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

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)-beta-D-glucans which increased the viscosity of the growth media. The (1,3)-beta-D-glucan consisted of two main molecular species, with masses of approximately 10(7) and 10(4) 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 foods.

A real-time PCR assay for detection and quantification of 2-branched (1,3)-beta-D-glucan producing lactic acid bacteria in cider.

Ropiness in natural cider is a relatively frequent alteration, mainly found after bottling, leading to consumer rejection. It is derived from the production of exopolysaccharides (EPS) by some lactic acid bacteria most of which synthesize a 2-branched (1,3)-beta-D-glucan and belong to the genera Pediococcus, Lactobacillus and Oenococcus. This polysaccharide synthesis is controlled by a single transmembrane glycosyltransferase (GTF). In this work, a method based on quantitative PCR (qPCR) and targeting the gtf gene was developed for detection and quantification of these bacteria in cider. The newly designed primers GTF3/GTF4 delimit a 151bp fragment within the 417bp amplicon previously designed for conventional PCR. The inclusivity and exclusivity of the qPCR assay were assessed with 33 cider isolates belonging to genus Lactobacillus, Oenoccocus and Pedioccocus, together with reference strains of 16 species and five genera including beta-glucan, alpha-glucan and heteropolysaccharide (HePS) producing strains and non-EPS producers. The qPCR assay, followed by the melting curve analysis, confirmed the generation of a single PCR product from the beta-glucan producers with a T(m) of 74.28+/-0.08 and C(T) values (10ng DNA) ranging between 8.46 and 16.88 (average 12.67+/-3.5). Some EPS(-) LAB strains rendered C(T) values ranging from 28.04 to 37.75 but they were significantly higher (P(C(T)<28.54)=0.05) than those of the beta-glucan producers. The assay showed a wide quantification range of 5 log units using calibrated cell suspensions of Pediococcus parvulus 2.6 and Oenococcus oeni I4. The linearity was extended over 7 log orders when calibration curves were obtained from DNA. The detection limit for beta-glucan producing LAB in artificially contaminated cider was about 3x10(2)CFU per ml. The newly developed qPCR assay was successfully applied to monitor the cidermaking process, in 13 tanks from two cider factories, revealing a decrease in C(T) values derived from an increase in beta-glucan producing LAB populations. In addition, 8 naturally spoiled bottled cider were tested for the quantification of these organisms using the five standard curves constructed: P. parvulus 2.6 genomic DNA and gtf amplicon (417bp), calibrated cell suspensions of Pediococcus parvulus 2.6, Lactobacillus diolivorans G77 and Oenococcus oeni I4 and results were compared to LAB total counts on MRS. Levels obtained from the different approaches were within a log range and showed no significant differences. Therefore, the amplicon-derived standard curve is proposed for the routine estimation of gtf(+)populations in cider.

Screening and selection of 2-branched (1,3)-beta-D-glucan producing lactic acid bacteria and exopolysaccharide characterization.

The ability to produce a 2-branched (1,3)-beta-D-glucan was screened in 147 lactic acid bacteria strains recovered from cider. Among them, 32 identified as Pediococcus parvulus exhibited a ropy character and were PCR positive for the presence of the gtf gene, related to the synthesis of the beta-glucan. Half of the strains produced more than 100 mg L(-1) of exopolysaccharide (EPS). (1)H NMR spectra of the crude EPSs were identical to that previously described for P. parvulus 2.6, indicating that all are 2-branched (1,3)-beta-D-glucans. The EPSs from two of the isolates were subjected to acid hydrolysis and methylation analysis, confirming the NMR results. Size exclusion chromatography (SEC) showed in all crude EPSs the presence of two different molecular mass fractions of about 10(7) Da and 10(4) Da, whose relative proportions varied among strains. EPS amounts and concentrations of high molecular fraction are linearly correlated. Intraspecific diversity of isolates was determined by RAPD profiles. Based on genotypic and phenotypic characteristics, two strains were selected to be further studied as probiotics.

Supramolecular structure and conformation of a (1-->3)(1-->2)-beta-D-glucan from Lactobacillus suebicus CUPV221 as observed by tapping mode atomic force microscopy.

Tapping mode atomic force microscopy (TM-AFM) has been used to analyze the supramolecular structure and conformation of the (1-->3)(1-->2)-beta-D-glucan produced by Lactobacillus suebicus CUPV221 isolated from cider. Solutions for TM-AFM observation were prepared by dispersing the solid glucan in distilled water and in alkaline aqueous solutions. It was found that from the distilled water at 10 mg/L or higher concentrations, the (1-->3)(1-->2)-beta-D-glucan forms networks. The heat resistance of the networks depends on the concentration. From the alkaline aqueous solutions, different supramolecular structures were observed depending on the pH. From the weakest alkaline solution, a fairly rough morphology with a high density of spikelike growth features was revealed. As the ionic force of the medium increased, the sizes of the spikelike growth features diminished, and even many disaggregated fibers could be found. At 0.4 M NaOH (pH 13.16), the aggregates had disappeared almost totally. NaOH aqueous solutions (0.1 and 0.4 M) were used to carry out the study of conformation. At 0.1 M NaOH, the aggregates were partially detached, and many free microfibers were found to which a helical conformation could be assigned due to their stiffness and rodlike character. At 0.4 M NaOH, the beginning of the dissociation of the helical structures was seen.

Probiotic properties of the 2-substituted (1,3)-beta-D-glucan-producing bacterium Pediococcus parvulus 2.6.

Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the 2-substituted (1,3)-beta-D-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages.

Chemical and rheological properties of the beta-glucan produced by Pediococcus parvulus 2.6.

