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Lactic acid bacteria are used for food improvement in a wide range of fermented foods and some strains can also produce antimicrobial peptides, named bacteriocins, active against food pathogens such as Listeria monocytogenes and Staphyloccus aureus.The aim of this study was to characterize antimicrobial peptides produced by two Enterococcus faecium strains, a Lactobacillus plantarum, a Lactococcus lactis ssp. cremoris and five Lactococcus lactis ssp. lactis. All bacteria were isolated from Italian cheeses. Lactococci and enterococci were grown in M17 broth at 37C while Lactobacillus plantarum was grown in MRS broth at 30C. All cultures were performed both in microaerophylia and in presence of O2 to test whether stress (presence of oxigen) stimulates bacteriocins production. The whole culture and the cell-free supernatant of both strains were tested against pathogens using well-diffusion assay and their antimicrobial activity was shown by the formation of an inhibition zone around the well on the plate. In order to evaluate the time of maximum production of bacteriocins, cultures at different growth phases were tested. Two different peptide purification protocols were tested in order to recover a larger amount of bateriocins. A biochemical characterization was performed: molecular size determination by Tricine-SDS-PAGE, thermostability at 70C, 80C and 90C and proteinase k sensitivity. Molecular targeting experiments of bacteriocins encoding genes by PCR were performed in order to confirm the bacteriocins identification. This study demonstrated that all lactococcal strains produced nisin A, the two enterecoccus strain produced enterocin A, while the L. plantarum was able to inhibit pathogen by means of large amounts of lactic acid. The most interesting strains (L. lactis L5 e L7) possess both extracellular and surface-bound bacteriocins and their maximum production occurs during the middle logarithmic phase.

Selenium is a nutritionally essential element important for men, animals and microorganisms. The human recommended intake is about 55g/day. Selenium deficiency may be associated with various chronic diseases such as oxidative stress, cardiovascular diseases and some kinds of cancer. Since selenium content in plantbased food depends on its availability from soil, the level of this element in food and feeds varies among regions. There are several potential options to improve human selenium intake such as soil fertilization and supplementation of food. Development and commercialization of organic forms of selenium have initiated a new era in the availability of selenium-enriched products. Selenium enriched chicken, pork and beef can be produced using organic selenium in the diet of farm animals. The use of Se-enriched yeasts and lactobacilli is also an interesting approach to solve seleniumdeficiency: when these microorganisms are added to ice-creams or yogurt, organic form of Se is then released at gut level. In this studywefocused our attention on a Lactobacillus buchneri strain, already commercialized as probiotic strain, able to overaccumulate selenium during its growth in aMRS modified medium supplemented with sodium selenite (2.19 g/l). Our purpose was to understand, by means of a comparative proteomic approach, if selenium addition could modify protein expression causing variations in the metabolism of the strain. Two-dimensional electrophoresis followed by MALDI-TOF/TOF mass spectrometry was performed on Lactobacillus buchneri 26 grown in a MRS medium fortified with selenium and in a control condition (same medium without selenium). The analysis was developed both on the cytosolic and the membrane fraction in the acidic pI range (4-7). From the cytosolic fraction 20 differentially expressed proteins were detected: 15 upregulated and 5 down-regulated. Image analysis on the membrane gels is actually in progress.

Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline isoelectric points, because of the limits inherent in 2DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pIs, the most crowded areas lying between pI 4-7 and pI 9-11. The aim of the present research was to set up a general, simple and standardizable 2DE protocol suitable for studying alkaline proteome of L. hilgardii, a Gram-positive bacillus isolated from wine. The method has been also tested on a Gram-negative bacterium able to degrade aromatic pollutants, A. radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup-loading), and different reducing agents (DTT and HED) were tested and compared. The proposed protocol was found to resolve alkaline proteins from both our Lactobacillus and Acinetobacter strains efficiently, in spite of their different external layers, thus enabling more .......................

