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Several strains of Enterococcus spp. are capable of producing bacteriocins with antimicrobial activity against important bacterial pathogens in dairy products. In this study, the bacteriocins produced by two Enterococcus strains (Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch), isolated from cheeses, were characterized and tested for their capability to control growth of Listeria monocytogenes 426 in experimentally contaminated fresh Minas cheese during refrigerated storage. Both strains were active against a variety of pathogenic and non-pathogenic microorganisms and bacteriocin absorption to various L. monocytogenes, Enterococcus faecalis ATCC 19443 and Lactobacillus sakei ATCC 15521 varied according to the strain and the testing conditions (pH, temperature, presence of salts and surfactants). Growth of L. monocytogenes 426 was inhibited in cheeses containing E. mundtii CRL35 up to 12 days at 8 C, evidencing a bacteriostatic effect. E. faecium ST88Ch was less effective, as the bacteriostatic affect occurred only after 6 days at 8 C. In cheeses containing nisin (12.5 mg/kg), less than one log reduction was observed. This research underlines the potential application of E. mundtii CRL35 in the control of L. monocytogenes in Minas cheese. Fil: Vera Pingitore, Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Universidade de Sao Paulo; Brasil Fil: Todorov, Svetoslav Dimitrov. Universidade de Sao Paulo; Brasil Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Franco, Bernadette Dora Gombossy de Melo. Universidade de Sao Paulo; Brasil

Listeria monocytogenes is a foodborne pathogen causative of opportunistic infections. Listeriosis is associated with severe infections in pregnant women causing abortion or neonatal listeriosis. An alternative to antibiotics are safe novel bacteriocins peptides such as enterocin CRL35 with strong antilisterial activity produced by Enterococcus mundtii CRL35. In the present paper, our goal is to study the effectiveness of this peptide and the producer strain in a murine model of pregnancy-associated listeriosis. A single dose of 5×10(9) colony-forming unit of L. monocytogenes FBUNT (Faculty of Biochemistry-University of Tucumán) resulted in translocation of pathogen to liver and spleen of BALB/c pregnant mice. The maximum level of Listeria was observed on day 3 postinfection. Interestingly, the intragastric administration of enterocin CRL35 significantly reduced the translocation of the pathogen to vital organs. On the other hand, the preadministration of E. mundtii CRL35 slightly inhibited this translocation. Listeria infection caused a significant increase in polymorphonuclear leukocytes at day 3 postinfection compared to the noninfected group. This value was reduced after the administration of enterocin CRL35. No significant changes were observed in either white blood cells or lymphocytes counts. Based on the data presented in the present work enterocin CRL35 would be a promising alternative for the prevention of Listeria infections.

Graphical abstract Highlights ► A chimerical gene encoding a hybrid bacteriocin–microcin was generated by PCR. ► Ent35–MccV is a hybrid bacteriocin containing enterocin CRL35 and microcin V. ► Ent35–MccV is active against Gram-positive and Gram-negative bacteria. ► Enterohemorrhagic Escherichia coli and Listeriamonocytogenes are sensitive to Ent35–MccV., Bacteriocins and microcins are ribosomally synthesized antimicrobial peptides that are usually active against phylogenetically related bacteria. Thus, bacteriocins are active against Gram-positive while microcins are active against Gram-negative bacteria. The narrow spectrum of action generally displayed by bacteriocins from lactic acid bacteria represents an important limitation for the application of these peptides as clinical drugs or as food biopreservatives. The present study describes the design and expression of a novel recombinant hybrid peptide combining enterocin CRL35 and microcin V named Ent35–MccV. The chimerical bacteriocin displayed antimicrobial activity against enterohemorrhagic Escherichia coli and Listeria monocytogenes clinical isolates, among other pathogenic bacteria. Therefore, Ent35–MccV may find important applications in food or pharmaceutical industries.

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B12 amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.

Some strains of Lb. reuteri produce cobalamin (vitamin B12). Cobalamin biosynthesis relies on the sequential action of more than 25 enzymes in a complex metabolic pathway. We have cloned, expressed and characterized the gene in Lb. reuteri that codes for the S-adenosy L-methionine uroprophyrinogen III methyltransferase/synthase (CobA/HemD), a key bifunctional enzyme in the biosynthesis of cobalamin and other tetrapyrrols. Fil: Vannini, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Rodríguez, Adriana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Vera, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: de Valdéz, Graciela F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Taranto, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Reactive oxygen species are involved in various aspects of intestinal inflammation and tumor development. Decreasing their levels using antioxidant enzymes, such as catalase (CAT) or superoxide dismutase (SOD) could therefore be useful in the prevention of certain diseases. Lactic acid bacteria (LAB) are ideal candidates to deliver these enzymes in the gut. In this study, the anti-inflammatory effects of CAT or SOD producing Lwere evaluated using a trinitrobenzenesulfonic acid (TNBS) induced Crohn's disease murine model. Engineered Lactobacillus casei BL23 strains producing either CAT or SOD, or the native strain were given to mice before and after intrarectal administration of TNBS. Animal survival, live weight, intestinal morphology and histology, enzymatic activities, microbial translocation to the liver and cytokines released in the intestinal fluid were evaluated. The mice that received CAT or SOD-producing Lshowed a faster recovery of initial weight loss, increased enzymatic activities in the gut and lesser extent of intestinal inflammation compared to animals that received the wild-type strain or those that did not receive bacterial supplementation. Our findings suggest that genetically engineered Lthat produce antioxidant enzymes could be used to prevent or decrease the severity of certain intestinal pathologies. (C) 2010 Elsevier B.V. All rights reserved.

