Supercritical fluid extraction and characterization of lipids from algae scenedesmus obliquus

Supercritical carbon dioxide (SC‐CO₂) extractions (with and without ethanol as an entrainer) were carried out to remove lipids and pigments from protein concentrate of green algae (Scenedesmus obliquus) cultivated under controlled conditions. The content and fatty acid composition of algal lipids using column, thin‐layer (TLC) and gas‐liquid chromatography (GLC) were determined. Absorption spectra of extracted fractions showed the predominance of chlorophyll A (λ_max at 410nm). Single step supercritical carbon dioxide (SC‐CO₂) extraction resulted mostly in removal of neutral lipids and a part of glycolipids, but phospholipids were not extracted. Addition of ethanol to SC‐CO₂ increased the amount of glycolipids and phospholipids in the extract. TLC pattern of algal lipids showed that the main part of neutral lipids consisted of diglycerides, triglycerides, hydrocarbons, free sterols, and sterol esters. The glycolipids were mostly monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. In phospholipids, phosphatidyl choline, phosphatidyl glycerol, and phosphatidyl ethanolamine were the main compounds. Fatty acid composition patterns indicated the main fatty acids to be 16:0, 16:1, 16:2, 16:3, 16:4, 18:1, 18:2 and 18:3(a). Relatively high recovery of polyunsaturated fatty acids and essential fatty acids in supercritical fluid extracted algal lipids and protein isolates were observed.     

Production of phospholipids from spray-dried egg yolk by consecutive in situ solid phase extraction with acetone and ethanol

Egg yolk lipids were extracted and fractionated into neutral lipids and phospholipids using acetone followed by ethanol as eluting solvents to a column consisting of spray-dried egg yolk. This technique, named in situ solid phase extraction, can be performed with spray-dried egg yolk due to the properties of this material. The effect of temperature, flowrate and scale has been studied. Results suggest that the methodology is suitable for industrial implementation.     

Supercritical fluid extraction and characterization of lipids from algae Scenedesmus obliquus

Supercritical carbon dioxide (SC-CO2) extractions (with and without ethanol as an entrainer) were carried out to remove lipids and pigments from protein concentrate of green algae (Scenedesmus obliquus) cultivated under controlled conditions. The content and fatty acid composition of algal lipids using column, thin-layer (TLC) and gas-liquid chromatography (GLC) were determined. Absorption spectra of extracted fractions showed the predominance of chlorophyll A (lambda max at 410 nm). Single step supercritical carbon dioxide (SC-CO2) extraction resulted mostly in removal of neutral lipids and a part of glycolipids, but phospholipids were not extracted. Addition of ethanol to SC-CO2 increased the amount of glycolipids and phospholipids in the extract. TLC pattern of algal lipids showed that the main part of neutral lipids consisted of diglycerides, triglycerides, hydrocarbons, free sterols, and sterol esters. The glycolipids were mostly monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. In phospholipids, phosphatidyl choline, phosphatidyl glycerol, and phosphatidyl ethanolamine were the main compounds. Fatty acid composition patterns indicated the main fatty acids to be 16:0, 16:1, 16:2, 16:3, 16:4, 18:1, 18:2, and 18:3(a). Relatively high recovery of polyunsaturated fatty acids and essential fatty acids in supercritical fluid extracted algal lipids and proteins isolates were observed.     

Selective extraction of phospholipids from whey protein phospholipid concentrate using supercritical carbon dioxide and ethanol as a co-solvent

In recent years, using dairy phospholipids (PL) as functional ingredients has increased because PL have nutritional benefits and functional properties. In this study, a novel 2-step supercritical fluid extraction (SFE) process was used to extract whey protein phospholipid concentrate (WPPC), a dairy co-product obtained during the manufacture of whey protein isolate, for PL enrichment. In the first step, nonpolar lipids in WPPC were removed using neat supercritical carbon dioxide (S-CO₂) at 41.4 MPa and 60°C. In the second stage, the feasibility of using the polar solvent ethanol as a co-solvent to increase the solubility of S-CO₂ extraction solvent was explored. A 3 × 3 × 2 factorial design with extraction pressure (35.0, 41.4, and 55.0 MPa), temperature (40 and 60°C), and concentration of ethanol (10, 15, and 20%) as independent factors was used to evaluate the extraction efficiency providing the most total PL, and the best proportion of each individual PL from the spent solids collected during S-CO₂ SFE. All lipid fractions were analyzed using thin-layer chromatography and high-performance lipid chromatography. The total amount of PL extracted from WPPC was significantly affected by ethanol concentration; the extraction pressure and temperature were nonsignificant. The optimal SFE condition for generating a concentrated PL lipid fraction was 35.0 MPa, 40°C, and 15% ethanol concentration; the highest amount of extracted PL averaged 26.26 g/100 g of fat. Moreover, adjusting SFE condition allowed successful recovery of a high concentration of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, giving averages of 11.07, 10.07, and 7.2 g/100 g of fat, respectively, 2 to 3 times more than conventional solvent extraction. In addition, exhausted solids obtained after the SFE process were enriched with denatured proteins (72% on dry basis) with significantly more water-holding capacity and emulsifying capacity than untreated WPPC. Overall, this 2-stage SFE process using neat S-CO₂ and ethanol has the greatest potential to produce a PL-rich lipid fraction from WPPC.     

