Under optimized conditions, the lactic acid bacterium Lactobacillus fermentum Lf2 secretes up to 2 gL-1 of a mixture of polysaccharides into the fermentation medium when grown on sucrose. Earlier studies had shown that the mixture is biologically active and work was undertaken to characterise the polysaccharides. Preparative size exclusion chromatography was used to separate a high molecular mass β-glucan (weight average mass of 1.23 × 106 gmol-1) from two medium molecular mass polysaccharides (weight average mass of 8.8 × 104 gmol-1). Under optimized growth conditions, the medium molecular mass polysaccharides accounted for more than 75% of the mixture by weight. Monomer, linkage analysis and NMR spectroscopy of the medium molecular mass polysaccharides, and material isolated after their Smith degradation, was used to identify the structure of the component polysaccharides. The mixture contains two novel polysaccharides. The first has a main chain of β-1,6-linked galactofuranoses which is non-stoichiometrically 2-O-glucosylated. The degree of substitution at the 2-position, with α-D-Glcp, depends on the fermentation conditions; under optimized conditions greater than 80% 2-O-α-D-glucosylation was observed. The second polysaccharide is a heteroglycan with four monosaccharides in the repeat unit: residual signals in the NMR suggest that the sample also contains trace amounts (<3%) of cell wall polysaccharides.

Polysaccharides have been isolated at pH 6.0 or 2.0 from fruits and leaves of two varieties of baobab (Adansonia digitata L.). The isolation protocol was designed to study the influence of pH on the structural and functional characteristics of polysaccharides. The polysaccharides were examined by means of sugar composition analysis, NMR spectroscopy, size exclusion chromatography (SEC) and dilute solution viscometry. Polysaccharide yield was substantially higher in fruits (~20% w/w) than in leaves (~4% w/w) with all samples having low protein content (<~0.5% w/w). Monosaccharide and NMR analysis revealed that polysaccharides extracted from leaves have characteristics of “linear” and “branched” pectins (~71 mol% uronic acids, ~12 mol% rhamnose and ~9 mol% galactose) whereas those from fruits were primarily xylogalacturonans (~69 mol% uronic acids, ~13 mol% xylose). Polysaccharides from leaves had greater molecular weight (>300 × 103 g mol−1) than fruits (<200 × 103 g mol−1) presenting distinct solution behaviour. Polysaccharides from leaves were only dispersed at low concentrations (~1% w/v) whereas those from fruits may be dispersed at >20% w/v, representing a clear divide in their functional properties and potential applications. Overall, baobab fruits and leaves offer a novel source of polysaccharides for the food or pharmaceutical industries.

Lime manufacture in the UK has resulted in the generation of a number of alkaline sites (>pH 11.0) with complex indigenous microbial populations. Within the present study, retrievable cotton samples were used to investigate the fate of cellulose, the primary carbon source, within three sites aged from approximate to 25 to 140years. Following 3 months incubation in situ, biofilms had formed on all cotton samples in these extreme pH conditions; with matrices comprised of carbohydrates, proteins, lipids and eDNA. Biofilms from the older sites contained greater amounts of eDNA, a structural component that aids the production of a denser biofilm. The age of the sites correlated with a shift from polysaccharides composed of 1,4 and 1,3 linked sugars to those composed of pyranosyl sugars within the older sites. These changes were reflected in the active biofilm communities which shifted from being Clostridiales dominated in the youngest site to Proteobacteria dominated in the older sites. The study demonstrates that the microbial communities resident in anthropogenic alkaline sites are able to form biofilms at pH values>pH 11.0 and that these biofilms evolve toward Proteobacteria dominated communities employing eDNA and pyranosyl sugar based polysaccharides to build the biofilm matrix.

