Continuous centrifugal separation of selectively precipitated α-lactalbumin

Molecular Analytical Assessment of Thermally Precipitated α-Lactalbumin after Resolubilization

“…Major changes in the environment, however, which could be caused by different pH values, temperature or solvent conditions, can affect the folding of the β-sheet domain and the structure of the loops in the molten globule [ 53 ]. Furthermore, apo-α-la is not only more susceptible to thermal treatment, but also to hydrostatic and mechanic stress compared to its holo-form [ 18 , 54 ]. It is likely that the applied conditions, such as heating to 50 °C for several hours and the mechanical stress through stirring and centrifugation are the root cause for the deviating conformation after refolding.…”

“…The suspension was transferred to 50 mL tubes and centrifuged at 6000× g for 30 min (Multifuge 1 S-R, Heraeus Holding GmbH, Hanau, Germany) to separate precipitates from β-lg enriched supernatant. In a previous study, it was shown that a considerable amount of native β-lg remains in the sediment phase [ 18 ]. Two washing steps of the sediment using DIW with adjusted pH to 3.4 using 0.1 M HCl were performed to remove β-lg residues from the sediment.…”

“…The quantification as well as determination of the degree of resolubilization of α-la was performed by RP-HPLC analysis as described by in [ 18 ]. For calculation of resolubilization degree RD (Equation (1)), it was required to prepare one sample that was clarified from insoluble protein by a pH 4.6 isoelectric precipitation and centrifugation ( c α-la, soluble ), and another one that was left untreated and in original composition ( c α-la, total ).…”

“…In this study, the method of acidic precipitation of α-la with gentle heat treatment and subsequent separation from soluble and native β-lg was used. A previous study demonstrated that this process in combination with a separation by a decanter centrifuge provides high yields, easy scalability, and high purities of the obtained fractions [ 18 ]. With a comparably low heat load and a good stability at acidic pH values, the nativity of β-lg remains virtually unaffected by this method as demonstrated by Kella and Kinsella [ 19 ].…”

“…Under these conditions, α-la forms precipitates, most likely caused by a combination of hydrophobic and electrostatic interactions [26]. The formed precipitates enable a size-based separation either by microfiltration [10,27] or centrifugation [18,28,29].…”