Detection of the whey proteins by RP-HPLC

Abstract

The aim of this study was to detect whey proteins α-lactalbumin (α-LA), β-lactoglobulin (β-LG) and bovine serum albumin (BSA) by reversed-phase high-performance liquid chromatography (RP-HPLC). In total 120 milk samples were collected individually from normal lactating dairy cows. Milk samples were collected once during the spring, summer and autumn (10 cows/40 quarter samples). For evaluation of the content of whey proteins, i.e. alpha-LA, beta-LG and BSA, samples from cow milk were prepared as follows: 25 mL of raw milk was adjusted to pH 4.6 with 0.1 mol·L–1 HCl and allowed to stand at room temperature for about one hour for acid precipitation of caseins. Consequently, whey (7 mL) was taken from each of the samples separately and then centrifuged at 10000 rpm for 15 min. Finally, whey solutions were filtered through quality filters and then through 0.20 μm disposable sterile filters (Millipore). The supernatants in vials were refrigerated until further analysis and, when appropriate, injected into the chromatograph (20 μL). A reverse-phase analytical column C18 (Nucleosil C18, 300 Å, 5 μm particle size, 250 X 4.6, Macherey-Nagel, Düren, Germany) and UV detector were used for the analysis. The separation was carried out at 37oC using the gradient system. alpha-LA, beta-LG and BSA were detected and separated in 92.31 min, 98.47 min and 93.99 min respectively; purified bovine milk protein genetic variants were employed in calibration. A linear relationship (R2 > 0.99%) between concentration and peak areas of alpha-LA, beta-LG and BSA were observed 0.9997, 0.9975 and 0.9996 respectively. Calibration curve designed over a concentration range for alpha-LA 0.300-1.500 mg/ml, for beta-LG 0.241-1.207 mg/ml and for BSA 0.030-0.253 mg/ml. Data collection and evaluation was performed by using LG Solution (Shimadzu Corp., Kyoto, Japan) operating system.Calibration of the chromatographic system for determinat[...].