Mesenteroides B-512F Research Articles

Dextran sulfate from Leuconostoc mesenteroides B512F exerts potent antiviral activity against SARS-CoV-2 in vitro and in vivo.

The emergent human coronavirus SARS-CoV-2 and its resistance to current drugs makes the need for new potent treatments for COVID-19 patients strongly necessary. Dextran sulfate (DS) polysaccharides have long demonstrated antiviral activity against different enveloped viruses in vitro. However, their poor bioavailability has led to their abandonment as antiviral candidates. Here, we report for the first time the broad-spectrum antiviral activity of a DS-based extrapolymeric substance produced by the lactic acid bacterium Leuconostoc mesenteroides B512F. Time of addition assays with SARS-CoV-2 pseudoviruses in in vitro models confirm the inhibitory activity of DSs in the early stages of viral infection (viral entry). In addition, this exopolysaccharide substance also reports broad-spectrum antiviral activity against several enveloped viruses such as SARS-CoV-2, HCoV229E, HSV-1, in in vitro models and in human lung tissue. The toxicity and antiviral capacity of DS from L. mesenteroides was tested in vivo in mouse models which are susceptible to SARS-CoV-2 infection. The described DS, administered by inhalation, a new route of administration for these types of polymers, shows strong inhibition of SARS-CoV-2 infection in vivo, significantly reducing animal mortality and morbidity at non-toxic doses. Therefore, we suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Structure-Function Relationship Studies of Multidomain Levansucrases from Leuconostocaceae Family.

Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis.

Enzymatic synthesis and biological characterization of a novel mangiferin glucoside

Mangiferin, a major constituent of Mangifera indica L., has attracted substantial attention due to its anti-oxidant, anti-diabetic, anti-inflammatory, and anti-microbial activities. However, its poor solubility in water limits its use in food and pharmaceutical industries. In this study, novel mangiferin-(1→6)-α-d-glucopyranoside (Mg-G1) was enzymatically synthesized from mangiferin and sucrose using glucansucrase from Leuconostoc mesenteroides B-512F/KM, and optimized using response surface methodology. The water solubility of Mg-G1 was found to be 824.7 mM, which is more than 2300-fold higher than that of mangiferin. Mg-G1 also showed DPPH radical scavenging activity and superoxide dismutase (SOD)-like scavenging activity, which were 4.77- and 3.71-fold higher than that of mangiferin, respectively. Mg-G1 displayed inhibitory activity against human intestinal maltase and COX-2. Thus, the novel glucosylated mangiferin may be used as an ingredient in functional food and pharmaceutical application.

Co-immobilization of dextransucrase and dextranase in epoxy-agarose- tailoring oligosaccharides synthesis

Abstract The best support reported for dextransucrase (DS) and dextranase (DN) immobilization was Eupergit C, but this carrier was discontinued and is no longer available in the market. Thus, epoxy-agarose support is suggested as an alternative to co-immobilize DS from Leuconostoc mesenteroides B-512F and DN from Chaetomium erraticum. The co-immobilization approach improved DS performance, compared to immobilized DS, due to the removal of the dextran layer accumulated on DS by DN, and enhanced both enzymes catalytic activities under a wide range of pH and temperature. Since DS and DN have the same optimum pH and temperature, the co-immobilization was done in a single step. The EPA-DS-DN0.5 (co-immobilized DS (26.16 UI) and DN (2.76 UI) in epoxy-agarose) showed the highest recovered activity (59.54%). The biocatalyst was stable at 4 °C, retaining above 70% of activity for 60 days and was efficient producing oligosaccharides, yielding 12.68 ± 0.09 g/L of oligosaccharides with a degree of polymerization up to 5.

Preparation and characterization of cross-linked enzyme aggregates of dextransucrase from Leuconostoc mesenteroides B-512F

Abstract We describe the development of cross-linked enzyme aggregates (CLEA) of dextransucrase of Leuconostoc mesenteroides B-512 F. Treatments of enzyme preparation using dextranase were evaluated varying incubation time to remove the dextran layer involving the enzyme molecule, turning it suitable for immobilization. Results showed 21 days of treatment as the best outcome. Subsequently, we tested different water-miscible organic solvents as precipitants, the reaction and centrifugation times, and the concentration of cross-linker agent (glutaraldehyde) in the preparation of CLEA. Optimal conditions were: the use of isopropanol as solvent, 30 min of centrifugation time, 3 h of cross-linking time, and 100 mM of glutaraldehyde. A central composite design was carried out to optimize conditions to obtain the highest enzymatic activity, testing the pH (3.0–7.0) and temperature (20 °C-60 °C). Results showed that dextransucrase CLEA operate at optimal pH of 3.0 and temperature of 60 °C. The operational stability of the immobilized biocatalyst showed up to 30% of residual activity after 10 cycles of reuse, in a solution of 100 mM of sucrose and 600 mM of maltose. The preparation of dextransucrase CLEA is described for the first time and results suggest that this novel immobilized biocatalyst has potential in many industrial applications.

