WHAT CAN WE OFFER?

• VECTORS WITH FLUORESCENT MARKERS

  In collaboration with the Group led by Dr. Paloma López from the Biological Research Centre (CIB, CSIC), we have developed transcriptional fusion vectors that allow detecting promoter uni- and bi-directional promoter regions in lactic bacteria . These vectors hold a patent (Patent P201130356, PCT\ES2012\070163) and are commercialized through the Solmeglas (www.http://www.solmeglas.com/gb/118-expression-vectors) to research groups or companies interested in them.

  

  The vectors contain as a marker a synthetic mRFP gene that codifies the mCherry red fluorescent protein (pTLR) and the GFP gene that codifies the green fluorescent GFP (pTLGR) protein. Plasmids are useful for characterizing promoter regions and for the expression of divergent genes, because they have the information to codify two proteins with completely separate fluorescence emission spectra. The fluorescence can be detected in real time, which allows establishing the environmental conditions (e.g., nutrients) intervening in the genetic regulation, as well as in the mechanisms of bacterial interaction, potentially probiotic as well as pathogenic, and with the eukaryotic cells of the host.

  The vectors developed by our group are:

  • pTLR to assess transcriptional promoters in Lactococcus lactis, Enterococcus faecalis, and Escherichia coli. It includes a synthetic mrfp reporter gene that codifies the mCherry red fluorescent protein.

  • pTLGR to assess transcriptional divergent promoters in L. lactis, E. faecalis, and E. coli. Includes the gfp and mrfp genes as fluorescent reporters.

  
  References:
  

  • Patent P201130356.
    Inventors (by order of signature): García-Cayuela, T., Mohedano, M.L., Pérez-Gómez de Cadiñanos, M.L., Fernández de Palencia, P., Boden, D., Wells, J., Peláez, C., López, P., Requena, T.
    Title: Vectores de fusión transcripcional para regiones promotoras uni- y bidireccionales para su uso en bacterias lácticas. (Transcriptional fusion vectors for uni- and bi-directional promoter regions for their use in lactic bacteria.)
    Priority country: Spain.      Priority date: 2011
    Holder entity: CSIC
    Companies making use of it: MYGEN. Licence agreement for non exclusive exploitation in the national territory.
    

  • International patent PCT 2012: PCT\ES2012\070163
    Title: Vectores de fusión transcripcional para regiones promotoras uni- y bidireccionales para su uso en bacterias lácticas.(Transcriptional fusion vectors for uni- and bi-directional promoter regions for their use in lactic bacteria)
    Priority date: 2012
    

  
  Articles:
  
  • García-Cayuela, T., Gómez de Cadiñanos, L.P., Mohedano, M.L., Fernández de Palencia, P., Boden, D., Wells, J., Peláez, C., López, P. and Requena, T. Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli. Appl. Microbiol. Biotechnol. 96:171-181, 2012.
  
  
  

  PROCEDURE FOR THE DIFFERENTIATION AND QUANTIFICATION OF LACTIC BACTERIA AND BIFIDOBACTERIA

  We have developed procedures that allow selectively and differentially identifying and quantifying four lactic bacteria species (Streptococcus thermophilus; Lactobacillus bulgaricus; Lactobacillus casei; Lactobacillus acidophilus) as well as Bifidobacterium lactis, in mixed cultures present in fermented milks. These procedures are registered in patents 200601788 and 200601789. We offer the commercialization licence of the procedures to companies specialized in culture media for microbiological analysis of food and diagnostic kits.

  One of the novelties in these techniques is they allow to distinguish simultaneously three different species of lactobacilli between them and against other lactic bacteria such as S. thermophilus, as well as bifidobacteria, providing advantages in comparison to existing techniques. All this is based on different incubation conditions and/or differentiation according to colony morphology, and without the need of adding antibiotics to the culture media, which could compromise the viability of the studied species.

  The validation of the procedure was carried out studying media selectivity, accuracy and precision in the recovery of the assayed population, reproducibility of the procedure, and analysis of possible competition between species. The matrix effect was also analyzed by quantifying the populations in acidified milk in comparison with the reference culture media.

