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Levan produced by Gluconobacter spp. has great potential in biotechnological applications. However, Gluconobacter spp. can synthesize organic acids during fermentation, resulting in environmental acidification. Few studies have focused on the effects of environmental acidification on levan synthesis. This study revealed that the organic acids, mainly gluconic acid (GA) and 2-keto-gluconic acid (2KGA) secreted by Gluconobacter sp. MP2116 created a highly acidic environment (pH < 3) that inhibited levan biosynthesis. The levansucrase derived from strain MP2116 had high enzyme activity at pH 4.0 âˆ¼ pH 6.5. When the ambient pH was less than 3, the enzyme activity decreased by 67 %. Knocking out the mgdh gene of membrane-bound glucose dehydrogenase (mGDH) in the GA and 2KGA synthesis pathway in strain MP2116 eliminated the inhibitory effect of high acid levels on levansucrase function. As a result, the levan yield increased from 7.4 g/l (wild-type) to 18.8 g/l (Δmgdh) during fermentation without pH control. This study provides a new strategy for improving levan production by preventing the inhibition of polysaccharide synthesis by environmental acidification.

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Levan is a fructose polysaccharide with multiple applications in different fields, but its obtaining in powdered form with a narrow particle size distribution is a complicated task. Two techniques, electrospraying and supercritical antisolvent (SAS) precipitation, were used to process levan that was first obtained enzymatically. The SAS process was able to micronize the polymer (at experimental conditions far above the mixture critical point of the solvent-antisolvent system) to obtain spherical particles between 0.30 and 0.50 µm with a proper particle size distribution. In this case, the Peng-Robinson equation of state was used to theoretically determine the mixture critical point. Bigger and elongated particles were obtained with electrospraying (0.60 µm). According to solution properties, mainly rheology, solubility and conductivity, the best solvent for levan electrospraying, in order to avoid problems of solvent evaporation and jet formation, was a mixture of water and ethanol with a polymer concentration of 50 mg·cm-3. Indeed, that solution has a viscous behavior (according to the oscillatory analysis), a low degree of pseudo-plasticity (based on the shear flow analysis), and the highest value of conductivity. Therefore, the particle size distribution of levan in powdered form can be tuned depending on the technique used.

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This review article delves into the intricate relationship between levan, a versatile polysaccharide, and its role in enhancing plant resistance against pathogens. By exploring the potential applications of levan in agriculture and biotechnology, such as crop protection, stress tolerance enhancement, and biotechnological innovations, significant advancements in sustainable agriculture are uncovered. Despite challenges in optimizing application methods and addressing regulatory hurdles, understanding the mechanisms of levan-mediated plant immunity offers promising avenues for future research. This review underscores the implications of utilizing levan to develop eco-friendly solutions, reduce reliance on chemical pesticides, and promote sustainable agricultural practices. Ultimately, by unraveling the pivotal role of levan in plant-pathogen interactions, this review sets the stage for transformative innovations in agriculture and highlights the path towards a more resilient and sustainable agricultural future.

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Nanoparticles composed of Levan and Dolutegravir (DTG) have been successfully synthesized using a spray drying procedure specifically designed for milk/food admixture applications. Levan, obtained from the microorganism Bacillus subtilis, was thoroughly characterized using MALDI-TOF and solid-state NMR technique to confirm its properties. In the present study, this isolated Levan was utilized as a carrier for drug delivery applications. The optimized spray-dried nanoparticles exhibited a smooth surface morphology with particle sizes ranging from 195 to 329 nm. In the in-vitro drug release experiments conducted in water media, the spray-dried nanoparticles showed 100 % release, whereas the unprocessed drug exhibited only 50 % release at the end of 24 h. Notably, the drug release in milk was comparable to that in plain media, indicating the compatibility. The improved dissolution rate observed for the nanoparticles could be attributed to the solid-state conversion (confirmed by XRD analysis) of DTG from its crystalline to amorphous state. The stability of the drug was verified using Fourier Transform Infra-Red Spectroscopy and Thermogravimetry-Differential Scanning Calorimetry analysis. To evaluate the in-vitro cellular toxicity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was conducted, which revealed the CC50 value of 88.88 ± 5.10 µg/mL for unprocessed DTG and 101.08 ± 37.37 µg/mL for DTG nanoparticles. These results indicated that the toxicity of the nanoparticles was comparable to the unprocessed drug. Furthermore, the anti-HIV activity of the nanoparticles in human cell lines was found to be similar to that of the pure drug, emphasizing the therapeutic efficacy of DTG in combating HIV.

