Levan Production Research Articles

Optimization of Levan Production by Cold-Active Bacillus licheniformis ANT 179 and Fructooligosaccharide Synthesis by Its Levansucrase.

Fructooligosaccharides (FOS) and levan attract much attention due to a wide range of applications in food technology and pharmaceutical and cosmetic industry. Bacillus licheniformis ANT 179, isolated from Antarctica soil, produced levansucrase and levan in a medium containing sucrose as carbon substrate. In this study, characterization of levansucrase and production of short-chain FOS and levan were investigated. Temperature and pH optimum of the enzyme were found to be 60°C and pH 6.0, respectively. The optimization of fermentation conditions for levan production using sugarcane juice by response surface methodology (RSM) was carried out. Central composite rotatable design was used to study the main and the interactive effects of medium components: sugarcane juice and casein peptone concentration on levan production by the bacterium. The optimized medium with sugarcane juice at 20% (v/v) and casein peptone at 2% (w/v) was found to be optimal at an initial pH of 7.0 and incubation temperature of 35°C for 48h. Under these conditions, the maximum levan concentration was 50.25g/L on wet weight basis and 16.35g/L on dry weight basis. The produced inulin type FOS (kestose and neokestose) and levan were characterized by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) analysis. The study revealed that the levansucrase could form FOS from sucrose. The locally available low-cost substrate such as sugarcane juice in the form of a renewable substrate is proposed to be suitable even for scale-up production of enzyme and FOS for industrial applications. The levan and FOS synthesized by the bacterium are suitable for food applications and biomedical uses as the bacterium has GRAS status and devoid of endotoxin as compared to other Gram-negative bacteria.

Development of a cost-effective microbial levan production process by Halomonas cultures

Development of a cost-effective microbial levan production process by Halomonas cultures

<b>Ethanol and Levan production by sequential bath using <i>Zymomonas mobilis</i> immobilized on alginate and chitosan beads

Current study evaluates ethanol and levan production using Zymomonas mobilis immobilized on alginate and chitosan beads and assesses the capacity of the beads for reuse in sequential fermentations. Two experiments were carried out: In the first experiment 2 (5-2) design evaluated the following independent variables: sucrose concentration; pH; incubation time; temperature and agitation. In the second experiment, based on the best conditions observed in 2 (5-2) , the capacity of the immobilization support reuse in subsequent fermentations was evaluated for 12 days. In experiments using fractionated factorial experimental design 2 (5-2) the best results by immobilized biomass (2.6 g L -1 ) and immobilization efficiency was shown by alginate and chitosan beads showed only 0.22 g L -1 . Chitosan support provided the best rates to levan production (8.90 g L -1 ). Alginate support showed the highest values by ethanol production (93.4 g L -1 ). In batch sequential fermentation, re-using immobilization support, alginate support was efficient by maintaining the cellular viability on immobilized support (0.94 g L -1 ), levan production (21.11 g L -1 ) and ethanol production (87.21 g L -1 ). Chitosan support was inadequate for sequential batches since the beads dissolved after the second cycle.

Production of extracellular fructans by Gluconobacter nephelii P1464.

Bacterial extracellular fructans, known as levans, have potential applications in food, pharmaceutical and cosmetic industries and high fructan producing strains could contribute into the cost reduction and more extensive commercial usage of them. An acetic acid bacteria (AAB) isolate P1464 was obtained from the Microbial Strain Collection of Institute of Microbiology and Biotechnology, University of Latvia and identified as Gluconobacter nephelii by DNA-DNA hybridization and the formation of extracellular fructans by this strain was confirmed. Isolated extracellular fructose polymers were characterized using FT-IR spectroscopy and the structural features of fructan appeared as similar to the reference sample of bacterial levan. Molecular mass estimates showed that the isolated G.nephelii P1464 fructose polymer has a relatively small molecular weight (Mw 1122·939±153·453kDa) and a sizeable polydispersity (Mw/Mn=21·57±1·60), as compared with other AAB, which could promote their physiological activity, including the prebiotic effects. Obtained at different cultivation conditions characteristics of fructan production, including the biotechnological indices such as the productivity (Qp) and yield (Yp/s) ranging from 0·774 to 1·244gl(-1) h and from 0·181 to 0·436gg(-1) , respectively, confirmed, that G.nephelii P1464 could be used as promising strain for commercial production of levan. Bacterial fructans, known as levans, have extensive options for practical usage, however, actually limited due to high production costs. Therefore, the searches for efficient producer strains should be an urgent task to reduce costs. This study is the first report on the formation of fructans by a novel strain of acetic acid bacteria (AAB) Gluconobacter nephelii P1464. Characteristics obtained at different cultivation conditions confirmed the operation of a competitive and perspective producer strain. Isolated extracellular fructans are characterized by a lower molecular weight as compared with other AAB which could promote their physiological activity, including the prebiotic effects.

Levan-Producing Leuconostoc citreum Strain BD1707 and Its Growth in Tomato Juice Supplemented with Sucrose.