A novel strain of Lactobacillus mucosae was isolated from a faecal sample of an individual who had adhered to a strict vegetarian diet for nine years. The strain displayed a ropy character when grown on plates and generated a relatively small amount (62 mg/L) of an exopolysaccharide (EPS) when grown in broth culture. The EPS eluted from a size exclusion chromatography column as a single band with a weight average molecular mass of 1.51 x 10(4) g/mol. Monomer analysis and sugar absolute configuration analysis confirmed that the EPS was a D-galactan. Using linkage analysis in combination with 1D and 2D-NMR spectroscopy, with spectra being recorded for both the native EPS and for the products generated by Smith degradation of the EPS, the following structure was determined for the repeat unit of the polysaccharide: [GRAPHICS] . This is a novel D-galactan and represents the first structure for an EPS produced by a strain of Lactobacillus mucosae to be reported.

When grown in a semi-defined medium, L. fermentum Lf2 synthesizes significant quantities (∼2 g/L) of two exopolysaccharides (EPS). The two EPS were separated by preparative size exclusion chromatography to give a high molecular mass β-glucan (1.23 × 106 Da) and a medium molecular mass heteroglycan (8.8 × 104 Da). The structure of the high molecular mass β-glucan was determined using a combination of NMR spectroscopy, monomer and linkage analysis. The EPS has the following structure: The immunomodulatory activity of the high molecular mass EPS was studied in peripheral blood mononuclear cells (PBMC). Exposure of PBMC to an aqueous solution of the EPS for 24 h led to increased cell proliferation, changes in expression of the cytokines CD14 and TLR2, and to an increase in production of TNF-α compared to controls. In contrast, when cells that had been treated with EPS for 24 h and from which the EPS had been removed, were subsequently exposed to the bacterial antigen LPS very low levels of TNF-α production were observed. This result indicates that the EPS imparts immunotolerance in PBMC. An ability to modulate the release of the proinflammatory mediators, such as TNF-α, is an important goal in the development of therapies for the treatment of diseases, such as Crohn's disease and ulcerative colitis, associated with excessive release of inflammatory mediators.

Lactobacillus fermentum Lf2 produces high amounts of exopolysaccharides (EPS) (~1 g/L) with demonstrated functional and technological roles when applied as a food ingredient in dairy matrices, properties that made these EPS interesting in comparison with other similar molecules from lactic acid bacteria (LAB). Those characteristics encouraged us to optimise the production. The EPS extract is composed of a high molecular mass β‐glucan and a medium molecular mass heteroglycan. In the present work, the optimal conditions that doubled the EPS yield using a semidefined medium (SDM, 0.63% yeast nitrogen base, 0.53% bacto casitone, 0.53% ammonium citrate, 6.25% sucrose, pH 6.5) were found by means of response surface methodology (RSM). The chemical characterisation indicated that under optimised conditions the synthesis of the heteroglycan was favoured compared with that of the β‐glucan.

14C is an important consideration within safety assessments for proposed geological disposal facilities for radioactive wastes, since it is capable of re-entering the biosphere through the generation of 14C bearing gases. The irradiation of graphite moderators in the UK gas-cooled nuclear power stations has led to the generation of a significant volume of 14C-containing intermediate level wastes. Some of this 14C is present as a carbonaceous deposit on channel wall surfaces. Within this study, the potential of biofilm growth upon irradiated and 13C doped graphite at alkaline pH was investigated. Complex biofilms were established on both active and simulant samples. High throughput sequencing showed the biofilms to be dominated by Alcaligenes sp at pH 9.5 and Dietzia sp at pH 11.0. Surface characterisation revealed that the biofilms were limited to growth upon the graphite surface with no penetration of the deeper porosity. Biofilm formation resulted in the generation of a low porosity surface layer without the removal or modification of the surface deposits or the release of the associated 14C/13C. Our results indicated that biofilm formation upon irradiated graphite is likely to occur at the pH values studied, without any additional release of the associated 14C.