Some physicochemical and rheological properties of the exopolysaccharide (EPS) produced by Pediococcus parvulus 2.6 were examined. Structural characterization by NMR ((1)H and 2D-COSY) showed that the same EPS, a 2-substituted (1,3)-beta-D-glucan, was synthesized irrespective of sugar source used for growth (glucose, fructose, or maltose). The molecular masses of these beta-glucans were always very high (>10(6) Da) and influenced by the culture medium or sugar source. The steady shear rheological experiments showed that all concentrations of the beta-glucan aqueous solutions exhibited a pseudoplastic behavior at high shear rates. Viscoelastic behavior of beta-glucan solutions was determined by dynamic oscillatory analysis. A critical concentration of 0.35% associated with the appearance of entanglements was calculated. The beta-glucan adopts an ordered hydrogen bond dependent helical conformation in neutral and slightly alkaline aqueous solutions, which was partly denatured under more alkaline conditions.

Development of alcoholic and malolactic fermentations in highly acidic and phenolic apple musts.

This work reports the influence of the high acidity and high phenolic content in apple musts on the development of alcoholic and malolactic fermentations and on the final chemical and microbiological composition of the ciders. Four different musts were obtained by pressing several varieties and proportions of cider apples from the Basque Country (Northern Spain). Specially acidic and phenolic varieties were selected. Three musts were obtained in experimental stations and the fourth one, in a cider factory following usual procedures. The evolution of these musts was monitored during five months by measuring 18 parameters throughout eight samplings. In the most acidic of the three experimental musts, yeasts were added to complete the alcoholic fermentation. In the rest of the musts, alcoholic and malolactic fermentations took place spontaneously due to natural microflora and no chemical was added to control these processes. Malolactic fermentation (MLF) finished before alcoholic fermentation in the three tanks obtained in experimental stations, even in the most acidic and phenolic one (pH 3.18, 1.78 g tannic acid/l). After four months, these ciders maintained low levels of lactic acid bacteria (10(4)CFU/ml) and low content of acetic acid (<0.60 g/l). Both fermentations began simultaneously in the must obtained in the cider factory, but MLF finished 10 days after alcoholic fermentation. Subsequently, this must maintained a high population of lactic acid bacteria (>10(6)CFU/ml), causing a higher production of acetic acid (>1.00 g/l) than in the other ciders. These results show the possible advantages of MLF finishing before alcoholic fermentation.

Pediococcus parvulus gtf gene encoding the GTF glycosyltransferase and its application for specific PCR detection of beta-D-glucan-producing bacteria in foods and beverages.

Exopolysaccharide production by lactic acid bacteria is beneficial in the dairy and oat-based food industries and is used to improve the texture of the fermented products. However, beta-D-glucan-producing bacteria are considered spoilage microorganisms in alcoholic beverages because their secreted exopolysaccharides alter the viscosity of cider, wine, and beer, rendering them unpalatable. The plasmidic glycosyltransferase (gtf) gene of the Pediococcus parvulus 2.6 strain isolated from ropy cider has been cloned and sequenced, and its GTF product was functionally expressed in Streptococcus pneumoniae. The GTF protein, which has glycosyltransferase activity, belongs to the COG1215 membrane-bound glycosyltransferase family, and agglutination tests revealed that the enzyme enables S. pneumoniae to synthesize beta-D-glucan. PCR amplification and Southern blot hybridization showed that the gtf gene is also present at different genomic locations in the beta-D-glucan producers Lactobacillus diolivorans G77 and Oenococcus oeni I4 strains, also isolated from ropy cider. A PCR assay has been developed for the detection of exopolysaccharide-producing bacteria. Forward and reverse primers, included respectively in the coding sequences of the putative glycosyltransferase domain and the fifth trans-membrane segment of the GTF, were designed. Analysis of 76 ropy and nonropy lactic acid bacteria validated the method for specific detection of beta-D-glucan homopolysaccharide producer Pediococcus, Lactobacillus, and Oenococcus strains. The limit of the assay in cider was 3 X 10(2) CFU/ml. This molecular method can be useful for the detection of ropy bacteria in cider before spoilage occurs, as well as for isolation of new exopolysaccharide-producing strains of industrial interest.

Exopolysaccharide production by Pediococcus damnosus 2.6 in a semidefined medium under different growth conditions.

Ropy Pediococcus damnosus (strain 2.6) was used for production of exopolysaccharide (EPS) in a semidefined medium. From a kinetic point of view, an experiment conducted in SMD medium containing 30 g l(-1) glucose and 5 g l(-1) Bacto casamino acids (Difco Laboratories, Detroit, MI), without pH control, showed that EPS production took place mainly during the growth phase. The viscosity of the cultures developed in parallel to the EPS synthesis until 94 h of incubation; after 200 h of fermentation, viscosity decreased. The effect of glucose, Bacto casamino acid concentrations and temperature on growth and EPS production was determined by using a full factorial design. Within the domain of experimental conditions considered, the concentration of glucose and Bacto casamino acids has a significant effect on the production of exopolysaccharide. The incubation at 12 degrees C produced a prolonged lag phase and due to the lower growth yield, higher specific EPS production was found at this temperature. At 25 degrees C the EPS production was mainly enhanced by the increase in glucose concentration. The increase in nitrogen concentration from 5 to 15 g l(-1) did not yield greater EPS production. However, at 12 degrees C optimal EPS production was obtained when both higher glucose and nitrogen concentrations were used.

El destino del sintoma en Freud

Publicación del: Centro de Investigación y Estudios Clínicos, asociado con el Instituto del Campo Freudiano y el Area de Psicoanálisis del Ex Hospital Neuro - Psiquiátrico Provincial de Córdoba