Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Efficiencies of Solvent Systems for Extraction of Legume Lipids

Ten solvent systems representing a wide range in polarity were highly variable in their abilities to extract the total lipids from field pea and lentil flours and starches. Propanol/water (3 : 1v/v) extracted the most lipid from lentil flour and both legume starches. Chloroform/methanol (2:1 v/v) and benzene/ethanol (4:1 v/v) gave consistently high recoveries of total lipids from both flours but appeared to be less effective with starches. Fractionation of the purified flour lipids demonstrated that propanol/water extracted phospholipids and glycolipids quite efficiently but chloroform/methanol and benzene/ethanol gave high recoveries of both nonpolar and polar lipids. These neutral, phospho- and glycolipids from the two legume flurs were highly polyunsaturated, containing 35–50% linoleic and 5–21% linolenic acid, and would be susceptible to oxidative rancidity. Purified lipids from legume starches were recovered as neutral lipids primarily and yields were very low.     

Characterization of porcine omental lipids

Porcine omental lipid extracts were fractionated and the major lipid components characterized. Approximately 97% of the chloroform/methanol extract consisted of triglycerides containing primarily 16∶0, 18∶0, 18∶1, and 18∶2 fatty acids. Small quantities of free fatty acids, cholesterol, di- and monoglycerides were also detected. The phospholipid fraction, obtained by solvent partition and Unisil column chromatography and characterized by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry, was found to consist primarily of phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol. The neutral glycolipids, isolated by solvent partition and Unisil column chromatography and identified by HPTLC and HPLC, were found to consist primarily of di-, tri-and tetraosylceramides. The complex glycolipid fraction, obtained from Folch upper phase solvent partition and characterized by HPTLC and immunoblotting, was found to consist primarily of ganglio-, globo-, and neolacto- neutral glycolipids and ganglio-, globo-, neolacto- and fucosylated gangliosides.     

Composition of Glycolipids and Phospholipids of Desilked Silkworm Pupae Oil (Bombyx mori L.)

The desilked silkworm pupae (Bombyx mori L.) collected from Kollegala, Malavalli and Ramanagaram belt of Karnataka State (South India) were extracted with chloroform : methanol = 2:1 (v/v). The oil was fractionated on activated silica gel column into neutral, glyco- and phospholipids. The fractions of glyco- and phospholipid;; were saponified seperately. The saponifiable fractions were analysed for fatty acids and un-saponifiable fractions for sterols. The glycolipid fraction contains mainly oleic, palmitic, stearic, palmitoleic, linolenic, linoleic, arachidic, myristic and lauric acids. Phospholipids had similar fatty acids, however their proportions were varying. Traces of cholesterol were observed in the unsaponifiables of glycolipids, which was absent in phospholipids.     

Lipid composition of millet (Pennisetum americanum) seeds

The composition of lipids extracted from a sample of millet seeds by each of 8 solvent systems is reported. Lipid components were separated by silicic acid column and thin layer chromatography (TLC) and quantitated by analysis of fatty acid methyl esters by gas liquid chromatography (GLC), with heptadecanoic acid as internal standard. Best results were obtained by extraction with hot water-saturated butanol. Lipids extracted amounted to 7.2% of the seed dry weight and consisted of 85% neutral lipids, 12% phospholipids and 3% glycolipids. Neutral lipids contained mostly (85%) triacylglycerols and small amounts of mono- and diacylglycerols, sterols and free fatty acids. Sterols consisted of campesterol, stigmasterol and 2 unidentified sterols, occurring in the same proportions in free and esterified forms. Ten glycolipid and 10 phospholipid components were separated and characterized. Contrary to previously published observations, lysophosphatidylcholine was the major phospholipid (42%) in millet seeds; smaller amounts of phosphatidylcholine (24%), lysophosphatidylethanolamine (21%) and trace amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine and phosphatidylinositol also were present. The major glycolipids were esterified sterol glycoside, sterol glycoside, monogalactosyldiacylglycerol, digalactosyldiacylglycerol and cerebrosides (ceramide monohexosides).     

Lipid composition of rat superior cervical ganglion

Excised rat superior cervical ganglion were incubated in Krebs-Ringer solution, freeze-dried, and lipids extracted with chloroform: methanol (2∶1) v/v. Chromatography of lipid extracts in three separate thin layer chromatography solvents was accomplished on a single thin layer chromatography plate coated with acid-washed Silica Gel H. Individual lipids were eluted from the silica gel using a modified Swinny filter holder technique and transesterified with methanolic-BF₃. Fatty acid methyl esters were separated and quantitated by gas liquid chromatography, while phospholipids were determined with a Malachite green dye organic phosphorus assay. Quantitation of neutral lipids was accomplished with a micro Liebermann-Burchard technique and sphingolipids estimated colorimetrically as free sphingosine. The composition of lipids from freezedried rat ganglia resembles the lipid composition of rat brain. Phosphatidylcholine and phosphatidylethanolamine represent 76% of the glycerolphosphatides. The sphingolipids were comprised primarily of sphingomyelin with moderate levels of cerebroside and sulfatide. Cholesterol was the predominant neutral lipid. The major phospholipid fatty acids included palmitic, stearic, and oleic acids. Sumbitted in part as a partial requirement for the Ph.D. degree, State University of New York at Buffalo, Buffalo, N.Y.     