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

Salivaricin CRL 1328 is a heat-stable bacteriocin produced by Lactobacillus salivarius CRL 1328, a strain isolated from healthy human vagina, with potential applications for preventing urogenital infections. The objective of this study was to characterize the locus responsible for salivaricin CRL 1328 production and its mechanism of action against Enterococcus faecalis MP97 as the sensitive strain. Oligonucleotides were designed based on sequences of antimicrobial peptides previously described in the literature. The salivaricin CRL 1328 cluster was identified, sequenced and analyzed. This cluster was similar to the previously described ABP118 which codified for a two-peptide bacteriocin. The putative mature peptides of salivaricin CRL 1328, Salα and Salβ were chemically synthesized. These peptides did not show bacteriocin activity when assayed individually. Both peptides exhibited optimal antimicrobial activity at an equimolar ratio. Spectroscopic fluorescence assays were carried out using the synthetic peptides to study the effect of salivaricin on proton motive force. This bacteriocin was shown to dissipate membrane potential and the transmembrane proton gradient, both components of proton motive force. E. faecalis MP97 cells treated with salivaricin CRL 1328 peptides were observed in transmission electron microscopy which revealed ultrastructural modifications of the cell wall. Fil: Vera Pingitore, Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Bovine mastitis (BM) is a costly disease in dairy cattle production. The prevention and treatment of mastitis is performed by applying antimicrobial products that negatively affect milk quality. In the last years, the use of probiotic microorganisms to prevent infections in humans and animals has being aggressively studied. Samples from teat canal and milk (foremilk and stripping) were taken from healthy and mastitic mammary quarters. A screening of the surface properties and antagonistic substances production of lactic acid bacteria (LAB) isolated from the mammary gland was performed to select potential probiotic strains to prevent mastitis. Somatic cell count, physico-chemical and microbiological studies were carried out. Pre-selected microorganisms were genetically identified. Compared with stripping milk, foremilk showed lower levels of fat and higher levels of pH, density, microorganism numbers, lower percentage of strains with mean and high hydrophobicity and mean autoaggregation and higher number of strains able to produce hydrogen peroxide and bacteriocins. The other parameters analyzed were not statistically significant. One hundred and two LAB strains were isolated. Most of them had low degrees of hydrophobicity and autoaggregation. No correlation between these properties was found. Antagonistic metabolites were mainly produced by strains isolated from healthy quarters. Most of the pre-selected strains were identified as Streptococcus bovis and Weissella paramesenteroides. Three bacteriocin-producers were found and their products partially characterized. The results of this work are the basis for the further design of a specie-specific probiotic product able to prevent BM. Fil: Espeche, Maria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Otero, María Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Nader, Maria Elena Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Objectives: Subclass IIa bacteriocins are characterized by a hydrophilic N-terminal domain that shares a YGNGVxCxxxxC consensus and a variable hydrophobic C-terminus. Enterocin CRL35 is a 43- amino-acid heat stable peptide with antilisterial activity. Short synthetic peptides derived from the N-terminal half of enterocin CRL35 and other subclass IIa bacteriocins were evaluated for antimicrobial properties. Methods: In vitro activities of synthetic peptides were evaluated in complex, chemically defined and minimal media. MIC assays were performed by the agar well-diffusion method. Fluorescence assays to evaluate the dissipation of membrane potentials in intact cells were carried out. Time-kill kinetics of Listeria innocua cells with the active peptide were performed. Results and conclusions: A 15-mer peptide derived from enterocin CRL35 inhibited the growth of L. innocua and Listeria monocytogenes in synthetic/minimal media and dissipated the membrane potential of sensitive cells, with MICs of 10 and 50 mM, respectively. 15-mer derivatives from other class IIa bacteriocins (mesentericin Y105, pediocin PA-1 and piscicolin 126) also showed antimicrobial activities. Fil: Salvucci, Emiliano Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

The three-dimensional crystal structure of the PduO-type corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) has been solved to 1.68-Å resolution. The functional assignment of LrPduO as a corrinoid adenosyltransferase was confirmed by in vivo and in vitro evidence. The enzyme has an apparent [Formula: see text] of 2.2 μM and [Formula: see text] of 0.13 μM and a k(cat) of 0.025 s(−1). Co-crystallization of the enzyme with Mg-ATP resulted in well-defined electron density for an N-terminal loop that had been disordered in other PduO-type enzyme structures. This newly defined N-terminal loop makes up the lower portion of the enzyme active site with the other half being contributed from an adjacent subunit. These results provide the first detailed description of the enzyme active site for a PduO-type adenosyltransferase and identify a unique ATP binding motif at the protein N terminus. The molecular architecture at the active site offers valuable new insight into the role of various residues responsible for the human disease methylmalonic aciduria.

