Determination of the proteolytic activity of lactic acid bacteria and identification of proteinase genes

Background. Bioactive peptides are described as amino acid sequences of proteins that have a positive effect on biological processes and/or human health in general. They can be formed in high-protein animal and vegetable raw materials under the action of enzymes of starter and starter cultures in situ. These enzymes are proteinases with a molecular weight of about 200 kDa belonging to the PrtP family.
Purpose. The aim of the study was to determine the genes of proteinases in lactic acid and denitrifying microorganisms, to evaluate their proteolytic activity and to determine the relationship between proteolytic activity and genetic determinants.
Materials and methods. The proteolytic potential of microorganisms of the genera Latilactobacillus and Pediococcus, as well as the genus Staphylococcus from the university collection was evaluated. The TNBS method was used to measure the released amino groups. The sizes of the enlightenment zones around the colonies of microorganisms were determined by the cup method on milk agar. 5 pairs of genomic DNA primers (PrtP700/PrtM700; P15C/P06C; PRTB10/PRTB20; Jp23/Jp25; prti2/IP6Xba) were used to determine the presence of PRTP, PRTM, PRTB, PRTH and PRTR genes responsible for proteolytic activity.
Results. On milk agar, all the studied microorganisms formed zones of enlightenment around the colonies, which indicates their proteolytic activity. The largest growth retardation zones were around colonies of the strain Latilactobacillus curvatus 2, as well as Pediococcus acidilactici 28 and Pediococcus acidilactici 38. According to the TNBS method, Latilactobacillus curvatus 1 and Staphylococcus carnosus 108 were the least productive in amine nitrogen accumulation. PCR results showed that the maximum number of proteinase genes is present in the strain L. sakei 105. But this strain did not show high proteolytic activity on the medium by qualitative and quantitative methods. Therefore, an integrated approach is needed to select strains with high proteolytic activity.
Conclusions. Using an integrated approach, strains with high proteolytic activity, potentially capable of forming bioactive peptides in fermented foods, were selected.