Quantitative real-time PCR method with internal amplification control to quantify cyclopiazonic acid producing molds in foods

A quantitative TaqMan real-time PCR (qPCR) method that includes an internal amplification control (IAC) to quantify cyclopiazonic acid (CPA)-producing molds in foods has been developed. A specific primer pair (dmaTF/dmaTR) and a TaqMan probe (dmaTp) were designed on the basis of dmaT gene which encodes the enzyme dimethylallyl tryptophan synthase involved in the biosynthesis of CPA. The IAC consisted of a 105 bp chimeric DNA fragment containing a region of the hly gene of Listeria monocytogenes. Thirty-two mold reference strains representing CPA producers and non-producers of different mold species were used in this study. All strains were tested for CPA production by high-performance liquid chromatography-mass spectrometry (HPLC-MS). The functionality of the designed qPCR method was demonstrated by the high linear relationship of the standard curves relating to the dmaT gene copy numbers and the Ct values obtained from the different CPA producers tested. The ability of the qPCR protocol to quantify CPA-producing molds was evaluated in different artificially inoculated foods. A good linear correlation was obtained over the range 1-4 log cfu/g in the different food matrices. The detection limit in all inoculated foods ranged from 1 to 2 log cfu/g. This qPCR protocol including an IAC showed good efficiency to quantify CPA-producing molds in naturally contaminated foods avoiding false negative results. This method could be used to monitor the CPA producers in the HACCP programs to prevent the risk of CPA formation throughout the food chain.Fil: Rodríguez, Alicia. Universidad de Extremadura. Facultad de Veterinaria; EspañaFil: Werning, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rodríguez, Mar. Universidad de Extremadura. Facultad de Veterinaria; EspañaFil: Bermúdez, Elena. Universidad de Extremadura. Facultad de Veterinaria; EspañaFil: Córdoba, Juan J.. Universidad de Extremadura. Facultad de Veterinaria; Españ

Biosynthesis, Purification and Biotechnological Use of Exopolysaccharides Produced by Lactic Acid Bacteria

32 p.-6 fig.-1 tab.This work was supported by the Spanish Ministry of Science and Innovation (grant AGL2009-12998-C03-01). Sara Notararigo and Montserrat Nácher are recipients of predoctoral fellowships from Consejo Superior de Investigaciones CientíficasPeer reviewe

Procedimiento de detección molecular de bacterias ácido lácticas productoras de β-glucanos.

Esta invención presenta un nuevo método de detección e identificación rápida de bacterias ácido lácticas productoras de exopolisacáridos por amplificación de su gen gtf codificante de una glicosil transferasa. La utilización de este método podría permitir la prevención del ahilamiento de bebidas alcohólicas y el aislamiento de nuevas estirpes que podrían ser utilizadas para la producción de alimentos fermentados.Solicitud: 200402176 (11.09.2004)Nº Pub. de Solicitud: ES2315038A1 (16.03.2009)Nº de Patente: ES2315038B1 (29.12.2009

Caracterización de la síntesis del (1,3)(1,2)-β-D-Glucano y de las bacterias lácticas productoras

