Jone Rossi

Arginine Catabolism by Sourdough Lactic Acid Bacteria: Purification and Characterization of the Arginine Deiminase Pathway Enzymes from Lactobacillus sanfranciscensis CB1

ABSTRACT 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°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°C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

ABSTRACT A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.

Lactic Acid Bacteria Isolated from Dairy Products Inhibit Genotoxic Effect of 4-Nitroquinoline-1-oxide in SOS-Chromotest

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.

Production of amylase(s) by Schwanniomyces castellii and Endomycopsis fibuligera

Schwanniomyces castellii and Endomycopsis fibuligera Produced extracellular amylase(s) when grown on various carbon sources and at different pH values. Both yeast species showed significant amylase synthesis in the presence of either maltose or soluble starch. On the other substrates tested (glucose, cellobiose, sucrose, trehalose, melezitose, raffinose, ethanol, glycerol) differences were found regarding growth and amylase production. Free glucose in the culture medium apparently inhibited enzyme synthesis. The pH range allowing maximal growth and amylase production was 4.5–6.0 for E. fibuligera and 5.5–7.0 for S. castellii.

Accelerated ripening of Pecorino Umbro cheese

We have investigated accelerating the ripening of Pecorino Umbro cheese by adding crude cytoplasmic extract from Pseudomonas fluorescens, non-starter lactic acid bacteria (NSLAB) or cheese slurry. Microbiological and biochemical analyses and sensory evaluation were carried out on control and experimental cheeses over 28 d ripening. In the cheeses containing NSLAB or slurry, counts of mesophilic lactobacilli ranged from log 7.6 at day 1 to ∼ log 8.6 cfu/g after 28 d ripening. ∼ 2 log cycles higher than in the control cheese. All the experimental cheeses contained higher levels than the control of total free amino acids and N soluble at pH 4.6 and in 120 g trichloroacetic acid/l. Compared with the control, higher aminopeptidase and dipeptidase activities were found in the cheeses containing NSLAB and slurry, and especially in those containing the Pseudomonas enzyme. The cheeses containing NSLAB or slurry were characterized by an accumulation of short peptides (M r < 2000) detected by FPLC. Although the cheese containing enzyme had an atypical flavour, the addition of mesophilic lactobacilli reduced from 60 to 28 d the ripening period of Pecorino Umbro cheese, without the appearance of off flavour.

α-Amylase and glucoamylase production by Schwanniomyces castellii

A chromogenic substrate (Cibachron blue-amylose), and soluble starch and maltose were used to characterize the amylolytic system from Schwanniomyces castellii 3754. The strain was able to produce inducible α-amylase (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3) when grown on different C sources. The effect of the C source was slightly different for α-amylase and glucoamylase production. Melezitose, maltose and soluble starch enhanced both α-amylase and glucoamylase synthesis to nearly the same extent; amylose, trehalose and cellobiose particularly induced α-amylase synthesis. The optimal pH for the release of both amylases was 5.5–7.0; maximal α-amylase synthesis, on the other hand, was observed in the medium buffered at pH 6.0. The optimal pH for α-amylase and glucoamylase activity was in the range of 4.5–7.2 and 4.2–5.5, respectively. Temperatures allowing maximal activity were 45°C for α-amylase and 45–52°C for glucoamylase; a rapid decline of both activities was observed just above these temperatures.

Effect of medium composition on microbial utilisation of citrus waste by mixed fungal culture

SummaryThe growth behaviour of Memnoniella echinata and Fusarium roseum was examined in slurry fermentation systems using untreated orange peel as substrate. A composite experiment was then designed to study the effect of orange peel initial concentration and the effect of the nitrogen: peel ratio on crude protein yidld (Yp) and protein enrichment (Zp) of the final biomass.The more concentrated the peel slurry, the greater the substrate inhibitory effect on microbial growth becomes.Finally, multiple regression technique allowed both the experimental values of Yp and Zp to be reconstructed with mean percentage errors smaller than 4% and 8%, respectively, and the optimal operating strategy for such SCP production process to be determined.