CHARACTERIZATION OF BIOACTIVE PEPTIDES WITH CALCIUM-BINDING ACTIVITY RELEASED BY SPECIALLY DESIGNED CHEESE STARTER

A total number of 210 strains of lactobacilli were isolated from different sources - homemade yoghurts and cheeses, plants and human fecal samples. The species affiliation of the strains was analyzed by Amplified Ribosomal DNA Restriction Analysis (ARDRA) followed by 16S rDNA sequencing. The strain differentiation was obtained by methods of Pulsed Field Gel Electrophoresis (PFGE) and Amplified Fragment Length Polymorphism (AFLP), which led to strain-specific genetic profiles. The general proteolytic activity for every strain was measured through derivatization of low-molecular weight peptide matter with ortho- phtalic aldehyde (OPA) and the strains with the best proteolytic activity were selected. Special peptidase activities were studied applying colored substrates. The ability of the selected strains to produce peptides possessing calcium and magnesium binding properties was evaluated by help of ion-selective electrode, measuring the concentration of the Ca ions non- bonded by peptides. Peptides with Ca binding effect from the strains demonstrating the highest Ca attraction were subjected to fractionating on different HPLC columns. Such peptides were isolated, purified and sequenced. Finally, two strains which were capable to produce bioactive peptides with the strongest Ca- and Mg-binding activities were selected for development of cheese starters. After pilot production of functional cheeses with the developed starters the functional effect of bioactive peptides was confirmed during the cheese ripening.