Obtaining of Β-Lactoglobulin by Gel Filtration of Cow Milk Whey

Abstract

Milk whey proteins carry out a number of important biological functions and also they are precursors of many biologically active peptides (antihypertensive peptides, antagonists of opioid receptors, regulators of intestinal motility, immunomodulatory, anti-microbial and anti-cancer peptides, appetite regulators and so on.). An important stage in natural bioactive peptides obtaining from milk whey proteins is the isolation of homogeneous proteins-precursors. Considering the significant difference in the molecular masses of whey proteins, a promising method for their selection is gel filtration. The purpose of the research was the fractionation of bioactive peptides precursors from milk whey using gel filtration on Sephadex G-150. The whey was obtained from fresh skimmed milk after isoelectric precipitation of casein. Gel filtration was carried out on the columns from a liquid chromatography kit by the “Reanal” company. The fractional composition and the degree of homogeneity of milk whey proteins were determined by disc-electrophoresis in the plates of a polyacrylamide gel. A repeated gel filtration of fractions from the chromatographic peaks, separated into sections, was performed to increase the fractionation efficiency. While choosing a dextran gel for gel filtration of precursors of biologically active peptides from milk whey proteins, we have taken into account the range of their molecular weights (from 10000 to 150000 Da), the ability to form supramolecular structures (β-LG), as well as the previously obtained results of gel filtration. As a result, it was shown that repeated gel filtration of milk whey on Sephadex G-150 allows efficiently fractionate the proteins-precursors of bioactive peptides. The range of peptides and proteins molecular weights that can be fractionated on this Sephadex is from 5000 to 300 000 Da. The usage of repeated gel filtration on Sephadex G-150 with the chromatogram separation into sectors allows to effectively fractionate proteins-precursors of bioactive peptides from milk whey. In particular, homogeneous β-lactoglobulin (degree of homogeneity > 95 %) and partially purified α-lactalbumin, as well as a group of immunoglobulins and a proteose-peptone fraction were obtained.

Improvement of the Method of Comparative Study of Milk Whey Proteins Enzymatic Hydrolysis

“…It is important that the obtained lyophilized preparation is well soluble in water. Taking into account the previously obtained results [12], chromatographic fractions from β-LG after the second gel filtration of milk whey on a column with Sephadex G-150 were combined and selected. The numbers of the combined fractions are shown in Fig.…”

“…The isolation of β-LG was carried out by gel filtration of the milk whey on a column with the Sephadex G-150 from «Pharmacia» (Sweden) as described earlier [12].The columns (1.5×70 cm) from the «Reanal» (Hungary) company of liquid chromatography were used for gel filtration. The speed of the elution was set at 20 ml/h.…”

A combination of Sephadexes for the milk whey protein fractions isolation

“…Деякою мірою це питання було вирішено за рахунок проведення повторної гель-фільтрації на певному виді сефадексу. Проте і в цьому випадку було досягнуто високого ступеня очистки лише для однієї фракції за рахунок низького виходу препарату (Yukalo & Datsyshyn, 2018a;Yukalo et al, 2019). Враховуючи діапазон молекулярних мас протеїнів сироватки молока, перспективним може бути використання різних сефадексів при повторній гель-фільтрації.…”

Comparison of products of whey proteins concentrate proteolysis, obtained by different proteolytic preparations

“…At the same tein fractions on electrophoregrams. Homogeneous β-LG was obtained from milk whey by repeated gel filtration [18]. To isolate the whey of milk, the pH of fresh skim milk was adjusted by 0.1 N HCl with stirring to 4.6 achieving the isoelectric precipitation of caseins.…”

“…To determine the concentration (mg/cm 3 ) of whey proteins and products of its proteolysis, the known absorption coefficient (D 1% ) -12.3 has been used. An absorption coefficient of 9.6 was used to determine the β-LG concentration and 6.7 for the BSA [18].…”