial Nuclei of Rat Neocortex

Lipid composition of homogenate and neuronal and glial nuclei of the brain cortex of Wistar rats was studied under normal conditions and after seizures induced by injection of picrotoxin. Seizures increased contents of lysophosphatidylcholine, sphingomyelin, and total phospholipids in the homogenate. In neuronal nuclei contents of total phospholipids, sphingomyelin, phosphatidylcholine, and phosphatidylserine decreased, and contents of free fatty acids and lysophosphatidylcholine increased. In glial nuclei content of total phospholipids decreased and content of free fatty acids increased. The role of changes in the lipid composition of the neocortex cells during seizures and the involvement of lipid messengers in signal mechanisms are discussed.     

超临界CO_2萃取当归挥发油及其在烟草中的应用研究

为开发新的天然烟用香料,研究了超临界CO2萃取当归挥发油的工艺,用GC/MS分析了挥发油中的挥发性和半挥发性成分,并用该挥发油进行了卷烟加香试验。结果表明:①超临界CO2萃取当归挥发油的优化工艺条件为:萃取温度40℃、萃取压力45Mpa、萃取时间1.5 h、CO2流量20 L.h-1,分离釜压力8 Mpa,分离釜温度45℃。②当归挥发油共鉴定出29种成分,主要有藁本内酯、丁烯基芙内酯、亚油酸、棕榈酸、油酸以及萜烯类化合物等。③卷烟加香评价结果表明,当归挥发油在丰富烟香、掩盖杂气、改善口感方面有较好的作用。     

Rapid techniques for the extraction of vitamin E isomers from Amaranthus caudatus seeds: ultrasonic and supercritical fluid extraction

Supercritical fluid extraction (SFE) of seeds of Amaranthus caudatus (Amaranthaceae) and the use of ultrasound as a co-adjuvant in the extraction process were compared with methods traditionally used in the extraction of tocopherols and fatty acids. The use of readily available ultrasound equipment as an adjunct to the classical methods employed for the extraction of tocols provided qualitatively acceptable results more rapidly and more economically. SFE gave quantitatively better yields in shorter times, with solvent-free extracts obtained under conditions that minimised the degradation of thermolabile components. No significant variations were observed in the profile of the fatty acids extracted from amaranth oil by SFE or other methods, thus confirming the qualitative comparability of the faster supercritical extraction with the more time-consuming classical techniques even when processed with the aid of ultrasound.     

Supercritical fluid extraction of lipids from the heterotrophic microalga Crypthecodinium cohnii

Microalgae biomass can be a feasible source of ω‐3 fatty acids due to its stable and reliable composition. In the present study, the Crypthecodinium cohnii growth and docosahexaenoic acid (DHA, 22:6ω3) production in a 100 L glucose‐fed batch fermentation was evaluated. The lipid compounds were extracted by supercritical carbon dioxide (SC‐CO₂) from C. cohnii CCMP 316 biomas, was and their fatty acid composition was analysed. Supercritical fluid extraction runs were performed at temperatures of 313 and 323 K and pressures of 20.0, 25.0 and 30.0 MPa. The optimum extraction conditions were found to be 30.0 MPa and 323 K. Under those conditions, almost 50% of the total oil contained in the raw material was extracted after 3 h and the DHA composition attained 72% w/w of total fatty acids. The high DHA percentage of total fatty acids obtained by SC‐CO₂ suggested that this extraction method may be suitable for the production of C. cohnii value added products directed towards pharmaceutical purposes. Furthermore, the fatty acid composition of the remaining lipid fraction from the residual biomass with lower content in polyunsaturated fatty acids could be adequate for further uses as feedstock for biodiesel, contributing to the economy of the overall process suggesting an integrated biorefinery approach.     