Phospholipid Composition of Karanja (Pongamia glabra) Seeds

The Karanja (Pongamia glabra) seed kernels were extracted with a solvent mixture of chloroform and methanol (2:1, v/v). The extract was dissolved in chloroform and precipitated with acetone. Acetone insolubles (0.78% wt. of the kernels) contained 2.9 percent of phosphorus. Major constituent phospholipids were identified as phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol by spraying with characteristic spray reagents on thin-layer chromatograms (TLC) and by comparing the Rf values with those of standards and literature values. A quantitative TLC method using a colorimetric determination of phosphorus without acid digestion was used for studying the phospholipid composition of the acetone insolubles. The composition was found to be phosphatidylcholine, 43.1%; phosphatidyl-ethanolamine, 18.8%, phosphatidylinositol, 33.3% and unidentified, 4.8%. The fatty acid composition of the individual phospholipids is also reported.     

Lipid extraction from natural plant source of Adenanthera pavonina using mixed solvent by superheated extractor

The lipid content was extracted from the saga seed by superheated condition and soxhlet apparatus. The mixture of hexane, chloroform and methanol was utilized as a mixed solvent for these extraction operations. Different parameters such as different solvent, temperature, mean particle size and solvent flow have been examined. The optimized lipid extraction was achieved as 26.2 wt% by using superheated condition from the saga seed powder at 90 °C for 120min. Then the fatty acids profile of the optimized Adenanthera pavonina oil were analyzed by gas chromatography. Unsaturated fatty acid was high as 83.7% compared with saturated fatty acid barely 15.4% by relative.     

Gewinnung von pflanzlichen Phospholipiden in präparativen Mengen mittels Flashchromatographie

Gram-Scale Preparation of Plant Phospholipids by Flash Chromatography Dietary phospholipids are usually described as healthy compounds but their prophylactic and therapeutic effects are not completely clear. Therefore the aim of this study was to prepare phospholipids with different physicochemical and physiological behaviour caused by variation of polar region and fatty acid pattern as basis for biochemical experiments. The flash chromatography had been investigated and used for rapid separation of natural phospholipid mixtures. First lecithin from rape seed was extracted with acetone, ethanol and chloroform/ethanol. Then the different unsoluble residues and extracts were separated into the individual phospholipids (PC, PE, PI). The fractionation of 10 g preextracted sample was carried out on a short silicagel column by an elution gradient of chloroform/methanol. The composition of the phospholipids was determined by high performance thin layer chromatography (individual phospholipids) and capillary gas chromatography (fatty acid composition).     

Lipids in sea water

Acidified and filtered sea water samples which were extracted with petroleum ether and ethyl acetate have been shown to contain a variety of lipid compounds in trace amounts. Concentrations of these solvent-soluble substances ranged from 0.5 to 6.0 mg/liter, the lower concentrations being found in offshore waters. The solvent extracts of the sea water were separated into eight lipid classes by column chromatography on silicic acid. The fractions eluted with solvents of increasing polarity were characterized by thin-layer chromatography, infrared and ultraviolet absorption and gas chromatography. These techniques revealed a complex mixture of alkanes, alkenes, fatty acids, steroids, phospholipids and many as yet unidentified components. Twenty to thirty alkanes were present as indicated by gas chromatography. No aromatic hydrocarbons were detected. Chromatography of the methyl esters of the fatty acids indicated the presence of acids with chain lengths varying from 14 to 22 carbons, both saturated and unsaturated. In many samples the unsaturated fatty acids containing 18 to 22 carbons predominated. The lipid components varied somewhat in composition as well as concentration from location to location and with season and depth.     

Varietal difference in lipid content and fatty acid composition of highbush blueberries

Lipids from five cultivars of highbush blueberries (Vaccinium corymbosum L.) were extracted and fractionated into neutral lipids (60–66%), glycolipids (20–22%) and phospholipids (14–18%). The major fatty acids in all fractions were palmitic (16∶0), oleic (18∶1), linoleic (18∶2), and linolenic (18∶3) acids. All lipid classes had a large concentration of C₁₈ polyunsaturated acids (84–92%), indicating that blueberries are a rich source of linoleic and linolenic acids. Changes in the fatty acid composition of neutral lipids and phospholipids were not significantly different among the five cultivars, but significant differences were noted in the ratios of linoleic and linolenic acids in the glycolipids fraction.     