The potential of blackcurrant pomace as a raw material for the extraction of dietary fibre was evaluated using two pomaces one sourced from the UK and one from Poland. A fractionation protocol was designed to isolate and subsequently quantify the soluble and insoluble dietary fibre fractions. Blackcurrant pomace and isolated pectins, hemicelluloses and celluloses were assessed by means of sugar compositional analysis, spectroscopy, size exclusion chromatography and dilute solution viscometry. The blackcurrant pomaces presented considerable amounts of dietary fibre with soluble fibre ranging from 25 to 30% w/w and insoluble dietary fibre accounting for about 47% w/w for both pomaces. Blackcurrant pomaces differed in the amount of extracted pectins with an almost two times higher pectin yield obtained from blackcurrant pomace sourced from Poland. The hemicellulosic polysaccharide content was 15% w/w whereas the amount of cellulosic fraction varied from 14 to 17% w/w. Pectins isolated from both blackcurrant pomaces were LM pectins with a degree of esterification in the range of 11–38%. The work has identified that dietary fibres obtained from blackcurrant pomace had desirable ratio of insoluble to soluble fibre and are a potential new source of dietary fibre.

Three strains of Bifidobacterium breve (JCM 7017, JCM 7019 and JCM 2258) and two strains of Bifidobacterium animalis subsp. lactis (AD011 and A1dOxR) were grown in broth cultures or on plates, and a standard exopolysaccharide extraction method was used in an attempt to recover exocellular polysaccharides. When the extracted materials were analysed by NMR it was clear that mixtures of polysaccharides were being isolated including exopolysaccharides (EPS) cell wall polysaccharides and intracellular polysaccharides. Treatment of the cell biomass from the B. breve strains, or the B. animalis subsp. lactis AD011 strain, with aqueous sodium hydroxide provided a very similar mixture of polysaccharides but without the EPS. The different polysaccharides were partially fractionated by selective precipitation from an aqueous solution upon the addition of increasing percentages of ethanol. The polysaccharides extracted from B. breve JCM 7017 grown in HBM media supplemented with glucose (or isotopically labelled D-glucose-1-13C) were characterised using 1D and 2D-NMR spectroscopy. Addition of one volume of ethanol generated a medium molecular weight glycogen (Mw=1×105 Da, yield 200 mg/l). The addition of two volumes of ethanol precipitated an intimate mixture of a low molecular weight β-(1→6)-glucan and a low molecular weight β-(1→6)-galactofuranan which could not be separated (combined yield 46 mg/l). When labelled D-glucose-1-13C was used as a carbon supplement, the label was incorporated into >95% of the anomeric carbons of each polysaccharide confirming they were being synthesised in situ. Similar 1H NMR profiles were obtained for polysaccharides recovered from the cells of B. animalis subsp. lactis AD011and A1dOxR (in combination with an EPS), B. breve JCM 7017, B. breve JCM 7019, B. breve JCM 2258 and from an EPS (-ve) mutant of B. breve 7017 (a non-EPS producer).

Lactobacillus fermentum Lf 2 produces large amounts of exopolysaccharides under optimized conditions (∼2 g/L, EPS) which have been shown to possess immunomodulatory activity. In this study, the crude EPS was fractionated to give a high molecular weight (HMw) homoglycan and a mixture of medium molecular weight heteroglycans. The HMw EPS was isolated and identified as a β-glucan.Peripheral blood mononuclear cells (PBMC) were pre-treated with purified polysaccharide to determine if the HMw β-glucan is responsible for the immunomodulatory activity. Cells were also stimulated with either lipopolysaccharide (LPS) or phytohemagglutinin (PHA) and their effects, both with and without β-glucan pre-treatment, compared.Exposure of the cells to β-glucan increased their metabolic activity and whilst a small but statistically significant drop in CD14 expression was observed at Day 1, the levels were significantly elevated at Day 2. High levels of CD14 expression were observed in cells initially exposed to the β-glucan and subsequently stimulated with either LPS or PHA. In contrast, reduced levels of TLR-2 expression were observed for cells initially exposed to the β-glucan and subsequently stimulated with LPS.TNF-α levels were elevated in β-glucan treated cells (Day1) with the levels dropping back once the β-glucan had been removed (Day 2). The stimulants LPS and PHA both induced significant rises in TNF-α levels, however, this induction was completely (LPS) or partially blocked (PHA) in β-glucan pre-treated cells.The results indicate a role for the bacterial β-glucan in modulating the immune response following exposure to agonists such as bacterial LPS.

