Mesenteroides NRRL Research Articles

Biophysical characterization of α-glucan nanoparticles encapsulating feruloylated soy glycerides (FSG)

Water insoluble α-glucans that were enzymatically synthesized using glucansucrase that was cloned from Leuconostoc mesenteroides NRRL B-1118 were previously shown to form nanoparticles via high pressure homogenization. These α-glucan nanoparticles were previously shown capable of encapsulating a small hydrophobic molecule. This work demonstrates that the same α-glucan can be formed into nanoparticles that encapsulate feruloylated soy glycerides from modified soybean oil, a product of interest to the cosmetic and skin care industries because of the UV absorbance and antioxidant properties of the feruloyl moiety. It is demonstrated that the feruloylated soy glyceride/α-glucan nanoparticles have distinct size, zeta potential and thermal profiles from that of nanoparticles made from α-glucan alone or feruloylated soy glyceride alone. Thermal analysis also demonstrates the release of feruloylated soy glycerides from the α-glucan nanoparticles.

Comprehensive structural characterization of water-soluble and water-insoluble homoexopolysaccharides from seven lactic acid bacteria

Several lactic acid bacteria are able to produce water-soluble and water-insoluble homoexopolysaccharides (HoEPS) from sucrose. In this study, structures of all HoEPS which were fermentatively produced by Leuconostoc mesenteroides subsp. dextranicum NRRL B-1121 and B-1144, Leuconostoc mesenteroides subsp. mesenteroides NRRL B-1149, B-1438 and B-1118, Leuconostoc suionicum DSM 20241, and Liquorilactobacillus satsumensis DSM 16230 were systematically analyzed. Monosaccharide analysis, methylation analysis, NMR spectroscopy, size-exclusion chromatography, and different enzymatic fingerprinting methods were used to obtain detailed structural information. All strains produced water-soluble dextrans and/or levans as well as water-insoluble glucans. Levans showed different degrees of branching and high molecular weights, whereas dextrans had comparable structures and broader size distributions. Fine structures of water-soluble HoEPS were analyzed after endo-dextranase and endo-levanase hydrolysis. Water-insoluble glucans were composed of different portions of 1,3-linkages (5 to 40 %). Hydrolysis with endo-dextranase and endo-mutanase yielded further information on block sizes and varying fine structures. Overall, clear differences between HoEPS yields and structures were observed.

Biochemical characterization of synthesized fisetin glucoside by dextransucrase from Leuconostoc mesenteroides NRRL B-1299CB4 with enhanced water solubility

Fisetin (7,3',4'-flavon-3-ol) is a flavonol found in plants, fruits, and vegetables. It exhibits diverse biological activities, including antioxidant, anti-inflammatory, and anti-cancer effects. However, the low water solubility and bioavailability of fisetin restrict its pharmaceutical applications. In this work, we synthesized a novel fisetin-4'-O-α-D-glucopyranoside (FST-G1) using transglucosylation with sucrose, fisetin, and dextransucrase from Leuconostoc mesenteroides NRRL B-1299CB4. The water solubility of FST-G1 (109.8 ± 6.3 mg/L) was enhanced compared to fisetin (13.6 ± 1.3 mg/L). The antioxidant activities of FST-G1 in non-cellular assays, including ORAC, ABTS•+, and FRAP assays, were lower compared to fisetin. However, FST-G1 exhibited higher nitric oxide inhibition (62.5 µM; 92.3 %) in lipopolysaccharide-stimulated RAW 264.7 murine macrophage cells compared to fisetin (81.4 %). Anti-lipid accumulation in mouse 3T3-L1 cells treated with FST-G1 was similar to that in cells treated with fisetin. Taken together, the novel synthesized FST-G1 is expected to become a promising functional material for using in the pharmaceutical industry.