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Levan, a ß-(2,6)-linked fructose polymer, exhibits diverse properties that impart versatility, rendering it a highly sought-after biopolymer with various industrial applications. Levan can be produced by various microorganisms using sucrose, food industry byproducts and agricultural wastes. Microbial levan represents the most potent cost-effective process for commercial-scale levan production. This study reviews the optimization of levan production by understanding its biosynthesis, physicochemical properties and the fermentation process. In addition, genetic and protein engineering for its increased production and emerging methods for its detection are introduced and discussed. All of these comprehensive studies could serve as powerful tools to optimize levan production and broaden its applications across various industries.

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In this paper, the preparation of three new polysaccharide-type chiral stationary phases (CSPs) based on levan carbamates (3,5-dimethylphenyl, 4-methylphenyl, and 1-naphthyl) is described. The enantioseparation of (±)-trans-ß-lactam ureas 1a-h was investigated by high-performance liquid chromatography (HPLC) on six different chiral columns (Chiralpak AD-3, Chiralcel OD-3, Chirallica PST-7, Chirallica PST-8, Chirallica PST-9, and Chirallica PST-10) in the polar organic mode, using pure methanol (MeOH), ethanol (EtOH), and acetonitrile (ACN). Apart from the Chirallica PST-9 column (based on levan tris(1-naphthylcarbamate), the columns exhibited a satisfactory chiral recognition ability for the tested trans-ß-lactam ureas 1a-h.

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The global demand for therapeutic prebiotics persuades the quest for novel exopolysaccharides that can retard the growth of pathobionts and healthcare-associated pathogens. In this regard, an exopolysaccharide (3.69 mg/mL) producing strain showing prebiotic and antibiofilm activity was isolated from indigenous pineapple pomace of Tripura and identified as Bacillus subtilis PR-C18. Zymogram analysis revealed EPS PR-C18 was synthesized by levansucrase (∼57 kDa) with a maximal activity of 4.62 U/mg. Chromatography techniques, FTIR, and NMR spectral data revealed the homopolymeric nature of purified EPS with a molecular weight of 3.40 × 104 Da. SEM and rheological study unveiled its microporous structure and shear-thinning effect. Furthermore, EPS PR-C18 showed remarkable emulsification, flocculation, water retention, water solubilization, and antioxidant activity. DSC-TGA data demonstrated its high thermostability and cytotoxicity analysis verified its nontoxic biocompatible nature. In addition, the antibiofilm activity of EPS PR-C18 was validated using molecular docking, molecular simulation, MM-GBSA and PCA studies, which exhibited its strong binding affinity (-20.79 kcal/moL) with PelD, a virulence factor from Pseudomonas aeruginosa. Together, these findings support the future exploitation of EPS PR-C18 as an additive or adjuvant in food and pharmaceutical sectors.

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Kombucha is prepared by fermenting sugared green or black tea with a symbiotic culture of bacteria and yeast (SCOBY). Some of the bacteria within the SCOBY are known to form exopolysaccharides (EPS) from sucrose. However, it is yet unknown whether water-soluble EPS are formed in kombucha, and if so, which specific EPS are present. Therefore, different kombucha samples were prepared by fermentation of green and black tea with SCOBYs from different manufacturers. Subsequently, the EPS were isolated and characterized by using various chromatographic methods, partial enzymatic hydrolyses and NMR spectroscopy. It was demonstrated that levans with a varying degree of branching at position O1 (4.3-7.9 %) are present, while only trace amounts of glucans were detected. Furthermore, levans isolated from kombucha had a comparably low molecular weight and the content of levan within the kombucha samples varied from 33 to 562 mg levan/L kombucha. Therefore, our study demonstrated that levans are the main EPS type in kombucha and that levan amounts and structures varied when different starter cultures and ingredients were used. Furthermore, we provide a comprehensive data set on the structural variability of levans from kombucha.

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In this study, we synthesized levan shell hydrophobic silica nanoclusters encapsulating doxorubicin (L-HSi-Dox) and evaluated their potential as ultrasound-responsive drug delivery systems for cancer treatment. L-HSi-Dox nanoclusters were successfully fabricated by integrating a hydrophobic silica nanoparticle-doxorubicin complex as the core and an amphiphilic levan carbohydrate polymer as the shell by using an electrospray technique. Characterization analyses confirmed the stability, size, and composition of the nanoclusters. In particular, the nanoclusters exhibited a controlled release of Dox under aqueous conditions, demonstrating their potential as efficient drug carriers. The levanic groups of the nanoclusters enhanced the targeted delivery of Dox to specific cancer cells. Furthermore, the synergism between the nanoclusters and ultrasound effectively reduced cell viability and induced cell death, particularly in the GLUT5-overexpressing MDA-MB-231 cells. In a tumor xenograft mouse model, treatment with the nanoclusters and ultrasound significantly reduced the tumor volume and weight without affecting the body weight. Collectively, these results highlight the potential of the L-HSi-Dox nanoclusters and ultrasound as promising drug delivery systems with an enhanced therapeutic efficacy for biomedical applications.