Alpha and beta-glucoisosaccharinic acids ((2S,4S)-2,4,5-trihydroxy-2-(hydroxymethyl)pentanoic acid and (2R,4S)-2,4,5-trihydroxy-2-(hydroxymethyl)pentanoic acid) which are produced when cellulosic materials are treated with aqueous alkali are potentially valuable platform chemicals. Their highly functionalised carbon skeleton, with fixed chirality at C-2 and C-4, makes them ideal starting materials for use in synthesis. In order to assess the potential of these saccharinic acids as platform chemicals we have explored the protecting group chemistry of the lactone form of alpha-glucoisosaccharinic acid (α-GISAL). We report here the use of single and multiple step reaction pathways leading to the regioselective protection of the three different hydroxyl groups of α-GISAL. We report strategies for protecting the three different hydroxyl groups individually or in pairs. We also report the synthesis of a range of tri-O-protected α-GISAL derivatives where a number of the products contain orthogonal protecting groups.

Alkaline environments represent a significant challenge to the growth of micro-organisms. Despite this, there are a number of alkaline environments which contain active microbial communities. Here we describe the genome of a diazotrophic, alkalitolerant strain of Azonexus, which was isolated from a microcosm seeded with hyperalkaline soils resulting from lime depositions. The isolate has a genome size 3.60 Mb with 3431 protein coding genes. The proteome indicated the presence of genes associated with the cycling of nitrogen, in particular the fixation of atmospheric nitrogen. Although closely related to Azonexus hydrophilus strain d8-1 by both 16S (97.9%) and in silico gDNA (84.1%) relatedness, the isolate demonstrates a pH tolerance above that reported for this strain. The proteome contained genes for the complete Na+/H+ antiporter (subunits A to G) for cytoplasmic pH regulation; this may account for the phenotypic characteristics of this strain which exhibited optimal growth conditions of pH 9 and 30°C.

The ability of biofilms to form on a range of materials (cementious backfill (Nirex Reference Vault Backfill (NRVB)), graphite, and stainless steel) relevant to potential UK intermediate level radioactive waste (ILW) disposal concepts was investigated by exposing these surfaces to alkaliphilic flocs generated by mature biofilm communities. Flocs are aggregates of biofilm material that are able to act as a transport vector for the propagation of biofilms. In systems where biofilm formation was observed there was also a decrease in the sorption of isosaccharinic acids to the NRVB. The biofilms were composed of cells, extracellular DNA (eDNA), proteins, and lipids with a smaller polysaccharide fraction, which was biased towards mannopyranosyl linked carbohydrates. The same trend was seen with the graphite and stainless steel surfaces at these pH values, but in this case the biofilms associated with the stainless steel surfaces had a distinct eDNA basal layer that anchored the biofilm to the surface. At pH 13, no structured biofilm was observed, rather all the surfaces accumulated an indistinct organic layer composed of biofilm materials. This was particularly the case for the stainless steel coupons which accumulated relatively large quantities of eDNA. The results demonstrate that there is the potential for biofilm formation in an ILW-GDF provided an initiation source for the microbial biofilm is present. They also suggest that even when conditions are too harsh for biofilm formation, exposed surfaces may accumulate organic material such as eDNA.