  
  References:
  

  • Patent nº 200601788.
    Inventors (by order of signature): Tabasco, R., Paarup, T., Janer, C., Peláez, C., Requena, T.
    Title: Procedimiento para la detección e identificación simultánea y específica de bacterias lácticas y bifidobacterias en leches fermentadas y en cultivos iniciadores para leches fermentadas. (Procedure to differentiate and quantify lactic bacteria and bifidobacteria in fermented milks using antibiotic-free culture media.)
    Priority country: Spain.      Priority date: 2006
    Holder entity: CSIC
    

  • Patent nº 200601789.
    Inventors (by order of signature): Tabasco, R., Paarup, T., Janer, C., Peláez, C., Requena, T.
    Title: Procedimiento para diferenciar y cuantificar bacterias lácticas y bifidobacterias en leches fermentadas que emplea medios de cultivo selectivos libres de antibióticos. (Procedure for simultaneously and specifically detecting and identifying lactic bacteria and bifidobacteria in fermented milks and started cultures for fermented milks)
    Priority country: Spain.      Priority date: 2006
    Holder entity: CSIC
    

  
  Articles
  
  • Tabasco, R. Paarup, T., Janer, C., Peláez, C. and Requena, T. 2007. Selective enumeration and identification of mixed cultures of S. thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus acidophilus, Lactobacillus paracasei and Bifidobacterium lactis in fermented milk. International Dairy Journal 17: 1107-1114. 2007.

  
  

  
  
  

  BACTERIOCIN-PRODUCING LACTOCOCCUS

  We offer microorganisms of the Lactococcus lactis species (IFPL 3593 and IFPL105) that produce the bacteriocin lacticin 3147 with broad spectrum of action and a lytic mechanism against sensitive bacteria. L. lactis IFPL 3593 can be used as part of a started culture for cheese. The procedure for its use is protected under patent nº 2.170.723 (ES2001100090). On the other hand, the high production of lacticin 3147 by L. lactis IFPL105 can favour the use of the bacteriocin as a biopreservative in ready-to-eat foods, e.g., meat and vegetables.

  The commercialization of both microorganisms is offered to companies in the lactic, meat, or vegetable field, as well as to firms that commercialize culture starters.

  L. lactis IFPL3593 contains a 46 kD non-conjugative plasmid (pBAC105), originally present in Lactococcus lactis IFL105, which codifies the production and immunity of bacteriocin. The acidifying capacity of L. lactis IFPL3593 allows it to act as a starter in cheese making. The bacteriocin produced during its growth induces lysis of other pre-existing or added lactic bacteria without interfering in the initial milk fermentation process, and thus without causing curd acidification problems.

  The result is the acceleration of cheese ripening and an earlier development of the organoleptic characteristics. Furthermore, this microorganism has the benefit of being active against Clostridium tyrobutyricum spores, preventing their germination, thus acting as a bioprotective culture preventing late blowing of semi-hard cheeses often caused by clostridia.

  
  References:
  

  • Patent nº 2.170.723 (ES2001100090).
    Inventors (by order of signature): Martínez-Cuesta, M.C., Requena, T., Peláez, C.
    Title: Lactococcus lactis productor de bacteriocina utilizable como cultivo iniciador para acelerar la maduración de queso.
    Priority country: Spain.      Priority date: 2001
    Holder entity: CSIC
    

  
  Artículos
  
  • Martínez Cuesta, M.C., Peláez, C. Juárez, M. and Requena, T. Autolysis of certain lactococci and lactobacilli strains. Cell lysis induced by a bacteriocin. International Journal of Food Microbiology 38: 125 131. 1997.
    
    
  • Martínez Cuesta, M.C.; Fernández de Palencia, P.; Requena, T. and Peláez, C. Enhancement of proteolysis by a Lactococcus lactis bacteriocin producer in a cheese model system. Journal of Agricultural and Food Chemistry 46: 3863 3867. 1998.
    
    
  • Martínez Cuesta, M.C., Kok, J., Herranz, E., Peláez, C. Requena, T. and Buist, G. Requirement of autolytic activity for bacteriocin induced lysis. Applied Environmental Microbiology. 66: 3174 - 3179. 2000.
    
