Anna Chojnacka

Enzymatic enrichment of egg-yolk phosphatidylcholine with α-linolenic acid

Abstractα-Linolenic acid (ALA) was incorporated at 28% into the sn-1 position of egg-yolk phospatidylcholine using Novozyme 435 in one-step transesterification process. Using phospholipase A2 in a two-step process gave 25% incorporation of ALA into the sn-2 position.

An LC method for the analysis of phosphatidylcholine hydrolysis products and its application to the monitoring of the acyl migration process

An assay for quantitative analysis of phosphatidylcholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and its hydrolysis products: 1-hydroxy-2-palmitoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, sn-glycero-3-phosphocholine and palmitic acid using high-performance liquid chromatography with charge aerosol detector (CAD) was developed. The separation of the compounds of interest was achieved on a reversed-phase/hydrophilic interaction stationary phase with a mobile phase consisting of acetonitrile:methanol:10mM ammonium acetate solution. The method was applied to control the acyl migration process of LPC regioisomers in the most common solvents used in the synthesis or modification of PC.

A simple method for positional analysis of phosphatidylcholine.

Simple and fast method of positional analysis of fatty acid composition of phosphatidylcholine (PC) from egg-yolk and soy has been elaborated. The key step of the procedure was complete ethanolysis of PC catalyzed by sn-1,3 specific lipase from Mucor miehei (Lipozyme). 2-Acyl-lysophosphatidylcholine (2-acyl LPC), fatty acids ethyl esters (FAEEs) and free fatty acids (FAs) were formed in this process. No acyl migration was observed during the reaction. The products were entirely separated from the products mixture by simple extraction in water:hexane (2:3 v/v) system. The hexane fraction containing free FAs and FAEEs was treated with BF(3)/Et(2)O in ethanol to obtain only FAEEs. The analysis of FAEEs by GC gave the composition of the FAs in the sn-1 position of the PC. 2-Acyl LPC from water fraction after precipitation in cold (-20°C) acetone was converted into FAEEs and analyzed by gas chromatography (GC) to determine FAs composition in the sn-2 position of the PC.

Lipase-Catalyzed Transesterification of Egg-Yolk Phophatidylcholine with Concentrate of n-3 Polyunsaturated Fatty Acids from Cod Liver Oil

Phospholipids containing PUFAs are important vehicles for their delivering to the targeted tissues. In our research project we established enzymatic methods for the enrichment of natural egg-yolk PC with n-3 PUFAs. Instead of synthetic PUFA ethyl esters, the new strategy was developed using polyunsaturated fatty acids enriched fraction (PUFA-EF) from cod liver oil as the natural acyl donors. PUFA-EF was produced by urea-complexation and contained 86.9% PUFA including 8.5% stearidonic acid (SDA; 18:4(n-3)), 26.7% EPA, and 45.2% DHA. The transesterification of PC with PUFA was catalyzed by lipases. After screening of enzymes the effect of reaction medium; molar ratio of substrates and etc. was investigated. The highest incorporation of PUFA was 45.6%; including 36.8% DHA and 5.8% EPA at the following reaction conditions: hexane; 55 °C; PUFA-EF/PC acyl ratio of 10; 48 h of reaction time and lipase B from Candida antarctica as a biocatalyst (20% of enzyme load).

Positional analysis of phosphatidylcholine and phosphatidylethanolamine via LC with a charged aerosol detector.

A new method for the positional analysis of egg yolk phospholipids (PLs) (phosphatidylcholine-PC, phosphatidylethanolamine-PE) using liquid chromatography with charge aerosol detector (CAD) is described. The method is based on six-step procedure: 1) extraction of phospholipids from tissue sample, 2) separation of lipid classes by solid phase extraction (SPE), 3) complete regiospecific hydrolysis of phospholipids by phospholipase A2 (PLA2), 4) separation of reaction products (fatty acids from sn-2 position and 1-acyl lysophospholipids) by SPE, 5) chemical hydrolysis of 1-acyl lysophospholipids, and 6) analysis of obtained fatty acids by LC with charge aerosol detection (CAD). Total time of enzymatic hydrolysis of PLs ranged from 10-30min. The reaction products were separated by SPE in three-step gradient elution procedure. Chloroform: methanol mixtures were used as eluents to obtain pure fractions of FAs from sn-2 position of PL and 1-acyl lysoPL (chemically hydrolyzed to FAs). FAs were separated by reversed-phase LC using a gradient elution and detected using CAD detector. This combination enables determination of all fatty acids in a single analysis, and without the sample derivatization. The method was optimized and the response of CAD, linearity, precision and sensitivity of the method were studied.