In accordance with the worldwide trend of increased use of probiotics, scientific interest has increased, as well as the aspect of legislative framework and clinical guidelines. The aim of this thesis is the phenotypic characterization of bacterial strains from different microenvironments following the requirements of the probiotic concept with a focus on the fermentation profile. The metabolic activity of bacterial strains was tested by controlled cultivation for 48 hours at 37 °C in skimmed milk. The most statistically significant acidification was determined after the growth of Lactobacillus fermentum D12 in skimmed milk and in skimmed milk with different concentrations of salt. Coagulation capacity was confirmed phenotypically for all tested strains after 24 and 48 hours of cultivation in skimmed milk. By applying the protocol for the targeted extraction of proteinases, extracts of proteins showing caseinolytic activity were prepared and analyzed by the SDS-PAGE method. Partially purified proteinases of L. lactis strains ZGBP5-32 and ZGZA7-10 showed caseinolytic activity even in very low concentrations. The modification of the extraction protocol by implementing ultrafiltration contributed to the purification of proteinases, which was confirmed by the SDS-PAGE method for protein extracts of strains L. brevis D6 and SF9B, L. fermentum D12 and L. plantarum D13. Further research will be focused on the characterization of bioactive peptides due to the protease activity of selected caseinolytic lactic acid bacteria. The characterization of caseinolytic lactic acid bacteria in this work may contribute to the selection of proteolytic strains as lactic starter cultures.