Characteristics of proteolytic processes during the isolation of natural casein phosphopeptides

Abstract

Introduction. The aim of this work is studying of the proteolytic processes features in the production of natural phosphopeptides of the milk casein complex. Materials and methods. Casein substrate was isolated by isoelectric sedimentation from skimmed milk. Proteolysis was performed using pancreatin. The degree of proteolysis was determined spectrophotometrically by the absorption of low molecular weight products of proteolysis at λ = 280 nm. The yield of phosphopeptides was determined gravimetrically after precipitation with ethanol in the presence of calcium ions. Electrophoresis of phosphoproteins of the substrate and products of proteolysis was carried out in an alkaline system of homogeneous polyacrylamide gel in the presence of urea. Results and discussion. Natural phosphopeptides were obtained by proteolysis of the phosphoproteins of the casein complex with pancreatin (E:S = 1:100) under physiological conditions (37° C, pH 7.9). At different stages of proteolysis, the yield of phosphopeptides and peptides soluble in 10% trichloroacetic acid was determined. Degree of proteolysis has increased monotonically throughout the studied period. The yield of phosphopeptides reaches its maximum in the 90th minute of proteolysis and then decreases continuously. The yield of phosphopeptides is lower than it was used proteolytic preparations of microbial origin. The results obtained by gel filtration and electrophoresis in a polyacrylamide gel indicate that a decrease in the yield of phosphopheptides after the 90th minute of proteolysis may be caused by the phosphopeptide's molecular weight decrease. Most of phosphopeptides obtained in the 90th minute of proteolysis have a molecular weight up to 2000 Da, which is characteristic for the already known biologically active phosphopeptides. Conclusions. During the proteolysis of casein with pancreatin in physiological conditions, the total yield of proteolysis products increases monotonously. The yield of phosphopeptides has maximum. Gel filtration and electrophoresis data indicate that this is due to a decrease in the molecular mass.