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Levan is a fructose-based biopolymer with diverse applications in the medicinal, pharmaceutical, and food industries. However, despite its extensive biological and pharmacological actions, including antioxidant, anti-inflammatory, and antidiabetic properties, research on its anti-aging potential is limited. This study explored levan's impact on the chronological lifespan (CLS) of yeast Saccharomyces cerevisiae for the first time. The results show that levan treatment significantly extended the CLS of wild-type (WT) yeast by preventing the accumulation of oxidative stress markers (reactive oxygen species, malondialdehyde, and protein carbonyl content) and ameliorating apoptotic features such as reduced mitochondrial membrane potential, loss of plasma membrane integrity, and externalization of phosphatidylserine. By day 40 of the CLS, a significant increase in yeast viability of 6.8 % (p < 0.01), 11.9 % (p < 0.01), and 20.8 % (p < 0.01) was observed at 0.25, 0.5, and 1 mg/mL of levan concentrations, respectively, compared to control (0 %). This study's results indicate that levan treatment substantially modulates the expression of genes involved in the TORC1/Sch9 pathway. Moreover, levan treatment significantly extended the CLS of yeast antioxidant-deficient mutant sod2Δ and antiapoptotic gene-deficient mutant pep4Δ. Levan also extended the CLS of signaling pathway gene-deficient mutants such as pkh2Δ, rim15Δ, atg1, and ras2Δ, while not affecting the CLS of tor1Δ and sch9Δ.

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Pantoea, a gram-negative, rod-shaped, anaerobic bacterium, is a rare cause of human disease. Pantoea species have been known to mostly cause pulmonary disease in agricultural workers as they are native to select crops and wild animal furs. However, in very few documented cases, Pantoea has been discovered as the source of nosocomial infections, usually in the setting of an immunocompromised host. This case report details the clinical course of a 62-year-old immunocompromised female with stage 3 breast cancer presenting with acute cholecystitis and bacteremia and the unexpected discovery of Pantoea in peripheral and chemotherapy port blood cultures. After appropriate management and susceptibility testing, the patient fortunately recovered with initial cefepime and eventual levofloxacin to target the Pantoea species. To our knowledge, this is the third documented case worldwide of Pantoea isolated from cholecystitis with associated bacteremia and the first documented case in North America. Of special interest, a few months after her infection, the patient was found to be free of breast cancer. Pantoea species are known to contain levan, an exopolysaccharide, that has been seen to upregulate tumor suppressor genes. This should be considered in the future management and research of Pantoea infections.

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The valorization of byproducts from the sugarcane industry represents a potential alternative method with a low energy cost for the production of metabolites that are of commercial and industrial interest. The production of exopolysaccharides (EPSs) was carried out using the yeast Suhomyces kilbournensis isolated from agro-industrial sugarcane, and the products and byproducts of this agro-industrial sugarcane were used as carbon sources for their recovery. The effect of pH, temperature, and carbon and nitrogen sources and their concentration in EPS production by submerged fermentation (SmF) was studied in 170 mL glass containers of uniform geometry at 30 °C with an initial pH of 6.5. The resulting EPSs were characterized with Fourier-transform infrared spectroscopy (FT-IR). The results showed that the highest EPS production yields were 4.26 and 44.33 g/L after 6 h of fermentation using sucrose and molasses as carbon sources, respectively. Finally, an FT-IR analysis of the EPSs produced by S. kilbournensis corresponded to levan, corroborating its origin. It is important to mention that this is the first work that reports the production of levan using this yeast. This is relevant because, currently, most studies are focused on the use of recombinant and genetically modified microorganisms; in this scenario, Suhomyces kilbournensis is a native yeast isolated from the sugar production process, giving it a great advantage in the incorporation of carbon sources into their metabolic processes in order to produce levan sucrose, which uses fructose to polymerize levan.