Vitamin production by lactic acid bacteria (LAB) has gained the attention of the scientific community. It has been shown that certain foods fermented with LAB contain high levels of B‐group vitamins as a result of microbial biosynthesis. Recently, it has also been reported that LAB and other beneficial microorganisms have the potential to produce vitamins in situ in the gastrointestinal (GI) tract. This chapter reviews the newest studies describing B‐group vitamin‐producing LAB, and shows that increasing the B‐group vitamin concentrations in yogurts and fermented milks is possible through judicious selection of the microbial species and cultivation conditions. The B‐group (or B‐complex) vitamins include thiamin, riboflavin, niacin, pyridoxin, pantothenic acid, biotin, folate, and cobalamin. The chapter discusses the most recent applications of beneficial microorganisms in order to increase the concentration of vitamins through microbial biosynthesis as an economically attractive alternative to mandatory fortification of foods. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Laiño, Jonathan Emiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Juarez del Valle, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Savoy, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Taranto, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Lactobacillus fermentum CECT 5716, isolated from human milk, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays, which suggests a strong potential as a probiotic strain. In this work, some phenotypic properties of L. fermentum CECT 5716 were evaluated, and the genetic basis for the obtained results was searched for in the strain genome. L. fermentum CECT 5716 does not contain plasmids and showed neither bacteriocin nor biogenic amine biosynthesis ability but was able to produce organic acids, glutathione, riboflavin, and folates and to moderately stimulate the maturation of mouse dendritic cells. No prophages could be induced, and the strain was sensitive to all antibiotics proposed by European Food Safety Authority (EFSA) standards, while no transmissible genes potentially involved in antibiotic resistance were detected in its genome. Globally, there was an agreement between the phenotype properties of L. fermentum CECT 5716 and the genetic information contained in its genome. © 2015, Springer-Verlag Berlin Heidelberg., This work was supported by CSD2007-00063 (FUN-C-FOOD, Consolider-Ingenio 2010), CYTED-IBEROFUN, and AGL2013-41980-P projects from the Ministerio de Ciencia e Innovación (Spain), a research contract funded by Biosearch Life (Granada, Spain) and by funding from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), Argentina.

Lactobacillus rhamnosus ATCC 7469 was able to grow in glycerol as the sole source of energy in aerobic conditions, producing lactate, acetate, and diacetyl. A biphasic growth was observed in the presence of glucose. In this condition, glycerol consumption began after glucose was exhausted from the culture medium. Glycerol kinase activity was detected in L. rhamnosus ATCC 7469, a characteristic of microorganisms which catabolize glycerol in aerobic conditions. Genetic analysis revealed that this strain possesses two glycerol kinase genes: gykA and glpK, that encode for two different glycerol kinases GykA and GlpK, respectively. The glpK geneis associated in an operon with α-glycerophosphate oxidase (glpO) and glycerol facilitator (glpF) genes. Transcriptional analysis revealed that only glpK is expressed when L. rhamnosus was grown on glycerol.

We found thatLactobacillus reuteriCRL1098, a lactic acid bacterium isolated from sourdough, is able to produce cobalamin. The sugar-glycerol cofermentation in vitamin B12-free medium showed that this strain was able to reduce glycerol through a well-known cobalamin-dependent reaction with the formation of 1,3-propanediol as a final product. The cell extract ofL. reutericorrected the coenzyme B12requirement ofLactobacillus delbrueckiisubsp.lactisATCC 7830 and allowed the growth ofSalmonella entericaserovar Typhimurium (metE cbiB) andEscherichia coli(metE) in minimal medium. Preliminary genetic studies of cobalamin biosynthesis genes fromL. reuteriallowed the identification ofcobgenes which encode the CobA, CbiJ, and CbiK enzymes involved in the cobalamin pathway. The cobamide produced byL. reuteri, isolated in its cyanide form by using reverse-phase high-pressure liquid chromatography, showed a UV-visible spectrum identical to that of standard cyanocobalamin (vitamin B12).

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705alpha and Lac705beta. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705alpha/Lac705beta peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705beta peptide alone. Furthermore, Lac705beta increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705alpha protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Deltapsi and DeltapH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705alpha peptide is responsible for receptor recognition, and the Lac705beta peptide is the active component on the cell membrane of CRL691 cells.

The microbial adhesion process includes passive forces; electrostatic interactions; hydrophobic, steric forces; lipoteichoic acids; and specific structures, such as external appendages (lectins) and (or) extracellular polymers. In a previous work, we showed that Lactobacillus animalis, L. fermentum, and L. fermentum ssp. cellobiosus had lectinlike proteic structures on their surfaces and high hydrophobicity values on the cell surface of L. fermentum ssp. cellobiosus. Here, we examined the presence of the bacterial forces or structures that could be involved in the interaction between bacteria and epithelial cells. Lactobacillus animalis and L. fermentum possessed a net negative surface charge, whereas L. fermentum ssp. cellobiosus showed similar affinity to both cationic and anionic exchange resins, aggregated in the presence of ammonium sulfate, and had high affinity (75.4%) to a hydrophobic matrix. Only L. animalis was shown to have ribitol teichoic acids in the cell wall. The amount of polysaccharides from cell walls varied between different strains, with L. fermentum ssp. cellobiosus having the highest concentration. Lectin extracts obtained from lactobacilli did not possess sugar residues, thereby demonstrating the proteic nature of the superficial surface structures of three strains. The lactic acid bacteria studied here showed different surface determinants, which could be involved in the interactions between these lactobacilli and intestinal epithelial cells.Key words: adhesion, lactobacilli, probiotics, chickens.

Lactic acid bacteria display a relatively simple and well-described metabolism where the sugar source is converted mainly to lactic acid. Here we will shortly describe metabolic engineering strategies on the level of sugar metabolism, that lead to either the efficient re-routing of the lactococcal sugar metabolism to nutritional end-products other than lactic acid such as L-alanine, several low-calorie sugars and oligosaccharides or to enhancement of sugar metabolism for complete removal of (undesirable) sugars from food materials. Moreover, we will review current metabolic engineering approaches that aim at increasing the flux through complex biosynthetic pathways, leading to the production of the B-vitamins folate and riboflavin. An overview of these metabolic engineering activities can be found on the website of the Nutra Cells 5th Framework EU-project (www.nutracells.com). Finally, the impact of the developments in the area of genomics and corresponding high-throughput technologies on nutraceutical production will be discussed.

