Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048.

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Bacteriocin production by Leuconostoc citreum ST110LD isolated from organic farm soil, a promising biopreservative.

AIMS: The objective of this study was to isolate a bacteriocin-producing strain and to characterize the expressed bacteriocin for the control of Listeria monocytogenes with aim of biopreservation application. METHODS AND RESULTS: Soil samples from a Korean organic farm were subjected to microbiological analysis for isolation of potential bacteriocinogenic LAB, based on a three-level approach, using L. monocytogenes ATCC 15313 as an indicator test micro-organism. From a total of 17 isolates with inhibitory potential, seven were confirmed to be bacteriocin producers. The selected isolates were differentiated based on their morphology, catalase reaction, sugar fermentation profile obtained by API50CHL and by RAPD-PCR generating two unique profiles. One of the isolates, ST110LD, a specific strong producer of anti-Listeria bacteriocins (12 800 AU ml-1 ) was identified as Leuconostoc citreum. The proteinaceous nature of the inhibitory compound produced by Leuc. citreum ST110LD was confirmed through treatment with pepsin and α-chymotrypsin. Bacteriocin activity was observed to be not affected by the presence of milk, NaCl, SDS, Tween 80 or glycerol. Bacteriocin ST110LD effectively inhibited the growth of exponentially growing L. monocytogenes ATCC 15313 during a 10-h incubation period in BHI at 37°C. In addition, this bacteriocin showed specific inhibition of only Listeria spp., but did not inhibit the growth of beneficial cultures included in the microbial test panel for assessment of the spectrum of activity. CONCLUSIONS: Leuconostoc citreum ST110LD was evaluated as safe bacterium strain, producing bacteriocin with high specificity against listerial and enterococcal species. Specificity of producer strain and expressed bacteriocin can be explored in biopreservation of different fermented food products or applied in biotherapy of antibiotic resistant listerial or enterococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of bacteriocin produced by Leuc. citreum strain with highly specific antimicrobial activity against Listeria sp. and Enterococcus sp.

Investigation of microorganisms involved in kefir biofilm formation.

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.

Response of Leuconostoc strains against technological stress factors: Growth performance and volatile profiles.

The ability of twelve strains belonging to three Leuconostoc species (Leuconostoc mesenteroides, Leuconostoc lactis and Leuconostoc pseudomesenteroides) to grow under diverse sub-lethal technological stress conditions (cold, acidic, alkaline and osmotic) was evaluated in MRS broth. Two strains, Leuconostoc lactis Ln N6 and Leuconostoc mesenteroides Ln MB7, were selected based on their growth under sub-lethal conditions, and volatile profiles in RSM (reconstituted skim milk) at optimal and under stress conditions were analyzed. Growth rates under sub-lethal conditions were strain- and not species-dependent. Volatilomes obtained from the two strains studied were rather diverse. Particularly, Ln N6 (Ln. lactis) produced more ethanol and acetic acid than Ln MB7 (Ln. mesenteroides) and higher amounts and diversity of the rest of volatile compounds as well, at all times of incubation. For the two strains studied, most of stress conditions applied diminished the amounts of ethanol and acetic acid produced and the diversity and levels of the rest of volatile compounds. These results were consequence of the different capacity of the strains to grow under each stress condition tested.

Lactobacillus and Leuconostoc volatilomes in cheese conditions.

New strains are desirable to diversify flavour of fermented dairy products. The objective of this study was to evaluate the potential of Leuconostoc spp. and Lactobacillus spp. in the production of aroma compounds by metabolic fingerprints of volatiles. Eighteen strains, including five Lactobacillus species (Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus sakei) and three Leuconostoc species (Leuconostoc citreum, Leuconostoc lactis, and Leuconostoc mesenteroides) were incubated for 5 weeks in a curd-based slurry medium under conditions mimicking cheese ripening. Populations were enumerated and volatile compounds were analysed by headspace trap gas chromatography-mass spectrometry (GC-MS). A metabolomics approach followed by multivariate statistical analysis was applied for data processing and analysis. In total, 12 alcohols, 10 aldehydes, 7 esters, 11 ketones, 5 acids and 2 sulphur compounds were identified. Very large differences in concentration of volatile compounds between the highest producing strains and the control medium were observed in particular for diacetyl, 2-butanol, ethyl acetate, 3-methylbutanol, 3-methylbutanoic acid and 2-methylbutanoic acid. Some of the characterized strains demonstrated an interesting aromatizing potential to be used as adjunct culture.