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Currently, levan is attracting attention due to its promising applications in the food and biomedical fields. Levansucrase synthesizes levan by polymerizing the fructosyl unit in sucrose. However, a large amount of the byproduct glucose is produced during this process. In this paper, an engineered oleaginous yeast (Yarrowia lipolytica) strain was constructed using a surface display plasmid containing the LevS gene of Gluconobacter sp. MP2116. The levansucrase activity of the engineered yeast strain reached 327.8 U/g of cell dry weight. The maximal levan concentration (58.9 g/l) was achieved within 156 h in the 5-liter fermentation. Over 81.2 % of the sucrose was enzymolyzed by the levansucrase, and the byproduct glucose was converted to 21.8 g/l biomass with an intracellular oil content of 25.5 % (w/w). The obtained oil was comprised of 91.3 % long-chain fatty acids (C16-C18). This study provides new insight for levan production and comprehensive utilization of the byproduct in levan biosynthesis.

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Levan is a fructan polymer with many industrial applications such as the formulation of hydrogels, drug delivery, and wound healing, among others. To this end, metabolic systems engineering is a valuable method to improve the yield of a specific metabolite in a wide range of bacterial and eukaryotic organisms. In this study, we report a systems biology approach integrating genomics data for the Bacillus subtilis model, wherein the metabolic pathway for levan biosynthesis is unpacked. We analyzed a revised genome-scale enzyme-constrained metabolic model (ecGEM) and performed simulations to increase levan biopolymer production capacity in B. subtilis. We used the model ec_iYO844_lvn to (1) identify the essential genes and bottlenecks in levan production, and (2) specifically design an engineered B. subtilis strain capable of producing higher levan yields. The FBA and FVA analysis showed the maximal growth rate of the organism up to 0.624 hr-1 at 20 mmol gDw-1 hr-1 of sucrose intake. Gene knockout analyses were performed to identify gene knockout targets to increase the levan flux in B. subtilis. Importantly, we found that the pgk and ctaD genes are the two target genes for the knockout. The perturbation of these two genes has flux gains for levan production reactions with 1.3- and 1.4-fold the relative flux span in the mutant strains, respectively, compared to the wild type. In all, this work identifies the bottlenecks in the production of levan and possible ways to overcome them. Our results provide deeper insights on the bacterium's physiology and new avenues for strain engineering.

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Soybean tempeh contains bioactive carbohydrate that can reduce the severity of diarrhea by inhibiting enterotoxigenic Escherichia coli (ETEC) adhesion to mammalian epithelial cells. Lactic acid bacteria (LAB) are known to be present abundantly in soybean tempeh. Some LAB species can produce exopolysaccharides (EPS) with anti-adhesion bioactivity against ETEC but there has been no report of anti-adhesion bioactive EPS from tempeh-associated LAB. We isolated EPS-producing LAB from tempeh-related sources, identified them, unambiguously elucidated their EPS structure and assessed the bioactivity of their EPS against ETEC. Pediococcus pentosaceus TL, Leuconostoc mesenteroides WA and L. mesenteroides WN produced both dextran (α-1,6 linked glucan; >1000 kDa) and levan (ß-2,6 linked fructan; 650-760 kDa) in varying amounts and Leuconostoc citreum TR produced gel-forming α-1,6-mixed linkage dextran (829 kDa). All four isolates produced EPS that could adhere to ETEC cells and inhibit auto-aggregation of ETEC. EPS-PpTL, EPS-LmWA and EPS-LmWN were more bioactive towards pig-associated ETEC K88 while EPS-LcTR was more bioactive against human-associated ETEC H10407. Our finding is the first to report on the bioactivity of dextran against ETEC. Tempeh is a promising source of LAB isolates that can produce bioactive EPS against ETEC adhesion and aggregation.

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Fructans have long been known with their role in protecting organisms against various stress factors due to their ability to induce controlled dehydration and support membrane stability. Considering the vital importance of such features in cryo-technologies, this study aimed to explore the cryoprotective efficacy of fructans in mammalian cell systems where structurally different fructan polymers were examined on in vitro cell models derived from organs such as the liver, frequently used in transplantation, osteoblast, and cord cells, commonly employed in cell banking, as well as human seminal fluids that are of vital importance in assisted reproductive technology. To gain insights into the fructan/membrane interplay, structural differences were linked to rheological properties as well as to lipid membrane interactions where both fluorescein leakage from unilamellar liposomes and membrane integrity of osteoblast cells were monitored. High survival rates obtained with human endothelial, osteoblast and liver cells for up to two months clearly showed that fructans could be considered as effective non-permeating cryoprotectants, especially for extended periods of cryopreservation. In trials with human seminal fluid, short chained levan in combination with human serum albumin and glycerol proved very effective in preserving semen samples across multiple patients without any morphological abnormalities.

