Extraction of Phospholipids from Structured Dry Egg Yolk

Chicken egg yolk is a concentrated source of phospholipids (PL). Extracting egg PL with high efficiency is vital to the availability and economics of this high-valued lipid product. In this study, two types of structured dry egg yolk materials, yolk flakes and pellets, were prepared. Two commonly used solvents, hexane and ethanol, were tested on the extraction of total yolk lipids including the PL. The PL fraction was obtained by the conventional cold acetone precipitation. The drum-dried yolk flakes were shown to be an ideal starting material for total lipid and PL extraction. Anhydrous ethanol can extract almost all the neutral lipids and PL with little change to the individual components of the native PL. A PL product with a purity of more than 90 % and a yield of 99 % can be prepared using this method.     

Extraction of Phospholipids from Egg Yolk Flakes Using Aqueous Alcohols

Two alcohols, ethanol and butanol, with different water contents were evaluated for phospholipids (PL) sequential extraction from drum dried egg yolk flakes. It showed that butanol was more effective in extracting total yolk lipids compared to ethanol, but the PL in the extract had the same concentration as in the original yolk total lipid. The use of aqueous ethanol of 95 and 75% resulted in lipid extracts with higher PL concentration during the initial stages of the sequential extraction. When ethanol was further diluted to a concentration of 55%, the solvent lost its PL extraction ability, and the total lipid recovery also decreased dramatically. When both the PL purity and recovery were considered, 75% ethanol was the most effective aqueous alcohol for PL extraction and enrichment from the yolk flakes. In the first stage of extraction using such a solvent, 67% of the total PL in the original yolk was recovered in a lipid fraction with a PL purity of 75%. This study identified the optimal ethanol concentration for PL extraction from dried egg yolk. With this information, the best solid:solvent ratio can be designed to extract and enrich the polar lipids from lipid-bearing materials with known moisture content using a renewable or “green” solvent, ethanol.     

The Effects of Biopolymer Encapsulation on Total Lipids and Cholesterol in Egg Yolk during in Vitro Human Digestion

The purpose of this study was to examine the effect of biopolymer encapsulation on the digestion of total lipids and cholesterol in egg yolk using an in vitro human digestion model. Egg yolks were encapsulated with 1% cellulose, pectin, or chitosan. The samples were then passed through an in vitro human digestion model that simulated the composition of mouth saliva, stomach acid, and the intestinal juice of the small intestine by using a dialysis tubing system. The change in digestion of total lipids was monitored by confocal fluorescence microscopy. The digestion rate of total lipids and cholesterol in all egg yolk samples dramatically increased after in vitro human digestion. The digestion rate of total lipids and cholesterol in egg yolks encapsulated with chitosan or pectin was reduced compared to the digestion rate of total lipids and cholesterol in other egg yolk samples. Egg yolks encapsulated with pectin or chitosan had lower free fatty acid content, and lipid oxidation values than samples without biopolymer encapsulation. Moreover, the lipase activity decreased, after in vitro digestion, in egg yolks encapsulated with biopolymers. These results improve our understanding of the effects of digestion on total lipids and cholesterol in egg yolk within the gastrointestinal tract.     

Egg-yolk lipid fractionation and lecithin characterization

Egg-yolk lecithin has phospholipid (PL) classes and a FA composition that differ from soybean lecithin and may have unique functional properties. The purposes of this research were to develop an effective method for extracting a sufficient amount to lecithin from fresh egg yolks and to evaluate its functional properties. Ethanol was used to dehydrate and partially extract the PL, after which hexane was used to extract the total lipids. A phase separation of the combined extracts resulted in neutral and polar lipid fractions. An acetone precipitation of PL from the final polar lipid fraction was necessary to remove the residual neutral lipids, especially cholesterol. The purity of PL in the lecithin product was 95%. Surface tension reduction, emulsion stability, and oxidative stability studies were conducted to characterize the functional properties of egg-yolk lecithin. Egg-yolk lecithin and soy lecithin had similar surface activities, as evaluated by the surface tension reduction in an aqueous system and the critical micelle concentration. Soybean lecithin created a more stable emulsion than egg-yolk lecithin. However, egg-yolk lecithin was more oxidatively stable than soybean lecithin.     