Interactions between candidate probiotics and the immune and antioxidative responses of European sea bass (Dicentrarchus labrax) larvae.

The use of lactic acid bacteria (LAB) as probiotics in aquaculture may improve the quality of seed production and limit the use of antibiotics in fish hatcheries. This study attempted to further characterize the candidate probiotic Lactobacillus casei X2, and the immune and physiological responses of the sea bass larvae. L. casei X2 was confirmed as a good candidate, due to its wide antibacterial spectrum against both Gram-positive and Gram-negative bacteria, and its free radical scavenging activity. In addition, if the strain did not seem able to form biofilm on abiotic surfaces, it adhered strongly to Hep-2 cells. However, these characteristics did not seem efficient in vivo. At 20 days post-hatch (dph), the expression level of CAT gene was significantly different between group fed without probiotic and the two groups treated with either Pediococcus acidilactici or L. casei. This gene was upregulated in the group treated with strain X2 and downregulated in the group with a commercial probiotic strain P. acidilactici, suggesting a better antioxidant activity with the later strain. At the same sampling date, the IL-1ß gene was upregulated in the group treated with P. acidilactici, and the HSP70 gene was overexpressed at 41 dph. As the stimulation of these two last genes, such transcriptomic indicators must be cautiously interpreted.

The potential of dextran-based glycoconjugates for development of Helicobacter pylori vaccine.

We have recently demonstrated that synthetic glycoconjugates based on delipidated lipopolysaccharide (LPS) of Helicobacter pylori and containing an α(1-6)-glucan chain induced broadly cross-reactive functional antibodies in immunized animals. To investigate the candidacy of α(1-6)-glucan as an alternative vaccine strategy we prepared glycoconjugates based on dextrans produced by lactic acid bacteria Leuconostoc mesenteroides B512F and consisting of linear α(1-6)-glucan chains with limited branching. Three dextrans with averaged molecular masses of 5,000 Da, 3,500 Da and 1,500 Da, respectively, were modified with a diamino group-containing linker and conjugated to a carrier protein, tetanus toxoid (TT) or diphtheria toxoid (DT), and their immunological properties investigated. The conjugates were immunogenic in both rabbits and mice and induced specific IgG responses against α(1-6)-glucan-expressing H. pylori LPS. Studies performed with post-immune sera of mice and rabbits immunized with dextran-based conjugates demonstrated cross-reactivity with LPS from typeable and non-typeable strains of H. pylori and selected mutants. The post-immune sera from rabbits that received the conjugates exhibited functional activity against α(1-6)-glucan-positive strains of H. pylori. These data provide evidence that dextran-based conjugates may offer a simplified approach to the development of carbohydrate-based vaccines against H. pylori.

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase.

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-D-glucopyranosyl units and 29 mol% 1,6-disubstituted α-D-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-D-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-D-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-D-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-D-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.

Structural and functional characterization of exopolysaccharide from Leuconostoc citreum BH10 discovered in birch sap.

In this study, Leuconostoc citreum BH10, an endophytic strain, was isolated from aseptically collected xylem sap of birch for the first time, and its exopolysaccharide (LCEPS) production was up to 46.31 g/L in glucan producing medium. The produced LCEPS was purified to obtain two water-soluble fractions, named as LCEPS-1 and LCEPS-2, respectively. The major fraction LCEPS-1 was characterized to be comprised of glucose with average molecular weight of 6.34 × 106 Da. The structure of LCEPS-1 was investigated by spectroscopy analysis, which revealed that LCEPS-1 was identified with containing 90.45 % α-(1,6) linkages in the main chains and 9.55 % α-(1,3) branch linkages. The scanning electron microscope results demonstrated that the dried LCEPS-1 appeared porous surface overlaid with an irregular glittering. The water solubility index (WSI) and water holding capacity (WHC) of LCEPS-1 were 88.02 ± 1.69 % and 241.43 ± 6.38 %, respectively. Besides, it exhibited high thermal stability as well as fine antioxidant activities. Taken together, the results indicated that LCEPS-1 could have good potentiality to be applied in fields of foods, cosmetics, nutraceuticals and pharmaceutical industries as the natural agent.

The exopolysaccharides produced by Leuconostoc mesenteroides XR1 and its effect on silk drawing phenomenon of yoghurt.