Extraction of lipids from Mortierella alpina and enrichment of arachidonic acid from the fungal lipids

Mortierella alpina is known as an arachidonic acid (AA) producing oleaginous fungus. Extraction of lipids from wet and dry M. alpina biomass was compared. Lipids yield of extraction from dry cells was higher than that of extraction from wet. Wet extraction mainly extracted lipid bodies and lipids in membranes did not extract effectively. Enrichment of AA from the fungal lipids by a urea inclusion method was studied. Most of the saturated and monounsaturated fatty acids, 93.0% and 84.6%, respectively, were removed by forming urea inclusion compounds. AA was concentrated after urea inclusion. Its content in total fatty acids increased 6.2-folds and reached 57.1% with a recovery of 81.9%.     

Alterations of the composition and size of the free fatty acid pool of Tetrahymena responding to low-temperature stress

Cells of Tetrahymena mimbres (formerly T. pyriformis NT-1) in midlogarithmic growth under isothermal conditions (at 39 degrees C) contained a very small, compositionally discrete pool of free fatty acids, principally (60.6% of the total free fatty acid mass) palmitic and stearic acids. The composition, degree of unsaturation, and size of this free fatty acid pool were rapidly (15 min or less) altered in response to chilling. During the acclimation period following chilling to 15 degrees C, the size of the free fatty acid pool increased from a mean value of 15.5 nmol free fatty acid/mumol lipid phosphorus in 39 degrees C cells to 24.2 nmol free fatty acid/mumol lipid phosphorus. The degree of free fatty acid saturation rapidly increased over the initial hour following the onset of hypothermal conditions, but by 24 h the unsaturated free fatty acid/saturated free fatty acid ratio was 0.35 (equivalent to a 2.7-fold increase in unsaturation relative to 39 degrees C controls (unsaturated/saturated ratio = 0.13) and 4.4-fold greater than cells acclimated for 1 h (unsaturated/saturated ratio = 0.08)). By 24 h the percentage of palmitic and stearic acids had decreased to 45.6%. Similar, and in some instances more pronounced, changes were observed to occur in triacylglycerol-bound fatty acids. Modulation of steady-state free fatty acid composition could also be achieved by the addition of exogenous fatty acids to the growth medium. The ability to manipulate the level of intracellular free fatty acids should prove to be a valuable experimental tool in determining how specific fatty acids regulate various lipid-modifying enzymes.     

Optimizing a continuous flow lipid extraction system (CFLES) used for extracting microalgal lipids

A laboratory‐made continuous flow lipid extraction system (CFLES) was devised to extract lipids from microalgae Nannochloropsis sp., a potential feedstock for biodiesel fuel, with a focus to assess the workable temperatures and pressures for future industrial applications. Using conventional solvents, the CFLES recovered 100% of the lipids extracted with conventional Soxhlet extraction. The optimum temperature and pressure were found to be 100 °C and 50 psi, respectively; conditions significantly lower than those normally used in pressurized liquid extractions requiring specialized equipment. Approximately 87% of the extracted oil was successfully transesterified into biodiesel fuel (fatty acid methyl esters). Preliminary calculations based on the tested lab‐scale system indicated savings in energy, solvent consumption, and extraction time as 96%, 80%, and more than 90%, respectively, as compared to Soxhlet extraction. However, the true cost savings can only be assessed at scaled up level. Energy efficiency of CFLES was calculated as 48.9%. Residual water (~70%) in the biomass had no effect on the extraction performance of CFLES, which is expected to help the process economics at scaled up application. The effect of temperature and pressure on the fatty acids profile of Nannochloropsis sp. is also discussed. Based on the existing literature, the authors believe that a pressurized liquid extraction system with continuous solvent flow has not been reported for lipid extraction from Nannochloropsis sp.     

Effect of the exogenous lipids of complex media on the fatty acid composition of mycelial microorganisms

The effect of exogenous lipid sources on the composition of fatty acids was studied in actinomycetes of the Streptomyces genus and in fungi belonging to the genera Blakeslea, Cunninghamella and Penicillium. The following sources of exogenous lipids were used: soybean and maize flour, sunflower by-products, chicken droppings, maize extract, yeast extract, peptone, sperm whale fat, sunflower and palm oil. The composition of fatty acids in total extracted lipids of the studied mycelial microorganisms was shown to reflect two processes: lipid synthesis de novo and assimilation of exogenous fatty acids. This fact ought to be taken into account both in the chemotaxonomic interpretation of fatty acid composition and in practical recommendations for the utilization of microbial lipids. It is of particular interest to study the physiological role of exogenous lipid metabolism in the cells of microorganisms.     