Lipid composition of coconut cake oil

Lipid classes and fatty acids were analyzed in expeller and rotary extracted coconut oils and corresponding solvent extracted cake oils. Triacylglycerols (84.0 to 93.1%); 1,2 diacylglycerols (1.5 to 5.1%); 1,3 diacylglycerols (1.2 to 2.1%); monoacylglycerols (1.0 to 7.0%); free fatty acids (1.0 to 2.6%); phospholipids (0.03 to 0.4%) and glycolipids (0.2 to 0.35%) were present in these oils. Fatty acid composition of triacylglycerols, phospholipids and glycolipids resembled each other and differed from those of 1,2 and 1,3 diacylglycerols which in turn were similar.     

Säulenchromatographische Abtrennung von Phospholipiden aus Gesamtlipidextrakten

Column Chromatographic Separation of Phospholipids from Total Lipid Extracts The separation of phospholipids out of total lipid extracts is described by using a column chromatographic method. Up to 800 mg total lipids in a mixture of chloroform and methanol (19 : 1, v/v) may be applied to dry-column-chromatography using 5 g silica gel as a matrix. Neutral lipids and free fatty acids are elutet by propanol-2. Following extraction of phospholipids is achieved by methanol, containing 0.5 ml/100 ml ammonia solution (25%). In the course of the preparation no lyso-phospholipids are formed and the composition of the phospholipidfraction is not altered. The recovery of applied phospholipids amounts 98-99.5%.     

Lipids of seven cereal grains

Grain samples of representative varieties of barley, corn, oats, rye, sorghum, triticale, and wheat grown commercially in the north central US were analyzed. Chemical constituents of the varieties studied are presented to provide an overview of their characteristics. Lipids of the milled grain samples were solvent extracted, classified by silicic acid column chromatography, and separated by thin layer chromatography. Fatty acid composition of the total lipid was determined by gas liquid chromatography and the fatty acid content was determined by saponification and extraction. Total lipid content of the grains ranged from 2.3% for ‘Polk’ wheat to 6.6% for ‘Chief’ oats. Lipid composition varied considerably. The row crops, corn and sorghum, have a high neutral lipid and low glycolipid content. The small grain varieties have a more balanced distribution among neutral lipids, glycolipids, and phospholipids. Fatty acid composition of the total lipid was similar for all grains. Minor qualitative differences were noted among the lipid classes of the 7 cereals.     

Lipid changes in maturing oil-bearing plants

Seeds ofCrambe abyssinica C.D. 6619 and theBrassica napus varieties Golden and Zero-erucic were collected at different stages of maturity and the free lipid extracted with hexane. The lipid thus obtained was separated into lipid classes by silicic acid column chromatography. The lipid classes were further examined by thin-layer chromatography and the component fatty acids and sterols by gas-liquid chromatography. The relative amounts of the lipid classes in crambe and both rape varieties varied as the seed matured and a period of great change occurred about 10 days after fertilization. The greatest change was in triglycerides and phospholipids plus glycolipids. Free fatty acids, present in immature seeds, has almost disappeared at maturity. The lipid classes of crambe and both types of rape were in similar proportion at maturity. Differences in phospholipid and glycolipid composition were found between crambe and rape and between immature and mature rape. The fatty acid composition differred between lipid classes and changed with maturity. Changes in 18-carbon acids of Zero-erucic rape were concurrent with the development of erucic and eicosenoic acids in Golden rape. Contribution No. 36 of the Food Research Institute. Presented at the AOCS Meeting, Cincinnati, October 1965.     

The effect of temperature on the fatty acid and phospholipid composition ofCephalosporium falciforme andCephalosporium kiliense

The effect of temperature on the lipid composition ofCephalosporium falciforme andCephalosporium kiliense, causative agents of maduromycosis, was investigated. The fungi were grown at 28.5 C and 37 C in a chemically defined medium. The lipids were solvent extracted, purified on Sephadex, and separated into their component classes by silicic acid column chromatography. Five lipid classes were found: (a) sterol esters, (b) triacylglycerides, (c) free fatty acids, (d) sterols, and (e) phospholipids. Fatty acids were analyzed by gas liquid chromatography and phospholipids by thin layer chromatography. Temperature induced changes of varying degrees occurred in both the fatty acid phospholipid fractions of each organism.     

