Detection and quantification of the milk proteins by RP-HPLC method

Abstract

Protein analysis is very important both in terms of milk quality and technological properties. Despite milk proteins having been extensively studied, there still is a substantial lack of characterization, and particularly of quantitative information, about whey proteins from cow’s milk in Lithuania. The aim of this study was to detect major whey proteins α-lactalbumin (α-LA), β-lactoglobulin (β-LG) and bovine serum albumin (BSA) by reversed-phase high-performance liquid chromatography (RP-HPLC). A reverse-phase analytical column C18 (Nucleosil C18, 300 Å, 5 μm particle size, 250×4.6, Macherey-Nagel, Düren, Germany) and UV detector were used for the analysis. The separation was carried out at 25 C using the gradient system. All chosen proteins were detected and separated in 90 min; purified bovine milk protein genetic variants were employed in calibration. A linear relationship (R2>0.99%) between concentration and peak areas of individual milk protein variants was observed. The sample injection volume was of 20 μL. Data collection and evaluation was performed by using LG Solution (Shimadzu Corp.,Kyoto, Japan) operating system. In total 120 milk samples were collected individually from normal lactating dairy cows. Milk samples from individual quarters were collected once during the spring, summer and autumn (10 cows / 40 quarter samples / each season). The average concentrations mg mL-1 (mean±SEM) of α-LA, β-LG and BSA in cow‘s milk were 0.898±0.036, 3.330±0.121 and 0.371±0.023 in spring, 0.979±0.029, 2.942±0.097 and 0.273±0.030 in summer, 0.835±0.020, 3.744±0.156 and 0.349±0.028 in autumn, respectively. The final results of one-way analysis of variance indicate a significant effect of season on the content of all the proteins analysed α-LA (p<0.01), β-LG (p<0.001) and BSA (p<0.05). In this study was achieved simple and sensitive method for proteins detection and quantification in cow’s milk.