Raúl R. Raya

Isolation and Characterization of a New T-Even Bacteriophage, CEV1, and Determination of Its Potential To Reduce Escherichia coli O157:H7 Levels in Sheep

ABSTRACT Bacteriophage CEV1 was isolated from sheep resistant to Escherichia coli O157:H7 colonization. In vitro, CEV1 efficiently infected E. coli O157:H7 grown both aerobically and anaerobically. In vivo, sheep receiving a single oral dose of CEV1 showed a 2-log-unit reduction in intestinal E. coli O157:H7 levels within 2 days compared to levels in the controls.

Biotechnology of lactic acid bacteria: novel applications.

1. Updates in the Metabolism of Lactic Acid Bacteria. (BaltasarMayo, Tamara Aleksandrzak-Piekarczyk, Maria Fernandez,Magdalena Kowalczyk, Pablo Alvarez-Martin and JacekBardowski). 2. Genomics of Lactic Acid Bacteria. The Post-GenomicsChallenge: from Sequence to Function. (M. Andrea Azcarate-Peril andTodd R. Klaenhammer). 3. Proteomics: A Tool for Understanding Lactic Acid BacteriaAdaptation to Stressful Environments. (Marie-ChristineChampomier-Verges, Monique Zagorec and Silvina Fadda). 4. Lactic Acid Bacteria: Comparative Genomic Analyses ofTransport Systems. (Graciela Lorca, Lakshmi Reddy, Anphong Nguyen,Eric I. Sun, John Tseng, Ming-Ren Yen and Milton H. Saier Jr). 5. Applications of Lactic Acid Bacteria-produced Bacteriocins.(Barry Collins, Paul D. Cotter, Colin Hill and R. Paul Ross). 6. Bacteriophages of Lactic Acid Bacteria. (Ana RodriguezGonzalez, Pilar Garcia and Raul R. Raya). 7. Lactic Acid Bacteria as Immunomodulators of the GutAssociated Immune System. (Carolina Maldonado Galdeano, Alejandrade Moreno de LeBlanc, Cecilia Dogi and Gabriela Perdigon). 8. Lactic Acid Bacteria in Prevention of the Urogenital andRespiratory Tracts. (Maria E. Fatima Nader-Macias,Gladis Susana Alvarez, Clara Silva de Ruiz, Marcela Medina andMaria Silvina Juarez Tomas). 9. Lactic Acid Bacteria as Live Vectors: Heterologous ProteinProduction and Delivery Systems. (Anderson Miyoshi, Luis G.Bermudez-Humaran, Marcela Santiago Pacheco de Azevedo,Philippe Langella and Vasco Azevedo). 10. Advances and Trends in Starter Cultures for DairyFermentations. (Domenico Carminati, Giorgio Giraffa, AndreaQuiberoni, Ana Binetti, Viviana Suarez and JorgeReinheimer). 11. Low-Calorie Sugars Produced by Lactic Acid Bacteria. (GinoVrancken, Tom Rimaux, Luc De Vuyst and Fernanda Mozzi). 12. B-Group Vitamins Production by Probiotic Lactic AcidBacteria. (Jean Guy LeBlanc, Maria Pia Taranto,Veronica Molina and Fernando Sesma). 13. Bioactive Peptides Derived from Casein and Whey Proteins.(Elvira Maria Hebert, Lucila Saavedra and PasqualeFerranti). 14. New Approaches for the Study of Lactic Acid BacteriaBiodiversity. A Focus on Meat Ecosystems. (Graciela M. Vignolo,Cecilia Fontana and Pier S. Cocconcelli). 15. New Trends in Cereal-Based Products Using Lactic AcidBacteria. (Graciela Font de Valdez, Carla L. Gerez, MariaInes Torino and Graciela Rollan). 16. An Overview of Lactic Acid Bacteria Applications forHealthful Soy Foods Development. (Graciela Savoy de Giori, LauraAguirre, Jose Marazza and Marisa S. Garro). 17. The Functional Role of Lactic Acid Bacteria in Cocoa BeanFermentation. (Luc De Vuyst, Timothy Lefeber, Zoe Papalexandratou,and Nicholas Camu). 18. Microbial Interactions in Kefir: A Natural Probiotic Drink.(Graciela L. Garrote, Analia G. Abraham and Graciela L. DeAntoni). 19. Safety of Lactic Acid Bacteria. (Charles M.A.P. Franz,Gyu-Sung Cho, Wilhelm H. Holzapfel and Antonio Galvez). 20. Genetically Modified Lactic Acid Bacteria. (PierreRenault).

Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

ABSTRACT The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM. The growth rate and the synthesis of their cell membrane-bound serine proteinases, but not of their intracellular leucyl-aminopeptidases, were influenced by the peptide content of the medium. The highest proteinase levels were found during cell growth in basal SCDM, while the synthesis of this enzyme was inhibited in SCDM supplemented with Casitone, Casamino Acids, or β-casein. Low-molecular-mass peptides (<3,000 Da), extracted from Casitone, and the dipeptide leucylproline (final concentration, 5 mM) play important roles in the medium-dependent regulation of proteinase activity. The addition of the dipeptide leucylproline (5 mM) to SCDM reduced proteinase activity by 25%.