Embryonic fatty acid composition as a function of yolk fatty acid composition in eggs of the lesser spotted dogfish (Scyliorhinus canicula L.)

Yolk and embryonic total lipids were extracted from spotted dogfish eggs at two developmental stages. Total lipids were fractionated into neutral lipids (NL) and polar lipids (PL), and the fatty acid composition of each group was determined. Yolk lipid composition was found to be quantitatively different (NL/PL≊1) from embryo lipid composition (NL/PL≊0.5), for both stages of development. However, individual fatty acid composition did not differ from younger to older eggs for either yolk or embryo. There were significant differences (p<0.05) in major fatty acid groups from yolk and embryonic PL for saturated fatty acids, monounsaturated fatty acids (MUFA) and n−3 polyunsaturated fatty acids (PUFA) for younger eggs, and for MUFA and n−3 PUFA for older eggs. For NL, only MUFA composition from the oldest eggs showed differences between yolk and embryo. Results are discussed in terms of embryonic needs for highly unsaturated fatty acid (HUFA) biosynthesis, as well as to provide some explanations for the unusually high levels of 20∶4n−6 in both yolk and embryonic neutral lipids and polar lipids.     

Novel method to administer radiolabeled lipid to juvenile oysters

Particles prepared from egg yolk were shown to encapsulate protein and to be in a size range that would be filtered by the oyster. A radiotracer study involving the addition of radiolabeled phosphatidylcholine to egg yolk demonstrated that the egg yolk particles were taken up and metabolized by juvenile oysters (Crassostrea gigas). Catabolism of the radiolabeled lipid and subsequent resynthesis into non-lipid components occurred to a slight extent. The main factor responsible for the distribution of radioactivity amongst the lipids in the stomach tissue was believed to be transacylation.     

Quantification of Lipidomics Profiling using UPLC-QE-HESI- Lipid Analysis on the Salted Duck Egg Incorporated with Clove Extract

The salted egg yolks supplemented with clove extract are compared with the non-supplemented group, and subsequently the lipidomics profiles are characterized and identified by UPLC-QE-MS/MS in positive and negative ion modes respectively. A total of 315 lipids are detected in the control group and the clove extract treated group. In the positive ion mode, the 10 subclasses of glycerolipids in the treated samples are significantly different (p < 0.05). The lipid types with most varied compositions are triglyceride and ceramides, followed by CerG1 (glucosylceramide), LPE (lyso-phosphatidylethanolamine), diglyceride, and monosialodihexosyl ganglioside. In the negative ion mode, the authors identified 35 subclasses which are significantly different (p < 0.05), that include glycerolphospholipids, glycosphingolipids, and neutralglycosphingolipid. The lipid types with most varied compositions in negative mode are PE (phosphatidylethanolamine), followed by PC (phosphatidylcholine), LPC (lyso-phosphatidylcholine), and GM3 (monosialo-dihexosyl ganglioside). The contents of phosphatidylcholine, lyso-phosphatidylcholine, phosphatidylethanolamine, and monosialo-dihexosyl ganglioside in egg yolks treated with 0.5% (w/v) clove are significantly higher than those in the control group (p < 0.05), in which phosphatidylcholine and phosphatidylethanolamine are the main components of glycerolphospholipid. Practical Application : The lipidomics analyses of the salted egg yolk added with and without clove extract are compared to reveal the beneficial effect of clove extracts on the lipid profiles, and further exploring the types and relative content of the lipid components. A total of 315 lipids are detected in all samples. In the positive ion mode, the 10 subclasses of glycerolipids are found to be significantly different in the treated samples. In the negative ion mode, the lipid content of 35 subclasses are significantly different. From the nutrition fact value, salted duck egg enriched with clove extract can serve as one of the alternative egg products rich in essential lipids.     