ABSTRACT Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the ability of strain DG to interact with the host is at least partly associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in the DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon nuclear magnetic resonance (NMR) and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of l-rhamnose, d-galactose, and N-acetyl-d-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that DG-EPS displays immunostimulating properties by enhancing the gene expression of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and particularly that of the chemokines IL-8 and CCL20, in the human monocytic cell line THP-1. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, DG-EPS is a bacterial macromolecule with the ability to boost the immune system either as a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross talk between L. paracasei DG and the host. IMPORTANCE The consumption of food products and supplements called probiotics (i.e., containing live microbial cells) to potentially prevent or treat specific diseases is constantly gaining popularity. The lack of knowledge on the precise mechanisms supporting their potential health-promoting properties, however, greatly limits a more appropriate use of each single probiotic strain. In this context, we studied a well-known probiotic, Lactobacillus paracasei DG, in order to identify the constitutive molecules that can explain the documented health-promoting properties of this bacterium. We found a novel polysaccharide molecule, named DG-EPS, that is secreted by and covers the bacterium. We demonstrated that this molecule, which has a chemical structure never identified before, has immunostimulatory properties and therefore may contribute to the ability of the probiotic L. paracasei DG to interact with the immune system.

The survival of microorganisms within a cementitious geological disposal facility for radioactive wastes heavily depends on their ability to survive the calcium-dominated, hyperalkaline conditions resulting from the dissolution of the cementitious materials. The results from this study show that the formation of flocs, composed of a complex mixture of extracellular polymeric substances (EPS), provides protection against alkaline pH values up to 13.0. The flocs were dominated by Alishewanella and Dietzia spp., producing a mannose-rich carbohydrate fraction incorporating extracellular DNA, resulting in Ca2+ sequestration. EPS provided a ∼10-μm thick layer around the cells within the center of the flocs, which were capable of growth at pH values of 11.0 and 11.5, maintaining internal pH values of 10.4 and 10.7, respectively. Microorganisms survived at a pH of 12.0, where an internal floc pH of 11.6 was observed, as was a reduced associated biomass. We observed limited floc survival (<2 weeks) at a pH of 13.0. This study demonstrates that flocs maintain lower internal pHs in response to the hyperalkaline conditions expected to occur within a cementitious geological disposal facility for radioactive wastes and indicates that floc communities within such a facility can survive at pHs up to 12.0.IMPORTANCE The role of extracellular polymeric substances (EPS) in the survival of microorganisms in hyperalkaline conditions is poorly understood. Here, we present the taxonomy, morphology, and chemical characteristics of an EPS-based microbial floc, formed by a consortium isolated from an anthropogenic hyperalkaline site. Short-term (<2 weeks) survival of the flocs at a pH of 13 was observed, with indefinite survival observed at a pH of 12.0. Measurements from micro-pH electrodes (10-μm-diameter tip) demonstrated that flocs maintain lower internal pHs in response to hyperalkaline conditions (pH 11.0, 11.5, and 12.0), demonstrating that floc formation and EPS production are survival strategies under hyperalkaline conditions. The results indicate how microbial communities may survive and propagate within the hyperalkaline environment that is expected to prevail in a cementitious geological disposal facility for radioactive wastes; the results are also relevant to the wider extremophile community.

The stoichiometry of the metal complexes formed between nickel and the ligand β-glucoisosaccharinic acid (β-GISA) and a racemic mixture of enantiomers of xyloisosaccharinic acid (XISA) has been determined at both neutral and alkaline pHs. Bjerrum plots, Job's plots and conductance measurements indicated that for each of the systems one to one Ni(ligand) complexes were formed at near neutral pHs (<7.5). At intermediate alkaline pHs (7.5-13) there is evidence to support the formation and precipitation of Ni2(ligand)(OH)3 complexes, finally, at high pH (>13) sparingly soluble Ni2(ligand)(OH)4 complexes were formed. The stability constants for the Ni(β-GISA), Ni(α-GISA) and Ni(XISA) complexes formed at neutral pH were determined under identical conditions using polarographic studies. The measured stability constants for Ni(β-GISA) (log10 β = 1.94 ± 0.15) and for Ni(α-GISA)(log10 β = 2.07 ± 0.13) are very similar; the value measured for the Ni(XISA) complex (log10 β = 0.83) was an order of magnitude smaller. The stability constants for the Ni2(Ligand)(OH)4 complexes formed at highly alkaline pHs were determined using the Schubert method. The measured stability constant for Ni2(β-GISA)(OH)4 (log10 β = 30.6 ± 0.5) was an order of magnitude bigger than the value for Ni2(α-GISA)(OH)4 (log10 β = 29.0 ± 0.5) measured under identical conditions. Attempts to measure the stability constant for Ni2(XISA)(OH)4 were unsuccessful; Ni2(XISA)(OH)4 complexes were not present in significant amounts at high pH to allow the log10β value to be determined by the Schubert method.

