Для протеїнів сироватки молока притаманна фракційна специфічність їх біологічної дії та здатності утворювати в процесах протеолізу і травлення біоактивні пептиди, які позитивно впливають на різні фізіологічні системи організму. Перспективи виробництва і застосування протеїнових фракцій з сироватки молока пов'язані з необхідністю контролю за їх складом. Для створення методики експрес-аналізу фракційного складу протеїнів сироватки молока проведено порівняльний аналіз електрофоретичних систем, які раніше використовувалися для аналізу протеїнів молока. Це анодна система диск-електрофорезу в присутності додецилсульфату натрію, система дискелектрофорезу Девіса для кислих протеїнів у нативних умовах, система в однорідному поліакриламідному гелі в присутності сечовини. За основу для аналізу протеїнів сироватки молока була взята система диск-електрофорезу Девіса для кислих протеїнів в нативних умовах. Для адаптації цієї системи до вимог експрес-аналізу з її складу було вилучено концентруючий поліакриламідний гель і зменшено концентрацію розділяючого гелю. Для забезпечення високої ефективності розділення протеїнових фракцій була використана різниця у складі іонів електродного буферу і буферу для поліакриламідного гелю. Це дозволяє зберегти ефект концентрування протеїнів зразку сироватки на перших етапах електрофорезу. З допомогою гомогенних маркерних протеїнів (β-лактоглобулін і альбумін сироватки), встановлено розміщення основних фракцій протеїнів сироватки молока на електрофореграмах. В результаті проведених досліджень запропонована доступна електрофоретична система в пластинках однорідного поліакриламідного гелю для серійного експрес-аналізу фракційного складу протеїнів сироватки молока. Система дозволяє надійно ідентифікувати чотири протеїнові фракції (α-LA, β-LG, BSA та IG). Про хорошу відтворюваність методу свідчать близькі середні значення і стандартні відхилення вмісту цих фракцій у 15-ти пробах сироватки однієї партії молока, отримані на основі денситометрії трьох електрофореграм: β-LG (. Запропонований метод може бути корисним для оперативної ідентифікації основних протеїнових фракцій сироватки молока, які є попередниками біологічно активних пептидівКлючові слова: фракції протеїнів сироватки молока, електрофорез в поліакриламідному гелі, експрес-аналіз, денситометрія UDC 577.112.083

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.

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%.

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.

The article considers the possibility of obtaining purified fractions-precursors of bioactive peptides from milk proteins by the method of preparative electrophoresis. To choose an electrophoretic system, a comparative study has been carried out of four methods of electrophoresis in polyacrylamide gel that are used to analyse milk proteins (disc-electrophoresis without disaggregating agents, and disc-electrophoresis in the presence of sodium dodecylsulfate in homogeneous and gradient gel, and electrophoresis in homogeneous gel with urea). Electrophoresis of the total milk protein has shown that none of these systems allows separating effectively all protein precursors of bioactive peptides. The next stage was obtaining two main groups of milk proteins – caseins and serum proteins for electrophoretic fractionation. With the help of analytical electrophoresis, it has been established that each of the obtained groups had a typical proteins composition. Then, the proteins groups obtained were fractionated by preparative electrophoresis using the four electrophoretic systems listed above. In this case, the casein proteins that differ in the primary structure (αS1-, αS2-, β-, and ϰ-caseins) can be effectively separated by preparative electrophoresis on the basis of a homogeneous gel system in the presence of urea. The composition of this electrophoretic system was simplified. Unlike the analytical variant of a homogeneous polyacrylamide gel system, the toxic 2-mercaptoethanol was excluded, and the urea concentration was reduced. For the fractionation of serum proteins, a disc-electrophoresis without disaggregating agents can be used as a basis. It allows obtaining the main precursors of bioactive peptides from milk serum proteins: β-lactoglobulin, α-lactalbumin, serum albumin, and immunoglobulins. The protein precursors obtained by preparative electrophoresis were used to develop the biotechnology of obtaining bioactive phosphopeptides and inhibitors of the angiotensin-converting enzyme.