Composition of sugarcane waxes in rum factory wastes

Wastes produced during fermentation and distillation of crude sugarcane juice in rum factories were evaluated as a new source of waxes. The chemical composition of the crude wax extracted from adsorbat of the wastes on fuller's earth was studied by GC-mass spectrometry. Series of linear alkanes (C19-C33), and wax esters constitute the main components. In addition, phytosterols, triterpene methyl ethers, ethyl and methyl esters of fatty acids, and free fatty acids were found as minor components. Acid (predominance of C16 and C18) and alcohol portions (C26-C32) of the wax esters were analysed after saponification.     

Lipidomics analysis of essential fatty acids in macrophages

The Lipid Metabolites and Pathway Strategy (LIPID MAPS) Consortium is a nationwide initiative that has taken on the task of employing lipidomics to advance our understanding of lipid metabolism at the molecular and mechanistic level in living organisms. An important step toward this goal is to craft enabling analytical procedures to comprehensively measure all lipid species, to establish the precise structural identity of the lipid molecules analyzed, and to generate accurate quantitative information. The LIPID MAPS Consortium has succeeded in the implementation of a complete infrastructure that now provides tools for analysis of the global lipidome in cultured and primary cells. Here we illustrate the advancement of a gas chromatography mass spectrometry (GC/MS) procedure for the analysis of essential fatty acids in RAW 264.7 cells. Our method allows for the specific identification and quantification of over 30 fatty acids present in cells in their free form in a single analytical GC/MS run. Free fatty acids are selectively extracted in the presence of deuterated internal standards, which permit subsequent estimation of extraction efficiencies and quantification with high accuracy. Mass spectrometer conditions were optimized for single-ion monitoring, which provides an extremely sensitive technology to measure fatty acids from biological samples in trace amounts. These methods will be presented in the context of our broader effort to analyze all fatty acids as well as their metabolites in inflammatory cells.     

Catalytic hydrothermal processing of microalgae: decomposition and upgrading of lipids

Hydrothermal processing of high lipid feedstock such as microalgae is an alternative method of oil extraction which has obvious benefits for high moisture containing biomass. A range of microalgae and lipids extracted from terrestrial oil seed have been processed at 350 °C, at pressures of 150-200 bar in water. Hydrothermal liquefaction is shown to convert the triglycerides to fatty acids and alkanes in the presence of certain heterogeneous catalysts. This investigation has compared the composition of lipids and free fatty acids from solvent extraction to those from hydrothermal processing. The initial decomposition products include free fatty acids and glycerol, and the potential for de-oxygenation using heterogeneous catalysts has been investigated. The results indicate that the bio-crude yields from the liquefaction of microalgae were increased slightly with the use of heterogeneous catalysts but the higher heating value (HHV) and the level of de-oxygenation increased, by up to 10%.     