Enterococcus mundtii CRL35 is a bacteriocinogenic strain isolated from an artisanal cheese of northwestern Argentina. Here we report its draft genome sequence, consisting of 82 contigs. In silico genomic analysis of biotechnological properties was performed to determine the potential of this microorganism to be used in a food model system. Fil: Bonacina, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Suárez, Nadia Elina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina

Aims: The objective of this work was to evaluate the fermentation pattern of and the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in milk batch cultures under controlled pH (4·5, 5·0 and 6·2). Methods and Results: EPS concentration was estimated by the phenol/sulphuric acid method and the chemical composition of purified EPS by HPLC. Fermentation products and residual sugars were determined by HPLC and enzymatic methods. The micro-organism shifted from a homofermentative to a heterofermentative pattern, producing acetate (9·5 and 5·8 mmol l–1) at pH 5·0 and 6·2, respectively, and acetate (7·1 mmol l–1) plus succinate (1·2 mmol l–1) at pH 4·5. At pH 5·0 and 6·2, acetate derived from citrate while at pH 4·5 it came from both citrate and pyruvate splitting. The EPS has a MW of 105–106 and contains phosphate (81% in average), rhamnose (traces), and glucose and galactose in a ratio of 1 : 1 (pH 6·2) and 2 : 1 (pH 4·5 and 5·0). The highest production (549 mg l–1) corresponded to pH 5·0 and the lowest (49 mg l–1) to pH 6·2. Conclusions: The heterofermentative pattern of Lact. helveticus ATCC 15807 was linked to alternative pyruvate pathways and/or citrate metabolism according to the environmental pH. The EPS production was improved under low environmental pH conditions. Significance and Impact of the Study: This work provides relevant information of the effect of pH on the metabolism of citrate and EPS production by Lact. helveticus. It may contribute to improve technological aspects of ropy and citrate-utilizing lactic acid bacteria.

The antimicrobial peptide Enterocin CRL35, a class II bacteriocin, produces at high concentrations (8 microg ml(-1)) localized holes in the wall and cellular membrane of Listeria monocytogenes, reflected in the efflux of macromolecules such as proteins and other ultraviolet-absorbing materials. At lower concentrations (0.5 microg ml(-1)), neither ultra structural changes nor macromolecules efflux were observed, however potassium and phosphate ions were released, dissipating the proton motive force. As a result the bacteria were killed.

Lactobacillus casei CRL 705, isolated from a dry fermented sausage, produces an antibacterial peptide which is active against Listeria monocytogenes. Previous studies have shown that this compound is potentially useful to control food-borne pathogens in ground meat. In view of the potential application of this antimicrobial substance in food fermentation, a detailed biochemical analysis of this peptide is required. In this work, the purification and amino acid sequence of this bacteriocin is presented. The adsorption-desorption pH-dependent property of lactocin 705 was exploited for purification. The active extract was further subjected to RP-HPLC and SDS-PAGE. The active antimicrobial band was electroeluted from an SDS-PAGE gel and its amino acid sequence determined. Lactocin 705 had an estimated molecular weight of 3357.80 and an isoelectric point of 10.03. The peptide contains a high ratio of glycine residues and does not show any modified amino acids, like lanthionine or beta-methyllanthionine. The sequence was unique when compared to several databases.

The bile salt hydrolase (BSH) of Lactobacillus reuteri CRL 1098 is a single, constitutive, intracellular enzyme which is only detectable in stationary phase cells. It has optimal activity at pH 4.5–5.5 and 37–45 °C. The enzyme (80 kDa apparent mass) has sulphydryl groups in the catalytic active site and hydrolyzes both glycine and taurine conjugated bile acids with higher affinity for glyco-conjugates.

Beta-lactamase activity was studied in Neisseria gonorrhoeae strains. Optimum temperature was found to be 37 degrees C. The enzyme was inactivated at temperatures higher than 60 degrees C, but remained active during storage at low temperatures (4 degrees C, -30 degrees C and -70 degrees C) for two months. Enzyme activity was observed within a pH range of 5.8-8.0, while the optimum pH was 7.0-7.2. Addition of Ni2+, Fe2+, Mn2+ and p-chloromercurybenzoate to the reaction buffer exerted a negative effect upon the activity, whereas Hg2+ and ethylene diamine tetra-acetic acid produced complete inhibition. These results would indicate the presence of -SH groups at the catalytic site of the enzyme.

