Identification and Quantitative Analysis of Phospholipids in Cocoa Beans

SUMMARY: The lipids extracted from several common cocoa bean varieties were separated into neutral lipid, glycolipid, and phospholipid fractions. The composition of the total lipid extract was 98% neutral lipid and 1 to 2% oolar lioid of which aporoximatelv 70% was glycolipid and 30% was phospholipids. Two-dimensional thin-layer chromatography was used to separate all of the known major phospholipids. The relative distribution of the phospholipids was determined by quantitative phosphorus analyses of individual spots scraped from two-dimensional thin-layer plates. The major components were lysophosphatidyl choline, phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl inositol. Phosphatidyl choline was found to contribute 36 to 40% of the phospholipids of cocoa beans. The phospholipid composition of Accra, Arriba, and Bahia beans was shown to be quite similar although minor variations were observed.     

Lipid content and fatty acid composition of green algae Scenedesmus obliquus grown in a constant cell density apparatus

The lipids of alga Scenedesmus obliquus grown under controlled conditions were separated and fractionated by column and thin-layer chromatography, and fatty acid composition of each lipid component was studied by gas-liquid chromatography (GLC). Total lipids were 11.17%, and neutral lipid, glycolipid and phospholipid fractions were 7.24%, 2.45% and 1.48% on a dry weight basis, respectively. The major neutral lipids were diglycerides, triglycerides, free sterols, hydrocarbons and sterol esters. The glycolipids were: monogalactosyl diglyceride, digalactosyl diglyceride, esterified sterol glycoside, and sterol glycoside. The phospholipids included: phosphatidyl choline, phosphatidyl glycerol and phosphatidyl ethanolamine. Fourteen fatty acids were identified in the four lipid fractions by GLC. The main fatty acids were C18:2, C16:0, C18:3(alpha), C18:1, C16:3, C16:1, and C16:4. Total unsaturated fatty acid and essential fatty acid compositions of the total algal lipids were 80% and 38%, respectively.     

Seasonal changes in phospholipids of mussel (Mytilus edulis Linne)

The phospholipids of mussels (Mytilus edulis Linne) from the coast of Qingdao were extracted, fractionated and analysed over a 12 month period. The contents of total lipids, neutral lipids, polar lipids and phospholipids were measured. The composition of phospholipids was determined by high‐performance liquid chromatography, and their fatty acid composition was analysed by gas chromatography. The phospholipid content ranged from 3.6 to 6.4 g kg^?1 soft tissue. PE (phosphatidyl ethanolamine) and PC (phosphatidyl choline) were the major constituents. C16:0, C20:5 and C22:6 were the major fatty acids. C20:5 (5.25–23.10%) and C22:6 (6.05–20.42%) varied regularly with the seasonal factors. Their total amounts were high from January to June, which would be an optimal time for the utilisation of the phospholipids of mussels. © 2002 Society of Chemical Industry     

The lipids of young cassava (Manihot esculenta,Crantz) leaves

Lipids were extracted quantitatively from young cassava (Manihot esculenta Crantz) leaves with a chloroform-methanol mixture. Total lipids were purified by the Folch procedure and separated into non-polar lipid, glycolipid and phospholipid fractions by column chromatography. Lipids of each fraction were further subjected to thin layer chromatography and gas-liquid chromatography. Young cassava leaves were found to have low content of lipids (3.02%) of which 22.4, 25.1 and 48.2 were non-polar lipids, glycolipids and phospholipids, respectively. Pigments (11.5%), wax and hydrocarbons (1.2%), steryl esters (2.9%), methyl esters of fatty acids (2.0%), trigly-cerides (1.5%), fatty acids (2.1%), diglycerides (1.1%) and sterols (0.1%) constituted the leaf non-polar lipids. The leaf glycolipids were made up of esterified steryl glycosides (2.1%), monogalactosyl diglycerides (12.5%), steryl glycosides (1.1%), cerebrosides (4.2%) and digalactosyl diglycerides (5.2%). The leaf phospholipids were found to include cardiolipin (3.6%), phosphatidylglycerol (21.5%), phosphatidylethanolamine (16.4%), phosphatidylserine (0.7%), phosphatidylinositol (4.0%) and other unidentified phospholipids (2.5%). Phosphatidylcholine was present only in trace quantity. Analysis of the fatty acid composition of each of the leaf lipids revealed that, with the exception of steryl esters, all leaf lipids have high content of polyunsaturated fatty acids.     