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During raw sugarcane processing, a significant portion of lost sucrose is attributable to microbial degradation. Sucrose consumption by many bacteria is also linked to the production of exopolysaccharides (EPS) such as dextrans and fructans. These resulting EPS cause operational challenges during raw sugar manufacturing. Here, we report the characterization of EPS from a fructan-forming Gluconobacter japonicus bacterium that we previously isolated from a Louisiana sugarcane factory. The genome sequencing revealed the presence of two encoded levansucrase genes, lsrA and lsrB. One levansucrase, LsrB, was detected in the secreted protein fraction of G. japonicus LASM12 by QTOF LC-MS. The spotting assays indicated that G. japonicus produces EPS using sucrose and raffinose as substrates. The G. japonicus EPS correlated with levan fructan commercial standards by 1H-NMR, and with the characteristic carbohydrate fingerprint region for FTIR spectra, confirming that the G. japonicus EPS is levan fructan. The glycosyl composition and glycosyl linkage analysis revealed a linear ß-2,6-fructofuranosyl polysaccharide with occasional (5.7%) ß-2,1-fructofuranosyl branches. The gel permeation chromatography of the levan fructan EPS showed two main peaks at 4.5 kDa and 8 kDa and a very minor peak at 500 kDa. G. japonicus was identified as a producer of levan fructan. These findings will be useful for future studies aimed at evaluating the impact of levan fructans on sugar crop processing, which have been historically underestimated in industry.

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Due to its bioactivity and versatile applications, levan has appeared as a promising biomaterial. Levansucrase is responsible for the conversion of sucrose into levan. With the goal of enhancing levan production, the strategy for enhancing the stability of levansucrase is being intensively studied. To make proteins more stable under high temperatures, proline, the most rigid residue, can be introduced into previously flexible regions. Herein, G249, D250, N251, and H252 on the flexible coil close to the calcium binding site of Bacillus licheniformis levansucrase were replaced with proline. Mutations at G249P greatly enhance both the enzyme's thermodynamic and kinetic stability, while those at H252P improve solely the enzyme's kinetic stability. GPC analysis revealed that G249P synthesize more levan, but H252P generate primarily oligosaccharides. Molecular dynamics simulations (MD) and MM/GBSA analysis revealed that G249P mutation increased not only the stability of levansucrase, but also affinity toward fructan.

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AIMS: Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. METHODS AND RESULTS: Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l-1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106-107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. CONCLUSIONS: The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

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Considering the documented health benefits of bacterial exopolysaccharides (EPSs), specifically of bacterial levan (BL), including its intrinsic antimicrobial activity against certain pathogenic species, the current study concentrated on the development of active pharmaceutical ingredients (APIs) in the form of colloid systems (CoSs) containing silver nanoparticles (AgNPs) employing in-house biosynthesized BL as a reducing and capping agent. The established protocol of fermentation conditions implicating two species of lactic acid bacteria (LAB), i.e., Streptococcus salivarius K12 and Leuconostoc mesenteroides DSM 20343, ensured a yield of up to 25.7 and 13.7 g L-1 of BL within 72 h, respectively. An analytical approach accomplished by Fourier-transform infrared (FT-IR) spectroscopy allowed for the verification of structural features attributed to biosynthesized BL. Furthermore, scanning electron microscopy (SEM) revealed the crystalline morphology of biosynthesized BL with a smooth and glossy surface and highly porous structure. Molecular weight (Mw) estimated by multi-detector size-exclusion chromatography (SEC) indicated that BL biosynthesized using S. salivarius K12 has an impressively high Mw, corresponding to 15.435 × 104 kilodaltons (kDa). In turn, BL isolated from L. mesenteroides DSM 20343 was found to have an Mw of only 26.6 kDa. Polydispersity index estimation (PD = Mw/Mn) of produced BL displayed a monodispersed molecule isolated from S. salivarius K12, corresponding to 1.08, while this was 2.17 for L. mesenteroides DSM 20343 isolate. The presence of fructose as the main backbone and, to a lesser extent, glucose and galactose as side chain molecules in EPS hydrolysates was supported by HPLC-RID detection. In producing CoS-BL@AgNPs within green biosynthesis, the presence of nanostructured objects with a size distribution from 12.67 ± 5.56 nm to 46.97 ± 20.23 was confirmed by SEM and energy-dispersive X-ray spectroscopy (EDX). The prominent inhibitory potency of elaborated CoS-BL@AgNPs against both reference test cultures, i.e., Pseudomonas aeruginosa, Escherichia coli, Enterobacter aerogenes, and Staphylococcus aureus and those of clinical origin with multi-drug resistance (MDR), was confirmed by disc and well diffusion tests and supported by the values of the minimum inhibitory and bactericidal concentrations. CoS-BL@AgNPs can be treated as APIs suitable for designing new antimicrobial agents and modifying therapies in controlling MDR pathogens.