Mathematical modeling characterization of mannitol production by three heterofermentative lactic acid bacteria

This study developed a non-structured mathematical model for describing the effect of carbon sources and culture pH on the mannitol production by three heterofermentative lactic acid bacteria (LAB), including Leuconostoc mesenteroides NRRL B-512F and NRRL B-523 as well as Lactobacillus fermentum NRRL B-1840. Mannitol production was studied in batch culture using four culture media containing the same carbon sources (glucose and fructose, but different glucose concentration) and different nitrogen sources (yeast extract, peptone, and meat extract) with diverse concentrations. The results obtained in the early stages of the estimation algorithm suggested that cell growth on fructose could be neglected. Therefore, the initially proposed mathematical model was redefined and subsequently confirmed. The strains NRRL B-512 F, NRRL B-523, and NRRL B-1840 reached the highest volumetric productivities (2.26 ± 0.05, 2.30 ± 0.05, and 1.95 ± 0.05 g L−1h−1; respectively) and mannitol yields (0.95 ± 0.01, 0.98 ± 0.04, and 1.00 ± 0.01 mol mannitol mol initial fructose−1; respectively) in medium A. The mannitol and biomass yields suggested a higher carbon concentration favored mannitol production and growth, but a lower nitrogen concentration directly impacted both yields. Mannitol production was associated with cell growth according to the estimated parameters.

The bifidogenic effects and dental plaque deformation of non-digestible isomaltooligosaccharides synthesized by dextransucrase and alternansucrase

Non-digestible isomaltooligosaccharides (NDIMOS) are functional food and beverage ingredients that contributed to human health benefits through metabolism of gastrointestinal microorganism. In this study, NDIMOS were synthesized by combine dextransucrase from Leuconostoc mesenteroides B512F/KM and alternansucrase from L. mesenteroides NRRL 1355CF10/KM using sucrose as substrate and maltose as acceptor. Their digestibility was confirmed by using digestive enzymes including α-amylase and amyloglucosidase. NDIMOS inhibited insoluble glucan formation through mutansucrase from Streptococcus mutans. The bifidogenic effect of NDIMOS was investigated by growth of four strains of Bifidobacterium in MRS broth containing NDIMOS, compared with MRS broth contain glucose and negative control. Additionally, Bifidobacterium bifidum or Bifidobacterium adolescentis inhibited the growth of Salmonella enterica serovar typhimurium when they were co-cultivation in MRS broth containing NDIMOS. These results suggested that NDIMOS is potential functional ingredient for food, beverage, and pharmaceutical application.

Dextran: Sources, Structures, and Properties

Dextran is an exopolysaccharide (EPS) synthesized by lactic acid bacteria (LAB) or their enzymes in the presence of sucrose. Dextran is composed of a linear chain of d-glucoses linked by α-(1→6) bonds, with possible branches of d-glucoses linked by α-(1→4), α-(1→3), or α-(1→2) bonds, which can be low (<40 kDa) or high molecular weight (>40 kDa). The characteristics of dextran in terms of molecular weight and branches depend on the producing strain, so there is a great variety in its properties. Dextran has commercial interest because its solubility, viscosity, and thermal and rheological properties allow it to be used in food, pharmaceutical, and research areas. The aim of this review article is to compile the latest research (in the past decade) using LAB to synthesize high or low molecular weight dextran. In addition, studies using modified enzymes to produce dextran with specific structural characteristics (molecular weights and branches) are addressed. On the other hand, special attention is paid to LAB extracted from unconventional sources to expose their capacities as dextran producers and their possible application to compete with the only commercial strain (Leuconostoc mesenteroides NRRL B512).

Development and Physical Characterization of α-Glucan Nanoparticles.

α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from Leuconostoc mesenteroides NRRL B-1118 were found to have a glass transition temperature of approximately 80 °C. Using high-pressure homogenization (~70 MPa), the α-glucans were converted into nanoparticles of ~120 nm in diameter with a surface potential of ~−3 mV. Fluorescence measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the α-glucan nanoparticles have a hydrophobic core that remains intact from 10 to 85 °C. α-Glucan nanoparticles were found to be stable for over 220 days and able to form at three pH levels. Accelerated exposure measurements demonstrated that the α-glucan nanoparticles can endure exposure to elevated temperatures up to 60 °C for 6 h intervals.