Short communication: Evaluation of supercritical fluid extraction aids for optimum extraction of nonpolar lipids from buttermilk powder

The milk fat globule membrane, present in buttermilk, contains complex lipids such as phospholipids. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method of enriching these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid enrichment needs to be optimized for lipid removal effectiveness. The role of matrix additions to the buttermilk powder for extraction efficiency was evaluated. Diatomaceous earth (biosilicates), Teflon beads, and physical vibration were tested and shown to reduce total lipid by 86, 78, and 70%, respectively. Four consecutive treatments were shown to exhaust the system; however, similar extraction efficiencies were noted for 1 treatment with biosilicate addition, 2 treatments with physical vibration, or 3 treatments with added Teflon beads. The extracted lipid material consisted of the nonpolar fraction, and protein concentration was observed to increase slightly compared with the control. Although higher lipid extraction was achieved from the powder with addition of diatomaceous earth, a removable aid is ideal for an edible product.     

Concentration of polar MFGM lipids from buttermilk by microfiltration and supercritical fluid extraction

Buttermilk contains the milk fat globule membrane (MFGM), a material that possesses many complex lipids that function as nutritionally valuable molecules. Milk-derived sphingolipids and phospholipids affect numerous cell functions, including regulating growth and development, molecular transport systems, stress responses, cross membrane trafficking, and absorption processes. We developed a two-step method to produce buttermilk derivative ingredients containing increased concentrations of the polar MFGM lipids by microfiltration and supercritical fluid extraction (SFE). These processes offer environmentally benign alternatives to conventional lipid fractionation methods that rely on toxic solvents. Firstly, using a ceramic tubular membrane with 0.8-micron pore size, we evaluated the cross flow microfiltration system that maximally concentrated the polar MFGM lipids using a 2n factorial design; the experimental factors were buttermilk source (fresh, or reconstituted from powder) and temperature (50 degrees C, and 4 degrees C). Secondly, a SFE process using supercritical carbon dioxide removed exclusively nonpolar lipid material from the microfiltered buttermilk product. Lipid analysis showed that after SFE, the product contained a significantly reduced concentration of nonpolar lipids, and a significantly increased concentration of polar lipids derived from the MFGM. Particle size analysis revealed an impact of SFE on the product structure. The efficiency of the SFE system using the microfiltration-processed powder was compared much more favorably to using buttermilk powder.     

Egg yolk protein modification by controlled enzymatic hydrolysis for improved functionalities

Delipidated egg yolk protein (EYP) is produced as a co-product of egg yolk lecithin extraction. This EYP is expected to have poor functionality because of protein denaturation caused by ethanol treatment during lecithin extraction. Two food grade endo-proteases were used to produce EYP hydrolysates (EYPh) with two degrees of hydrolysis (DH), 3% and 6%. Protein solubility was improved as DH increased. Solubility profiles for both EYP and EYPh were relatively less pH-dependent compared with soy protein. Except for foaming capacity, EYPh showed improvement in foam stability, foaming speed, and foam density. Emulsion stability was improved for all EYPhs. Treatments at DH of 6% showed significant increase in emulsification capacity. We have shown quantitatively how controlled enzymatic hydrolysis can be applied to ethanol-treated lipid-free EYP to increase protein solubility, and thus to improve foaming and emulsification properties.     