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.     

Changes in the composition of the fatty acids and aldehydes of meat lipids after heating

The effects of heating at 132°C on the fatty acids and fatty aldehydes of neutral lipids and phospholipids of lean beef, veal, lamb, pork and chicken were studied. Heating caused hydrolysis of the plasmalogens in the phospholipids, and varying amounts of the liberated fatty aldehydes were recovered in the neutral lipid fractions. Beef phosphatidyl choline lost more polyunsaturated fatty acids than that of the other meats. Beef and veal phosphatidyl ethanolamine lost more polyunsaturated fatty acid than that of lamb, pork or chicken, but the effect was obscured by the influx of fatty acids from elsewhere into this fraction after heating.     

Isolation and characterization of whey phospholipids

A freeze-dried whey powder was produced by microfiltration of Cheddar cheese whey. A 0.2-micron ceramic membrane in a stainless steel housing unit was used to concentrate components > 400 kDa present in the whey. The experimental whey powder, derived from Cheddar cheese whey, and a commercial whey powder were subjected to proximate analysis, lipid classes, phospholipid classes, and fatty acid compositional analyses. Commercial whey powder and commercial soybean lecithin were subjected to an alcohol fractionation procedure in an effort to alter the ratio of phosphatidyl choline to phosphatidyl ethanolamine and the functionality of dairy phospholipids. The fractionation procedure produced an alcohol-insoluble fraction containing 84% phosphatidyl ethanolamine, whereas the alcohol-soluble fraction resulted in a decrease in the phosphatidyl choline to phosphatidyl ethanolamine ratio. The commercial whey contained a higher ratio of phospholipids to neutral lipids compared with the experimental whey. The classes of phospholipids present within the two wheys were similar, whereas the experimental whey contained a phosphatidyl choline content twice that of the commercial whey, and the phospholipids composition of both wheys differed from the milk fat globule membrane. Comparison of the phospholipids and fatty acid composition of the wheys with the soy lecithin revealed that although the wheys were similar to each other, they differed from the soy lecithin in both the classes of phospholipids present and in the fatty acid composition. These compositional differences may influence the functionality of whey phospholipids.     

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.     

Isolation and purification of egg yolk phospholipids using liquid extraction and pilot-scale supercritical fluid techniques

Egg yolk is an excellent source of phospholipids. Egg yolk powder (EYP) contains about 60% lipids, which consist of, on average, neutral lipids (65%), phospholipids (31%) and cholesterol (4%). The utilization of supercritical fluid techniques is a new way to selectively extract and fractionate non-polar and slightly polar components from foods and food products. In this study, we developed pilot-scale production methods for the isolation of high-purity egg yolk phospholipids. The method involves either liquid ethanol or supercritical fluid extraction (SFE) as isolation method and supercritical antisolvent process as precipitation method. EYP was fractionated to lipid- and protein-rich fractions using liquid ethanol or supercritical fluid as an extraction medium. In both cases, the target fraction was phospholipids dissolved in ethanol. From this solution, phospholipids were precipitated using supercritical carbon dioxide as antisolvent. Depending on the process conditions, 72–99% of the precipitate consisted of phosphatidyl choline and phosphatidyl ethanolamine, which appeared in ratios 6:1–7:1. The highest purity of phospholipids was obtained via precipitation of the isolate obtained using two-step SFE.     