不同提取溶剂和方法组合从落叶松毛虫蛹中提取的脂肪酸成分和含量的比较

落叶松毛虫Dendrolimus superans (Butler)蛹个体较大, 具有很高利用价值。为明确东北落叶松毛虫蛹中脂肪酸成分, 探讨最佳提取溶剂和提取方法的组合, 分别以正己烷、 石油醚和乙醚为提取溶剂, 结合超声波振荡萃取法、 索氏萃取法及溶剂萃取方法热浸和冷浸4种提取方法提取落叶松毛虫蛹油, 并采用毛细管色谱-质谱法分析提取物的脂肪酸种类和相对含量。结果表明： 正己烷溶剂与4种提取方法的组合中, 溶剂萃取热浸法提取率最高, 为25.60%。索氏萃取及溶剂萃取方法热浸和冷浸均检测到10种脂肪酸, 正己烷-超声波振荡萃取组合检测到9种脂肪酸。石油醚溶剂与4种提取方法的组合中, 索氏萃取提取率最高, 为29.31%, 均检测到10种脂肪酸。乙醚溶剂与4种提取方法的组合中, 溶剂萃取冷浸法提取率最高, 为29.11%, 检测到的脂肪酸种类为溶剂萃取冷浸法（13种）＞索氏萃取法（12种）＞溶剂萃取热浸法（11种）＞超声波振荡萃取法（9种）。在检测到的总脂肪酸中, 63%以上为不饱和脂肪酸, 其含量受提取溶剂和方法的影响不大。因此, 适合东北落叶松毛虫蛹中脂肪酸提取的最佳组合为石油醚溶剂 索氏萃取法。     

Efficacy of extraction methods for lipid and fatty acid composition from fungal cultures

The efficacy of three extraction methods for determining the lipid and fatty acid composition of six fungal cultures was studied. The extraction methods were: chloroform/methanol (2:1), hexane/isopropanol (3:2) and Soxhlet extraction by using hexane. The total lipid and fatty acid composition varied in fungal cultures depending on the extraction conditions. Of the three methods, chloroform/methanol (2:1) was found to be the best for extraction of lipid and fatty acids from fungal cultures.     

The effects of delipidation on the major antigenic determinant of purified human intestinal mucin

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidylethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to less than 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.     

碳源对粉核油球藻生长和脂肪酸组成特性的影响

研究了不同碳源类型（CO2、NaHCO3和葡萄糖）及其浓度对粉核油球藻（Pinguiococcus pyrenoidosus CCMP 2078）生长及脂肪酸组成的影响。结果表明：（1）培养液中适量添加碳源促进了粉核油球藻的生长,三种碳源的适宜添加浓度分别是0.5% CO2,5mmol/L NaHCO3和20g/L葡萄糖,对数生长末期的细胞密度分别是对照的3.10倍、1.47倍和2.78倍;（2）除了低浓度葡萄糖外,其他碳源类型和浓度均降低了TPUFA和EPA占总脂肪酸的比例,提高了TSFA的比例,胞内EPA和TSFA含量均下降;（3）低浓度碳源提高了TSFA和EPA产量。通入0.5% CO2培养的EPA和TSFA产量分别是对照的2.30倍和2.69倍,5mmol/L NaHCO3培养的TSFA产量是对照的1.85倍,5g/L和10g/L葡萄糖培养的EPA和TSFA产量最高分别可达对照的2.11倍和1.58倍。因此,通入低浓度CO2最有利于粉核油球藻的生长以及EPA和饱和脂肪酸的生产,EPA和饱和脂肪酸含量的提高主要是通过生物量的增大来实现的。     

响应面法优化微波辅助提取山苍子核仁油的研究

通过微波辅助提取技术结合响应面法优化山苍子核仁油提取条件,以期建立更高产率的提取方法。该研究在单因素设计基础上,选取液料比、微波功率、萃取时间、萃取温度4个主要因素,分析这4个因素对山苍子核仁油提取率的影响。结果表明:通过建立多元回归拟合分析,得出山苍子核仁油提取最佳工艺条件为液料比1∶16,萃取温度为69℃,微波功率为337 W,萃取时间为63 min,在此条件下山苍子核仁油提取率为37.42%,与环己烷溶剂回流法相比较提取率提高了30.11%。气质联用仪分析结果显示,山苍子核仁油主要成分有16种占总成分的88.21%,鉴定出10种脂肪酸占总成分的78.24%,饱和脂肪酸有4种占总成分的43.23%,不饱和脂肪酸有6种占总成分的35.01%,脂肪酸中含量最高的为月桂酸(31.36%)。该研究结果表明该方法严谨、可靠,采用微波辅助提取山苍子核仁油是可行的。     