Even though many sequences and structures of bacteriocins from lactic acid bacteria have been fully characterized so far, little information is currently available about bacteriocins heterologously produced by Escherichia coli. For this purpose, the structural gene of enterocin CRL35, munA, was PCR-amplified using specific primers and cloned downstream PelB sequence in the pET22b (+) expression vector. E. coli Rosetta (DE3) pLysS was chosen as the host for production and enterocin was purified by an easy two-step protocol. The bacteriocin was correctly expressed with the expected intramolecular disulfide bond. Nevertheless, it was found that a variant of the enterocin, differing by 12 Da from the native polypeptide, was co-expressed by E. coli Rosetta in comparable amount. Indeed, the mutant bacteriocin contained two amino acid substitutions that were characterized by matrix assisted laser desorption ionization-time of flight (MALDI-TOF) and HPLC-electrospray (ESI)-Q-TOF tandem mass spectrometry (MS/MS) sequencing. This is the first report regarding the production of mutants of pediocin-like bacteriocins in the E. coli expression system. Fil: Masias, Ruth Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Picariello, Gianluca. Consiglio Nazionale delle Ricerche (CNR). Istituto di Scienze dell’Alimentazione; Italia Fil: Acuña, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); Argentina Fil: Morero, Roberto Dionisio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Saavedra, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Centro de Referencia Para Lactobacilos (i); Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina Fil: Minahk, Carlos Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentina

We report the draft genome sequence of the bacteriocin producer Enterococcus faecium strain CRL 1879, isolated from a northwestern Argentinian artisanal cheese. The draft genome sequence is composed of 73 contigs for 2,886,747 bp, with 3,140 proteincoding genes. Six biosynthetic clusters for bacteriocin class II production were found. Typical virulence determinants, which have relevance in food safety, were not present. Fil: Suárez, Nadia Elina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Složilová, Ivana. Institute of Chemical Technology; República Checa Fil: Bonacina, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Demnerová, Kateřina. Institute of Chemical Technology; República Checa Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Lactobacillus rhamnosus ATCC 7469 exhibited diauxie when grown in a medium containing both glucose and citrate as energy source. Glucose was used as the primary energy source during the glucose-citrate diauxie. Uptake of citrate was carried out by an inducible citrate transport system. The induction of citrate uptake system was repressed in the presence of glucose. This repression was reversible and mediated by cAMP.

Food-related lactic acid bacteria (LAB) as well as human gut commensals such as bifidobacteria can de novo synthesize and supply vitamins. This is important since humans lack the biosynthetic capacity for most vitamins and these must thus be provided exogenously. Although vitamins are present in a variety of foods, deficiencies still occur, mainly due to malnutrition as a result of insufficient food intake and because of poor eating habits. Fermented milks with high levels of B-group vitamins (such as folate and riboflavin) can be produced by LAB-promoted and possibly bifidobacteria-promoted biosynthesis. Moreover, certain strains of LAB produce the complex vitamin cobalamin (or vitamin B12). In this review, fermented foods with elevated levels of B-group vitamins produced by LAB used as starter cultures will be covered. In addition, genetic abilities for vitamin biosynthesis by selected human gut commensals will be discussed. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Milani, Christian. University of Parma. Department of Genetics, Biology of Microorganisms, Anthropology and Evolution. Laboratory of Probiogenomics; Italia; Fil: Savoy, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: van Sinderen, Douwe. National University of Ireland Cork. Bioscience Institute. Department of Microbiology & Alimentary Pharmabiotic Centre; Irlanda; Fil: Ventura, Marco. University of Parma. Department of Genetics, Biology of Microorganisms, Anthropology and Evolution. Laboratory of Probiogenomics; Italia

We report the draft genome sequence of Enterococcus mundtii CRL1656, which was isolated from the stripping milk of a clinically healthy adult Holstein dairy cow from a dairy farm of the northwestern region of Tucumán (Argentina). The 3.10-Mb genome sequence consists of 450 large contigs and contains 2,741 predicted protein-coding genes.

The Lactococcus lactis subsp. lactis biovar diacetylactis citrate permease P is encoded by the citP gene borne by an 8.3-kb Cit+ plasmid (F. Sesma, Gardiol, D., de Ruiz Holgado, A.P. and de Mendoza, D., Appl. Environ. Microbiol. 56 (1990) 2099–2103). By subcloning different DNA fragments from the Cit+ plasmid it was found that citP is the only plasmidic gene required for the transport of citrate. Northern blot analysis revealed that citP is transcribed in two mRNA species with lengths of 2.9 and 1.9 kb. The transcriptional pattern, the total amount of citP mRNAs and the transport of citrate were unaffected by the presence of citrate in the growth media. Our results support the conclusion that transcription of citP as well as citrate transport are not induced by citrate in Lactococcus lactis subsp. lactis biovar diacetylactis.

Lactic acid bacteria (LAB) are widely used as probiotics in humans and animals to restore the ecological balance of different mucosa. They help in the physiological functions of newborn calves that are susceptible to a variety of syndromes. The criteria for the selection of strains for the design of probiotic products are not available. Based in the host-specificity of the indigenous microbiota, 96 LAB isolates from faeces and oral cavity of calves were obtained. The surface properties were screened showing a small number of highly hydrophobic or autoagglutinating isolates. Also, a group produced H(2)O(2) and were able to inhibit pathogens, and two strains were bacteriocin-producers. Some grew at very low pH and high bile concentrations. The strains sharing some of the specific properties evaluated were identified genetically, assayed their compatibility and exopolysaccharide production. The results allow going further in the establishment of criteria to select strains to be included in a multi-strain-probiotic-product to be further assayed in animals.

