A methodology for the selection and characterization of riboflavin-overproducing Weissella cibaria strains after treatment with roseoflavin.

Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype.

Exopolysaccharide-producing Lacticaseibacillus paracasei strains isolated from kefir as starter for functional dairy products.

Exopolysaccharides (EPS) produced by lactic acid bacteria are molecules of great interest for the dairy food industry. Lacticaseibacillus paracasei CIDCA 8339, CIDCA 83123, and CIDCA 83124 are potentially probiotic strains isolated from kefir grains whose EPS-production on MRS broth is dependent on incubation temperature. The aim of the present work is to evaluate the effect of fermentation temperature on the characteristics of EPS produced in milk by L. paracasei strains and the consequent impact on the rheological properties of the fermented products. Additionally, the protective effect of these EPS against Salmonella infection was evaluated in vitro. Acid gels with each strain were obtained by milk fermentation at 20°C, 30°C, and 37°C evidencing for all the strains a reduction in growth and acidification rate at lower temperature. Lacticaseibacillus paracasei CIDCA 83123 showed low fermentation rate at all temperatures requiring between 3 and 8 days to obtain acids gels, whereas CIDCA 8339 and 83124 needed between 24 and 48 h even when the temperature was 20°C. Fermentation temperature led to changes in crude EPS characteristics of the three strains, observing an increase in the relative amount of the high molecular weight fraction when the fermentation temperature diminished. Additionally, EPS83124 and EPS83123 presented modifications in monosaccharide composition, with a reduction of rhamnose and an increase of amino-sugars as temperature rise. These changes in the structure of EPS83124 resulted in an increase of the apparent viscosity of milks fermented at 20°C (223 mPa.s) and 30°C (217 mPa.s) with respect to acid gels obtained at 37°C (167 mPa.s). In order to deepen the knowledge on EPS characteristics, monosaccharide composition of low and high molecular weight EPS fractions were evaluated. Finally, it was evidenced that the preincubation of intestinal epithelial cells Caco-2/TC-7 with EPS8339 and EPS83124 partially inhibit the association and invasion of Salmonella. In light of these results, it can be concluded that the selection of the EPS-producing strain along with the appropriate fermentation conditions could be an interesting strategy to improve the technological properties of these L. paracasei fermented milks with potential protective effects against intestinal pathogens.

Biotechnological Potential and Safety Evaluation of Dextran- and Riboflavin-Producing Weisella cibaria Strains for Gluten-Free Baking.

Gluten consumption causes several immunological and non-immunological intolerances in susceptible individuals. In this study, the dextran-producing Weissella cibaria BAL3C-5 and its derivative, the riboflavin-overproducing strain BAL3C-5 C120T, together with a commercial bakery yeast, were used to ferment gluten-free (GF)-doughs obtained from corn and rice flours at two different concentrations and supplemented with either quinoa, buckwheat, or chickpea to obtain laboratory-scale GF bread. The levels of dextran, riboflavin, and total flavins were determined in the fermented and breads. Both strains grew in fermented doughs and contributed dextran, especially to those made with corn plus quinoa (~1 g/100 g). The highest riboflavin (350-150 µg/100 g) and total flavin (2.3-1.75 mg/100 g) levels were observed with BAL3C-5 C120T, though some differences were detected between the various doughs or breads, suggesting an impact of the type of flour used. The safety assessment confirmed the lack of pathogenic factors in the bacterial strains, such as hemolysin and gelatinase activity, as well as the genetic determinants for biogenic amine production. Some intrinsic resistance to antibiotics, including vancomycin and kanamycin, was found. These results indicated the microbiological safety of both W. cibaria strains and indicated their potential application in baking to produce GF bread.

Functional Analysis of Lactic Acid Bacteria and Bifidobacteria and Their Effects on Human Health.

Many lactic acid bacteria (LAB) and Bifidobacteria are beneficial components of human, animal, foods, and beverage microbiota [...].