Phospholipids of marine origin. V. The crab--a comparative study of a marine species (Cyclograpsus punctatus) and a fresh water species (Potamon)

A comparison has been made between phospholipids extracted from a marine crab and from a fresh water crab. Marine crab (body and viscera) contained a phospholipid fraction which liberated 2-aminoethylphos-phonic acid upon hydrolysis, while this substance was absent from the fresh water crab. Marine crab phospholipids had a higher content of phosphatidyl choline (57%), a higher content of phosphatidyl serine (5%) but a lower content of phosphatidyl ethanolamine (22%) than phospholipids from the fresh water crab (respective values: 52, 2 and 27%). Marine crab phospholipids and non-phosphorylated lipids were richer in C20 plus C22 fatty acids but poorer in C18: 2 acid, than were the corresponding lipids from the fresh water crab.     

Characterization of Shrimp Lipids

Edible shrimp (Penaeus aztecus) tissue contains approximately 1.2% extractable lipids, the majority of which are phospholipids. Data from the gravimetric quantitation of lipid classes isolated by column chromatography indicated that phosphatidyl choline was the predominant phospholipid and cholesterol the predominant neutral lipid in edible shrimp tissue. Fatty acid distribution data indicated that sphingomyelins contained the greatest percent by weight of unsaturated fatty acids while cholesterol esters contained the greatest proportion of saturated fatty acids. Enzymatic hydrolysis followed by gas liquid chromatography of phosphatidyl choline, phosphatidyl ethanolamine, and phosphatidyl serine indicated that fatty acids located at the β position were more highly unsaturated than those at the α position.     

Changes in the composition of the rumen and abomasum lipids of sheep from birth to maturity

The composition of the lipids of the rumen and abomasum tissues of foetal (at term), 1 month, 2 month and 1-2-year old sheep grazed on pasture has been determined. The lipid content of the rumen tissues increased from 2.0% at birth to 3.4% in 1-2-year old sheep While that of the abomasum tissues increased from 2.6 to 5.7%. The main change in the neutral lipid fraction was a decrease in the hydrocarbon content from 0.13 % in the rumen tissues and 0.08 % in the abomasum tissues at birth to 0.003% and 0.006% respectively in the 1-2-year old sheep. The main components of the phospholipid fraction of the rumen and abomasum tissues were 34.9- 46.8% phosphatidyl choline; 14.623.5 % phosphatidyl ethanolamine; 14.3-21.1 % sphingomyelin; with smaller amounts of lysophosphatidyl choline (4 13-12.6 %), acidic phospholipids (cardiolipin) (4.1-8 -5 %) and phosphatidyl inositol/phosphaticlyl serine (3.0-5.5 %). No marked changes in phos- pholipid composition with age were noted. The amounts of phosphatidyl choline and phosphatidyl ethanolamine tended to be higher in the abomasum than in the rumen whereas the sphingomyelin content of the rumen tissue phospholipids was generally greater than that of the abomasurn tissue phospholipids. From the fatty acid composition of the triglycerides of the rumen and abomasum tissues it appeared that the foetal triglycerides were largely, though not entirely, of endogenous origin. In contrast the phospholipids of the foetal rumen and abomasum tissues contained di- and poly-unsaturated as well as branched-chain fatty acids in proportions similar to those found in older animals having access to pasture. From these results it is suggested that the phospholipids of the foetus are derived to a considerable extent from the maternal phospholipids.     

Phospholipids of marine origin. The lantern fish (Lampanyctodes hectoris)

Lantern fish of the species Lampanyctodes hectoris were shown to contain phospholipids (10 g kg^?1) and non-phosphorylated lipids (140 g kg^?1). The phospholipid fraction consisted of phosphatidyl choline (47% of total phospholipids), phosphatidyl ethanolamine (42%), phosphatidyl serine (3%), phosphatidyl inositol (1%), sphingomyelin (4%), lyso-phosphatidyl choline (1%) and cardiolipins (2%). Lantern fish (L hectoris) meals normally contain unacceptably high lipid contents (150 g kg^?1 and over); this characteristic was found not to be due to a high phospholipid level in the lantern fish. The major fatty acid of the phospholipids was C22:6n-3 (25% total fatty acids) followed by C16:0 (18%), C18:ln-9 (16%) and C20:5n-3 (8%). This distribution was different from that of the non-phosphorylated lipids where the major fatty acid was C16:0 (21%) followed by C18:ln-9 (19%), C20:5n-3 (11%), C20:l (7%) and C22:6n-3 (7%). The lantern fish press oil and residual meal lipids had fatty acid distributions similar to those of the non-phosphorylated lipids.     