Lipid profile and fatty acid composition of two silurid fish eggs

The lipid content and the composition pattern of the lipid class including fatty acid composition in the eggs of two different Indian silurid cat fishes Ompok pabda and Wallagu attu have been examined. The lipid content of O. pabda and W. attu (on dry basis) are about 14.7% and 17.8% respectively. The major lipid classes are phospolipid (PL) and triacylglycerol (TAG). The O. pabda egg lipid contains more PL while the W. attu egg lipid contains more TAG. Phosphatidylcholine (PC) constitutes the major phospholipid followed by phosphatidylinositol (PI). PI represents in about 31.7% and 21.3% of total PC in O. pabda and W. attu respectively while phosphatidylethanolamine (PE) (about 28.0%) is significantly higher in the egg of W. attu than O. pabda (9.6%). Cholesterol content in egg of O. pabda is also higher (about 9.6%) than W. attu (4.1%). The lipids are rich in polyunsaturated fatty acids (PUFAs) and they are mainly concentrated in the respective PL fractions. Among PUFAs the arachidonic acid (20:4 n-6 AA) is present at about 9.3% in both egg PL. Eicosapentaenoic acid (20:5 n-3 EPA) is significantly lower in egg lipids of both W. attu (1.8%) and O. pabda (3.2%), whereas docosahexaenoic acid (22:6 n-3 DHA) is predominantly higher (14.6% and 18.1% in W. attu and O. pabda respectively) in their PL fractions.     

Destabilization of Egg Yolk Emulsion After IgY Removal Through Enzymatic Treatments

The objective of this study was to destabilize the protein–lipid complex in egg yolk precipitate obtained after the removal of soluble proteins, referred to as the pellet, through enzymatic treatment for further phospholipids extraction. A combination of proteolytic and lipolytic enzymes was applied to release the lipids from the pellet or weaken the pellet emulsion. Emulsions prepared using Protease P/Lipase AY30, Protease II/Lipase AY30 and Protease M/Lipase AY30 treated pellets had larger oil droplets (78, 65, 56 µm) and higher coalescence rates (51, 41, 35 %) than those of Protex 51FP, pellet, Protex 7L and Protease A with oil droplet size of 20, 18, 15 and 13 µm and coalescence rates of 31, 8, 7.5 and 8 %, respectively. Cream and liquid subnatant fractions obtained after further centrifugation of hydrolysates were subjected to lipid analyses. Over 90 % of phosphatidylcholine (PC) present in the pellet and 80 % of that in the original egg yolk were recovered in the cream from Protease P/Lipase AY30 treatment, while the recovery of PC from the egg yolk was significantly lower in creams from Protex 7L or Protease 51FP treatments (12 and 10 %, respectively). Pellets treated with Protease M, Protex 7L or Protex 51FP in combination with Lipase AY30 led to a significant loss of PC due to the conversion of PC to lysophosphatidylcholine or its degradation. Cream fractions obtained from the study represented a better material for the recovery of PL than intact egg yolk using environmentally-friendly techniques such as supercritical carbon dioxide (SC-CO₂) extraction.     

Methoden zur Phosphatidanalytik am Beispiel der Lipide aus Seren und Eigelb

Techniques of Phospholipid Analyses Applied to Sera and Egg Yolk Thin layer (TLC) and gas-liquid-chromatography (GLC) were used to analyse phospholipids and diglycerides prepared from phospholipids. TLC of lipids is carried out on silica gel and for diglycerides on silica gel impregnated with 10% AgNO₃. Fatty acids of individual lipids were identified by GLC. The recovery of lipids was evaluated after chromatography of phospholipids and diglycerides and after digestion with phospholipase-C from Bacillus cereus. Two techniques of acetylation with two different catalysts are compared. The calculations were referred to phosphorus or glycerol in lipids. The precise amount of phospholipids was determined after charring as inorganic phosphorus. Diglycerides were assayed after saponification by an enzymatic determination of glycerol. The reliability of some lipid techniques could be shown. The values of phospholipids for egg yolk and sera are in correspondence to literature.     