Biological Functions of Exopolysaccharides from Lactic Acid Bacteria and Their Potential Benefits for Humans and Farmed Animals.

Lactic acid bacteria (LAB) synthesize exopolysaccharides (EPS), which are structurally diverse biopolymers with a broad range of technological properties and bioactivities. There is scientific evidence that these polymers have health-promoting properties. Most commercialized probiotic microorganisms for consumption by humans and farmed animals are LAB and some of them are EPS-producers indicating that some of their beneficial properties could be due to these polymers. Probiotic LAB are currently used to improve human health and for the prevention and treatment of specific pathologic conditions. They are also used in food-producing animal husbandry, mainly due to their abilities to promote growth and inhibit pathogens via different mechanisms, among which the production of EPS could be involved. Thus, the aim of this review is to discuss the current knowledge of the characteristics, usage and biological role of EPS from LAB, as well as their postbiotic action in humans and animals, and to predict the future contribution that they could have on the diet of food animals to improve productivity, animal health status and impact on public health.

Lactic Acid Bacteria Isolated from Fermented Doughs in Spain Produce Dextrans and Riboflavin.

Many lactic acid bacteria (LAB) produce metabolites with applications in the food industry, such as dextran-type exopolysaccharides (EPS) and riboflavin (vitamin B2). Here, 72 bacteria were isolated from sourdoughs made by Spanish bread-makers. In the presence of sucrose, colonies of 22 isolates showed a ropy phenotype, and NMR analysis of their EPS supported that 21 of them were dextran producers. These isolates were identified by their random amplified polymorphic DNA (RAPD) patterns and their rrs and pheS gene sequences as LAB belonging to four species (Weissella cibaria, Leuconostoc citreum, Leuconostoc falkenbergense and Leuconostoc mesenteroides). Six selected strains from the Leuconostoc (3) and Weissella (3) genera grew in the absence of riboflavin and synthesized vitamin B2. The EPS produced by these strains were characterized as dextrans by physicochemical analysis, and the L. citreum polymer showed an unusually high degree of branching. Quantification of the riboflavin and the EPS productions showed that the W. cibaria strains produce the highest levels (585-685 µg/and 6.5-7.4 g/L, respectively). Therefore, these new LAB strains would be good candidates for the development of fermented foods bio-fortified with both dextrans and riboflavin. Moreover, this is the first report of riboflavin and dextran production by L. falkenbergense.

Quantitative Determination of Acrolein in Cider by 1H NMR Spectrometry.

Acrolein occasionally appears in cider, completely spoiling its quality due to its bitter taste. It is crucial to detect it in the early steps, before the taste is severely affected, to apply the appropriate treatment. A simple and rapid analytical method to determine this compound in cider is therefore desirable. In this work, a quantitative determination method of acrolein in cider is proposed using the proton nuclear magnetic resonance technique (1H NMR). Acrolein produces a doublet signal in the spectrum at 9.49 ppm, whose area is used to determine the concentration of this compound. Importantly, 3-(Trimethylsilyl)-2,2,3,3-d4-propionic acid sodium salt is added to the cider as a reference for 0.00 ppm and 1,3,5-benzenetricarboxylic acid as an internal standard for acrolein determination. The method is validated by gas chromatography (GC). There is a good correlation between the acrolein concentrations obtained by 1H NMR and by gas chromatography in different commercial ciders (Pearson coefficient 0.9994). The 95% confidence interval for the intercept is 0.15 ± 0.49 (includes 0) and for the slope is 0.98 ± 0.03 (includes 1). When applying the paired t test, no significant difference is observed. The proposed method is direct, and no prior derivatization is needed.

Compression Molded Soy Protein Films with Exopolysaccharides Produced by Cider Lactic Acid Bacteria.

