Enrichment of Phospholipids by Enzymatic Hydrolysis and Membrane Filtration of Whey Protein Phospholipid Concentrate

Whey protein phospholipid concentrate (WPPC) contains high amounts of phospholipids (PL) (16.4 ± 3.6 % PL as % lipid) but there is interest in further enriching the PL content for nutritional and functional applications. Enzymatic hydrolysis, in combination with microfiltration (MF), was explored for the pilot scale production of an enriched PL concentrate derived from WPPC. This was achieved by hydrolyzing proteins to facilitate passage of low molecular weight peptides through the MF membrane, while concentrating the fat and PL in the MF retentate. Bench top experiments were performed to select the proteolytic enzyme that resulted in the most extensive hydrolysis from among five different commercial proteases. SDS-PAGE analysis was performed to measure the extent of protein hydrolysis over a period of 4 h with samples drawn every 30 min. Alcalase enzyme was found to exhibit highest proteolytic activity at conditions of pH 8 and temperature of ⁓50°C. The intensity of major protein bands (milkfat globule membrane proteins, caseins, β-lactoglobulin) in WPPC decreased in SDS-PAGE profiles as hydrolysis progressed, along with the appearance of low molecular weight bands. Pilot scale MF production, coupled with diafiltration (DF), of the hydrolyzed sample yielded a final retentate with total PL content of 19.0 ± 3.0 % as % of final retentate in dry basis (db) with protein and fat contents at about 43.8 ± 0.4% (db) and 48.9 ± 1.2% (db), respectively. A two-fold increase in PL content (db) was therefore achieved through this process with an ~18% reduction in protein content. The MF permeate had minimal fat content indicating that there was no transmission of lipids or PL through the membrane during MF/DF process. Complete removal of proteins and peptides was not achieved by this process suggesting that either additional enzymes would be needed for further hydrolysis or possibly a more open MF membrane might be needed to remove more peptides and further increase the PL content.Dairy Management In

A Study of Various Chemical Pre-treatments to Fractionate Lipids from Whey Protein Phospholipid Concentrate

Dairy-derived lipids such as phospholipids (PL) have been gaining interest due to their functional and nutritional properties. Our research goal was to develop a separation process (non-solvent based) to produce an enriched dairy lipid fraction from whey protein phospholipid concentrate (WPPC). Various chemical pre-treatments (i.e., adjustment of pH, calcium, or temperature) were applied to rehydrated commercial WPPC solutions. These treatments were done on a bench top scale to aid in the precipitation of proteins or PL. The chemically treated solutions were centrifuged and fractionated into three layers: (1) top fat layer, (2) supernatant in the middle zone, and (3) sediment at the bottom of the centrifuge tubes. The thickness/size of the layers varied with the treatment parameters. Compositional analysis of each layer showed that the proteins, fat, and PL always appeared to fractionate in similar proportions. The proteins in each layer were characterized using SDS-PAGE under reducing and non-reducing conditions. Different proteins including whey proteins, caseins and milk fat globule membrane proteins/lipoproteins were identified and no specific type of protein had an affinity for either the top or bottom layer. All types of proteins were present in each of the layers after centrifugation and there were no major differences in fractionation of the proteins between layers with respect to the chemical treatment applied. The microstructure of protein and fat in WPPC was investigated using confocal laser scanning microscopy (CLSM). Dual staining of the rehydrated WPPC solution with Fast Green FCF (proteins) and Nile Red (lipids) showed the presence of very large protein aggregates that varied in size from 20 to 150 μm, with fat trapped within these aggregates. CLSM images of liquid WPPC revealed fine strands of a weak protein network surrounding the fat globules. This indicated that there were specific interactions between the proteins, as well as between the fat and proteins in WPPC. SDS treatment was performed to understand the nature of the interactions between protein and fat. It was found that about 35% of the fat present in WPPC was in the form of free fat, which was only physically entrapped within the protein aggregates. The remaining fat has some form of association with the proteins in WPPC. Other fractionation techniques would be needed to obtain an enriched dairy lipid fraction.National Dairy Counci