The search for new antimicrobial peptides produced by lactic acid bacteria and other Gram-positive microorganisms has become an interesting field of research in the past decades. The fact that bacteriocins are active against numerous foodborne and human pathogens, are produced by generally regarded as safe (GRAS) microorganisms, and are readily degraded by proteolytic host systems makes them attractive candidates for biotechnological applications. However, before suggesting or choosing a new bacteriocin for future technology developments, it is necessary to elucidate its biochemical structure and its mode of action, which may be carried out once the bacteriocin is purified to homogeneity. This chapter focuses on describing the main strategies used for the purification of numerous bacteriocins. Fil: Saavedra, Maria Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

The use of food-grade microorganisms such as lactic acid bacteria (LAB) is one of the most promising methods for delivering health promoting compounds. Since it is not always possible to obtain strains that have the ability to produce specific compounds naturally or that produce them in sufficient quantities to obtain physiological responses, genetic modifications can be performed to improve their output. The objective of this study was to evaluate if previously studied genetically modified LAB (GM-LAB), with proven in vivo beneficial effects, are just as safe as the progenitor strain from which they were derived. Mice received an elevated concentration of different GM-LAB or the native parental strain from which they were derived during a prolonged period of time, and different health parameters were evaluated. Similar growth rates, hematological values, and other physiological parameters were obtained in the animals that received the GM-LAB compared to those that were fed with the native strain. These results demonstrate that the GM-LAB used in this study are just as safe as the native strains from which they were derived and thus merit further studies to include them into the food chain. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: van Sinderen, Douwe. University College Cork; Irlanda Fil: Hugenholtz, Jeroen. Nizo Food Research, Ede,; Países Bajos Fil: Piard, Jean Christophe. Institut National de la Recherche Agronomique; Francia Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Savoy, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

Aims: To evaluate the inhibition effectiveness of enterocin CRL35 in combination with cell wall, membrane-acting antibiotics and muranolytic enzymes against the foodborne pathogen Listeria. Methods and Results: Synthetic enterocin CRL35 alone and in combination with monensin, bacitracin, gramicidin, mutanolysin and lysozyme were used in this study. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) index assays were performed using Listeria innocua 7 and Listeria monocytogenes FBUNT as sensitive strains. Antibiotics showed positive interactions with the bacteriocin in both strains tested. On the other hand, when mutanolysin and enterocin CRL35 were added to resting cells in a buffer system, the lytic effect of mutanolysin was enhanced. However, the addition of mutanolysin showed no effect on the growth of L. innocua 7 cells in a culture medium. Moreover, mutanolysin allowed the overgrowth of L. innocua 7 cells to an OD similar to control cells in the presence of inhibitory concentration of enterocin CRL35. In contrast, the combination of lysozyme and enterocin CRL35 resulted in a 50% inhibition of the L. innocua 7 growth. Conclusions: Based on our results, we conclude that the combination of synthetic enterocin CRL35 with some antibiotics is effective against L. innocua 7 and L. monocytogenes FBUNT cells, and more importantly the amount of these agents to be used was considerably reduced. The effectiveness of the combination of synthetic enterocin CRL35 with muramidases seems to depend on complex environments, and more detailed studies need to be performed to elucidate this issue. Significance and Impact of the Study: Enterocin CRL35 represents a promising agent that not only can ensure the quality and safety of food but it can also be combined with several antimicrobial agents important in the medical field.

Objective: The aim of this study was to establish the bioavailability of different folates produced by engineered Lactococcus lactis strains using a rodent depletion-repletion bioassay. Methods: Rats were fed a folate-deficient diet, which produces a reversible subclinical folate deficiency, supplemented with different L lactis cultures that were added as the only source of folate. Three bacterial strains that overexpressed the folC, folKE, or folC + KE genes were used. These strains produce folates with different poly glutamyl tail lengths. The growth response of the rats and the concentration of folates in different organs and blood samples were monitored. Results: The folate produced by the engineered strains was able to compensate the folate depletion in the diet and showed similar bioavailability compared with commercial folic acid that is normally used for food fortification. Folate concentrations in organ and blood samples increased significantly in animals that received the folate-producing strains compared with those that did not receive bacterial supplementation. Hematologic studies also showed that administration of the L towns strains was able to revert a partial megaloblastic anemia caused by folate deficiency. No significant differences were observed in the bioavailability of folates containing different glutamyl tail lengths. Conclusion: To our knowledge, this is the first study that demonstrated that folates produced by engineered lactic acid bacteria represent a bioavailable source of this essential vitamin. (C) 2010 Elsevier Inc. All rights reserved.

Publisher Summary This chapter discusses the role of probiotics in cancer prevention. The connection of probiotics in prevention of animal and human cancers has been extensively reviewed. The effects of probiotics on intestinal disorders are most extensively studied because they can beneficially affect the intestinal microbiota. Probiotics such as lactobacilli and bifidobacteria in fermented or culture-containing dairy foods such as yogurt may play a role in reducing the risk of colon cancer. The principal cause that can be attributed to the probiotic and/or fermented product against cancer is the immune surveillance, which differs according to the site where the tumor is present. In contrast to the results observed in the intestine, where the administration of the fermented products itself induces changes on the gut-associated immune cells and in other immune cells as peritoneal macrophages, in the mammary glands, changes on immune cell balances were observed only when immune cells have to act against a target-like tumor cells, avoiding an exacerbated immune response. This fact could be explained because at intestinal level, where there are constant stimuli on the immune system, the immune cell stimulation is maintained with the probiotic administration whereas at other sites, such as mammary glands, the immune system is maintained alert and when the target affects this site, the immune cells quickly react against the agent.