Supercritical fluid and solid–liquid extraction of phenolic antioxidants from grape pomace: a comparative study

Distilled white grape pomace (Vitis vinifera var. Garnacha) was subjected to extraction by using two different methods: (1) solid–liquid extraction (SLE) employing 96% ethanol and water and (2) supercritical extraction (SFE) by running carbon dioxide coupled with ethanol as a modifier. Higher phenolic concentrations of extracts were attained by SFE (∼400 ppm), doubling those obtained by SLE. In the latter, increasing values of both temperature (from 25 to 50 °C) and contact time (from 30 to 90 min) and lower solvent-to-solid ratios (from 5:1 to 1:1) resulted in an enhancement of extraction efficiency. In SFE, the addition of the modifier (8%) was also found to favour the release of phenolic compounds. Antiradical activity values—evaluated by the ability to scavenge DPPH radicals—were, as general, also higher for SFE extracts, although maximum values reached at were similar (73% inhibition versus 68%). Chromatographic profiles confirmed the diverse nature of phenolic species occurring in extracts obtained from both extraction methods. Extracts from SLE contained more proanthocyanidins, whereas SFE ones contained basically gallic acid, catechin and epicatechin. Protection against oil oxidation assayed with two samples confirmed these results.     

Phospholipid enrichment in sweet and whey cream buttermilk powders using supercritical fluid extraction

Milk fat globule membrane contains many complex lipids implicated in an assortment of biological processes. Microfiltration coupled with supercritical fluid extraction (SFE) has been shown to provide a method of concentrating these nutritionally valuable lipids into a novel ingredient. In the dairy industry there are several by-products that are rich in phospholipids (PL) such as buttermilk, whey, and whey cream. However, PL are present at low concentrations. To enrich PL in buttermilk powders, regular buttermilk and whey buttermilk (by-product of whey cream after making butter) were microfiltered and then treated with SFE after drying. The total fat, namely nonpolar lipids, in the powders was reduced by 38 to 55%, and phospholipids were concentrated by a factor of 5-fold. Characterization of the PL demonstrated specific molecular fatty amide combinations on the sphingosine (18:1) backbone of sphingomyelin with the greatest proportion being saturated; the most common were 16:0, 20:0, 21:0, 22:0, 23:0, and 24:0. Two unsaturated fatty amide chains, 23:1 and 24:1, were shown to be elevated in a whey cream buttermilk sample compared with the others. However, most unsaturated species were not as abundant.     

Supercritical CO2 extraction of Mortierella alpina single cell oil: Comparison with organic solvent extraction

Analysis of supercritical fluid extracted (SFE) and organic solvent extracted lipids from freeze dried Mortierella alpina biomass was carried out. The effect of biomass pre-treatment on effectual lipid recovery was also investigated. Results indicated that the methods employed viz., SFE and organic solvent (Sox Tec™) exhibited significant variation in lipid extraction efficacy. Efficacy of SFE on lipid recovery (38.73%) was significantly lower when compared to that of Sox Tec™ method (51.47%) because the organic solvent can also extract polar lipids. Biomass pretreatment by enzymatic method resulted in a substantial increase in lipid recovery. Maximum disruption of cell wall evidenced by scanning electron microscopy of treated cells warranted their higher mass transfer and efficient extraction. Characterisation of the extracted lipids by GC and GC–MS revealed considerable variation in the fatty acid profile. The FT-IR spectrum indicated a prominent cis-stretching vibration, no evident trans- stretching vibration and no major discrepancies between the SFE and Sox Tec™ extracts.     

Supercritical fluid extraction as a method for fat content determination and preparative technique for fatty acid analysis in mesh feed for pigs

Objective of this investigation was supercritical fluid extraction (SFE) with CO₂, as analytical technique for total fat extraction from food and feed samples. Most significant advantages of this technique, compared with the others, are safety, cleanness, and shorter analysis time. Its main limitation includes difficulty of extracting polar lipids, due to the non-polar character of the CO_2, which is used as a solvent. Influence of added absorbent and co-solvent on the SFE of mash pig feed was investigated in this paper. Total fat content was determined for ten commercially available mesh pig feeds. Optimization of absorbent weight and volume of added co-solvent showed that the maximum yield of extracted fat has been achieved at adsorbent mass of 1.8 g and ethanol volume of 0.54 ml. Yields of fat extracted with both Soxhlet method (AOCS Method Ba 3-38) and SFE were plotted one against the other. There were no significant (p > 0.05) differences between results of these two methods, which were confirmed by low value for standard deviation (SD). SFE appears to be reliable, faster, and less hazardous alternative for traditional extraction method. Fatty acid compositions from lipid extracts obtained by both methods were determined by gas chromatography—flame ionization detection (GC–FID). Results obtained by SFE were far more representative than results obtained by analyzing Soxhlet extracts, and in consistence with literature results. SFE method gave significantly higher (p < 0.001) contents of polyunsaturated fatty acids (PUFA) than Sohxlet method, especially of linoleic acid, C18:2 n-6.     