Leída en la Universidad Autónoma de Madrid. Facultad de Ciencias el 06-21-2010; 193 págs.Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented foods, and among them, some β-glucans have immunomodulating properties. Pediococcus parvulus 2.6 and other lactic acid bacteria (LAB) isolated from infected alcoholic beverages produce an identical (1,3)(1,2)-β-D-glucan. The characterization of the synthesis of this EPS is the main objective of this work. The study was performed with a combination of genetic engineering, molecular microbiological, biochemical and biophysical approaches. The results obtained as well as their interpretation are detailed below. We have identified and determine the nucleotide sequence of 6 glycosyltransferase (gtf) genes from LAB belonging to the Pediococcus, Lactococcus and Oenococus genera. Their genetic location in P. parvulus 2.6, L. diolivorans G77 and O. oeni I4 has been established. The gene is carried by two different plasmids of 35 kb and 5,5 kb in the first two hosts, whereas in O. oeni the gene is chromosomally located. Bioinformatic analysis of the gtf gene product indicated that it is an enzyme that belongs to the GT-2 membrane-bond glycosyltransferase family. Moreover, the topological prediction of the GTF protein indicates that a cytosolic glycosyl transferase domain is flanked by two and four trans-membrane segments. Detection of glycosyltransferase activity in membrane preparations of GTF overexpressor strains strongly supports the bioinformatic predictions for the enzyme activity and location. In addition, the heterologous expression of the P. parvulus 2.6R gtf in Streptococcus pneumoniae and in Lactoccus lactis enable these strains to synthesize a β-glucan immunologically indistinguishable from that of S. pneumoniae serotype 37. The overproduction of the P. parvulus 2.6 GTF glucosyltransferase in L. lactis resulted in synthesis and secretion of 300 mg/l of the (1,3)(1,2)-β-D-glucan in this host. The method used to purify the EPS allowed an 80% recovery of pure β-glucan with a high molecular mass (6,5 x 106 Da) similar to that of the biopolymer synthesized by P. parvulus (9,6 x 106 Da). An immunological method for detection of (1,3)(1,2)-β-D-glucans has been standardizedand validated in this work. The ELISA method is based in the use of anti-serotype 37 pneumococcal antibodies and allows direct quantification of the exopolysaccharide in the supernatant of its producing bacteria grown in complex media, which contain mono- and polysaccharides with a threshold of 18 ng/ml. The method is substantially specific for this β-Dglucan, only detecting (1,3)(1,6)-β-glucan at high concentrations (>10 μg/ml) and not interacting with linear (1,3)-β-D-glucan or heteropolysaccharides. The above described method has been also used for the in vitro enzymatic characterization of the GTF glucosyltransferase associated with membrane vesicles of L. lactis NZ9000. The results showed that the activity is optimal at 37ºC in the pH range of 6,5 to 7, it displays typical Michaelis Menten kinetics with respect to UDP-glucose (Km = 0,125 mM) and has a Vmax = 0,018 μg EPS/mg prot /min. Moreover, β-glucan synthesis proceeds linearly with time up to 4 h, and catalysis is inhibited in the presence of UTP, UDP or EDTA. Finally, the chemical characterization of the reaction product revealed that indeed it is the (1,3)(1,2)-β-D-glucan. Finally, a comparative analysis has been performed of the potential probiotic properties of the β-D-glucan producer P. parvulus 2.6R and its isogenic EPS-non-producer 2.6RN strain. After exposure to gastrointestinal stress, both strains showed the same pattern of resistance, indicating that the ability to produce EPS does not protect P. parvulus in this sense. By contrast, the adhesion of 2.6R to Caco-2 cells was twenty four-fold higher than that of 2.6NR strain. Moreover, the high adherence of the EPS producer (6%) to Caco-2 cells was partially abolished (decrease to 1.8%) after EPS removal by washing. Both strains induce the production of the anti-inflammatory cytokine IL-10 by polarized macrophages, however the levels of proinflammatory cytokines (TNF-α and IL-8) are higher in response to the 2.6NR strain, suggesting that EPS could be considered a beneficial immunomodulator.Peer reviewe

Caracterización de la síntesis del (1,3) (1,2)-[beta]D-Glucano y de las bacterias lácitcas productoras