Two exopolysaccharide (EPS)-producing lactic acid bacteria (LAB) strains, Liquorilactobacillus (L.) sp CUPV281 and Liquorilactobacillus (L.) mali CUPV271, were isolated from Spanish apple must. Each of the strains produced a dextran, with different branching degrees, to be incorporated into soy protein isolate (SPI) film-forming formulations. Films were prepared by compression molding, a more rapid processing method than solution casting and, thus, with a greater potential for scaling-up production. Thermal analysis showed that SPI and EPS start the degradation process at temperatures above 190 °C, confirming that the compression temperature selected (120 °C) was well below the corresponding degradation temperatures. Resulting films were transparent and homogeneous, as shown by UV-Vis spectroscopy and SEM, indicating the good compatibility between SPI and EPS. Furthermore, FTIR analysis showed that the interactions between SPI and EPS were physical interactions, probably by hydrogen bonding among the polar groups of SPI and EPS. Regarding antifungal/fungistatic activity, LAB strains used in this study showed an inhibitory effect on germination of fungal spores.

Effects of anti-TNF-alpha therapy on hemoglobin levels and anemia in patients with inflammatory bowel disease.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is involved in inducing inflammatory anemia. The potential effect of anti-TNF-α agents on anemia in inflammatory bowel diseases (IBD) is still unknown. METHODS: Analytical data and disease characteristics from 362 IBD patients [271 CD/91UC) treated with anti-TNF-α drugs were retrospectively collected. Effects on disease activity, blood markers and prevalence of anemia were assessed after 6 and 12 months of therapy. RESULTS: 29.3% patients presented anemia at baseline, and significantly reduced to 14.4% and 7.8% after 6 and 12 months of therapy, respectively. Mean ±â€¯SD Hb levels increased significantly at month 6, and this increase was sustained at 12 months. Serum markers of iron metabolism increased significantly compared to baseline, as disease activity measured by C-reactive protein (CRP) was reduced. All these effects were observed independently for CD and UC, and were independent of iron supplementation during treatment. Anemia at baseline (OR 4.09; 95%CI 1.98-8.45) and elevated CRP (OR 3.45; 95CI 1.29-9.22) were independently associated with risk of persistent anemia, as well as iron replacement during therapy (OR 4.36; 95%CI 2.07-9.16). CONCLUSIONS: Controlling disease activity with anti-TNF- α therapy significantly and independently associated with resolution of anemia in IBD, with no relevant role for iron replacement therapy.

Rheology and bioactivity of high molecular weight dextrans synthesised by lactic acid bacteria.

Dextrans synthesised by three Leuconostoc mesenteroides strains, isolated from mammalian milks, were studied and compared with dextrans produced by Lc. mesenteroides and Lactobacillus sakei strains isolated from meat products. Size exclusion chromatography coupled with multiangle laser light scattering detection analysis demonstrated that the dextrans have molecular masses between 1.74×108Da and 4.41×108Da. Rheological analysis of aqueous solutions of the polymer revealed that all had a pseudoplastic behaviour under shear conditions and a random, and flexible, coil structure. The dextrans showed at shear zero a difference in viscosity, which increased as the concentration increased. Also, the purified dextrans were able to immunomodulate in vitro human macrophages, partially counteracting the inflammatory effect of Escherichia coli O111:B4 lipopolysaccharide. During prolonged incubation on a solid medium containing sucrose, dextran-producing bacteria showed two distinct phenotypes not related to the genus or species to which they belonged. Colonies of Lc. mesenteroides CM9 from milk and Lb. sakei MN1 from meat formed stable and compact mucoid colonies, whereas the colonies of the other three Leuconostoc strains became diffuse after 72h. This differential behaviour was also observed in the ability of the corresponding strains to bind to Caco-2 cells. Strains forming compact mucoid colonies showed a high level of adhesion when grown in the presence of glucose, which decreased in the presence of sucrose (the condition required for dextran synthesis). However no influence of the carbon source was detected for the adhesion ability of the other Lc. mesenteroides strains, which showed variable levels of binding to the enterocytes.

Draft Genome Sequence of Lactobacillus collinoides CUPV237, an Exopolysaccharide and Riboflavin Producer Isolated from Cider.