Optimization of the expression of levansucrase L17 in recombinant E. coli

ABSTRACTLevansucrases synthesize levans and fructooligosaccharides, which are of interest in the food and pharmaceutical industry. Leuconostoc mesenteroides Lm 17 produces levansucrase of about 120 kDa. The encoding gene from this strain was cloned and expressed in Escherichia coli BL21(DE3). The cloned gene encodes a 1022 amino acids long levansucrase with 96% identity to levansucrase LevS from L. mesenteroides NRRL B-512F strain. The induction and expression of the levansucrase gene were performed at temperatures between 15 °C and 37 °C, and concentration of the inducer isopropyl-β-D-thiogalactopyranoside from 0.1 to 2.0 (mmol/L)-1. We report for the first time recombinant expression of a levansucrase gene at a low temperature of induction, after cell biomass accumulation at 37 °C. The highest enzyme activity of 1.90 (U/mg)-1 was measured in TB medium at 18 °C temperature of induction, and concentration of the inducer from 0.1 to 1.0 (mmol/L)-1. The in situ analysis of the purified enzyme showed an active band of about 120 kDa, similar to the one produced by the native strain. The purified enzyme has a temperature optimum at 35 °C, pH optimum at 5.5, and Km = 64 (mmol/L)-1 of sucrose.

Efficient Production of Prebiotic Gluco-oligosaccharides in Orange Juice Using Immobilized and Co-immobilized Dextransucrase.

Dextransucrase from Leuconostoc mesenteroides NRRL B-512F was subjected to immobilization and co-immobilization with dextranase from Chaetomium erraticum. Immobilization has enhanced the operational and storage stability of dextransucrase. Two hundred milligrammes (2.4IU/mg) of alginate beads (immobilized and co-immobilized) were found to be optimum for the production of gluco-oligosaccharides (GOS) in orange juice with a high degree of polymerization. The pulp of the orange juice did not interfere in the reaction. In the batch process, co-immobilized dextransucrase (41g/L) produced a significantly higher amount of GOS than immobilized dextransucrase (37g/L). Alginate entrapment enhanced the thermal stability of dextransucrase for up to 3days in orange juice at 30°C. The production of GOS in semi-continuous process was 39g/L in co-immobilized dextransucrase and 33g/L in immobilized dextransucrase. Thus, immobilization technology offers a great scope in terms of reusability and efficient production of a value added functional health drink.

Effect of a single point mutation on the interaction of glucans with a glucansucrase from Leuconostoc mesenteroides NRRL B-1118

Our previous work showed that substitution of an amino acid that is coupled with the +2 subsite adjacent to the transition stabilizer of a glucansucrase, which produces a water-insoluble glucan, resulted in significant changes in the structures and yields of the water-insoluble glucans produced. We now describe how these changes affect the ability of the glucansucrase to bind to exogenous glucans, and how these glucans can influence the yield, product structures, and kinetics of the mutant glucansucrases. The activity of the wild-type enzyme, with threonine at position 654, is not significantly activated by added dextran, and the yield of water-insoluble glucan from sucrose is only slightly increased by dextran. Mutant T654Y is not affected at all by the addition of dextran. However, several mutant enzymes exhibit markedly lower yields of glucan relative to the wild type; these lower yields can be partially or completely overcome by the addition of water-soluble dextran. Although evidence indicates that the soluble dextran is incorporated into water-insoluble glucan, the increased yields cannot be accounted for solely by incorporation of the dextran into insoluble product. Furthermore, these DsrI mutants are significantly activated by exogenous glucans. The addition of dextran does not markedly change the KM for sucrose in the mutant enzymes, but does increase the Vmax of the reaction. These effects apparently depend on the presence of unbranched sequences of α1→6-linked D-glucose units in the glucan.