Acinetobacter radioresistens S13 is able to grow on phenol or benzoate as the sole carbon and energy source: both these compounds are catabolized through the ²-ketoadipate pathway. Genes encoding the catabolic enzymes for degradation of aromatic compounds are localized on A. radioresistens S13 chromosome and organized in, at least, two distinct sets, one for benzoate degradation and another for phenol catabolism. In the present study, the growth and biodegradation kinetics for benzoate and phenol, and an easily metabolized substrate (acetate) were established. Benzoate was degraded slower and supports a less rapid and efficient growth than either acetate or phenol. A combined transcript-proteomic analysis of some of the major catabolic genes and their products nonetheless has shown that benzoate induces the expression of both benzoate and phenol catabolic operons. This result was confirmed by the fact that benzoate-acclimatized bacteria were rapidly able to degrade phenol too. Finally, the growth and biodegradation kinetics for different mixtures of acetate, benzoate and phenol were determined. Results indicate that a hierarchy of substrate utilization, ................

All fermented foods are subject to the risk of biogenic amine contamination. Histamine and tyramine are among the most toxic amines for consumers’ health, exerting undesirable effects on the central nervous and vascular systems, but putrescine and cadaverine can also compromise the organoleptic properties of contaminated foods. These compounds are produced by fermentino microbial flora that decarboxylate amino acids to amines. Little is known of the factors which induce biosynthesis of decarboxylating enzymes and/or which modulate their catalytic activity: the accumulation of amines is generally considered to be a mechanism that contrasts an acidic environment and/or that produces metabolic energy through coupling amino acid decarboxylation with electrogenic amino acid/amine antiporters. Two Lactobacillus strains, Lactobacillus sp. 30a (ATCC 33222), and a Lactobacillus sp. strain (w53) isolated from amine-contaminated wine, carrying genetic determinants for histidine decarboxylase (HDC) and ornithine decarboxylase (ODC), were studied and the influence of some environmental and nutritional parameters on amine production and protein biosynthesis was analyzed through a proteomic approach; this is the first report of a proteomic analysis of amine-producing bacteria. HDC and ODC biosynthesis were shown to be closely dependent on the presence of high concentrations of free amino acids in the growth medium and to be modulated by the growth phase. The stationary phase and high amounts of free amino acids also strongly induced the biosynthesis of an oligopeptide tran sport protein belonging to the proteolytic system of Lactic Acid Bacteria. At least two isoforms of glyceraldehyde-3-phosphate dehydrogenase, with different Mr, pI and expression profiles, were identified from Lactobacillus sp. w53: the biosynthesis of one isoform, in particular, is apparently repressed by high concentrations of free amino acids. Other proteins were identified from the Lactobacillus proteome, affording a global knowledge of protein biosynthesis modulation during biogenic amine production.

Two novel catechol 1,2-dioxygenase genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catAa and catAb , they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH2-terminal and COOH-terminal regions have no sequences in common, this may explain their different thermal and pH stability. Polyclonal antibodies raised agaist an amino-terminal catAa peptide or the whole catAb protein were used to establish their inducible and differetial expression patters upon bacterial growth in pheno or benzoate. Tha catAa protein (Iso a) was induced by both phenol and benzoate though with different kinetics, whereas the catAb product (iso b) was constitutively produced at low levels that increased only during growth in the presence of benzoate.

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of beta-phenylethylamine. Kinetics of tyramine and beta-phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine-enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to beta-phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2-DE and MALDI-TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and beta-phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down-regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy-supplying routes. The most interesting finding is a membrane-bound TDC highly over-expressed during amine production. This is the first evidence of a true membrane-bound TDC, longly suspected in bacteria on the basis of the gene sequence.

Selenium (Se) has received great attention in the last few years, as it is considered to be essential for human health (prevention of viral infections, heart diseases and ageing-related diseases). Se deficiency can be counteracted by the administration of selenium-enriched probiotics that are able to convert inorganic selenium into less toxic and more bio-available organic forms. This study was performed on Lactobacillus reuteri Lb2 BM DSM 16143, a probiotic LAB previously demonstrated to be able to fix Se into selenocysteines. The aim was to assess Se influence on its metabolism, by a 2-DE proteomic approach, on two different cellular districts: envelope-enriched and extracellular proteomes. While in the envelope-enriched fraction 15 differentially expressed proteins were identified, in the extracellular proteome no quantitative difference was detected. However, at a molecular level, we observed the insertion of Se into selenocysteine, exclusively under the stimulated conditions. The obtained results confirmed the possibility to use L. reuteri Lb2 BM DSM 16143 as a carrier of organic Se that can be easily released in the gut becoming available for the human host. © 2014 the Partner Organisations.