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

The effcient and rapid technique of ultrathin‐layer isoelectric focusing on 50 μm polyacrylamide gels was used for determination of heat‐induced changes of whey proteins (β‐lactoglobulin A and B as well as α‐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. Coefficients of variation of the integrated peak areas were approximately 3 % for β‐lactoglobulin A and B and about 6 % for α‐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 α‐lactalbumin was a first‐order reaction.