Glucooligosaccharide production by Leuconostoc mesenteroides fermentation with efficient pH control, using a calcium hydroxide-sucrose solution

95.3% of the sucrose in a feed batch fermentation (300 g/L) was hydrolyzed by Leuconostoc mesenteroides subp. mesenteroides NRRL B-23188 glucansucrase. Further, the glucose of sucrose formed glucooligosaccharides (GOS) of degree of polymerization (DP) over 2, together with 91.6% of the maltose (200 g/L). Lime saccharate (lime sucrate) was used to control the pH during fermentation. The GOS products had DP between 2 and 7. When Streptococcus mutans mutansucrase (0.1 U/mL) reacted with 0.1% sucrose, addition of 0.1 ~ 10% GOS to the mutansucrase reaction digest resulted in a 56 ~ 90% reduction of mutan formation. GOS also reduced E. coli (72.2%) and Salmonella sp. (over 40.0%) growth, when 2.5% GOS was used as a single carbon source, compared to growth using glucose. The calculated glycemic index and glycemic load of GOS was 8 and 1, respectively, based on a 10 g carbohydrate serving. GOS was calculated to have 2.43 kcal/g. After a glucose tolerance test was performed using C57BL/6 mice, we found that mice treated with GOS showed a 59.4% lower increase in plasma glucose than those treated with maltose.

Enzyme-resistant isomalto-oligosaccharides produced from Leuconostoc mesenteroides NRRL B-1426 dextran hydrolysis for functional food application.

The extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1426 was produced and purified using polyethylene glycol fractionation. In our earlier study, it was reported that L. mesenteroides dextransucrase synthesizes a high-molecular mass dextran (>2×10(6) Da) with ∼85.5% α-(1→6) linear and ∼14.5% α-(1→3) branched linkages. Isomalto-oligosaccharides (IMOs) were synthesized through depolymerization of dextran by the action of dextranase. The degree of polymerization of IMOs was 2-10 as confirmed by mass spectrometry. The nuclear magnetic resonance spectroscopic analysis revealed the presence of α-(1→3) linkages in the synthesized IMOs. The IMOs were resistant to dextranase, α-glucosidase, and α-amylase, and therefore can have potential application as food additives in the functional foods.

In vitro analysis of dextran from Leuconostoc mesenteroides NRRL B-1426 for functional food application

Abstract The present study describes physico-chemical properties, prebiotic potential and biocompatibility of dextran produced from Leuconostoc mesenteroides NRRL B-1426. Dextran displayed porous surface, pseudoplastic non-Newtonian behavior, 32% solubility, 290% water holding capacity and a degradation temperature of 290 °C. The dextran exhibited significantly higher non digestibility as compared with the standard prebiotic, inulin in simulated human gastric juice and α-amylase. The dextran significantly stimulated the growth of probiotics, bifidobacteria and lactobacilli and was comparable to that by inulin. The viability of human embryonic kidney (HEK 293), human cervical adenocarcinoma (INT-407) and human colon carcinoma (HT-29) cell lines by dextran treatment (50–1000 µg/ml) remained unaffected displaying the biocompatibility of the dextran. Therefore, the dextran from L. mesenteroides NRRL B-1426 can act as potential prebiotic, gelling agent and be used as food supplement for health benefits.

Lime application for the efficient production of nutraceutical glucooligosaccharides from Leuconostoc mesenteroides NRRL B-742 (ATCC13146).

We have previously demonstrated the production of glucooligosaccharides via a fermentation of sucrose with Leuconostoc mesenteroides NRRL B-742 using sodium hydroxide (NaOH) to control the pH. Because NaOH is expensive, we sought to minimize the cost of our process by substituting hydrated lime and saccharate of lime (lime sucrate) in its place. The yield of glucooligosaccharides using either 5 % lime (41.4 ± 0.5 g/100 g) or 5 % lime sucrate (40.0 ± 1.4 g/100 g) were both similar to the NaOH control (42.4 ± 1.5 g/100 g). Based on this, it appears that the cost associated with pH control in our process can be reduced by a factor of approximately 2.4 using lime instead of NaOH. Because our chromatographic stage is based on a Ca(2+)-form resin to separate glucooligosaccharides, the use of lime not only negates the need for costly de-salting via ion-exchange (elimination of two ion-exchange sections) prior to separation, but also greatly reduces the resin regeneration cost.