Phenolic and antioxidant potential of olive oil mill wastes

Olive oil mill waste was subjected to conventional liquid solvent extraction and supercritical fluid extraction using different solvents and carbon dioxide, respectively. The optimum solvent extraction conditions of phenols were 180 min using ethanol, at a solvent to sample ratio 5:1 v/w, and at pH 2. Solvent and SFE extracts were tested for their antioxidant activity by the DPPH radical scavenging method and by determination of peroxide value on virgin olive oil and sunflower oil. The ethanol extract exhibited the highest antiradical activity, and no correlation was found between antiradical activity and phenol content. The SFE extract exerted good antioxidant capacity although its phenolic yield was not quite high. Moreover, the ethanol extract appeared to be a stronger antioxidant than BHT, ascorbyl palmitate and vitamin E by the Rancimat method on sunflower oil. HPLC analysis of the extracts showed that the predominant phenolic compound was hydroxytyrosol. Various phenolic acids and flavonoids were also identified.     

Optimization of Solvent Extraction and Direct Transmethylation Methods for the Analysis of Egg Yolk Lipids

The composition of egg yolk lipids differs greatly from most other food lipids, as it combines high contents of triglycerides and phospholipids, as well as a high proportion of cholesterol. The lipids are organized in lipoproteins and are dispersed in aqueous phase. Classical extraction methods using chloroform and methanol have not been optimized for such a matrix, therefore quantitative extraction yields of egg lipids can be inaccurate. In this study, the original method of Bligh and Dyer is evaluated and adapted for egg lipids. Furthermore, a direct extraction/derivatization method is presented. Solvent extraction resulted in a lipid yield of 31.8 ± 0.89% and total fatty acid yield was 257 ± 2 mg/g yolk whereas the direct method yielded 250 ± 7 mg/g yolk. Gas chromatographic analyses confirmed that lipid recovery was complete for triglycerides and phospholipids as well.     

虾头中磷脂提取与组学分析

研究虾头中磷脂的提取制备方法,并分别利用气相色谱法和多维度串联质谱法对样品磷脂的脂肪酸链结构进行脂质组学分析。利用乙醇浸提法制备得到虾头中的磷脂,并从乙醇体积分数、提取温度和提取时间3?个因素对提取方法进行优化,得到较优条件为乙醇体积分数90%、提取温度50?℃、提取时间30?min,磷脂的提取效果最佳,实际提取量可达到（11.58±0.03）mg/g,与优化前相比提高了83.8%。磷脂样品经甲酯化反应后用气相色谱法分析其脂肪酸链组成,并用高效液相色谱-质谱联用技术经负离子全扫描对磷脂分子实现分离鉴定和定量分析。结果显示,磷脂的脂肪酸链中主要含有棕榈酸链、亚油酸链、二十碳五烯酸和硬脂酸链等23?种脂肪酸链,其中单不饱和脂肪酸链占9.51%,多不饱和脂肪酸链占35.33%;磷脂样品中共检出磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰肌醇和磷脂酰丝氨酸4?类共28?种磷脂分子,该提取方法所得各种类磷脂的脂肪酸链中存在许多不饱和度较高的脂肪酸链,如40∶8、36∶7、38∶7、O-40∶7、40∶7、34∶6、O-36∶6、36∶6、O-38∶6、38∶6、40∶6等。虾头中磷脂含量丰富,其脂肪酸链不饱和度较高,因此具有较高的开发利用价值。     

The influence of temperature and pressure factors in supercritical fluid extraction for optimizing nonpolar lipid extraction from buttermilk powder

The milk fat globule membrane, readily available in buttermilk, contains complex lipids claimed to be beneficial to humans. Phospholipids, including sphingolipids, exhibit antioxidative, anticarcinogenic, and antiatherogenic properties and have essential roles in numerous cell functions. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method for removing triacylglycerols while concentrating these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid concentration needs to be optimized for triacylglycerol removal in buttermilk. The SFE conditions were assessed using a general full factorial design; the experimental factors were pressure (15, 25, and 35 MPa) and temperature (40, 50, and 60°C). Particularly interesting is that only triacylglycerols were removed from buttermilk powder. Little to no protein loss or aggregation was observed compared with the untreated buttermilk powder. Calculated theoretical values showed a linear increase for lipid solubility as pressure, temperature, or both were increased; however, experimental values showed nonlinearity, as an effect of temperature. In addition, the particular SFE parameters of 35 MPa and 50°C displayed enhanced extraction efficiency (70% total lipid reduction).     