Studies on the lipid composition of developing soybeans

Studies are reported on changes in fatty acid and lipid class composition in developing soybeans picked at intervals from ca. nine days after flowering to maturity. In the early stages of development of the bean, the lipid was virtually devoid of triglyceride and the major constituents consisted of glycolipids and phospholipids. As the bean developed, there was a rapid synthesis of triglyceride that paralleled the deposition of lipid. Simultaneously, unknown substances which occurred in relatively large amounts in the neutral, as well as the glycolipid and phospholipid, fractions of the immature bean diminished to less than 2% of the total lipid in the mature bean. The glycolipids and phospholipids also increased as the bean developed but at a much slower rate than the triglycerides and became minor components in the mature bean. The major component of the phospholipids in the immature bean was phosphatidic acid. It decreased as the phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol, as well as triglyceride, increased. The major component of the glycolipid fraction in the early stages of the development of the bean had the same migration pattern on two-dimensional thin layer chromatography as phosphatidic acid and gave a positive test for phosphorus; it also gave a positive test for glycolipids and was separated completely from phosphatidic acid and other phospholipids by column chromatography. It also decreased as the bean developed. Changes also occurred in the fatty acid composition of the developing bean. The percentage of saturated fatty acids decreased rapidly in the early stages of the development of the bean; oleic and linoleic increased rapidly as the bean developed. Linolenic acid increased rapidly to a maximum concentration in the early stages of the development of the bean and then gradually decreased as the bean matured.     

The lipid components of white potato tubers (Solanum tuberosum)

Four Canadian varieties of potatoes were examined for their lipid composition. Lipids, extracted with chloroformmethanol, were shown by TLC and column chromatography to consist of 16.5% neutral lipids, 45.5% phospholipids and 38.1% glycolipids. Among the phospholipids and glycolipids, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, the galactolipids and the sterol glucosides were the major lipids. The predominant fatty acids were palmitic (19.5%), linoleic (44.8%) and linolenic (30.4%, in Kennebec). Analyses of the fatty acids of stored potatoes showed a marked decrease in linoleic acid and an increase in linolenic acid, in the Irish Cobbler and Sebago potatoes. β-sitosterol comprised 85.0% of total sterols. Nearly half of the carotenoids was lutein (xanthophyll), the others being α-carotene, β-carotene, an unidentified pigment and lutein epoxide. Contribution No. 101 of the Food Research Institute, Canada Department of Agriculture.     

Quantitative analysis of lipid classes by liquid chromatography via a flame ionization detector

A method is described for the direct quantitative analysis of the lipid classes of mammalian tissue lipids using high performance liquid chromatography (HPLC) with a flame ionization detector (FID). The lipid is extracted from the tissue with chloroform/methanol after deactivation of hydrolytic enzymes and removal of nonlipid substances by extraction with hot dilute acetic acid (0.05N). Separation of the lipid classes is performed with a column (45 cm × 0.2 cm id) of 8 μm silica (Spherisorb, Phase Sep, Hauppague, NY) treated with concentrated ammonium hydroxide at a solvent flow rate of 0.5 ml/min, which requires a pressure of ca. 900 psi. Cholesteryl esters (CE) and triglycerides (TG) are eluted first with Skellysolve B/methylene chloride (1∶1, v/v); cholesterol (CH) is eluted with chloroform/methylene chloride (1∶2, v/v) and the phospholipids with methanol containing 6% ammonium hydroxide added to the latter solvent in a linear gradient. The neutral lipids are eluted in ca. 12 min and the phospholipids in an additional 30 min. The relative amount of each lipid class was determined from standard curves of the peak areas obtained according to response factors using erucyl alcohol as an internal standard. The method was applied to samples of kidney, liver and serum of rats. Duplicate analyses were generally within ca. 1.0% and good agreement was obtained in the analysis of the lipid classes of Azolectin and liver mitochondria lipid compared to thin layer chromatography (TLC) via photodensitometry of charred spots or phosphorus analysis of recovered phospholipids.     

Preparation of Fatty Acid Methyl Esters by Selective Methanolysis of Polar Glycerolipids

KOH in aqueous methanol catalyzes selective methanolysis of polar glycerolipids with O-ester-linked acyl residues, while triacylglycerols and sterol esters are inert in the solution. Based on these findings, a convenient and reliable method was developed for the preparation of fatty acid methyl esters (FAMEs) from polar glycerolipids in lipid mixtures without prior isolation. Methanolysis of polar glycerolipids was completed within 2.5 min by vortexing or 20 min by shaking with 0.7 M KOH/70% (v/v) methanol in the presence of hexane at 30 °C. The yields of FAMEs obtained by the present method were greater than 95%. The method was applied successfully to gas chromatographic analysis of the fatty acid compositions of polar glycerolipids in seed oil and blood. No obvious differences were found between the fatty acid compositions determined by the present method and those determined by conventional methods, including lipid extraction with chloroform/methanol followed by isolation of polar lipids by chromatography. The fatty acid composition of polar glycerolipids, including phospholipids, can be determined readily in many crude samples.