Quantitative densitometry of heat‐induced changes in whey proteins following ultrathin‐layer isoelectric focusing

Abstract

The efficient and rapid technique of ultrathin-layer isoelectric focusing on 50 pm polyacrylamide gels was used for determination of heat-induced changes of whey proteins (P-lactoglobulin A and B as well as a-lactalbumin) and a method for quantitative densitometry was developed. Improved reproducibility could be achieved by (i) introducing an internal standard (carbonic anhydrase), allowing for compensation for minor irregularities in gel composition, sample application or staining, (ii)running each sample in triplicate, and (iii) multiple scanning of each sample at different positions of the focusing track. Coefticients of variation of the integrated peak areas were approximately 3 % for P-lactoglobulin A and B and about 6 % for a-lactalbumin. Skim milk was heated in a pilot heating plant especially designed for reaction kinetic studies. Using the initial content of native whey proteins in each milk sample as a reference value, degrees of denaturation of more than 90 % could be determined for each of the three protein fractions. Results obtained from reaction kinetic studies revealed that denaturation of a-lactalbumin was a first-order reaction. I IntroductionIt is not possible to determine the heat-induced denaturation of whey protein fractions in milk and whey directly by means of an analytical method. Usually, the extent of denaturation is calculated indirectly by evaluating the proportion of undenatured or native whey proteins remaining in the serum. A variety of methods has been described for the isolation and detection of individual milk proteins, including salt fractionation and determination of residual protein content by microkjeldahl analysis of nitrogen L11, gel chromatography [ 2 , 31, immunoelectrophoresis (4, 51, cellulose acetate electrophoresis [61, horizontal polyacrylamide gel electrophoresis (PAGE) 171, and more recently, high performance liquid chromatography (HPLC) [8- 101 and, in particular,. Hillier 141 gives a detailed description ofthe densitometric determination of whey protein fractions in heated milk samples separated by means of PAGE. Most of the published methods need complex equipment and are time consuming, which makes them inconvenient for processing a large number of samples or for routine analysis. Application of electrophoretic methods sometimes resulted in incomplete separation of individual whey protein fractions. Moreover, Levieux et al. ( technique of ultrathin-layer isoelectric focusing in 50 pm polyacrylamide gels on silanized polyester films [ 171 could be anticipated to be useful for the detection of whey proteins. The latter technique combines high resolution and operational simplicity. A quantitative densitometry of the separated fractions obtained by isoelectric focusing has not been described so far. The aim of this work was to develop a method, utilizing a laser densitometer, for the determination of undenatured plactoglobulin (p-lg) A and B and of a-lactalbumin (a-la), the three main whey protein fractions in milk or whey. For a study of the reaction kinetics of...

The use of “lab-on-a-chip” microfluidic SDS electrophoresis technology for the separation and quantification of milk proteins

“…Other PAGE techniques such as isoelectric focussing PAGE (Dannenberg and Kessler, 1986) and alkaline PAGE under native conditions (Hillier, 1976) have reported CVs of 2-6% for the determined concentrations of individual whey proteins. HPLC techniques have produced CVs, ranging from about 2 to 10% (Bobe et al, 1998b;Strange et al, 1991) whereas studies on capillary electrophoresis have reported CVs of about 2.5-4% for the determined concentrations of the individual milk proteins (de Jong et al, 1993).…”