Gram-scale preparative HPLC of phospholipids from soybean lecithins

A preparative procedure has been developed to isolate gram quantities of phospholipid classes from soybean lecithins. Various steps taken to accomplish the isolation are described—an analytical method with silica column and light scattering detector, alcohol fractionation of deoiled lecithins, and columns with increasing internal diameters but packed with the same stationary phase. The loading study showed that it was possible to inject 20 mg on a 100 × 8 mm RadialμPorasil colume. The separation was scaled up to a 25-mm i.d. column and finally to a 50-mm i.d. column. With the larger column, 2.1 g of phospholipids were separated. The collected fractions (phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid and phosphatidylcholine) were of high purity (>99%). The solvent consumption was 7.2 L (separation and column equilibration), and a minimum of 10 g of polar lipids can be separated daily. Presented at the 82nd American Oil Chemists’ Society Annual Meeting, May 13–15 1991, Chicago, IL.     

Extraction of egg-yolk lecithin

In this research, the extraction of egg-yolk lecithin with ethanol was studied. Extraction was performed with deoiled and undeoiled yolks and with heated and unheated yolks. The yield of the extracted fraction relative to the initial material, phospholipid (PL) purity, and cholesterol content of both the PC-enriched fractions and the remaining PL fractions were determined. The yield and PL purity of the PC-enriched fractions obtained from the undeoiled yolks were 23.9 and 35.7%, and those obtained from deoiled yolks were 13.5 and 53.3%. The recovery of total PL in the two fractions was higher from the undeoiled (70%) than from the deoiled yolks (60%). However, heating had a negligible effect on PL extraction. Better enrichment of PC was observed by extraction from the undeoiled than from the deoiled yolks. The cholesterol content of the PC-enriched fraction obtained from the undeoiled yolks was much higher than that from the deoiled yolks.     

Antioxidant effect of soy lecithins on vegetable oil stability and their synergism with tocopherols

The antioxidant effect of lecithins was tested on several oils and fats varying in FA composition and tocopherol content. Standard lecithins, when added at a level of 1% w/w, exhibited a good protective effect against oxidation. This effect was observed to depend on the phospholipid content of the tested lecithins and the FA composition of the tested oils. Better results were obtained with lecithin samples containing high proportions of PC and PE. Indeed, the main antioxidant mechanism of lecithins was due to a synergistic effect between amino-alcohol phospholipids and γ- and δ-tocopherols. No synergism was observed with α-tocopherols, especially when the tested oil was rich in linoleic acid. Therefore, the antioxidant protection of lecithins was not effective for sunflower oil. Finally, the use of fractionated or enriched lecithins was not clearly advantageous compared to standard oil lecithins.     

High-performance liquid chromatographic separations of soy phospholipids

High-performance liquid chromatography methods to quantitate soy phospholipids vary as to which phospholipids are analyzed, degree of method ruggedness, precision, time, and standards. Fluid and deoiled soy lecithins were analyzed by three different high-performance liquid chromatography methods, including the American Oil Chemists’ Society (AOCS) Method Ja 7b-91. The other methods include an isocratic mixed phase (normal-phase column and reverse-phase solvent) method with ultraviolet detection, and a binary gradient normal-phase (proposed International Lecithin and Phospholipid Society) method with light-scattering detection. A set of 20 analyses were repeated on three different days for fluid and deoiled product by the three methods. The statistical comparison involved the selected methods and the phosphatidylcholine and phosphatidylethanolamine measured data, which were the common analytes among the methods. The precision for the mixed phase method and the Proposed International Lecithin and Phospholipid Society method was better than that for the AOCS method. Selection of reference standards was an important issue in defining the results. Column conditioning varied by 2–3 h for the Proposed International Lecithin and Phospholipid Society method, 16 h for the mixed phase method, and 2–3 d for the AOCS method. The ruggedness for the methods showed the following descending order: proposed International Lecithin and Phospholipid Society, mixed phase, AOCS.     

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.     