Dextransucrase immobilized on activated-chitosan particles as a novel biocatalyst

Dextransucrase from Leuconostoc mesenteroides B-512F was covalently immobilized on glutaraldehyde-actived chitosan particles. The best initial protein loading (400mg/g of dried support) showed 197U/g of catalytic activity. The optimal reaction pH and temperature of this new biocatalyst were determined to be 4.5 and 20°C, respectively. Regarding the thermal stability, the immobilization enhanced enzyme protection against high temperatures, whereas glucose and maltose acted as stabilizers. The biocatalyst was stable under storage at 5°C for a month. The biocatalyst presented good operational stability, retaining up to 40% of its initial activity after ten batch cycles of reaction to obtain oligosaccharides. These results suggest the use of the immobilized dextransucrase on chitosan particles as a promising novel biocatalyst to produce dextran and oligosaccharides.

Synthesis and characterization of isomaltulose-derived oligosaccharides produced by transglucosylation reaction of Leuconostoc mesenteroides dextransucrase.

This paper reports the efficient enzymatic synthesis of a homologous series of isomaltulose-derived oligosaccharides with degrees of polymerization ranging from 3 to 9 through the transglucosylation reaction using a dextransucrase from Leuconostoc mesenteroides B-512F. The total oligosaccharide yield obtained under optimal conditions was 41-42% (in weight with respect to the initial amount of isomaltulose) after 24-48 h of reaction. Nuclear magnetic resonance (NMR) structural characterization indicated that dextransucrase specifically transferred glucose moieties of sucrose to the C-6 of the nonreducing glucose residue of isomaltulose. Likewise, monitoring the progression of the content of each individual oligosaccharide indicated that oligosaccharide acceptor products of low molecular weight acted in turn as acceptors for further transglucosylation to yield oligosaccharides of a higher degree of polymerization. The produced isomaltulose-derived oligosaccharides can be considered as isomalto-oligosaccharides (IMOs) because they are linked by only α-(1→6) bonds. In addition, having isomaltulose as the core structure, these IMO-like structures could possess appealing bioactive properties that could find potential applications as functional food ingredients.

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.

Dextran-like exopolysaccharide-producing Leuconostoc and Weissella from kimchi and its ingredients

Dextran-like exopolysaccharide (EPS)-producing lactic acid bacteria were isolated from kimchi and its ingredients. Analysis of 16S rRNA genes (500–600 bp) of 158 isolates revealed that dextran-like EPS-producing strains were most similar to either Leuconostoc or Weissella. The gene sequence analysis of the strains revealed a similarity to Leuconostoc mesenteroides and Weissella confusa in the samples. The molecular masses of dextranlike polymers from the most isolated Leuconostoc and Weissella strains were 1,176 and 1,158 kDa, respectively. Fourier transform infrared analysis of these polymers showed a similarity to the structure of commercial dextran from Leu. mesenteroides B-512F.

