Obtaining of β-LG, α-LA and BSA protein fractions from milk whey

Abstract

Introduction. The purpose of the work is to obtain the protein fractions β-LG, α-LA and BSA from the milk whey by preparative disc-electrophoresis. Materials and methods. The whey was obtained from the fresh cow's milk after isoelectric precipitation of casein complex proteins. The concentration of whey proteins was determined on a spectrophotometer by the absorption at λ = 280 nm. Gel filtration was carried out on liquid chromatography columns. Sephadexes G-25 and G-100 were used for the gel filtration. The fractional composition and homogeneity of whey proteins were analyzed by disc-electrophoresis in the polyacrylamide gel slabs. Results and discussion. A system of disc-electrophoresis in native conditions for acidic and neutral proteins has been selected for the preparative isolation of homogeneous fractions of bioactive peptides proteins-precursors from milk whey taking into account the effectivety and existence of denaturation factors. A sample of whey proteins, for preparative fractionation purified, from low molecular weight compounds had been obtained at the result of the gel filtration. An optimal whey proteins sample amount should not exceed 10 ml to provide an effective separation in a modified electrophoretic chamber of Studier type apparatus. Too large sample amount does not allow to separate the whey proteins effectively and obtain the homogeneous fractions extraction. The duration of the extraction process of individual protein fractions after preparative discelectrophoresis was about 55-60 min. The degree of homogeneity of the obtained protein fractions was established with the help of analytical disc-electrophoresis. It was 95-97% for β-LG and BSA fractions and 89-91% for α-LA fractions. It has been calculated that the total yield of homogeneous milk whey protein fractions was nearly 49-61% (from milk whey proteins taken for separation). Conclusions. The proposed variant of the disc-electrophoresis allows to isolate from milk whey the electrophoretically homogeneous fractions of β-LG, α-LA and BSA in native conditions. The duration of electrophoresis and proteins extraction is about 4 hours. The average yield of homogeneous fractions from whey proteins is 55±6%.

Adaptation of Stadier's apparatus for electrophoresis of main milk proteins

“…In express electrophoresis, in order to save reagents, the volume of the electrophoretic chamber can be reduced by shortening its length. In the case of preparative electrophoresis, one large starting cell is used for samples [15,20]. At the same time, in order to regulate the volume of the protein sample, PAG plates with a thickness of both 3 and 5 mm, as well as different chamber widths, are provided.…”

“…Such an electrophoresis system is useful if the preservation of the native protein structure is important. Express [11] and preparative [20] versions of whey protein electrophoresis have also been developed on its basis. However, using this type of electrophoresis, it is difficult to analyse products that contain denatured whey proteins.…”

“…1 shows a typical electrophoregram of a casein sample and its densitogram. With the use for homogeneous casein markers, the placement of casein fractions, which is characteristic of this electrophoretic system, was estab-lin (β-Lg) and α-lactalbumin (α-La) were isolated by preparative electrophoresis [20]. Homogeneous α S1 -CN and β-CN casein fractions were obtained by differential precipitation in the presence of urea followed by purification by ion exchange chromatography on DEAE cellulose [22].…”

“…Express analysis of the fractional composition of caseins was performed according to the method described previously in [14]. For analytical and preparative electrophoresis of milk whey proteins, a modified Davis disc electrophoresis method was used under native conditions for neutral and weakly acidic proteins [18,20]. The procedure of express electrophoresis of whey proteins is described in [11].…”

“…The apparatus proposed in the work also allows for preparative electrophoresis of milk whey proteins. To isolate two electrophoretically pure fractions, the disc electrophoresis technique was used under native conditions [20]. Preparative electrophoresis was performed in a chamber with dimensions of 3×81×90 mm.…”