The aggregates in heated bovine β-lactoglobulin solutions were
separated by high-performance size-exclusion chromatography, and molecular characteristics were measured
on-line using multiangle
laser-light scattering detection (SEC-MALLS). This technique was
proven to be a very useful and
valuable approach for characterization of heat-induced
β-lactoglobulin aggregates. Using TSK G2000
SWXL and TSK G4000 SWXL silica gel columns
connected in series, aggregates up to a molecular
mass of 4 × 106 Da could be separated, and complete
molecular mass distributions were derived.
Up to a molecular mass of 1.5 × 106 Da the molecular
masses calculated by the MALLS agreed
very well with the results obtained by conventional calibration.
The measured molecular mass
distributions of the heat-induced β-lactoglobulin aggregates varied
strongly with experimental
heating conditions (β-lactoglobulin concentration, pH, heating
temperature, ionic strength). The
results obtained with several β-lactoglobulin concentrations at
neutral pH were consistent with a
kinetic aggregation model based on thiol/disulfide exchange reactions.
The average molecular mass
and the radius of gyration of the heat-induced β-lactoglobulin
aggregates increased with increasing
initial β-lactoglobulin concentration.
Keywords: β-Lactoglobulin; aggregation; size-exclusion chromatography;
light scattering; molecular
size
The amount of heat-denatured serum proteins in heat-treated milk could be estimated by analyzing the casein fraction, obtained by isoelectric precipitation at pH 4.6, by capillary zone electrophoresis. A hydrophilically coated capillary was used in combination with 6 M urea in a citrate buffer at pH.3. Optimization of the sample and running buffer minimized adsorption of serum proteins, especially that of bovine serum albumin (BSA). This afforded a detection limit down to ca. 5-65 micrograms/mL of the three main serum proteins in milk. The detector response (UV at 214 nm) was linear in the range of 0.05-0.35 and 0.05-0.85 mg/mL for alpha-lactalbumin and beta-lactoglobulin, respectively. BSA showed a slightly less linear behavior, due to residual adsorption to the capillary wall. The recovery of serum proteins was in the range of 89-107%. The method was evaluated by analyzing Dutch commercial milks and cheese milk, which had received increasing heat loads. The addition of milk powder to pasteurized milk could be detected by this method as well as the serum protein to casein ratio in various products.
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