    
  • Martínez Cuesta, M.C., Buist, G., Kok, J., Hauge, H.H., Nissen Meyer, J., Peláez, C. and Requena, T. Biological and molecular characterization of a two peptide lantibiotic produced by Lactococcus lactis IFPL105. Journal Applied Microbiology 89: 249 260. 2000.
    
    
  • Martínez Cuesta, M.C., Requena, T. and Peláez, C. Use of a bacteriocin producing transconjugant as starter in acceleration of cheese ripening. International Journal of Food Microbiology 70:79-88. 2001.
    
    

  
  

  
  
  

  LACTIC ACID BACTERIA COLLECTION

  Over the years, the Group has created a collection of lactic bacteria made up by approximately 500 strains corresponding to the Lactococcus, Lactobacillus, Leuconostoc, and Enterococcus genera, most of which have been isolated from dairy products produced by artisanal fermentation with no previous contact with commercial cultures. This characteristic provides an important added value to the bacterial collection, as many characteristics present in wild type strains have been described, which are not found in industrial strains.

  

  An important part of these strains has been identified through molecular methods, mainly the sequence of the 16S- rDNA and RAPD technique.. More recently, MALDI-TOF/TOF mass spectrometry techniques have been applied for bacterial identification and functional characterization based on their peptidic profiles. Furthermore, in many strains of interest, the technological properties have been characterized at molecular level, e.g., the proteolytic system, catabolism of amino acids - aminotransferases, glutamate dehydrogenase, lyases, and keto acid decarboxylase - and bacteriocin production.

  We offer to companies innovating in design and development of new starter cultures the possibility of collaborating with the exploitation, taking advantage of the knowledge and the know-how developed by Group during its molecular characterization.

  
  
  

  UNIQUE LACTOBACILLUS STRAIN IN THE INTESTINAL METABOLISM OF BIOACTIVE COMPUNDS

  Lactobacillus plantarum IFPL935, from our collection, is a polyphenol-metabolizing microorganism, e.g., monomeric flavan-3-ol and galloyl-derived compounds generated by the action of galloyl esterase, decarboxylase, and benzyl alcohol dehydrogenase. Lactobacillus plantarum IFPL935 is also capable of breaking the C-ring (heterocycle) of monomeric flavan-3-ols to produce the corresponding diphenyl propan-2-ol. The interaction between this bacterium and phenolic compounds in gut epithelium has also been assessed, i.e., absorption and metabolism, using cellular lines. The ability of L. plantarum IFPL935 to produce diphenyl propan-2-ol from grape seed extracts has been reproduced by incubating the strain with human faecal microbiota. Thus Lactobacillus plantarum IFPL935 has the metabolic capacity not described until now for this species of other intestinal bacteria, and that potentially opens important doors for this microorganism in the sector of functional foods.

  On one hand, we offer research and clinical groups interested in probiotic research and intestinal health, the possibility of collaborating in scientific projects related to the potential beneficial activity of this microorganism in the gut and its capacity to produce bioactive metabolites from plant polyphenols.

  On the other hand, we offer wine-growing, juice, and derivative companies, as well as to companies engaged in the commercialization of probiotic cultures the possibility to collaborate with the commercial exploitation of this probiotic microorganism.

  
  References:
  
  • Tabasco, R., Sánchez-Patán, F., Monagas, M., Bartolomé, B., Moreno-Arribas, M.V., Peláez, C., Requena, T. Effect of grape polyphenols on lactic acid bacteria and bifidobacteria growth: resistance and metabolism. Food Microbiology 28: 1345-1352. 2011.
    
    
  • Sánchez-Patán, F., Tabasco, R., Monagas, M., Requena, T., Peláez, C., Moreno-Arribas, M.V., Bartolomé, B. Capability of Lactobacillus plantarum IFPL935 to catabolize flavan-3-ol compounds and complex phenolic extracts. Journal of Agricultural and Food Chemistry 60: 7142–7151. 2012.
    
    
  • Bustos, I., García Cayuela, T., Hernández-Ledesma, B., Requena, T., Martínez Cuesta, M.C. Effect of flavan-3-ols on the adhesion of potential probiotic lactobacilli to intestinal cells. Journal of Agricultural and Food Chemistry 60: 9082-9088. 2012.
    
    

  
  

  Degradation of flavan-3-oles by Lactobacillus plantarum IFPL935