Evaluation of surfactant-aided degumming of vegetable oils by membrane technology

The first step in the process of vegetable oil refining is degumming, in which phospholipids and mucilaginous gums are removed that otherwise result in a low-grade oil. A membrane process is remarkably simple yet potentially offers many advantages in degumming. Studies were conducted on surfactant-aided membrane degumming with soybean and rapeseed oils in a magnetically stirred flat membrane batch cell with different types of microfiltration membranes. The reduction of phospholipids in soybean oil was in the range of 85.8–92.8% during the membrane process. The phosphorus content of membrane permeates of soybean oil was in the range of 20–58 mg/kg. Crude rapeseed oil contained higher amount of nonhydratable phospholipids and hence resulted in lower reduction in phospholipids, in the range of 66.4–83.2%. Addition of hydratable phospholipids could improve the efficiency of degumming in the membrane process without using any electrolyte, resulting in improvement of quality as well as quantity of the phospholipids.     

Selective extraction of phosphatidylcholine from lecithin by supercritical carbon dioxide/ethanol mixture

Selective extraction of phosphatidylcholine (PC) from deoiled soybean lecithin using supercritical fluid (SCF) mixtures of carbon dioxide (CO₂) and ethanol was studied at moderate pressures. Temperature was varied between 60 and 80°C at pressures of 17.2 and 20.7 MPa. Ethanol was added as co-solvent to supercritical CO₂ at the levels of 10 and 12.5 wt%. Constant rate of extraction of the individual phospholipids (PL) was observed for 150 min during which the extractions were carried out. Pressure and ethanol fraction had a positive effect on the selective extraction of PC, whereas temperature had a negative effect. Under all the conditions studied, the extracts were mainly composed of PC while the extraction of the other PL was very low. Extraction at 60°C and 20.7 MPa with 10 wt% ethanol/90 wt% CO₂ SCF mixture resulted in 95% selectivity to PC.     

Phospholipids at the Interface: Current Trends and Challenges

Phospholipids are one of the major structural elements of biological membranes. Due to their amphiphilic character, they can adopt various molecular assemblies when dispersed in water, such as bilayer vesicles or micelles, which give them unique interfacial properties and render them very attractive in terms of foam or emulsion stabilization. This article aims at reviewing the properties of phospholipids at the air/water and oil/water interfaces, as well as the recent advances in using these natural components as stabilizers, alone or in combination with other compounds such as proteins. A discussion regarding the challenges and opportunities offered by phospholipids-stabilized structure concludes the review.     

Phospholipid class and FA compositions of modified soybeans processed with two extraction methods

Soybean lecithin is used as an emulsifier in food, cosmetic, and pharmaceutical industries. The proportion of individual phospholipids (PL) and their FA composition may affect the functional properties of lecithin. In this research, lecithins recovered from four modified soybeans and one commodity soybean, which were processed by extrusion-expelling and conventional solvent extraction, were analyzed for proportion of PL class and FA composition. HPLC with an ELSD analysis demonstrated that the percentage of PC in extrusion-expelled lecithin was higher than in solvent-extracted lecithin, whereas the PE content was lower. GC analysis showed that FA compositions of the PL varied with soybean type. The oil extraction method did not significantly affect FA composition. Critical micelle concentration tested with a tensiometer showed differences among the lecithins.     

Resistance of recombinant proteins to proteolysis during folding and in the folded state

Protein purification often involves the use of denaturing agents for solubilization. During refolding, following removal of the denaturants, the proteins of interest are exposed to proteases present in the expression system. Here the resistance of selected recombinant proteins to three widely used proteolytic enzymes, trypsin (EC 3.4.21.4), proteinase K (EC 3.4.21.14) and endoproteinase Glu-C (EC 3.4.21.19), was investigated during folding and in the folded state. Target proteins and protease mixtures were denatured in 8 mol dm^?3 urea and the proteins were allowed to refold by removal of the urea by dialysis. The proteolytic products were analyzed by sodium dodecyl sulfate–polyacrylamide gels and the protein digestion during folding was compared with the digestion under similar conditions in physiological buffer. Depending on the folding state of the proteins, the proteases had different effects on the substates. During folding, certain recombinant proteins were more efficiently digested by trypsin and, in particular, by proteinase K in comparison with digestion in the folded state, while other protein substrates were more resistant to proteolytic degradation in a denatured or partially denatured state than their folded counterparts. Incubation of most substrate proteins with endoproteinase Glu-C yielded kinetics of digestion that were essentially similar for both partially folded and unfolded substrates. The results reported may be useful for protection of sensitive proteins and in studies of protein folding mechanisms.