Control del flujo metabólico de α-cetoácidos mediante la inactivación del gen panE en estirpes de Lactococcus lactis con actividad GDH

Trabajo presentado al XVIII Congreso Nacional de Microbiología de los Alimentos celebrado en Logroño del 25 al 28 de septiembre de 2012.Este trabajo ha sido financiado por el Ministerio de Economía y Competitividad (Proyectos: AGL2009‐13361‐C02‐02, RM2011‐00003‐00‐00 y Consolider Ingenio 2010 FUN‐C‐FOOD‐CSD2007‐00063) y la Comunidad de Madrid (Proyecto: ALIBIRD P2009/AGR‐1469).Peer Reviewe

Expression in Lactococcus lactis of functional genes related to amino acid catabolism and cheese aroma formation is influenced by branched chain amino acids

Formation of cheese aroma compounds by Lactococcus lactis from amino acid catabolism depends on a complex network of reactions, which involve enzymes such as aminotransferases, dehydrogenases, lyases, and decarboxylases, among others. Based on the ability of some L. lactis strains to grow with low requirements of amino acids, we have studied in L. lactis IFPL730 the effect of the branched chain amino acid (BCAA) content on the expression of functional genes related to amino acid catabolism and aroma compound formation (araT, bcaT, kivD, ytjE and panE). L. lactis IFPL730 growth rate decreased under leucine, valine or isoleucine starvation but the strain reached similar viable counts at the stationary phase in all culture conditions studied. The level of expression of some genes encoding enzymes involved in amino acid catabolism changed significantly (P < 0.05) when those conditions were compared. Specially, α-ketoisovalerate decarboxylase (kivD), BCAA-specific aminotransferase (bcaT) and C-S lyase (yjtE) gene expressions increased markedly by both isoleucine and valine starvation. In addition to gene expression, formation of volatile compounds was determined in all growth conditions. The results showed that BCAA starvation conditions caused a significant increase (P < 0.05) in the formation of metabolic end products related to cheese aroma, such as 3-methylbutanal and 3-methylbutanol.This work was supported by the Spanish Ministry of Science and Innovation (grants: AGL2009-13361-C02-02, RM2011-00003-00-00 and Consolider Ingenio 2010 FUN-C-FOOD-CSD2007-00063) and the Comunidad de Madrid (grant: ALIBIRD P2009/AGR-1469).Peer Reviewe

Inactivation of the panE gene in Lactococcus lactis enhances formation of cheese aroma compounds

Hydroxyacid dehydrogenases limit the conversion of α-keto acids into aroma compounds. Here we report that inactivation of the panE gene, encoding the α-hydroxyacid dehydrogenase activity in Lactococcus lactis, enhanced the formation of 3-methylbutanal and 3-methylbutanol. L. lactis IFPL953ΔpanE was an efficient strain producing volatile compounds related to cheese aroma.This work was supported by the Spanish Ministry of Science and Innovation (grants AGL2006-12100, AGL2009-13361-C02-02, RM2011-00003-00-00, and Consolider Ingenio 2010 FUN-C-FOOD-CSD2007-00063) and Comunidad de Madrid (grant ALIBIRD P2009/AGR-1469).Peer Reviewe

Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli

29 p.- 4 tab.-2 fig.Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.This work was supported by the Spanish Ministry of Science and Innovation (grants AGL2009-12998-C03-01, AGL2009- 13361-C02-02, and CSD2007-00063 Consolider Ingenio 2010 FUN-CFOOD), the Comunidad de Madrid (grant ALIBIRD P2009/AGR-1469), and the European Union VII framework program (Initial Training Network, grant no. 238490). We thank Dr. Stephen Elson for the critical reading of the manuscript. We are grateful to M. Angeles Corrales for her technical assistance.Peer Reviewe

Vectores de fusión transcripcional para regiones promotoras uni- y bidireccionales para su uso en bacterias lácticas

La presente invención se refiere a una secuencia nucleotídica recombinante que comprende la secuencia del gen mrfp que codifica para una proteína autofluorescente roja optimizada para la expresión en bacterias lácticas así como a los vectores que la comprenden. La invención también se refiere a las células que comprenden la secuencia o el vector de la invención y sus usos. Así mismo, también se refiere al uso tanto de la secuencia como del vector para la detección y caracterización de una región promotora unidireccional o bidireccional, así como de su uso para transcripción. Además, se refiere al uso de las células que contienen la secuencia de la invención o el vector de la invención para la determinación de la capacidad colonizadora de células. También se refiere a los métodos relacionados con los usos de la invención, a un kit que comprende los elementos de la invención y su uso.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Identification and characterization of glutamate dehydrogenase activity in wild Lactococcus lactis isolated from raw milk cheeses

The glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate, which initiates amino acid transamination during cheese ripening. This work has investigated the GDH activity in 39 wild isolates of Lactococcus lactis from raw milk cheeses. Only 25% of the isolates were GDH positive with NAD as the preferred cofactor. L. lactis IFPL953 showed the highest NAD-GDH activity. The GDH activity at the genetic level in the lactococcal isolates was analysed by PCR amplification of the gdh gene in genomic and plasmid DNA. The gdh gene arrangement of L. lactis IFPL953 in its plasmid location was similar to that in the reference strain GDHL. lactis TiL504, suggesting that both lactococci could harbour the same plasmid pGdh442 containing the gdh gene. L. lactis IFPL953 has previously demonstrated a remarkable α-ketoisovalerate decarboxylase activity, which along with its high GDH activity makes the strain particularly useful in enhancing cheese flavour formation.The authors acknowledge the funding from the Spanish MINEICO (AGL2012-35814, AGL2016-75951-R and P916PTE0233).Peer Reviewe

