Improved method for the preparation of crystalline β-lactoglobulin and α-lactalbumin from cow's milk

Abstract

[beta]-Lactoglobulin and [alpha]-lactalbumin were separated from globulin, proteose peptone components and fat of milk by the addition of Na2SO4 (20 g/100 ml) to precipitate the latter. The precipitate was removed by filtration and all the protein constituents with the exception of [beta]-lactoglobulin precipitated from the filtrate F1 by reducing the pH to 2 with concentrated HC1 (1 ml/100 ml). [alpha]-Lactalbumin was isolated from this precipitate. [beta]-Lactoglobulin was precipitated by adjusting filtrate F2 to pH 6 with NH3 solution and adding (NH4)2SO4 (20 g/100 ml). The [beta]-lactoglobulin precipitate was filtered and solubilized by dialysis; the pH was adjusted to 5.8 and dialysis continued for 24 hours in a rotating bag. The pH was adjusted to 5.2 and the protein separated on further dialysis within a few hours in the form of orthorhombic plates or an oil that could be crystallized by seeding. [beta]2-Lactaglobulin was slow to form an oil and crystallize unaided. Yields of B2-lactoglobulin improve when it is crystallized in the orthorhombic rather than the monoclinic form. The precipitate containing [alpha]-lactalbumin was dissolved in dilute NH3 solution and residual [beta]-lactoglobulin removed by precipitation of the [alpha]-lactalbumin at pH 3.5. The centrifuged precipitate was dissolved in dilute NH3 solution and dilute HC1 added to pH 4. [alpha]-Lactalbumin precipitate slowly and is best stored in the cold overnight. Additional [alpha]-lactablumin can be obtained from the separated supernatant by adding (NH4)2SO4 (11.5 g/100 ml). The [alpha]-lactalbumin precipitates are dissolved in dilute NH3 solution, pH adjusted to 6.6 and an equal volume of saturated (NH4)2SO4 solution, pH 6.6, added with stirring. A discolored precipitate is discarded and [alpha]-lactalbumin crystallized by adding saturated (NH4)2SO4 to 1.5 times the volume for 0.5 saturation.