The Fractional Extraction of Lipids and Cholesterol from Dried Egg Yolk Using Supercritical Carbon Dioxide

Results from extraction of cholesterol and other lipid components from dried egg yolk using supercritical carbon dioxide at the range of temperature from 40°C to 60°C and pressure between 150 bar und 350 bar for 2.5 hours and 2.7 kg CO₂ consumption is described in this paper. The solubility of lipids and cholesterol increased with the increase of pressure at a constant temperature of 50°C, while at a constant pressure, more cholesterol was removed at 45°C than that at other temperatures. Nearly 60 percent cholesterol was removed at 45°C and 250 bar. Lipids were more efficiently extracted at 60°C than at 40°C at 250 and 350 bar, however, a decrease in the total extracted lipids was observed with the increase in temperature at 150 bar. The removed total lipids from dried egg yolk at 250 bar/55°C was over 80 %.     

Marine phospholipids as dietary carriers of long‐chain polyunsaturated fatty acids

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are polyunsaturated fatty acids (PUFA) of the n‐3 series. Fish oil is a classical source of n‐3 PUFA, where they occur in the form of triacylglycerols (TAG). However, new sources of n‐3 PUFA esterified in phospholipids (PL) are emerging. We prepared liposomes from a natural marine lipid extract and examined their behaviour under conditions mimicking that of the gastrointestinal tract. This physicochemical approach proved that liposomes could be used as an effective oral PUFA delivery system. In vivo studies in rats were performed to examine the metabolic fate of EPA (20:5 n‐3) and DHA (22:6 n‐3) delivered either in PL from liposomes or in TAG from oil. Liposome ingestion increased PUFA bioavailability in lymph compared with fish oil. The proportion of n‐3 PUFA esterified in the sn‐2 position of chylomicron TAG depended on the dietary lipid source. Complex time‐course profiles were observed for plasma lipids with liposome supplementation over a 2‐week period, suggesting time‐dependent regulations. Taken together, the type of PUFA, EPA or DHA, as well as its intramolecular distribution in chylomicron TAG seemed to influence the metabolic fate of the fatty acids and their physiological activities.     

High Saturated Fat Diet Alters the Lipid Composition of Triacylglycerol and Polar Lipids in the Femur of Dam and Offspring Rats

Previous work has shown that dietary lipids alter femur lipid composition. Specifically, we have shown that exposure to high saturated fatty acid (SFA) diets in utero, during suckling, or post‐weaning alters femur total lipid composition, resulting in higher percent bone mass in males and females and bone mineral density (BMD) in female offspring with no effect on bone mineral outcomes in dams. Comparatively, high n‐3 polyunsaturated fatty acid (PUFA) diets increase femur polar (PL) lipid n‐3 content, which has been associated with increased bone mineral content and strength. However, the extent that PL or triacylglycerol (TAG) lipids change with high SFA diets is unknown. The current investigation examined the influence of a high SFA diet (20 % lard by weight) on femur PL and TAG lipid composition in 5‐month old female Wistar rats (fed high SFA diet from age 28 days onwards; dams) and their 19‐day old offspring (exposed to high SFA in utero and during suckling; pups). High SFA exposure resulted in increased monounsaturates and decreased n‐3 and n‐6 PUFA in the TAG fraction in both dams and pups, and higher SFA and n‐6:n‐3 ratio in dams only. The PL fraction showed decreased n‐6 PUFA in both dams and pups. The magnitude of the diet‐mediated responses, specifically TAG 18:1 and PL n‐6 PUFA, may have contributed to the previously reported altered BMD, which was supported with correlation analysis. Future research should investigate the relationship of diet‐induced changes in bone lipids on bone structure, as quantified through micro‐computed tomography.     

Omega‐3 long‐chain polyunsaturated fatty acid enriched eggs by microalgal supplementation

The health promoting effects of omega‐3 polyunsaturated fatty acids (n‐3 PUFA) are mainly ascribed to the n‐3 long chain (LC)‐PUFA, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). However, their intake is mostly below the recommended daily intake. A possible way to raise their average intake is to enrich food products with n‐3 LC‐PUFA. Addition of autotrophic microalgae to the diet of the laying hens can increase the level of these fatty acids in the egg yolk. Moreover, depending on the microalgal species, other nutritionally interesting algal carotenoids can also be transferred to the egg yolk. As a consequence egg yolk colour changes may occur. A survey conducted among 511 people showed that they will buy n‐3 PUFA enriched products, such as enriched eggs, and are even prepared to pay more for these products. However, the change of the yolk colour must be taken into account, since consumer' acceptability decreases when a deeply red yolk colour is obtained.     