무 절임 제조 시 천일염과 스타터 첨가에 따른 품질특성 비교

본 연구는 국내산 천일염(장판염, 토판염)의 성분 비교와 이를 이용하여 무 절임을 제조할 때 첨가한 스타터와 함께 발효기간 동안의 변화를 알아보고자 수행하였다. 그 결과 두 소금간의 일반성분 중 수분함량은 차이를 보였지만 회분 함량은 그룹간의 차이를 보이지 않아, 생산시기가 동일한 점을 고려할 때 천일염 생산방식에 따른 차이라고 판단된다. 이들의 무기성분 함량은 소금의 주성분인 Na 함량은 큰 차이를 보이지 않았고, Ca, K 그리고 Mg 함량은 장판염이 높게 측정된 반면, Fe 그리고 Zn 함량은 토판염이 높게 측정되었지만, Ca 함량을 제외하고 크게 차이를 보이지 않았다. 천일염의 당도 측정 결과 10.70 Brix로 높은 함량을 나타내어 어떤 종류의 당으로 구성되었는지에 대한 정성 분석 연구가 요구된다. 무 절임 중 천일염과 스타터 첨가에 의한 발효 변화를 관찰하였을 때 스타터를 첨가한 그룹이 스타터를 첨가하지 않은 그룹보다 활발하게 유리당, 유기산 등 2차 대사산물을 생성하여, 우점종으로서 초기 미생물 조절에 영향을 끼쳤다고 판단된다. 그리하여 실험에 사용된 Leuconostoc mesenteroides B-512F 균주의 스타터 첨가가 발효과정 중 미생물 균총 변화에 큰 영향을 주므로, 발효 식품 제조 시 초기미생물 조절자로서의 명확한 역할 구명이 필요할 것으로 생각된다. In this study, the effects of the use of a starter on radish soaked at <TEX>$4^{\circ}C$</TEX> for 28 days using two kinds of domestic sun-dried salt (white and gray salt) were determined. As a result, the moisture contents of the radish soaked with white and gray salt were 7.93 and 4.50%, respectively. The salinity levels were found to be equal (90%). No significant difference was found between the two groups in terms of the mineral contents of the salts therein (<TEX>$37812.41{\pm}1922.95$</TEX> and <TEX>$39755.13{\pm}1205.70$</TEX> mg/100 g, respectively). However, iron and zinc contents of gray salt were higher than those of white salt while the calcium, potassium, and magnesium contents were lower than those of white salt. After the addition of the starter, the sugar content increased, the pH decreased, and the total acidity rapidly decreased compared with the non-starter group during fermentation. The total microorganism count of the two groups increased during the fermentation period of 28 days. From the initial fermentation for 7 days, the lactic acid bacteria in the non-starter group increased while those in the leuconostoc starter group steadily increased within the whole fermentation period. The Leuconostoc spp. in non-starter group increased, but that in the starter group decreased to pH 4.0 on day 14.

Substitution of yeast extract by ammonium sulfate for mannitol production in cashew apple juice

Abstract The use of agriculture excess as substrate in industrial fermentations became an interesting alternative to reduce production costs and to reduce negative environmental impact caused by the disposal of these products. In this work, the use of cashew apple juice supplemented with ammonium sulfate as substrate for mannitol production by two Leuconostoc strains was studied. The carbohydrates of cashew apple juice are glucose and fructose. Process pH was not significant on fructose conversion into mannitol by Leuconostoc citreum B-742. However, for L. mesenteroides B-512F the process pH showed a significant effect on fructose conversion into mannitol. The use of cashew apple juice supplemented with ammonium sulfate in replacement of yeast extract enhanced the yield L. mesenteroides B-512F was the best mannitol producer. The optimal pH for mannitol production from fructose by this strain in pH-controlled fermentation was at pH 5.0 with mannitol yield of 95% and productivity of 1.6 g/L h. Lactic acid bacteria are known as nutritional exigent microorganisms. Usually, they are cultivated in yeast extract and other complex nitrogen source. Herein, the combination of an inorganic exogenous nitrogen source and the amino acids from the cashew apple juice was enough to support LAB growth in a low cost substrate.

Efficient Synthesis and Characterization of Lactulosucrose by Leuconostoc mesenteroides B-512F Dextransucrase

This work describes an efficient enzymatic synthesis and NMR structural characterization of the trisaccharide β-D-galactopyranosyl-(1→4)-β-D-fructofuranosyl-(2→1)-α-D-glucopyranoside, also termed as lactulosucrose. This oligosaccharide was formed by the Leuconostoc mesenteroides B-512F dextransucrase-catalyzed transfer of the glucosyl residue from sucrose to the 2-hydroxyl group of the reducing unit of lactulose. The enzymatic reaction was carried out under optimal conditions, i.e., at 30 °C in 20 mM sodium acetate buffer with 0.34 mM CaCl(2) at pH 5.2, and the effect of factors such as reaction time (0-48 h), enzyme charge (0.8, 1.6, and 2.4 U mL(-1)), and sucrose:lactulose concentration ratios (20:40, 30:30, and 40:20, expressed in g/100 mL) on the formation of transfer products were studied. The highest formation in lactulosucrose was attained at 8 and 24-32 h by using 20%:40% and 30%:30% sucrose:lactulose mixtures, respectively, with 1.6 or 2.4 U mL(-1) dextransucrase, leading to lactulosucrose yields of 27-35% in weight respect to the initial amount of lactulose. Furthermore, minor tetra- and pentasaccharide, both probably derived from lactulose, were also detected and quantified. Likewise, the capacity of lactulosucrose to act as D-glucosyl donor once the sucrose was consumed, could explain its decrease from 16 to 24 h when the highest charge of dextransucrase was used. Considering the chemical structure of the synthesized oligosaccharides, lactulosucrose and its derivatives could potentially be excellent candidates for an emerging prebiotic ingredient.