Modification of Crude Canola Lecithin for Food Use

Crude canola lecithin was fractionated by removal of neutral lipids with acetone followed by ethanol extraction with or without supercritical carbon dioxide (SC-CO₂). Two extraction steus of 60 min each at 35°C with 500 mL acetone were optimum for maximum yield of acetone solubles and maximum phospholipid (PL) concentration (66.7%) in the acetone insoluble (AI) fraction. AI were separated into 26.5% ethanol soluble and 68.7% ethanol insoluble fractions. PL concentration in the extract increased with increasing amounts of ethanol used. Yields were low for SC-CO₂/ethanol extraction, of which extracts from 55.2 MPa/ 70°C contained 32% PL (83% phosphatidyl choline).     

Selective removal effect of subcritical fluid extraction on egg yolk lipids and characterization and enzymatic improvement of defatted egg yolk powder

This study investigated the effects of subcritical fluid extraction on lipid removal and the physicochemical properties of egg yolk powder. In addition, two-step enzymatic hydrolysis was used to improve the physicochemical properties. The content of total lipids and cholesterol decreased to 30.2% and 10.5 mg/g after subcritical propane-butane extraction, respectively, while phospholipids were retained. Protein solubility, surface hydrophobicity and total sulfhydryl results showed that this partial defatting avoided further quality deterioration of egg yolk powder. Scanning electron microscopy and confocal microscopy showed that the surface morphology and distribution of proteins and lipids changed with defatting degree. After two-step enzymatic hydrolysis, protein solubility of subcritical propane-butane extraction egg yolk powder significantly increased to 31.4% and soluble peptides with an average molecular weight lower than 1000 Da accounted for 70.0%. The improved defatted egg yolk powder is rich in protein, peptides and phospholipids and has great potential as a nutritional supplement.     

Supercritical CO2 extraction of resveratrol and its glycoside piceid from Chinese traditional medicinal herb Polygonum cuspidatum

Resveratrol (3,5,4′‐trihydroxystilbene) and its glycoside piceid are phenolic compounds found to be beneficial to health. In this paper, resveratrol and piceid were successfully extracted from the Chinese traditional medicinal herb Polygonum cuspidatum (Hu Zhang in Chinese), with supercritical CO₂ plus ethanol modifier. The effects of ethanol content, pressure and temperature on the yields of resveratrol and piceid were investigated. Results showed that the extraction pressure was the main factor for the effective extraction of the two substances in the presence of ethanol modifier, especially for piceid. The optimal extraction condition was obtained: 30 MPa, 50 °C, (100 ml l^?1), ethanol and 20 kg h^?1 of CO₂, at which the yields of resveratrol and piceid were 7.1 g kg^?1 and 16.0 g kg^?1, respectively. The yields obtained by supercritical fluid extraction (SFE) was comparable with those obtained by traditional organic solvent methods, indicating that SFE is an alternative method for extraction of the two compounds. Copyright © 2004 Society of Chemical Industry     

不同储存年限广陈皮的多甲氧基黄酮提取研究

采用水、95%乙醇、水蒸汽蒸馏和超临界CO2流体分别提取广陈皮中的多甲氧基黄酮（PMFs）,用紫外分光光度法（UV）测定所得提取物中PMFs的含量,并考察了广陈皮的储存年份对提取的影响。结果表明,在四种提取物中,超临界CO2流体萃取产物（SFE提取物）的PMFs含量（以提取物计算）最高,其次为醇提物,水蒸汽蒸馏产物（SD提取物）和水提物中PMFs的含量较低。值得注意的是,由于95%乙醇对广陈皮的提取率较高,醇提物中PMFs的提取率（以药材计算）最高。因此,95%乙醇和超临界CO2流体均能较好地将广陈皮中PMFs提取出来。但是,随着广陈皮的储存年份延长,其95%乙醇提取物和SFE提取物中PMFs的含量均呈下降趋势。     

超临界萃取技术在酿酒生产中的应用进展

超临界萃取技术作为一种环境友好、高效新型的分离技术,具有操作条件温和,无溶剂残留,分离效率高等特点。对该技术在酿酒生产过程中的白酒香味物质萃取、酒精脱水精制和玉米胚芽油提取等过程的应用研究进行了综述,并对该技术在酒精工业中的应用前景进行了展望。