Validation of Two Methods for Fatty Acids Analysis in Eggs

A comparative study between two methods (lipid extraction followed by saponification and methylation, and direct methylation) to determine the fatty acids in egg yolk was evaluated. Direct methylation of the samples resulted in lower fatty acid content and greater variation in the results than the lipid extraction followed by saponification and methylation. The low repeatability observed for the direct HCl methylation method was probably due to a less efficient extraction and conversion of the fatty acids into their methyl esters as compared to the same procedure starting with the lipid extract. As the lipid extraction followed by esterification method was shown to be more precise it was validated using powdered egg certified as reference material (RM 8415, NIST) and applied to samples of egg, egg enriched with polyunsaturated omega-3 fatty acids (n-3 PUFA), and commercial spray-dried whole egg powder.     

Separation and analysis of phospholipids in different foods with a light-scattering detector

A method for the separation of phospholipids (PL) from total lipids by solid-phase extraction (SPE) with reversephase C₈ cartridges is described. The method was validated with a standard mixture of PL and applied to natural food matrixes, such as egg, chicken meat, salami, and ripened cheese. The recovery of PL ranged between 93 and 99.7% and was evaluated by an organic phosphorus spectrophotometric determination. The egg powder PL fraction obtained by SPE contained about 20% (w/w) nonpolar PL material when 100–150 mg of lipids were loaded onto the cartridge. Higher percentages of nonphospholipid components (30–43%) were obtained when the amount of lipids loaded was below or above the 100–150 mg range. The purified PL fractions were analyzed by high-performance liquid chromatography (HPLC) with an evaporative light-scattering detector. Good HPLC performance was observed even with low-purity SPE fractions (43% nonphospholipid material).     

Simultaneous extraction and preparative fractionation of egg yolk lipids using the principle of adsorption

A new approach describing the simultaneous extraction and preparative fractionation of egg yolk lipids is described, a method which can be extended to other tissues. In this method, egg yolk is adsorbed on activated thin layer chromatography grade silicic acid and sequentially extracted with different solvents to get a crude fractionation of its lipids into nonpolar and polar components. These crude concentrates help achieve larger yields of high purity lipids per unit column load during subsequent chromatographic subfractionation.     

Quantification of oxysterols in dutch foods: Egg products and mixed diets

A sensitive and specific method is described for quantifying various cholesterol oxidation products in foodstuffs, including 7β-hydroxycholesterol, cholesterol-α-epoxide, cholestane-triol, 7-ketocholesterol and 25-hydroxycholesterol. A chloroform-methanol extract of the food was fractionated over two successive silica columns. Two fractions containing different classes of oxysterols were then analyzed as trimethylsilyl derivatives by capillary gas liquid chromatography, using on-column injection and a temperature gradient from 70 to 200°C. The detection limit was about 0.5 μg/g dry weight for egg yolk powder. Fresh egg yolk contained only 1.2 μg/g of total oxides per g dry weight, showing that artifactual oxidation during the procedure was minimal. Recovery of 5 pure oxysterols added to egg yolk at levels of 6.5 and 10 μg/g was between 93 and 102%. In commercial egg yolk and whole egg powder stored for one year, total amounts of oxysterols ranging from 21 to 137 μg/g dry weight were found. In duplicates of mixed Dutch diets, total amounts ranged from 3.6 to 6.2 μg/g dry weight. Duplicates containing mostly fried and baked foods did not have higher levels than duplicates in which foods had been prepared by boiling or left raw. We conclude that a normal mixed diet provides only minor amounts of cholesterol oxidation products.     