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-06-2010Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented foods, and among them, some β-glucans have immunomodulating properties. Pediococcus parvulus 2.6 and other lactic acid bacteria (LAB) isolated from infected alcoholic beverages produce an identical (1-3, 1-2)-β-D-glucan. The characterization of the synthesis of this EPS is the main objective of this work. The study was performed with a combination of genetic engineering, molecular microbiological, biochemical and biophysical approaches. The results obtained as well as their interpretation are detailed below. We have identified and determine the nucleotide sequence of 6 glycosyltransferase (gtf) genes from LAB belonging to the Pediococcus, Lactococcus and Oenococus genera. Their genetic location in P. parvulus 2.6, L. diolivorans G77 and O. oeni I4 has been established. The gene is carried by two different plasmids of 35 kb and 5,5 kb in the first two hosts, whereas in O. oeni the gene is chromosomally located. Bioinformatic analysis of the gtf gene product indicated that it is an enzyme that belongs to the GT-2 membrane-bond glycosyltransferase family. Moreover, the topological prediction of the GTF protein indicates that a cytosolic glycosyl transferase domain is flanked by two and four trans-membrane segments. Detection of glycosyltransferase activity in membrane preparations of GTF overexpressor strains strongly supports the bioinformatic predictions for the enzyme activity and location. In addition, the heterologous expression of the P. parvulus 2.6R gtf in Streptococcus pneumoniae and in Lactoccus lactis enable these strains to synthesize a β-glucan immunologically indistinguishable from that of S. pneumoniae serotype 37. The overproduction of the P. parvulus 2.6 GTF glucosyltransferase in L. lactis resulted in synthesis and secretion of 300 mg/l of the (1,3)(1,2)-β-D-glucan in this host. The method used to purify the EPS allowed an 80% recovery of pure β-glucan with a high molecular mass (6,5 x 106 Da) similar to that of the biopolymer synthesized by P. parvulus (9,6 x 106 Da). An immunological method for detection of (1,3)(1,2)-β-D-glucans has been standardized and validated in this work. The ELISA method is based in the use of anti-serotype 37 pneumococcal antibodies and allows direct quantification of the exopolysaccharide in the supernatant of its producing bacteria grown in complex media, which contain mono- and polysaccharides with a threshold of 18 ng/ml. The method is substantially specific for this β-Dglucan, only detecting (1,3)(1,6)-β-glucan at high concentrations (>10 μg/ml) and not interacting with linear (1,3)-β-D-glucan or heteropolysaccharides. The above described method has been also used for the in vitro enzymatic characterization of the GTF glucosyltransferase associated with membrane vesicles of L. lactis NZ9000. The results showed that the activity is optimal at 37ºC in the pH range of 6,5 to 7, it displays typical Michaelis Menten kinetics with respect to UDP-glucose (Km = 0,125 mM) and has a Vmax = 0,018 μg EPS/mg prot /min. Moreover, β-glucan synthesis proceeds linearly with time up to 4 h, and catalysis is inhibited in the presence of UTP, UDP or EDTA. Finally, the chemical characterization of the reaction product revealed that indeed it is the (1-3)(1-2)-β-D-glucan. Finally, a comparative analysis has been performed of the potential probiotic properties of the β-D-glucan producer P. parvulus 2.6R and its isogenic EPS-non-producer 2.6RN strain. After exposure to gastrointestinal stress, both strains showed the same pattern of resistance, indicating that the ability to produce EPS does not protect P. parvulus in this sense. By contrast, the adhesion of 2.6R to Caco-2 cells was twenty four-fold higher than that of 2.6NR strain. Moreover, the high adherence of the EPS producer (6%) to Caco-2 cells was partially abolished (decrease to 1.8%) after EPS removal by washing. Both strains induce the production of the anti-inflammatory cytokine IL-10 by polarized macrophages, however the levels of proinflammatory cytokines (TNF-α and IL-8) are higher in response to the 2.6NR strain, suggesting that EPS could be considered a beneficial immunomodulator

Secuencias, vectores y células GTF y sus aplicaciones en el sector alimentario.

Fecha de presentación internacional: 08.09.2005.- Titulares: Consejo Superior de Investigaciones Científicas (CSIC).- Universidad de Cantabria.- Universidad del País Vasco.[EN]The invention relates to GTF micro-organisms, vectors and sequences which are formed by or comprise gene gtf de P.damnosus 2.6 which encodes a glycosyltransferase that is located in the membrane and which enables the production of exopolysaccharides of the ss-glucan type by overexpression of said recombinant gene. The fact that only one gene is involved in the synthesis of the aforementioned enzyme facilitates the optimisation of the expression conditions which enable the production of ss-glucans on an industrial scale. Said polymers, which can be used as additives in numerous foods, also have properties that are beneficial to human health. Moreover, said transformed GTF micro-organisms can be used in processes for the fermentation of foods, such as dairy products, alcoholic drinks and oats.[ES]Esta invención presenta secuencias, vectores y microorganismos GTF que están constituidas o comprenden el gen gtfde P damnosus 2.6 que codifica una glicosiltransferasa localizada en la membrana y que permite la producción de exopolisacáridos del tipo B-glucano por sobreexpresión de dicho gen recombinante. El hecho de que sea un único gen el implicado en la síntesis de este enzima facilita la optimización de las condiciones de expresión que permitirán la producción de B-glucanos a escala industrial. Estos polímeros, que pueden ser utilizados como aditivos en numerosos alimentos, poseen además propiedades beneficiosas para la salud humana. Por otro lado, estos microorganismos GTF transformados pueden ser utilizado en procesos de fermentación de alimentos como productos lácteos, bebidas alcohólicas y avena.Peer reviewe

