Mild isolation procedure discloses new protein structural properties of beta-lactoglobulin.

To explore the potentially available functional properties of beta-lactoglobulin in, for example, the processing of food products, it is important to isolate the protein by a procedure that avoids all possible denaturing conditions, such as low pH, high ionic strength, or low or elevated temperatures that could cause the protein to undergo irreversible conformational changes. In this work, a mild isolation protocol for beta-lactoglobulin from bovine milk is presented, applicable to semi large-scale isolations (50 to 200 g). The protein could be isolated with a high efficiency (>80%) and a good purity (>98%). Biochemical characterization of the material demonstrated no lactosylation of the protein, nor the formation of irreversibly associated dimers. Also, no proteose peptones could be detected. The ability of beta-lactoglobulin to undergo conformational changes is studied by far and near-ultraviolet circular dichroism and differential scanning calorimetry. A "global" unfolding of the protein is detected around 72 (tertiary level) and 77 degrees C (secondary level). The dimer-monomer dissociation occurring around 52 degrees C could also be monitored at a secondary structural level. Remarkably, a low temperature transition around 30 degrees C was observed, where approximately 10 beta-stranded residues unfold cooperatively, not been reported previously. This low temperature transition is irreversible at temperatures higher than 35 degrees C or upon freezing the material at -20 degrees C. The addition of 20% glycerol could prevent this irreversible conformational change. The effect of the low temperature transition on the protein's functionality remains to be investigated.