Characterization of the Pattern of αs1- and β-Casein Breakdown and Release of a Bioactive Peptide by a Cell Envelope Proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581

ABSTRACT The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzymes production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEPI/III variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the αs1- and β-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of αs1- and β-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.

Nasal Immunization with Lactococcus lactis Expressing the Pneumococcal Protective Protein A Induces Protective Immunity in Mice

ABSTRACT Nisin-controlled gene expression was used to develop a recombinant strain of Lactococcus lactis that is able to express the pneumococcal protective protein A (PppA) on its surface. Immunodetection assays confirmed that after the induction with nisin, the PppA antigen was predictably and efficiently displayed on the cell surface of the recombinant strain, which was termed L. lactis PppA. The production of mucosal and systemically specific antibodies in adult and young mice was evaluated after mice were nasally immunized with L. lactis PppA. Immunoglobulin M (IgM), IgG, and IgA anti-PppA antibodies were detected in the serum and bronchoalveolar lavage fluid of adult and young mice, which showed that PppA expressed in L. lactis was able to induce a strong mucosal and systemic immune response. Challenge survival experiments demonstrated that immunization with L. lactis PppA was able to increase resistance to systemic and respiratory infection with different pneumococcal serotypes, and passive immunization assays of naïve young mice demonstrated a direct correlation between anti-PppA antibodies and protection. The results presented in this study demonstrate three major characteristics of the effectiveness of nasal immunization with PppA expressed as a protein anchored to the cell wall of L. lactis: it elicited cross-protective immunity against different pneumococcal serotypes, it afforded protection against both systemic and respiratory challenges, and it induced protective immunity in mice of different ages.

Genome Sequence of the Bacteriocin-Producing Lactobacillus curvatus Strain CRL705

Lactobacillus curvatus is one of the most prevalent lactic acid bacteria found in fermented meat products. Here, we present the draft genome sequence of Lactobacillus curvatus CRL705, a bacteriocin producer strain isolated from an Argentinean artisanal fermented sausage, which consists of 1,833,251 bp (GC content, 41.9%) and two circular plasmids of 12,342 bp (pRC12; GC, 43.9%) and 18,664 bp (pRC18; GC, 34.4%).

Isolation and characterization of a slowly milk-coagulating variant of Lactobacillus helveticus deficient in purine biosynthesis

ABSTRACT A slowly milk-coagulating variant (Fmc−) ofLactobacillus helveticus CRL 1062, designated S1, was isolated and characterized. Strain S1 possessed all the known essential components required to utilize casein as a nitrogen source, which include functional proteinase and peptidase activities as well as functional amino acid, di- and tripeptide, and oligopeptide transport systems. The amino acid requirements of strain S1 were similar to those of the parental strain. However, on a purine-free, chemically defined medium, the growth rate of the Fmc− strain was threefold lower than that of the wild-type strain. L. helveticusS1 was found to be defective in IMP dehydrogenase activity and therefore was deficient in the ability to synthesize XMP and GMP. This conclusion was further supported by the observation that the addition of guanine or xanthine to milk, a substrate poor in purine compounds, restored the Fmc+ phenotype of L. helveticusS1.

Draft Genome Sequence of Oenococcus oeni Strain X2L (CRL1947), Isolated from Red Wine of Northwest Argentina.

ABSTRACT We report the draft genome sequence of Oenococcus oeni strain X2L, a potential starter culture of malolactic fermentation, isolated from Malbec wine of Argentina. Genes encoding for enzymes involved in the metabolism of malate, citrate, and nitrogen compounds, as well as aroma compounds, were found in this genome, showing its ability to improve the sensorial characteristics of wines.

Global Analysis of Mannitol 2-Dehydrogenase in Lactobacillus reuteri CRL 1101 during Mannitol Production through Enzymatic, Genetic and Proteomic Approaches.

Several plants, fungi, algae, and certain bacteria produce mannitol, a polyol derived from fructose. Mannitol has multiple industrial applications in the food, pharmaceutical, and medical industries, being mainly used as a non-metabolizable sweetener in foods. Many heterofermentative lactic acid bacteria synthesize mannitol when an alternative electron acceptor such as fructose is present in the medium. In previous work, we reported the ability of Lactobacillus reuteri CRL 1101 to efficiently produce mannitol from sugarcane molasses as carbon source at constant pH of 5.0; the activity of the enzyme mannitol 2-dehydrogenase (MDH) responsible for the fructose conversion into mannitol being highest during the log cell growth phase. Here, a detailed assessment of the MDH activity and relative expression of the mdh gene during the growth of L. reuteri CRL 1101 in the presence of fructose is presented. It was observed that MDH was markedly induced by the presence of fructose. A direct correlation between the maximum MDH enzyme activity and a high level of mdh transcript expression during the log-phase of cells grown in a fructose-containing chemically defined medium was detected. Furthermore, two proteomic approaches (2DE and shotgun proteomics) applied in this study confirmed the inducible expression of MDH in L. reuteri. A global study of the effect of fructose on activity, mdh gene, and protein expressions of MDH in L. reuteri is thus for the first time presented. This work represents a deep insight into the polyol formation by a Lactobacillus strain with biotechnological potential in the nutraceutics and pharmaceutical areas.