Yoghurt fermented by Leuconostoc mesenteroides XR1 from Kefir grains was found to produce a unique silk drawing phenomenon. This property was found to be associated with the exopolysaccharides (EPS), X-EPS, produced by strain XR1. In order to better understand the mechanism that produced this phenomenon, the X-EPS was extracted, purified and characterized. The molecular weight and monosaccharide composition were determined by size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) and ion chromatography (IC) analysis, respectively. The results showed that its molecular weight was 4.183 × 106 g/mol and its monosaccharide composition was glucose, and glucuronic acid, with the contents of 567.6148 and 0.2096 µg/mg, respectively. FT-IR and NMR analyses showed that X-EPS was an α-pyranose polysaccharide and was composed of 92.22 % α-(1 â†’ 6) linked d-glucopyranose units and 7.77 % α-(1 â†’ 3) branching. Furthermore, it showed a chain-like microstructure with branches in atomic force microscopy (AFM) and scanning electron microscopy (SEM) experiments. These results suggested that the unique structure of X-EPS, gave the yoghurt a strong viscosity and cohesiveness, which resulted in the silk drawing phenomenon. This work suggested that X-EPS holds the potential for food and industrial applications.

Structural characterization of enzymatically synthesized dextran and oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase.

Leuconostoc mesenteroides NRRL B-1426 dextransucrase synthesized a high molecular mass dextran (>2 × 10(6) Da) with ~85.5% α-(1→6) linear and ~14.5% α-(1→3) branched linkages. This high molecular mass dextran containing branched α-(1→3) linkages can be readily hydrolyzed for the production of enzyme-resistant isomalto-oligosaccharides. The acceptor specificity of dextransucrase for the transglycosylation reaction was studied using sixteen different acceptors. Among the sixteen acceptors used, isomaltose was found to be the best, having 89% efficiency followed by gentiobiose (64%), glucose (30%), cellobiose (25%), lactose (22.5%), melibiose (17%), and trehalose (2.3%) with reference to maltose, a known best acceptor. The ß-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.

Design of chimeric levansucrases with improved transglycosylation activity.

Fructansucrases (FSs), including levansucrases and inulosucrases, are enzymes that synthesize fructose polymers from sucrose by the direct transfer of the fructosyl moiety to a growing polymer chain. These enzymes, particularly the single domain fructansucrases, also possess an important hydrolytic activity, which may account for as much as 70 to 80% of substrate conversion, depending on reaction conditions. Here, we report the construction of four chimeric levansucrases from SacB, a single domain levansucrase produced by Bacillus subtilis. Based on observations derived from the effect of domain deletion in both multidomain fructansucrases and glucansucrases, we attached different extensions to SacB. These extensions included the transitional domain and complete C-terminal domain of Leuconostoc citreum inulosucrase (IslA), Leuconostoc mesenteroides levansucrase (LevC), and a L. mesenteroides glucansucrase (DsrP). It was found that in some cases the hydrolytic activity was reduced to less than 10% of substrate conversion; however, all of the constructs were as stable as SacB. This shift in enzyme specificity was observed even when the SacB catalytic domain was extended only with the transitional region found in multidomain FSs. Specific kinetic analysis revealed that this change in specificity of the SacB chimeric constructs was derived from a 5-fold increase in the transfructosylation k(cat) and not from a reduction of the hydrolytic k(cat), which remained constant.

Polyphasic screening, homopolysaccharide composition, and viscoelastic behavior of wheat Sourdough from a Leuconostoc lactis and Lactobacillus curvatus exopolysaccharide-producing starter culture.

After isolation from different doughs and sourdoughs, 177 strains of lactic acid bacteria were screened at the phenotypic level for exopolysaccharide production on media containing different carbohydrate sources. Two exopolysaccharide-producing lactic acid bacteria (Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A) were selected through quantitative analysis on solid media containing sucrose and yeast extract. The PCR detection of homopolysaccharide (gtf and lev) and heteropolysaccharide (epsA, epsB, epsD and epsE, and epsEFG) genes showed different distributions within species and strains of the lactic acid bacteria studied. Moreover, in some strains both homopolysaccharide and heteropolysaccharide genes were detected. Proton nuclear magnetic resonance spectra suggest that Lactobacillus curvatus 69B2 and Leuconostoc lactis 95A produced the same exopolysaccharide, which was constituted by a single repeating glucopyranosyl unit linked by an α-(1→6) glycosidic bond in a dextran-type carbohydrate. Microbial growth, acidification, and viscoelastic properties of sourdoughs obtained by exopolysaccharide-producing and nonproducing lactic acid bacterial strains were evaluated. Sourdough obtained after 15 h at 30°C with exopolysaccharide-producing lactic acid bacteria reached higher total titratable acidity as well as elastic and dissipative modulus curves with respect to the starter not producing exopolysaccharide, but they showed similar levels of pH and microbial growth. On increasing the fermentation time, no difference in the viscoelastic properties of exopolysaccharide-producing and nonproducing samples was observed. This study suggests that dextran-producing Leuconostoc lactis 95A and Lactobacillus curvatus 69B2 can be employed to prepare sourdough, and this would be particularly useful to improve the quality of baked goods while avoiding the use of commercially available hydrocolloids as texturizing additives.