Gentio-oligosaccharides from Leuconostoc mesenteroides NRRL B-1426 dextransucrase as prebiotics and as a supplement for functional foods with anti-cancer properties.

Gentio-oligosaccharides (GnOS) were synthesized by the acceptor reaction of dextransucrase from Leuconostoc mesenteroides NRRL B-1426 with gentiobiose and sucrose. GnOS were purified by gel permeation chromatography using a Bio-Gel P-2 column and identified by mass spectrometry. The purified GnOS (degree of polymerization ≥3) were investigated for their in vitro prebiotic and cytotoxic activity. GnOS exhibited a significantly lower degree of digestibility of 18.1% by simulated human gastric juice (pH 1.0) and 7.1% by human α-amylase (pH 7.0) after 6 h, whereas inulin, a standard prebiotic, showed 39.7% and 12.8% of digestibility, respectively. The prebiotic score showed that GnOS significantly supported the growth of probiotics such as Bifidobacterium infantis and Lactobacillus acidophilus and was comparable to that of inulin. The selective inhibitory effect of GnOS on human colon carcinoma (HT-29) cells revealed its potential as an anti-cancer agent that can serve as a functional food additive for the benefit of human health.

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.

Combinatorial Engineering of Dextransucrase Specificity

We used combinatorial engineering to investigate the relationships between structure and linkage specificity of the dextransucrase DSR-S from Leuconostoc mesenteroides NRRL B-512F, and to generate variants with altered specificity. Sequence and structural analysis of glycoside-hydrolase family 70 enzymes led to eight amino acids (D306, F353, N404, W440, D460, H463, T464 and S512) being targeted, randomized by saturation mutagenesis and simultaneously recombined. Screening of two libraries totaling 3.6.104 clones allowed the isolation of a toolbox comprising 81 variants which synthesize high molecular weight α-glucans with different proportions of α(1→3) linkages ranging from 3 to 20 %. Mutant sequence analysis, biochemical characterization and molecular modelling studies revealed the previously unknown role of peptide 460DYVHT464 in DSR-S linkage specificity. This peptide sequence together with residue S512 contribute to defining +2 subsite topology, which may be critical for the enzyme regiospecificity.

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 β-linked disaccharide, gentiobiose, showed significant efficiency for oligosaccharide production that can be used as a potential prebiotic.

Cashew juice containing prebiotic oligosaccharides.

The enzyme dextransucrase in a medium containing sucrose and an acceptor as substrate synthesizes prebiotics oligosaccharides. The cashew apple juice works as a source of acceptors because it is rich in glucose and fructose (enzyme acceptors). The use of cashew apple juice becomes interesting because it aims at harnessing the peduncle of the cashew that is wasted during the nut processing, which is the product of greater economic expression. The production of dextransucrase enzyme was done by fermentative process by inoculating the bacterium Leuconostoc mesenteroides NRRL B512F into a culture medium containing sucrose as the only carbon source. Thus, the aim of this work was the production of prebiotic oligosaccharides by enzymatic process with addition of the dextransucrase enzyme to the clarified cashew apple juice. Dextran yield was favored by the combination of low concentrations of sucrose and reducing sugars. The formation of oligosaccharides was favored by increasing the concentration of reducing sugars and by the combination of high concentrations of sucrose and reducing sugars, the highest concentration of oligosaccharides obtained was 104.73g/L and the qualitative analysis showed that at concentrations of 25g/L and 75g/L of sucrose and reducing sugar, respectively, it is possible to obtain oligosaccharides of degree of polymerization up to 12. The juice containing prebiotic oligosaccharide is a potential new functional beverage.

Functional and Structural Characterization of α-(1→2) Branching Sucrase Derived from DSR-E Glucansucrase

ΔN(123)-glucan-binding domain-catalytic domain 2 (ΔN(123)-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN(123)-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite -1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN(123)-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN(123)-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN(123)-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.