Synthesis and Characterization of a Potential Prebiotic Trisaccharide from Cheese Whey Permeate and Sucrose by Leuconostoc mesenteroides Dextransucrase

The production of new bioactive oligosaccharides is currently garnering much attention for their potential use as functional ingredients. This work addresses the enzymatic synthesis and NMR structural characterization of 2-α-D-glucopyranosyl-lactose derived from sucrose:lactose and sucrose:cheese whey permeate mixtures by using a Leuconostoc mesenteroides B-512F dextransucrase. The effect of synthesis conditions, including concentration of substrates, molar ratio of donor/acceptor, enzyme concentration, reaction time, and temperature, on the formation of transfer products is evaluated. Results indicated that cheese whey permeate is a suitable material for the synthesis of 2-α-D-glucopyranosyl-lactose, giving rise to yields around 50% (in weight respect to the initial amount of lactose) under the optimum reaction conditions. According to its structure, this trisaccharide is an excellent candidate for a new prebiotic ingredient, due to the reported high resistance of α-(1→2) linkages to the digestive enzymes in humans and animals, as well as to its potential selective stimulation of beneficial bacteria in the large intestine mainly attributed to the two linked glucose units located at the reducing end that reflects the disaccharide kojibiose (2-α-D-glucopyranosyl-D-glucose). These findings could contribute to broadening the use of important agricultural raw materials, such as sucrose or cheese whey permeates, as renewable substrates for enzymatic synthesis of oligosaccharides of nutritional interest.

Monitoring of Leuconostoc Population During Sauerkraut Fermentation by Quantitative Real-Time Polymerase Chain Reaction

A real-time PCR assay method was established to monitor Leuconostoc spp. populations via specific amplification of the dextransucrase gene. Quantification of L. mesenteroides B-512F using both genomic DNA and cell suspensions yielded a log-linear correlation spanning approximately 5 log units. By using this method, monitoring changes of Leuconostoc spp. during sauerkraut fermentation was successfully accomplished with accuracy after inoculation of starter and sugars (sucrose and maltose).

Novel quantitative method for the degree of branching in dextran

A novel quantitative method for the determination of degree of branching in Leuconostoc mesenteroides B-512F dextran was developed by using the combination of 3 dextran-degrading enzymes. First, Paenibacillus sp. endo-dextranase was randomly degraded B-512F dextran into linear or branched isomalto-oligosaccharides with various degree of polymerization (2–8). Second, Streptococcus mutans dextran glucosidase hydrolyzed linear or branched isomalto-oligosaccharides into glucose and branched isomalto-penta-saccharides. Third, the branched isomaltopenta-saccharide was degraded into glucose by using Bacteroides thetaimicron α-glucosidase. The number of branching points in B-512F dextran (5.42%) was determined by the difference in the amount of glucose in the reaction digest between BTGase-PDex and DGase-PDex treatments.

Stability Study of Crude Dextransucrase from Leuconostoc citreum NRRL B-742

In the present work, the stability of crude dextransucrase from Leuconostoc citreum B-742 was evaluated in synthetic and in cashew apple juice culture broth. Optimum stability conditions for dextransucrase from L. citreum B-742 were different from the reported for its parental industrial strain enzyme (L. mesenteroides B-512F). Crude dextransucrase, from L. citreum B-742, produced using cashew apple juice as substrate, presented higher stability than the crude enzyme produced using synthetic culture medium, showing the same behavior previously reported for dextransucrase from L. mesenteroides B-512F. The crude enzyme presented good stability in cashew apple juice for 48h at 25°C and pH 6.5.