The synthesis of phosphorothioate oligonucleotides is often accomplished in the pharmaceutical industry by the sulfurisation of the nucleotide-phosphite using phenylacetyl disulfide (PADS) which has an optimal combination of properties. This is best achieved by an initial 'ageing' of PADS for 48 h in acetonitrile with 3-picoline to generate polysulfides. The initial base-catalysed degradation of PADS occurs by an E1cB-type elimination to generate a ketene and acyldisulfide anion. Proton abstraction to reversibly generate a carbanion is demonstrated by H/D exchange, the rate of which is greatly increased by electron-withdrawing substituents in the aromatic ring of PADS. The ketene can be trapped intramolecularly by an o-allyl group. The disulfide anion generated subsequently attacks unreacted PADS on sulfur to give polysulfides, the active sulfurising agent. The rate of degradation of PADS is decreased by less basic substituted pyridines and is only first order in PADS indicating that the rate-limiting step is formation of the disulfide anion from the carbanion.

Diasteriomeric isosaccharinic acid (ISA) is an important consideration within safety assessments for the disposal of the United Kingdoms' nuclear waste legacy, where it may potentially influence radionuclide migration. Since the intrusion of micro-organisms may occur within a disposal concept, the impact of ISA may be impacted by microbial metabolism. Within the present study we have established two polymicrobial consortia derived from a hyperalkaline soil. Here, α-ISA and a diatereomeric mix of ISAs' were used as a sole carbon source, reflecting two common substrates appearing within the literature. The metabolism of ISA within these two consortia was similar, where ISA degradation resulted in the acetogenesis and hydrogenotrophic methanogenesis. The chemical data obtained confirm that the diastereomeric nature of ISA is likely to have no impact on its metabolism within alkaline environments. High throughput sequencing of the original soil showed a diverse community which, in the presence of ISA allowed for the dominance the Clostridiales associated taxa with Clostridium clariflavum prevalent. Further taxonomic investigation at the genus level showed that there was in fact a significant difference (p = 0.004) between the two community profiles. Our study demonstrates that the selection of carbon substrate is likely to have a significant impact on microbial community composition estimations, which may have implications with respect to a safety assessment of an ILW-GDF.

NMR and anion exchange chromatography analysis of the waste streams generated during the commercial production of sophorolipids by the yeast Candida bombicola identified the presence of small but significant quantities (1% w/v) of free sophorose. Sophorose, a valuable disaccharide, was isolated from the aqueous wastes using a simple extraction procedure and was purified by chromatography on a carbon celite column providing easy access to large quantities of the disaccharide. Experiments were undertaken to identify the origin of sophorose and it is likely that acetylated sophorose derivatives were produced by an enzyme catalysed hydrolysis of the glucosyl-lipid bond of sophorolipids; the acetylated sophorose derivatives then undergo hydrolysis to release the parent disaccharide. Treatment of sophorose with aqueous alkali at elevated temperatures (0.1M NaOH at 50 °C) resulted in C2-epimerisation of the terminal reducing sugar and its conversion to the corresponding 2-O-β-D-glucopyranosyl-D-mannose which was isolated and characterised. In aqueous alkaline solution β-(1,2)-linked glycosidic bonds do not undergo either hydrolysis or peeling reactions.