Lipid Profiles in By‐Products and Muscles of Three Shrimp Species (Penaeus monodon,Penaeus vannamei,and Penaeus chinensis)

To better understand the health benefits of lipids in shrimp and evaluate their potential value, a comprehensive analysis of lipid profiles in by‐products (head and body carapace) and muscles of shrimps Penaeus monodon, Penaeus vannamei, and Penaeus chinensis is performed. Results show that freeze‐dried muscles of these shrimps contain 3.83%, 4.39%, and 2.93% of lipids, respectively, while the corresponding by‐products contain 4.69%, 5.89%, and 5.39% of lipids, respectively. The total lipids comprise glycerophospholipid (PL, 54.86–77.29%), cholesterol (12.67–18.79%), triacylglycerol (1.28–7.02%), diacylglycerol (0.27–1.58%), monoacylglycerol (0.32–3.04%), and free fatty acid (FFA, 2.84–20.58%). Further, PL contains cholineglycerophospholipid (PLCho, 49.75–66.99 mol%), ethanolamineglycerophospholipid (PLEtn, 14.02–28.16 mol%), serineglycerophospholipid (PLSer, 4.85–12.66 mol%), inositolglycerophospholipid (PLIns, 4.36–15.63 mol%), and cholinelysoglycerophospholipid (LPLCho, 2.07–6.35 mol%). Lipids are abundant in polyunsaturated FA (36.55–42.72% of total FA), among which eicosapentaenoic acid (6.38–11.59% of total FA), and docosahexaenoic acid (7.63–12.08% of total FA) are dominant. About 200 species of PL belonging to PLCho, PLEtn, PLIns, LPLCho, lysoglycerophosphoethanolamine (LPLEtn), and lysoglycerophosphoserine (LPLSer) are characterized. Moreover, the by‐products contain higher amounts of astaxanthin than the muscles. Considering high level of PUFA enriched PL, shrimp by‐products can serve as a source for nutritional lipids. Practical Applications: This study presented a comprehensive analysis of the lipid profiles in by‐products (head and body carapace) and muscles of shrimps Penaeus monodon, Penaeus vannamei, and Penaeus chinensis. The results obtained justified the use of by‐products in shrimp processing, indicating that by‐products can be used for commercial exploitation and production of value‐added products due to PUFA‐enriched PL, especially EPA and DHA.     

Fatty Acid and Lipid Profiles with Emphasis on n-3 Fatty Acids and Phospholipids from Ciona intestinalis

In order to establish Ciona intestinalis as a new bioresource for n‐3 fatty acids‐rich marine lipids, the animal was fractionated into tunic and inner body tissues prior to lipid extraction. The lipids obtained were further classified into neutral lipids (NL), glycolipids (GL) and phospholipids (PL) followed by qualitative and quantitative analysis using GC‐FID, GC–MS, ¹H NMR, 2D NMR, MALDI‐TOF‐MS and LC–ESI–MS methods. It was found that the tunic and inner body tissues contained 3.42–4.08 % and 15.9–23.4 % of lipids respectively. PL was the dominant lipid class (42–60 %) irrespective of the anatomic fractions. From all lipid fractions and classes, the major fatty acids were 16:0, 18:1n‐9, C20:1n‐9, C20:5n‐3 (EPA) and C22:6n‐3 (DHA). The highest amounts of long chain n‐3 fatty acids, mainly EPA and DHA, were located in PL from both body fractions. Cholestanol and cholesterol were the dominant sterols together with noticeable amounts of stellasterol, 22 (Z)‐dehydrocholesterol and lathosterol. Several other identified and two yet unidentified sterols were observed for the first time from C. intestinalis. Different molecular species of phosphatidylcholine (34 species), sphingomyelin (2 species), phosphatidylethanolamine (2 species), phosphatidylserine (10 species), phosphatidylglycerol (9 species), ceramide (38 species) and lysophospholipid (5 species) were identified, representing the most systematic PL profiling knowledge so far for the animal. It could be concluded that C. intestinalis lipids should be a good alternative for fish oil with high contents of n‐3 fatty acids. The lipids would be more bioavailable due to the presence of the fatty acids being mainly in the form of PL.