Lactobacillus collinoides CUPV237 is a strain isolated from a Basque cider. Lactobacillus collinoides is one of the most frequent species found in cider from Spain, France, or England. A notable feature of the L. collinoides CUPV237 strain is its ability to produce exopolysaccharides.

Polyphenolic profile in cider and antioxidant power.

BACKGROUND: The aim of this work was to find the effect of polyphenolic compounds in Basque ciders on the following parameters: antioxidant activity, browning, protein-precipitating capacity, turbidity and reduction potential. These five parameters are highly important, as they affect the taste, the visual aspect and the preservation of cider, and are mainly related to polyphenolic compounds. RESULTS: Procyanidin B1 and procyanidin B2 showed a significant positive effect on antioxidant activity. p-Coumaric acid, (-)-epicatechin and hyperin had a significant positive effect on protein-precipitating capacity. Tyrosol had a significant negative effect on reduction potential. CONCLUSION: Procyanidin B1 and procyanidin B2 are the most powerful antioxidants in Basque cider, while p-coumaric acid, (-)-epicatechin and hyperin are those with greatest capacity to precipitate proteins. Ciders with higher tyrosol concentration will have less reduction potential and higher antioxidant reservoir.

A specific immunological method to detect and quantify bacterial 2-substituted (1,3)-ß-D-glucan.

Exopolysaccharides synthesized by lactic acid bacteria have prebiotic properties and contribute to the rheology and texture of fermented foods. Here, we have standardized an immunological method for the specific detection of 2-substituted (1,3)-ß-D-glucans. The method allows direct detection and quantification of this exopolysaccharide in culture supernatants containing other mono- and poly-saccharides. Moreover, it allows specific detection of the biomolecules synthesized in vitro in enzymatic reactions. Thus, this method allows the fast identification of producing bacteria, as well as biochemical characterization of the glycosyltransferases responsible for their synthesis.

Riboflavin-overproducing strains of Lactobacillus fermentum for riboflavin-enriched bread.

Lactobacillus fermentum isolated from sourdough was able to produce riboflavin. Spontaneous roseoflavin-resistant mutants were obtained by exposing the wild strain (named L. fermentum PBCC11) to increasing concentrations of roseoflavin. Fifteen spontaneous roseoflavin-resistant mutants were isolated, and the level of vitamin B2 was quantified by HPLC. Seven mutant strains produced concentrations of vitamin B2 higher than 1 mg L⁻¹. Interestingly, three mutants were unable to overproduce riboflavin even though they were able to withstand the selective pressure of roseoflavin. Alignment of the rib leader region of PBCC11 and its derivatives showed only point mutations at two neighboring locations of the RFN element. In particular, the highest riboflavin-producing isolates possess an A to G mutation at position 240, while the lowest riboflavin producer carries a T to A substitution at position 236. No mutations were detected in the derivative strains that did not have an overproducing phenotype. The best riboflavin overproducing strain, named L. fermentum PBCC11.5, and its parental strain were used to fortify bread. The effect of two different periods of fermentation on the riboflavin level was compared. Bread produced using the coinoculum yeast and L. fermentum PBCC11.5 led to an approximately twofold increase of final vitamin B2 content.

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products.

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.

Beta-glucans improve growth, viability and colonization of probiotic microorganisms.

Probiotics, prebiotics and synbiotics are frequently-used components for the elaboration of functional food. Currently, most of the commercialized probiotics are limited to a few strains of the genera Bifidobacteria, Lactobacillus and Streptococcus, most of which produce exopolysaccharides (EPS). This suggests that the beneficial properties of these microorganisms may be related to the biological activities of these biopolymers. In this work we report that a 2-substituted-(1,3)-ß-d-glucan of non-dairy bacterial origin has a prebiotic effect on three probiotic strains. Moreover, the presence of this ß-d-glucan potentiates in vitro adhesion of the probiotic Lactobacillus plantarum WCFS1 to human intestinal epithelial cells.

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.

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.