Characterization of the cell wall associated proteinase activity from Lactobacillus delbrueckii strains.

ELEAN M; CATALDO, P; SAAVEDRA, L; HEBERT EM

Salta

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

SAIB

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented dairy products. During milk fermentation process, the proteolytic system of LAB plays a key role because it enables these bacteria to grow in milk. LAB are fastidious microorganisms that require an exogenous source of amino acids or peptides, which are provided by the proteolysis of casein, the most abundant protein in milk and the main source of amino acids. The proteolytic system of LAB consists of a cell envelope-associated proteinase (CEP), amino acid and peptide transport systems and various intracellular peptidases. The CEP is the key enzyme of the system and it is responsible for casein initial degradation. In the present work, the goal is to characterize the proteinase activity of 36 L. delbrueckii strains belonging to the CERELA culture collection considering the major economic importance of these species as dairy starters. All strains were subjected to genotyping using the rep-PCR technique to group those isolates corresponding to clones of the same strain. One representative of each profile group was selected to further characterize their CEP enzymes.The strains were grown in a chemically defined medium (CDM) and their proteolytic activities were evaluated by two methods: the degradation of the chromogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide; and by the degradation profiles of alpha- and beta-casein by SDS-PAGE. Results from the hydrolysis of a- and β-casein degradation evidenced six types of caseinolytic cleavage specificity. Since proteolytic activity is repressed under high peptide content, we next study the inhibitory effect of peptides concentration on the CEP activity by growing bacterial cells in CDM plus Casitone. The proteolytic activity was repressed in the presence of peptides; however the strength of repression was strain-dependent. Finally, the release of these CEPs from the cell envelope was observed after treatment with 2 M NaCl. These results contribute to enlarge the limited knowledge on thermophilic lactobacilli CEP and are important from an industrial point of view since during the manufacture of hard cheeses, high concentrations of NaCl are present, and CEPs would remain active either bound to the cell or released, maintaining the beneficial health effects of the fermented milk products.