Composition of wheat-flour lipids

Lipids were extracted from a single sample of wheat flour using three solvent systems: ethanol–diethyl ether–water (2:2:1 by vol.); chloroform–methanol (2:1 by vol.); and water-saturated n-butanol. Analysis of the extracts and of residual lipid in the extracted flour showed that water-saturated n-butanol was the most efficient solvent. Wheat-flour lipids were extracted with water-saturated n-butanol and separated by chromatographic procedures into individual components. The lipid classes which were isolated and studied were steryl ester, free sterol, 6-O-acyl steryl glucoside, steryl glucoside, triglyceride, diglyceride, monoglyceride, free fatty acid, monogalactosyl diglyceride, 6-O-acyl monogalactosyl diglyceride, digalactosyl diglyceride, digalactosyl monoglyceride, monoglycosyl ceramide, diglycosyl ceramide, N-acyl phosphatidyl ethanolamine, N-acyl lysophosphatidyl ethanolamine, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine, phosphatidyl choline, lysophosphatidyl choline, phosphatidyl serine and phosphatidyl inositol. Monogalactosyl monoglyceride was also tentatively identified. The quantitative distributions of the lipid classes were determined. Monoglycosyl ceramide contained small amounts of normal fatty acids (12:0–24:0) and large amounts of 2-hydroxy fatty acids (principally 16:0 and 20:0), with similar amounts of dihydroxy long-chain bases (18:0 and 18:1) and trihydroxy long-chain bases (18:0, 18:1, 19:0, 19:1, 20:0, 22:0). The principal sterols were identified as β-sitosterol, campesterol, and C₂₈ and C₂₉ saturated sterols. The fatty acids in the sterol lipids were principally 16:0 (50–60%) and 18:2 (28–30%) with small amounts of 16:1, 18:0, 18:1 and 18:3. The fatty acids in all the glycerides were principally 18:2 (51–84%) with lesser amounts of 16:0, 18:0, 18:1 and 18:3.     

Polar lipid fraction from oat (Avena sativa): characterization and use as an o/w emulsifier

Oat seeds have an oil content of up to 13?%, of which up to 34?% can be polar lipids (glycolipids and phospholipids). Because of their amphiphilic structure, these polar lipids are potential emulsifiers. In this study, polar lipid fraction from oat produced by a supercritical fluid extraction process was fractionated into different polar lipid classes by HPLC and the lipid classes in subfractions were identified by comparing retention times with reference compounds and performing co-injections. The oat polar lipid fraction contained monogalactosyldiacylglycerol, digalactosyldiacylglycerol, steryl glycoside, and phosphatidyl choline, and also possibly phosphatidyl inositol. The polar lipid fraction was also used as an emulsifier to produce oil-in-water emulsions with different amounts of emulsifier and oil, and the stability and other properties of emulsions were studied. Emulsions were formed quite easily, but they were prone to rapid creaming even after a couple of days of storage at ambient conditions. Droplet size and droplet size distribution of the emulsions seemed to be slightly smaller with smaller amount of oil and larger amount of emulsifier. Generally, the droplet size of the emulsions was in the range of 0.2?C4???m, and with the largest amount of oil (5?%, w/v) up to 10???m. The upper phase of creamed emulsions contained slightly larger droplets, up to 30???m, while the lower phase retained smaller droplets. Microscopic investigation revealed that the increase in droplet size of the upper phase was mainly due to aggregation, which implies that these emulsions may be stable against coalescence to some extent.     