Cloning, expression, and characterization of an insoluble glucan-producing glucansucrase from Leuconostoc mesenteroides NRRL B-1118.

We have cloned a glucansucrase from the type strain of Leuconostoc mesenteroides (NRRL B-1118; ATCC 8293) and successfully expressed the enzyme in Escherichia coli. The recombinant processed enzyme has a putative sequence identical to the predicted secreted native enzyme (1,473 amino acids; 161,468 Da). This enzyme catalyzed the synthesis of a water-insoluble α-D-glucan from sucrose (K(M) 12 mM) with a broad pH optimum between 5.0 and 5.7 in the presence of calcium. Removal of calcium with dialysis resulted in lower activity in the acidic pH range, effectively shifting the pH optimum to 6.0-6.2. The enzyme was quickly inactivated at temperatures above approximately 45°C. The presence of dextran offered some protection from thermal inactivation between room temperature and 40°C but had little effect above 45°C. NMR and methylation analysis of the water-insoluble α-D-glucan revealed that it had approximately equal amounts of α(1 → 3)-linked and α(1 → 6)-linked D-glucopyranosyl units and a low degree of branching.

Enhancement of the tyrosinase inhibitory activity of Mori Cortex Radicis extract by biotransformation using Leuconostoc paramesenteroides PR.

Mori Cortex Radicis (MCR), the root bark of Morus alba L., consists of various phytochemicals and exhibits a strong inhibitory effect on tyrosinase. To enhance the tyrosinase inhibitory activity of MCR extract without further purification of bioactive compounds, whole MCR extract was biotransformed with crude enzyme extract from a selected lactic acid bacterium, Leuconostoc paramesenteroides PR (LP). Mulberroside A (MA), a major stilbene glucoside of MCR, contains two ß-glucosyl residues at the C3 and C4' positions of oxyresveratrol (OXY). The crude enzyme of LP hydrolyzed the two glycosidic bonds of MA effectively, and 97.1% of MA was biotransformed into OXY within 2 h. Commercial almond ß-glucosidase hydrolyzed only one site of the two glycosidic bonds of MA, and 68.7% of MA was biotransformed to OXY-glucoside. The tyrosinase inhibitory activity of the crude extract of MCR was increased approximately 6.5-fold by biotransformation using LP, and the IC(50) value of the transformed MCR was 3.7 µg/mL.

Stable alpha-synuclein oligomers strongly inhibit chaperone activity of the Hsp70 system by weak interactions with J-domain co-chaperones.

α-Synuclein aggregation and accumulation in Lewy bodies are implicated in progressive loss of dopaminergic neurons in Parkinson disease and related disorders. In neurons, the Hsp70s and their Hsp40-like J-domain co-chaperones are the only known components of chaperone network that can use ATP to convert cytotoxic protein aggregates into harmless natively refolded polypeptides. Here we developed a protocol for preparing a homogeneous population of highly stable ß-sheet enriched toroid-shaped α-Syn oligomers with a diameter typical of toxic pore-forming oligomers. These oligomers were partially resistant to in vitro unfolding by the bacterial Hsp70 chaperone system (DnaK, DnaJ, GrpE). Moreover, both bacterial and human Hsp70/Hsp40 unfolding/refolding activities of model chaperone substrates were strongly inhibited by the oligomers but, remarkably, not by unstructured α-Syn monomers even in large excess. The oligomers acted as a specific competitive inhibitor of the J-domain co-chaperones, indicating that J-domain co-chaperones may preferably bind to exposed bulky misfolded structures in misfolded proteins and, thus, complement Hsp70s that bind to extended segments. Together, our findings suggest that inhibition of the Hsp70/Hsp40 chaperone system by α-Syn oligomers may contribute to the disruption of protein homeostasis in dopaminergic neurons, leading to apoptosis and tissue loss in Parkinson disease and related neurodegenerative diseases.