Avaliação da produção de ácido lático por Leuconostoc mesenteroides B512F em xarope de caju

The production of lactic acid by fermentation accounts for more than 50% of the world production. To meet the demand, efforts to lower the costs have been the objective of several studies. This work aimed to study the lactic acid production in cashew apple (Anacardium orcidentale) syrup. The microorganism used was the Leuconostoc mesenteroides B512F. In the experimental investigation, the ideal concentrations of yeast extract and initial sugar were determined. A second experimental investigation was carried out with cashew apple syrup by adding yeast extract and phosphate and by diluting the syrup according to the initial sugar concentrations to optimize the medium culture. A kinetic study was carried out with the optimized substrate obtained. The results showed that the studied strains can produce lactic acid in satisfactory concentrations provided the natural syrup substrate contains only yeast extract and phosphate.

Prebiotic effect of fermented cashew apple (Anacardium occidentale L) juice

Abstract Leuconostc mesenteroides B-512F and L. mesenteroides B-742 were cultivated in clarified cashew apple juice to produce prebiotic oligosaccharides. Yeast extract (20 g/L); K 2 HPO 4 (g/L) and sucrose (50 g/L) were added to the juice to promote the microbial growth and dextransucrase production. Initial pH was adjusted to 6.5 with H 3 PO 4 . Fermentations were carried out at 30 °C and 150 rpm for 24 h. The prebiotic effect of the fermented cashew apple juice, containing oligosaccharides, was evaluated through the Lactobacillus johnsonii B-2178 growth. L. johnsonii was incubated for 48 h using fermented cashew apple juice as substrate. Lactobacillus growth was compared to the microbial growth in non-fermented juice and in MRS broth. L. johnsonii growth in the fermented cashew apple juice was threefolds the observed growth in the non-fermented juice.

NMR spectroscopic analysis of exopolysaccharides produced by Leuconostoc citreum and Weissella confusa

Dextrans are the main exopolysaccharides produced by Leuconostoc species. Other dextran-producing lactic acid bacteria include Streptococci, Lactobacilli, and Weissella species. Commercial production and structural analysis has focused mainly on dextrans from Leuconostoc species, particularly on Leuconostoc mesenteroides strains. In this study, we used NMR spectroscopy techniques to analyze the structures of dextrans produced by Leuconostoc citreum E497 and Weissella confusa E392. The dextrans were compared to that of L. mesenteroides B512F produced under the same conditions. Generally, W. confusa E392 showed better growth and produced more EPS than did L. citreum E497 and L. mesenteroides B512F. Both L. citreum E497 and W. confusa E392 produced a class 1 dextran. Dextran from L. citreum E497 contained about 11% α-(1→2) and about 3.5% α-(1→3)-linked branches whereas dextran from W. confusa E392 was linear with only a few (2.7%) α-(1→3)-linked branches. Dextran from W. confusa E392 was found to be more linear than that of L. mesenteroides B512F, which, according to the present study, contained about 4.1% α-(1→3)-linked branches. Functionality, whether physiological or technological, depends on the structure of the polysaccharide. Dextran from L. citreum E497 may be useful as a source of prebiotic gluco-oligosaccharides with α-(1→2)-linked branches, whereas W. confusa E392 could be a suitable alternative to widely used L. mesenteroides B512F in the production of linear dextran.

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

The enzyme dextransucrase (sucrose:1, 6-α-D-glucan 6-α-glucosyltransferase, EC 2.4.1.5) catalyses the synthesis of exopolysaccharide, dextran from sucrose. This class of polysaccharide has been extensively exploited in pharmaceutical industry as blood volume expander, as stabiliser in food industry and as a chromatographic medium in fine chemical industry because of their nonionic nature and stability. Majority of the dextrans are synthesized from sucrose by dextransucrase secreted mainly by bacteria belonging to genera Leuconostoc, Streptococcus and Lactobacillus. Bulk of the information on purification of extracellular dextransucrase has been generated from Leuconostoc species. Various methods such as precipitation by ammonium sulphate, ethanol or polyethylene glycol, phase partitioning, ultrafiltration and chromatography have been used to purify the enzyme. Purification of dextransucrase is rendered difficult by the presence of viscous dextran in the medium. However, processes like ultra-filtration, salt and PEG precipitation, chromatography and phase partitioning have been standardized and successfully used for higher scale purification of the enzyme. A recombinant dextransucrase from Leuconostoc mesenteroides B-512F with a histidine tag has been expressed in E. coli cells and purifi ed by immobilized metal ion chromatography. This review reports the available information on purifi cation methods of dextransucrase from Leuconostoc mesenteroides strains.