Phospholipids c test wako

The concentration of phospholipids in the eluate was determined using a Phospholipids C-test Wako (Wako Pure Chemical Industry, Osaka, Japan). An aliquot of the stock solution was diluted in distilled water to a concentration of 0.2 mg total lipids per mL, and the particle size and zeta potential of the liposomes were measured in a Zetasizer Nano ZS (Malvern, Worcester, UK). On the other hand, another aliquot of the stock solution was diluted 40 times with methanol and vortexed for 3 min to disrupt liposomes. Following filtration of the samples using 0.45 μm filter, EVE entrapment efficiency of each liposome was determined using an HPLC system (Prominence UFLC, Shimadzu Corporation, Kyoto, Japan) equipped with a ZORBAX column (SB-C8 4.6 I.D. × 75 mm, 3.5 μm, Agilent Technologies, Santa Clara, CA, USA), which was maintained at 60 °C. The mobile phase comprised distilled water with 0.1% trifluoroacetic acid and acetonitrile with 20 mM triethylamine and 0.156% trifluoroacetic acid (40 : 60 v/v) and was maintained at a flow rate of 1 mL min⁻¹. The wavelength of detection of EVE was set to 278 nm.

Micellar lipid solubility in the presence of casein digest, EPL digest, and l-lysine was measured by the method of Nagaoka et al. (4 (link)) with some modifications. The micellar solutions were incubated at 37°C for 24 h and then 0.5 mL of digest products and l-lysine (0.05 g/mL) were individually mixed with 0.5 mL of micellar solutions. Micellar solutions (0.5 mL) containing 6.6 mmol/L sodium taurocholate, 1.5 mmol/L cholesterol, 1 mmol/L oleic acid, 0.5 mmol/L monoolein, 0.6 mmol/L phosphatidylcholine, 132 mmol/L NaCl, and 15 mmol/L sodium phosphate (pH 7.4) were treated by sonication. The mixture was incubated at 37°C for 1 h, and then ultracentrifuged at 100,000 g for 1 h at 37°C. The supernatant was collected for the determination of cholesterol, fatty acid, phospholipid, and bile acid concentrations using an enzymatic assay kit (Cholesterol-E-Test Wako, Wako NEFA C-test kit, Phospholipids C-test Wako, and Total Bile Acid Test Kit; Wako Pure Chemical Industries Ltd.).