Lactic acid bacteria (LAB) represent a heterogeneous group of microorganisms that are naturally present in many foods. Some selected strains are frequently added as probiotics in order to confer specific benefits to consumers. Many studies have shown that LAB possess therapeutic properties since they are able to prevent the development of some diseases, as shown mostly on animal models for cancer, infections and gastrointestinal disorders such as intestinal inflammation. They have been shown to regulate mucosal immune responses by modulating the production and liberation of regulatory agents such as cytokines by the host. Some of these cytokines, such as the anti-inflammatory interleukin-10 (IL-10), modulate the inflammatory immune response, thus immunomodulation is a mechanism by which LAB can prevent certain inflammatory bowel diseases (IBD). Since oxidative stress participates to the inflammatory processes and to the appearance of damages in pathologies of the gastrointestinal tract of humans such as IBD, LAB could also prevent inflammation by eliminating reactive oxygen species (ROS) through the activity of antioxidant enzymes. Engineering LAB to produce either antioxidant enzymes (such as catalases and superoxide dismutases) or anti-inflammatory cytokines (such as IL-10) is a strategy currently exploited by several groups. These novel strains have successfully been used to prevent inflammatory bowel diseases in animal models and could be evaluated in human clinical trials. Here, we present an overview of the current knowledge of the mechanisms by which LAB can be used to prevent undesired intestinal inflammatory responses. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Rochat, Tatiana. Institut National de la Recherche Agronomique; Francia Fil: Bermudez Humaran, Luis. Institut National de la Recherche Agronomique; Francia Fil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Azevedo, Vasco. Universidade Federal de Minas Gerais; Brasil

The coenzyme B(12) production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B(12) gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi, cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ, cobA/hemD, cbiKLMNQOP, sirA, hemACBL, and cobUSC, hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B(12) itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B(12) pathways previously characterized in a few representatives of the genera Listeria and Salmonella. This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B(12) biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B(12) biosynthesis and reuterin production, which might be implicated in Lb. reuteri's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B(12).

BackgroundSoya and its derivatives represent nutritionally high quality food products whose major drawback is their high content of α-galacto-oligosaccharides. These are not digested in the small intestine due to the natural absence of tissular α-galactosidase in mammals. The passage of these carbohydrates to the large intestine makes them available for fermentation by gas-producing bacteria leading to intestinal flatulence. The aim of the work reported here was to assess the ability of α-galactosidase-producing lactobacilli to improve the digestibility of α-galacto-oligosaccharidesin situ.ResultsGnotobiotic rats were orally fed with soy milk and placed in respiratory chambers designed to monitor fermentative gas excretion. The validity of the animal model was first checked using gnotobiotic rats monoassociated with aClostridium butyricumhydrogen (H2)-producing strain. Ingestion of native soy milk by these rats caused significant H2emission while ingestion of α-galacto-oligosaccharide-free soy milk did not, thus validating the experimental system. When native soy milk was fermented using the α-galactosidase-producingLactobacillus fermentumCRL722 strain, the resulting product failed to induce H2emission in rats thus validating the bacterial model. WhenL. fermentumCRL722 was coadministered with native soy milk, a significant reduction (50 %,P= 0.019) in H2emission was observed, showing that α-galactosidase fromL. fermentumCRL722 remained activein situ, in the gastrointestinal tract of rats monoassociated withC. butyricum. In human-microbiota associated rats,L. fermentumCRL722 also induced a significant reduction of H2emission (70 %,P= 0.004).ConclusionThese results strongly suggest thatL. fermentumα-galactosidase is able to partially alleviate α-galactosidase deficiency in rats. This offers interesting perspectives in various applications in which lactic acid bacteria could be used as a vector for delivery of digestive enzymes in man and animals.

Reactive oxygen species, such as hydrogen peroxide (H2O 2), are involved in various aspects of tumour development. Decreasing their levels can therefore be a promising approach for colon cancer prevention. The objective of this study was to evaluate the effect of catalase-producing Lactococcus lactis on the prevention of an experimental murine 1,2-dimethylhydrazine (DMH)-induced colon cancer. DMH-treated BALB/c mice received either a catalase-producing L. lactis strain or the isogenic non-catalase-producing strain as a control, whereas other untreated mice did not receive bacterial supplementation. Catalase activity and H2O 2 levels in intestinal fluids and blood samples were measured, and changes in the histology of the large intestines during tumour progression were evaluated. The catalase-producing L. lactis strain used in this study was able to slightly increase catalase activities in DMH-treated mice (1.19 ± 0.08 U ml-1) and reduce H2O2 levels (3.4 ± 1.1 μM) compared to (i) animals that received the non-catalase-producing strain (1.00 ± 0.09 U ml-1, 9.0 ± 0.8 μM), and (ii) those that did not receive bacterial supplementation (1.06 ± 0.07 U ml-1, 10.0 ± 1.1 μM). Using the histopathological grading scale of chemically induced colorectal cancer, animals that received the catalase-producing L. lactis had a significantly lesser extent of colonic damage and inflammation (2.0 ± 0.4) compared to animals that received the non-catalase-producing L. lactis (4.0 ± 0.3) or those that did not receive bacterial supplementation (4.7 ± 0.5). The catalase-producing L. lactis strain used in this study was able to prevent tumour appearance in an experimental DMH-induced colon cancer model. Fil: de Moreno, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Perdigon, Gabriela del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina Fil: Miyoshi, Anderson. Universidade Federal de Minas Gerais; Brasil Fil: Langella, Philippe. Institut National de la Recherche Agronomique; Francia Fil: Azevedo, Vaso. Universidade Federal de Minas Gerais; Brasil Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina

The citrate plasmid (Cit+ plasmid) from Lactococcus lactis subsp. lactis biovar diacetylactis was cloned into the EcoRI site of plasmid pUC18. This recombinant plasmid enabled Escherichia coli K-12 to transport and utilize citrate as a source of energy, indicating expression of the citrate permease from L. lactis biovar diacetylactis. The citrate permease was under the control of the lac promoter of pUC18. Genetic expression of the Cit+ plasmid in maxicells revealed that the plasmid encoded two polypeptides of 47 and 32 kilodaltons, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

We have reported previously on the ability of Lactobacillus reuteri to produce a compound with vitamin B12 activity. Here we report on the chemical characterisation of this corrinoid-like molecule. High performance liquid chromatography coupled to an ultraviolet diode array detector, mass spectrometry and nuclear magnetic resonance spectroscopy has enabled us to identify the compound as Coα-[α-(7-adenyl)]-Coβ-cyanocobamide or pseudovitamin B12. This molecule differs from cobalamin in the α-ligand, where it has adenine instead of 5,6-dimethylbenzimidazole bound in a α-glycosidic linkage to C-1 of ribose. L. reuteri is the first lactic acid bacterium in which the production of a cobalamin-like molecule has been identified and the first microorganism reported to produce exclusively pseudo-B12. © 2007 Federation of European Biochemical Societies. Fil: Santos, Felipe. NIZO Food Research; Países Bajos Fil: Vera, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Lamosa, Pedro. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica; Portugal Fil: Font, Graciela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: de Vos, Willem M.. Wageningen University and Research Centre.Laboratory of Microbiology ; Países Bajos Fil: Santos, Helena. Universidade Nova de Lisboa. Instituto de Tecnologia Química e Biológica; Portugal Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina Fil: Hugenholtz, Jeroen. NIZO Food Research; Países Bajos

Objective: Riboflavin deficiency is common in many parts of the world, particularly in developing countries. The use of riboflavin-producing strains in the production of dairy products such as fermented milk, yogurt, and cheese is feasible and economically attractive because it would decrease the costs involved during conventional vitamin fortification and satisfy consumer demands for healthier foods. The present study in a rat bioassay assessed the response of administration of yogurt containing a riboflavin-producing strain of Propionibacterium freudenreichii on the riboflavin status of deficient rats. Methods: Propionibacterium freudenreichii NIZO B2336 is a spontaneous roseoflavin-resistant mutant derived from P. freudenreichii B374 that produces larger amounts of riboflavin than the parental stain. Rats were fed a riboflavin-deficient diet for 21 d (depletion period), after which this same diet was supplemented with conventional yogurt, yogurt containing the riboflavin-producing strain (B2336), or the parental non-producing strain (B374) and fed to animals for 28 d (repletion period). As controls, rats were fed the same diet with different concentrations of commercial riboflavin. Results: The novel fermented product containing P. freudenreichii B2336, with increased levels of riboflavin, eliminated most physiologic manifestations of ariboflavinosis such as stunted growth, high erythrocyte glutathione reductase activation coefficient values, and hepatomegaly that were observed when using a riboflavin depletion-repletion model, whereas the product fermented with the non-riboflavin-producing strain did not show this beneficial effect. Conclusions: Consumption of such products with increased levels of riboflavin on a regular basis may help prevent deficiencies of this essential vitamin. Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Rutten, Ger. NIZO Food Research; Países Bajos Fil: Bruinenberg, Paul. Campina Innovation; Países Bajos Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Savoy, Graciela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Microbiología; Argentina Fil: Smid, Eddy J.. NIZO Food Research; Países Bajos

α-Galactosidase (α-Gal) enzyme, which is encoded by the melA gene hydrolyzes α-1,6 galactoside linkages found in sugars, such as raffinose and stachyose. These α-galacto-oligosaccharides (α-GOS), which are found in large quantities in vegetables, such as soy, can cause gastrointestinal disorders in sensitive individuals because monogastric animals (including humans) do not posses α-Gal in the gut. The use of microbial α-Gal is a promising alternative to eliminate α-GOS in soy-derived products. Using degenerate primers, the melA gene from Lactobacillus (L.) fermentum CRL722 was identified. The complete genomic sequence of melA (2223 bp), and of the genes flanking melA, were obtained using a combination of polymerase chain reaction-based techniques, and showed strong similarities with the α-Gal gene of thermophilic microorganisms. The α-Gal gene from L. fermentum CRL722 was cloned and the protein purified from cell-free extracts of the native and recombinant strains using various techniques (ion exchange chromatography, salt precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ultra-filtration); Its main biochemical properties were determined. The enzyme was active at moderately high temperatures (55°C) and stable at wide ranges of temperatures and pH. The thermostable α-Gal from L. fermentum CRL722 could thus be used for technological applications, such as the removal of α-GOS found in soy products. The complete melA gene could also be inserted in other micro-organisms, that can survive in the harsh conditions of the gut to degrade α-GOS in situ. Both strategies would improve the overall acceptability of soy-derived products by improving their nutritional value. © 2006 Springer Science+Business Media, Inc. Fil: Carrera Silva, Eugenio Antonio. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Silvestroni, A.. University of Washington. School of Medicine; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Leblanc, Jean Guy Joseph. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Piard, J. C.. Institut National de la Recherche Agronomique; Francia Fil: Savoy, Graciela. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina Fil: Sesma, Fernando Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; Argentina

The [ H]cholesterol removal by Lactobacillus reuteri was due to a co-precipitation together with unconjugated bile acids, which was linked to the bile salt hydrolase activity of the cells. No [ H]cholesterol was found inside the cells indicating that no assimilation occurred.