De Angelis, M; Mariotti, L; Rossi, J; Servili, M; Fox, PF; Rollán, G; Gobbetti, M
The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
Aromatic compounds; Cytology; Electrophoresis; Enzymes; Fermentation; Growth kinetics; pH effects; Purification; Cytoplasma; Bacteriology; arginine deiminase; carbamate kinase; ornithine carbamoyltransferase; bacterium; enzyme; amino acid sequence; article; bacterial strain; catabolism; cell growth; cell survival; chromatography; electrophoresis; enzyme activity; fermentation; lactic acid bacterium; Lactobacillus; Lactobacillus brevis; LActobacillus fructivorans; lactobacillus hilgardii; Lactobacillus plantarum; Lactobacillus sanfranciscensis; molecular weight; nonhuman; Adenosine Triphosphate; Amino Acid Sequence; Arginine; Biodegradation, Environmental; Bread; Fermentation; Hydrogen-Ion Concentration; Hydrolases; Lactobacillus; Molecular Weight; Bacteria (microorganisms); Lactobacillus; Lactobacillus brevis; Lactobacillus fructivorans; Lactobacillus hilgardii; Lactobacillus plantarum; Lactobacillus sakei; Lactobacillus sanfranciscensis; Posibacteria