Gel Filtration of Cow Milk Whey Proteins

Abstract

The work considers the isolation of homogeneous precursor proteins of biologically active peptides from milk whey by gel filtration, in conditions that maximally ensure the preservation of their structure, composition, and properties. Considering the range of molecular masses of the main precursor proteins, the sephadex G-100 was selected for the gel filtration. As a result of the gel filtration of milk whey on a column with this sephadex, three peaks have been obtained, one of which was asymmetric and divided into two sectors. In total, four sectors from the three peaks have been received. Further, an electrophoretic analysis in the polyacrylamide gel of the proteins composition of all sectors was carried out. Sector A (the first peak) included immunoglobulins, lactoferrin, serum albumin. Sectors B and C (the second peak) consisted of β-lactoglobulin and α-lactalbumin in different ratios. The components of sector D (the third peak) had a small molecular weight and did not contain proteins. The next stage of the work was obtaining homogeneous lactoproteins. To this end, another gel filtration of the combined fractions of sectors A, B, and C was performed. Each of the chromatographic peaks obtained was divided into three ranges for the analysis of the proteins composition. Analytical electrophoresis of the combined chromatographic fractions of each range has shown that in six ranges out of nine, homogeneous precursor proteins of bioactive peptides were present. As a result of this repeated gel filtration on sephadex G-100, two homogeneous fractions (β-lactoglobulin, immunoglobulins) were obtained, which together, based on the results of the three gel filtrations, composed 59% of the whole milk whey protein. The processing of electrophoregrams, with the use of the image reading function imread, has shown high homogeneity of the fractions obtained (immunoglobulins ˃ 90%, and β-lactoglobulin ˃94%). These fractions were used to develop a biotechnology for obtaining and studying bioactive antihypertensive and bactericidal peptides from milk whey proteins.

Electrophoretic system for express analysis of whey protein fractions

“…Blood serum albumin (BSA) from Sigma (Germany) company and cow's milk β-lactoglobulin were used as marker proteins in electrophoretic researches. Homogeneous β-LG was isolated from whey by repeated gel filtration on a column of Sephadex G-100 [18]. Sephadex from Pharmacia (Sweden) company was used for gel filtration.…”

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

“…Homogenous β-LG fractions and the IG fraction obtained by repeated gel filtration of the milk whey on Sephadex G-100, as previously described, were used to identify the electrophoretic fractions [12].…”

Декстранові Гелі Для Ексклюзивної Хроматографії Протеїнів Сироватки Молока

“…Також у препаративних кількостях можна отримати протеїни сироватки молока висолюванням та ексклюзивною хроматографією або гель-фільтрацією (McSweeney and Fox, 2013). Відкриття додаткової функції протеїнів сироватки молока, а саме здатності утворювати біологічно активні пептиди у процесі протеолізу в шлунковокишковому тракті стимулювало пошук методів отримання окремих протеїнових фракцій (Iukalo et al, 2013). На сьогоднішній день відомо багато лабораторних методик, які дозволяють виділити окремі очищені протеїни.…”

“…При виборі декстранового гелю враховували молекулярні маси протеїнів, а також особливості їх просторової структури і фізико-хімічних властивостей. Серед протеїнів сироватки молока до попередників біоактивних пептидів відносяться β-лактоглобулін (М = 18363 Да); α-лактальбумін (М = 14178 Да); альбумін сироватки крові (М = 66399 Да); лактоферин (М = 76110) і за даними окремих авторів імуноглобуліни (М від 150000 до 500000 Да) (Farrell et al, 2004;Iukalo et al, 2013). Всі вказані фракції відносяться до глобулярних протеїнів, розчинні при фізіологічних значеннях температури і рН.…”