Comparative analysis of production and purification of homo- and hetero-polysaccharides produced by lactic acid bacteria

2 figuras, 2 tablasLactic acid bacteria (LAB) produce homopolysaccharides (HoPS) and heteropolysaccharides (HePS) with potential functional properties. In this work, we have performed a comparative analysis of production and purification trials of these biopolymers from bacterial culture supernatants. LAB strains belonging to four different genera, both natural as well as recombinant, were used as model systems for the production of HoPS and HePS. Two well characterized strains carrying the gft gene were used for β-glucan production, Pediococcus parvulus 2.6 (P. parvulus 2.6) isolated from cider, and the recombinant strain Lactococcus lactis NZ9000[pGTF] (L. lactis NZ9000[pGTF]). In addition, another cider isolate, Lactobacillus suebicus CUPV225 (L. suebicus CUPV225), and Leuconostoc mesenteroides RTF10 (L. mesenteroides RTF10), isolated from meat products were included in the study. Chemical analysis of the EPS revealed that L. mesenteroides produces a dextran, L. suebicus a complex heteropolysaccharide, and the β-glucan producing-strains the expected 2-substituted (1,3)-β-glucanThis study was supported by grants AGL2009-12998-C03 and CSD2007-00063 from the Spanish Ministry of Science and Innovation, ACOMP/2009/257 from the Generalitat Valenciana, and IT335-10 from the Basque Government. M. N-V and S. N. are the recipients of JAE pre-doctor grants from the “Consejo Superior de Investigaciones Científicas” (CSIC)Peer reviewe

Naturally occurring 2-substituted (1,3)-β-D-glucan producing Lactobacillus suebicus and Pediococcus parvulus strains with potential utility in the production of functional foods

35 p.-4 fig.-2 fig. supl.We have isolated three lactic acid bacteria (Lactobacillus suebicus CUPV221, Pediococcus parvulus CUPV1 and P. parvulus CUPV22) that produced high levels of 2-substituted (1,3)-β-d-glucans which increased the viscosity of the growth media. The (1,3)-β-d-glucan consisted of two main molecular species, with masses of approximately 107 and 104 Da, whose proportions varied among the strains. The three strains survived exposure to saliva and simulated gastric conditions at pH 5, with P. parvulus CUPV22 surviving at pH 3.1, and L. suebicus CUPV221 surviving at pH 1.8. All strains were resistant to pancreatin and bile salts. P. parvulus CUPV22 exhibited the highest adhesion (10.5%) to Caco-2 cells, which decreased to 1.2% after washing the cells. Finally, P. parvulus CUPV22 and L. suebicus CUPV221 induced the production of inflammation-related cytokines by polarized macrophages, and interestingly, L. suebicus stimulated the production of cytokine IL-10. These results indicate that the three strains have potential utility for the production of functional foodsThis work was supported by the Ministerio de Ciencia e Innovación (Projects AGL2006-11932 and AGL2009-12998, Subprojects 01 and 02), the Universidad del País Vasco (UPV/EHU) (EHU08/37) and the Diputación Foral de Gipuzkoa, Programa Red Gipuzkoana de Ciencia, Tecnología e Innovación (co-financed by the European Union). Gaizka Garai-Ibabe acknowledges the Gobierno Vasco (Dpto. Agricultura, Pesca y Alimentación) for the pre-doctoral fellowshipPeer reviewe

Heterologous Expression of a Position 2-Substituted (1→3)-β-d-Glucan in Lactococcus lactis▿ †

Exopolysaccharides play an important role in the rheology and texture of fermented foods, and among these β-glucans have immunomodulating properties. We show that the overproduction of the Pediococcus parvulus GTF glycosyltransferase in an uncapsulated Lactococcus lactis strain results in synthesis and secretion (300 mg liter−1) of a position 2-substituted (1→3)-β-d-glucan that has potential use as a food additive