LIBERATION OF 5,8,11,14,17-EICOSAPENTAENOIC ACID AND OTHER POLYUNSATURATED FATTY ACIDS FROM LIPIDS AS A GRAZER DEFENSE REACTION IN EPILITHIC DIATOM BIOFILMS

Acute grazer toxicity of freshwater diatom biofilms was determined using Thamnocephalus platyurus Packard, an anostracan grazer, as the bioassay organism. The diatoms exhibited toxicity when the cells of the biofilm were freeze–thawed before the assay. The toxicity could be extracted from the biofilms with methanol and acetone, and only minimal toxicity was left in the insoluble residue. Bioassay-guided HPLC separation of the methanolic extract was performed to trace the most toxic components. Analysis by UV spectrometry, gas chromatography, and mass spectrometry showed that 5,8,11,14,17-eicosapentaenoic acid was responsible for most grazer toxicity. The 24-h LC₅₀ of this polyunsaturated fatty acid was 34 μM in the Thamnocephalus platyurus bioassay. The concentrations of other free fatty acids were not high enough to contribute significantly to the toxicity. Procedures that affected the integrity of the cells (e.g. solvent extraction, freezing and thawing, osmotic stress by addition of 20% NaCl, or grinding the cells in a mortar) were taken as model reactions for grazing and had the common effect of resulting in a dramatic increase of free polyunsaturated and saturated fatty acids. Under these conditions, about 30% of the total fatty acids of the diatoms was transformed from the bound into the free form. The time necessary for liberation was very short. With the exception of 5,8,11,14,17-eicosapentaenoic acid, which continued to be liberated, the hydrolysis of the other fatty acids was terminated less than 1 min after initiating the reaction. The classical extraction procedures using methanol and other solvents led to the appearance of a high percentage of free fatty acids in live cells. Treatment of biofilms with these solvents did not stop the hydrolysis of lipids initiated by the disintegration of the cells. However, boiling acetone completely suppressed the hydrolytic reactions, and free polyunsaturated fatty acids were not detected in live biofilm organisms, although nontoxic saturated fatty acids were present in moderate concentrations. These results were interpreted as an indication that the frequently reported existence of free polyunsaturated fatty acids in live biomass is an analytical artifact.     

Mathematical modeling and study of mass transfer parameters in supercritical fluid extraction of fatty acids from Trout powder

Mathematical modeling of fatty acids (FAs) extraction from Trout powder by supercritical carbon dioxide was performed in the present work. Trout powder with its low cost contains high amount of essential FAs and it is commonly available as a proper source of FAs. The effect of process parameters, such as pressure (25, 28, 31, 34 and 37 MPa) and temperature (310, 318 and 326 K) of extraction and void fraction of the bed (0.25, 0.35 and 0.45, v/v) on the yield of FAs extraction was examined in a series of experiments conducted in a laboratory scale apparatus. The results indicated a significant increase of extraction yield with an increase of pressure from 25 to 34 MPa, but working at the higher pressure (37 MPa) caused reduction of the extract. Increasing the temperature higher than 318 K revealed significant reduction of the FAs yield and increasing the bed void fraction from 0.25 to 0.45 showed enhancement of the extraction.

The mathematical model was developed considering diffusion-controlled regime in the particle and film mass transfer resistance around the particle with axial dispersion of the bulk phase at dynamic conditions. Henry law was used to describe the equilibrium state of solid and fluid phases. The proposed mass balance equations were numerically solved using implicit finite difference method and the model parameters were correlated using the experimental results of the outlet FAs concentration in the oil extracted at dynamic conditions. Well-known Nelder–Mead method was applied to estimate the four parameters of the model, namely, mass transfer coefficient (k_f), axial dispersion coefficient (D_ax) in the bulk phase, effective diffusivity (D_eff) into the pores and Henry coefficient (H). In the range of studied conditions, the higher extraction efficiency with higher pressure resulted lower correlated H, although the temperature increasing which showed a retrograde phenomena in the FAs yield, revealed H passing though a minimum.