Bìol. Tvarin, 2015, volume 17, issue 3, pp. 139–144


A. V. Iukalo1, O. I. Tsisaryk2

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1Ternopil Ivan Puluj National Technical University,
56 Ruska str., Ternopil 46001, Ukraine

2Lviv National University of Veterinary Medicine and Biotechnologies named after S. Z. Gzhytsky,
50 Pekarska str., Lviv 79010, Ukraine

The specificity of cell-envelop proteinases of lactococci toward the casein fractions plays an important role in formation the parameters of quality of fermented dairy products, as well as in the creation of natural origin bioactive casein peptides. Existing methods of establishing the specificity of these enzymes are based on long-term cultivation of lactococci in milk or complex obtaining and identification of cell-envelop proteases. The aim of our study was to determine the specificity of the proteinase of lactococci using a homogeneous casein fractions as substrate. Casein fractions (αS1-, β- and κ-caseins) were isolated using differential precipitation in isoelectric point in the presence of urea and preparative electrophoresis in polyacrylamide gel. To determine the total proteolytic activity and selection of proteinase-positive strains was held their short-term cultivation (12 hours) in sterile skim milk at 30оС. In case of development of proteinase-positive strains the concentration of proteolysis product after 12 hours of cultivation remains the same or increases. It depends on the activity and amount of the cell-envelop proteinase. It was also tested the ability of strains to form lactic acid and coagulate casein milk. These characteristics are the most important in lactic acid bacteria and are closely related to proteolytic activity. For the proteolysis protease-positive strains of lactococci, which were grown in the milk culture medium, were used. Cells were washed from the components of the culture medium and concentrated in condition while preserving their cell-envelop proteases. The specificity of cell-envelop proteases was established on the basis of electrophoretic analysis of undigestive casein substrates in alkaline system of homogeneous polyacrylamide gel. As a result of analysis of cell-envelop proteases types of four strains was defined. The proposed method can significantly reduce the time of the analysis and to identify the type and specificity of cell-envelop proteases of lactococci.


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