Phospholipids of marine origin. IV. The abalone (Haliotis midae)

Phospholipids of the abalone were separated into component fractions by chromatography on silicic acid. The phospholipids were remarkable for the presence (6%) of an unusual sphingolipid liberating 2-amino-ethylphosphonic acid on hydrolysis, and for the high proportion of plasmalogens (23%). The presence of phosphatidyl ethanolamine plus ethanolamine plasmalogen (32%), phosphatidyl serine (5%), phosphatidyl inositol (5%), phosphatidyl choline (41%) and sphingomyelin (1%) were also demonstrated. The fatty acid distribhion in the phospholipids, the non-phosphorylated lipids and the unusual sphingolipid was determined by gas chromatography. In general these results show a similarity between the phospholipid and the non-phosphorylated lipid fatty acids, the former being richer in long chain polyunsaturated fatty acids. Fatty acids of the unusual sphingolipid were outstanding for the high palmitic (53%) and stearic acid (15%) content.     

Oxidation of free and esterified linoleic and linolenic acids in bread doughs by wheat and soya lipoxygenases

Lipids containing ¹⁴C-labelled linoleic and linolenic acids were added to flour which was then made into bread doughs with or without 2% enzyme-active soya flour. The ¹⁴C-labelled lipids and their oxidation products were analysed as intact lipids or as ¹⁴C fatty acid methyl esters by thin-layer chromatography. In the control doughs wheat lipoxygenase oxidised free fatty acids and monoglycerides. In doughs containing soya flour, steryl esters, triglycerides, diglycerides, mono-galactosyl diglycerides, digalactosyl diglycerides and phosphatidyl choline were also oxidised, and the oxidation of these lipids is attributed to soya lipoxygenase.     

The effect of extractable lipid on the viscosity characteristics of corn and wheat starches

The defatting of both corn and wheat starch with 85% methanol yields starches which start to gelatinise at lower temperatures and have increased overall viscosities compared with untreated starches. The lipids extracted from wheat and corn starch with 85 % methanol were similar in total amount but showed very large differences in the proportions of neutral and phospholipids. The extracted corn lipids contained eight times as much neutral lipids (mainly free fatty acids) as phospholipids whereas the proportions of neutral and phospholipids in wheat starch were approximately equal. Addition of extracted lipids to defatted starches significantly modified the swelling and viscosity characteristics. Addition of wheat and corn lipids to potato starch (which contains almost no lipids) indicated that the higher percentage of phospholipids in wheat starch probably contributed towards the lower viscosity characteristics of wheat starch compared with corn starch.     

A Research Note Lipid Composition of Cumin (Cuminum cyminum L.) Seeds

Total lipids extracted from Cumin (Cuminum cyminum L., Umbelliferae) amounted to 14.5% of the dry seeds. The total lipids consisted of 84.8% neutral lipids, 10.1% glycolipids, and 5.1% phospholipids. Neutral lipids consisted mostly of triacylglycerols (89.4%) and small amounts of diacylglycerols, free fatty acids, sterols, sterolesters, and hydrocarbons. At least five glycolipids and five phospholipids were identified. Acylmonogalactosyldiacylglycerol and acylatedsterylglucoside were the major glycolipids, while monogalactosyldiacylglycerol, monogalactosylmonoaclycerol, and digalactosyldiacylglycerol were present in small quantities. Phosphatidylethanolamine and phosphatidylcholine were the major phospholipids, while phosphatidylinositol, lysophosphatidylethanolamine, and phosphatidylglycerol were present in small amounts. The fatty acid composition of these different neutral lipids, glycolipids, and phospholipids was determined     

Identification and quantitative determination of the lipids of dried Origanum dictamnus leaves

The Origanum dictamnus plant was examined for its lipid and fatty acid compositions. A combination of chromatographic techniques has been employed for the qualitative and quantitative determination of the lipids of dried leaves of O. dictamnus. The following polar lipids were identified: mono-, di- and poly-digalactosyl diglycerides, sulpholipids, cerebrosides, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl choline and phosphatidic acid. The non-polar lipids identified were sterols, steryl esters, fatty alcohols, free fatty acid, waxes, traces of triglyceride, triterpenic acids and essential oil. The predominant fatty acids were palmitic, oleic and linoleic acids.