Isolation of Milk Phospholipids and Determination of Their Polyunsaturated Fatty Acids

Several methods were compared for recovery and pm'ification of mixtures of phospholipids from dried buttermilk.Samples were analyzed for phosphorus, nitrogen, and unsaturated fatty acids.Evidence was obtained that phospholipids purifed by repeated acetone precipitation are not representative of the original milk phospholipids.An improved procedure was developed to extract the lipids from buttermilk with ethanol, ether, and chloroform.Most of the phospholipids were separated from the other lipids by precipitation with acetone.Acetone-soluble phospholipids were recovered by chromatography with silieie acid columns.The two fractions were combined and nonlipid contaminants were removed by washing.In an alternative procedure, the lipids were extracted from buttermilk with methanol and chloroform before the foregoing method was used to isolate the phospholipids.The amounts of unsaturated fatty acids were estimated from spectrophotometric data and iodine values.A representative sample of milk phospholipids (3.72% phosphorus, 2.23% nitrogen, iodine value 53.8) contained 26.3% monoene, 1.5% conjugated diene, and 5.8, 2.4, 1.3, and 1.5% nonconjugated diene, triene, tetraene, and pentaene acids, respectively.Percentages of each of these acids in the total fatty acids (with corresponding percentages for milk fat from the same source given in parentheses) were: 39.8 (.q2.1), 2.3 (1.4), 8.8 (1.6), 3.6 (0.9), 2.0 (0.3), and 2.2 (0.2), respectively.The phospholipids contained 41.3% saturated fatty acids and the milk fat 63.5%.These data show that there was a selective concentration of nonconjugated polyunsaturated acids in milk phospholipids.The lipids of milk include about 1% phospholipids, most of which are associated with milk proteins and partly concentrated at the surface of the fat globules.Many investigators (1,15,16,33) have estimated the amounts of phospholipids in milk and milk products by determining the phosphorus content of extracts obtained by various methods, including the Mojonnier modification of the RSse-Gottlieb procedure.In several studies (12,17,18,19,25,26, 31,32) phospholipids were actually separated from the other milk lipids and analyzed.However, these samples were not completely representative of the original phospholipids, except in the recent study by Rhodes and Lea (25).The reason is the difficulties involved in the extraction and purification of phospholipids (23).For example, purification procedures that depend on repeated precipitation of phospholipids with acetone, without recovery of those phospholipids soluble in acetone, result in selective losses.The Mojonier procedure has often been used for extracting lipids.Although this method does not extract significant amounts of inorganic phosphorus, it may not extract all the individual phospholipids, including lecithins, eephalins,