Expression of amino acid converting enzymes and production of volatile compounds by Lactococcus lactis IFPL953

Lactococcus lactis contains a large number of key enzymes responsible for the formation of volatile compounds characteristic of cheese flavour. In the present work we have investigated the expression of genes codifying amino acid converting enzymes (AACE) and the formation of volatile compounds by L. lactis IFPL953 and its mutant lacking the hydroxy acid dehydrogenase PanE (L. lactis IFPL953ΔpanE). The growth in absence of isoleucine was the main induction factor in the expression of araT, bcaT, panE, and kivD. The expression of the gdh gene of L. lactis IFPL953ΔpanE increased remarkably during the stationary growth phase, particularly under isoleucine and valine starvation conditions. L. lactis IFPL953ΔpanE showed an enhanced formation of 2- and 3-methylbutanal and their corresponding alcohols and an increase in the formation of ketones. These findings contribute to a better knowledge of L. lactis AACE regulation and its potential application in cheese flavour formation.This study was funded by Spanish Ministry of Science (Grants AGL2016-75951-R and PCIN-2017-003).Peer reviewe

Evaluación de la capacidad de adhesión de cepas de Lactobacillus plantarum con fenotipo de agregación

Resumen del trabajo presentado a las I Jornadas Científicas del CIAL celebradas el 5 de junio de 2014 en Madrid.La capacidad de adhesión a las células epiteliales del intestino es un criterio a tener en cuenta para la selección de cepas con potencial probiótico. Las propiedades de agregación (autoagregación y coagregación) y la hidrofobicidad de cepas probióticas parecen jugar un papel clave en la adhesión al epitelio intestinal y en la inhibición de la colonización de bacterias patógenas. En nuestro laboratorio, hemos podido observar cómo ciertas cepas de Lactobacillus plantarum muestran un fenotipo de agregación, es decir, la capacidad para formar agregados visibles tras la agitación vigorosa del cultivo. En este sentido, se ha evaluado la correlación entre las propiedades de autoagregación, coagregación e hidrofocidad, y la capacidad de adhesión y exclusión competitiva de cepas de L. plantarum que fueron previamente seleccionadas por poseer el fenotipo de agregación. Los resultados mostraron que las cepas con mayor capacidad de autoagregación (que se correlacionó con el fenotipo visualmente observado tras 24 h de incubación del cultivo) fueron las que mostraron los mayores niveles de coagregación con los microorganismos ensayados, Listeria monocytogenes, Staphylococcus aureus y Escherichia coli. Asimismo, se constató una relación positiva entre autoagregación y adhesión a células Caco-2, si bien se observó que otros factores, además de las propiedades estudiadas, deben influir en la capacidad de adhesión. Por otra parte, no se observó ninguna relación entre hidrofobicidad y capacidad de adhesión para todas las cepas evaluadas. Además, estas propiedades no están completamente correlacionadas con la capacidad de las cepas estudiadas para inhibir la adhesión de patógenos. Estos resultados sugieren que las capacidades de agregación y la hidrofobicidad de las cepas bacterianas pueden ser utilizadas a la hora de seleccionar cepas con potencial uso probiótico, aunque se necesitarían más estudios para identificar completamente todos los mecanismos implicados en la adhesión bacteriana y la exclusión de patógenos.Los autores agradecen la financiación otorgada por el Ministerio de Economía y Competitividad a través de los proyectos AGL2009-13361-C02-02, AGL2012-35814, RM2011-00003-00-00, y Consolider Ingenio 2010 FUN-C-FOOD-CSD2007-00063; y por la Comunidad de Madrid a través del proyecto ALIBIRD P2009/AGR-1469.Peer Reviewe

Adhesion abilities of dairy Lactobacillus plantarum strains showing an aggregation phenotype

Bacterial aggregation phenotype and cell surface hydrophobicity of dairy Lactobacillus plantarum strains were screened in order to assess a correlation with their adhesion and pathogen competitive exclusion abilities. Lactobacilli strains showing an aggregation phenotype, L. plantarum IFPL33, IFPL81, IFPL150, IFPL156 and IFPL162, also exhibited the highest percentages of autoaggregation (> 50%) at 24. h. In addition, autoaggregation abilities of the lactobacilli were highly correlated with their percentages of co-aggregation with all the pathogens tested, although co-aggregation properties were pathogen specific. Nevertheless, none of the autoaggregation and co-aggregation abilities correlated with the affinity to xylene (hydrophobicity) and the ability of adhesion to Caco-2 cells. Furthermore, these properties are not fully correlated with the ability of the lactobacilli strains for inhibiting pathogen adhesion. Aggregation abilities and cell surface hydrophobicity may not be the only components responsible for adhesion but some of the criteria to bear in mind of a complex mechanism that enables microorganisms to interact with the host and exert its beneficial effect. Further research is needed to identify remaining attributes related to adhesion and pathogen exclusion properties of potential probiotic strains. © 2014 Elsevier Ltd.This work has been funded by the Spanish Ministry for Science and Innovation (AGL2009-13361-C02-02, RM2011-00003-00-00, AGL2012-35814 and Consolider Ingenio 2010 FUN–C-FOOD CSD2007-00063), the Comunidad Autónoma de Madrid (ALIBIRD P2009/AGR-1469) and CYTED (IBEROFUN 110 AC0386). The authors are participants in the COST Action FA1005 INFOGEST.Peer Reviewe