Structural studies of the capsular polysaccharide produced by Leuconostoc mesenteroides ssp. cremoris PIA2.

The structure of the capsular polysaccharide (CPS) produced by Leuconostoc mesenteroides ssp. cremoris PIA2 has been determined using component analysis and NMR spectroscopy. (1)H and (13)C resonances were assigned using 2D NMR experiments, and sequential information was obtained by (1)H,(1)H-NOESY and (1)H,(13)C-HMBC experiments. The CPS consists of linear pentasaccharide repeating units with the following structure: →3)-ß-D-Galf-(1→6)-ß-D-Galf-(1→2)-ß-D-Galf-(1→6)-ß-D-Galf-(1→3)-ß-D-Galp-(1→, in which four out of the five sugar residues have the furanoid ring form, a structural entity found in bacteria but not in mammals. The analysis of the magnitude of the homonuclear three-bond coupling constants of the anomeric protons for the five-membered sugar rings indicates that the sugar residues substituted at a primary carbon atom show one kind of conformational preferences, whereas those substituted at a secondary carbon atom show another kind of conformational preferences.

Isolation of a gene from Leuconostoc citreum B/110-1-2 encoding a novel dextransucrase enzyme.

The amplicon encoding dextransucrase DSR-F from Leuconostoc citreum B/110-1-2, a novel sucrose glucosyltransferase (GTF)-specific for α-1,6 and α-1,3 glucosidic bond synthesis, with α-1,4 branching was cloned, sequenced, and expressed into Escherichia coli JM109. Recombinant enzyme catalyzed oligosaccharides synthesis from sucrose as donor and maltose acceptor. The dsrF gene encodes for a protein (DSR-F) of 1,528 amino acids, with a theoretical molecular mass of 170447.72 Da (~170 kDa). From amino acid sequence comparison, it appears that DSR-F possesses the same domains as those described for GTFs. However, the variable region is longer than in other GTFs (by 100 amino acids) and two APY repeats (a 79 residue long motif with a high number of conserved glycine and aromatic residues, characterized by the presence of the three consecutive residues Ala, Pro, and Tyr) were identified in the glucan binding domain. The DSR-F catalytic domain possesses the catalytic triad involved in the glucosyl enzyme formation. The amino acid sequence of this domain shares a 56% identity with catalytic domain of the alternansucrase ASR from L. citreum NRRL B-1355 and with the catalytic domain of a putative alternansucrase sequence found in the genome of L. citreum KM20. A truncated active variant DSR-F-∆SP-∆GBD of 1,251 amino acids, with a molecular mass of 145 544 Da (~145 kDa), was obtained.

Corrosion protection of low-carbon steel using exopolysaccharide coatings from Leuconostoc mesenteroides.

Corrosion of metals is a serious and challenging problem faced worldwide by industry. Purified Leuconostoc mesenteroides exopolysaccharide (EPS) coatings, cast from aqueous solution, inhibited the corrosion of low-carbon steel as determined by electrochemical impedance spectroscopy (EIS). There were two different corrosion behaviors exhibited when EPS films from different strains were cast onto the steel. One EPS coating reacted immediately with the steel substrate to form an iron (III) oxide layer ("rust") during the drying process while another did not. The samples that did not flash corrode had higher corrosion inhibition and formed an iron (II) passivation layer during EIS testing that persisted after the cells were disassembled. Corrosion inhibition was strain-specific as polysaccharides with similar structure did not have the same corrosion potential.

Carboxymethylation of (1 → 6)-α-dextran from Leuconostoc spp.: Effects on microstructural, thermal and antioxidant properties.

The carboxymethylated (1 â†’ 6)-α-dextran (CM-dex) was synthesized by introducing carboxymethyl groups at different degrees of substitution (DS). The resulting dex1-1, dex2-1, dex3-1, and dex4-1 products had degrees of substitution of 0.57, 0.78, 1.13, and 1.25, respectively. The dex3-1 showed the highest glass transition temperature (Tg) of 215.96 °C, whereas Tg of pure dextran was 149.83 °C. TGA results indicated that the residual loss was reduced along with the increase of DS in the high-temperature region (450-600 °C). Besides, the CM-dex had stronger scavenging capacity against OH radicals but lower scavenging capacity for DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals compared to that of pure dextran. The carboxymethylation of (1 â†’ 6)-α-dextran will extend the applications for modified dextran.