Application of the thin layer chromatography method for the determination of the degree of hydrolysis of soy lecithin
Abstract
The aim of this study was the determination of the degree of enzymatic hydrolysis of soy lecithin using thin layer chromatography in combination with a modern imaging data processing program. Hydrolysis of phospholipids was carried by the action of the enzyme phospholipase A2 (PLA2) in the presence of calcium ions for 2 h at a temperature of 50°C and pH 5.7. Chromatographic separation of the components of the reaction medium was carried out 10, 20, 30, 60, 90, and 120 min after adding the PLA2 in the chloroform: methanol: ammonium hydroxide system in a ratio of 6.5 : 2.5 : 0.4 (v/v/v). The following phospholipids and their lysoforms were identified: Rf = 0.41±0.03 for phosphatidylethanolamine (PE), Rf = 0.28±0.02 for phosphatidylcholine (PC), Rf = 0.09±0.01 for phosphatidylinositol (PI), Rf = 0.06±0.01 for phosphatidic acid (PA), Rf = 0.15±0.02 for lysophosphatidylethanolamine (LPE), Rf = 0.08±0.01 for lysophosphatidylcholine (LPC), Rf = 0.03±0.01 for lysophosphatidylinositol (LPI) and Rf = 0.02±0.01 for lysophosphatidic acid (LPA). It was shown that soy lecithin contained PC, PE, PI, and PA before the enzymatic reaction, the content of these compounds after the addition of the PLA2 decreases due to the formation of LPC, LPE, LPI, and LPA, respectively. The dependence of the degree of hydrolysis (α, %) of PC on the duration of the enzymatic reaction was obtained. It was established that the intensive conversion of PC to LPC under the action of the PLA2 occurred during 60 min (α = 82%). Further conversion of PC occurred insignificantly: 84% after 90 min and 87% after 120 min, which was due to the inhibition of enzyme activity by the final reaction product.
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