A levan-producing strain, BD1707, was isolated from Tibetan kefir and identified as Leuconostoc citreum. The effects of carbon sources on the growth of L. citreum BD1707 and levan production in tomato juice were measured. The changes in pH, viable cell count, sugar content, and levan yield in the cultured tomato juice supplemented with 15% (wt/vol) sucrose were also assayed. L. citreum BD1707 could synthesize more than 28 g/liter of levan in the tomato juice-sucrose medium when cultured at 30°C for 96 h. Based on the monosaccharide composition, molecular mass distribution, Fourier transform infrared (FTIR) spectra, and nuclear magnetic resonance (NMR) spectra, the levan synthesized by L. citreum BD1707 was composed of a linear backbone consisting of consecutive β-(2→6) linked d-fructofuranosyl units, with an estimated average molecular mass of 4.3 × 10(6) Da.

Identification of a seed-borne rice bacterium, Burkholderia glumae using cultural, morphological and biochemical methods

Burkholderia glumae is a seed-borne rice bacterium that causes bacterial panicle blight (BPB), which is a promising disease in many rice-growing areas around the world. The bacterium has been successfully grown on King’s B agar medium (KBM) at 280C for 48 hrs. It is Gram negative, non-spore-forming, rod-shaped with cell size 0.5 to 0.7 - 1.5 to 2.0 ?m with rounded ends. The colony appears as grayish white or yellow due to the pigment. The bacterium gave positive test in gelatin liquefaction, KOH solubility and nitrate reduction and while negative test in starch hydrolysis, oxidase reaction, levan production and arginine dihydrolase test. The bacterium utilized different carbon sources viz., sugars, amino acids, sugar alcohols and organic acids when examined through BiologTMGN2 Microplate System. This study would help in control and management of seed-borne bacterial plant pathogen B. glumae.

Immobilization and stabilization of levansucrase biocatalyst of high interest for the production of fructooligosaccharides and levan

AbstractBACKGROUNDLevansucrase (LS)‐catalyzed‐transfructosylation reaction is a potential approach for the synthesis of fructooligosaccharides (FOSs) and levan as health promoting compounds. This biocatalytic approach is hindered by low thermal stability of LS and its high rate of hydrolysis. In the present study, LS from Bacillus amyloliquefaciens was immobilized onto modified and unmodified epoxy‐activated supports (Eupergit® C; Sepabeads®) and on modified cross‐linked‐agarose beads, to increase its thermal stability and modulate its reaction selectivity (hydrolysis/transfructosylation).RESULTSLS bound to Sepabeads® HA (98.8%) and glyoxyl agarose‐IDA/Cu (67%) retained high initial activity along with good immobilization yields. The thermal stability results indicated that glyoxyl agarose‐IDA/Cu and glyoxyl agarose, provided the greatest thermal stability with factors of 14 and 106 times, respectively. Immobilization through Sepabeads® HA increased the ratio of transfructosylation/hydrolysis by 2.3 times, although it did not promote the stabilization of LS. Immobilization on glyoxyl agarose‐IDA/Cu provided a good compromise of all three properties: retention of activity (67.0%), transfructosylation/hydrolysis ratio (120%) and thermal stability (stability factor of 13.6).CONCLUSIONThe stabilization of LS through immobilization contributes to its potential use commercially. With an increasingly stable enzyme, further work will be directed towards altering LS reaction specificity towards levan and levan‐type FOSs. © 2015 Society of Chemical Industry

Recruiting a new strategy to improve levan production in Bacillus amyloliquefaciens.

Microbial levan is an important biopolymer with considerable potential in food and medical applications. Bacillus amyloliquefaciens NK-ΔLP strain can produce high-purity, low-molecular-weight levan, but production is relatively low. To enhance the production of levan, six extracellular protease genes (bpr, epr, mpr, vpr, nprE and aprE), together with the tasA gene (encoding the major biofilm matrix protein TasA) and the pgsBCA cluster (responsible for poly-γ-glutamic acid (γ-PGA) synthesis), were intentionally knocked out in the Bacillus amyloliquefaciens NK-1 strain. The highest levan production (31.1 g/L) was obtained from the NK-Q-7 strain (ΔtasA, Δbpr, Δepr, Δmpr, Δvpr, ΔnprE, ΔaprE and ΔpgsBCA), which was 103% higher than that of the NK-ΔLP strain (ΔpgsBCA) (15.3 g/L). Furthermore, the NK-Q-7 strain also showed a 94.1% increase in α-amylase production compared with NK-ΔLP strain, suggesting a positive effect of extracellular protease genes deficient on the production of endogenously secreted proteins. This is the first report of the improvement of levan production in microbes deficient in extracellular proteases and TasA, and the NK-Q-7 strain exhibits outstanding characteristics for extracellular protein production or extracellular protein related product synthesis.

Unifying bacteria from decaying wood with various ubiquitous Gibbsiella species as G. acetica sp. nov. based on nucleotide sequence similarities and their acetic acid secretion

Bacteria were isolated from necrotic apple and pear tree tissue and from dead wood in Germany and Austria as well as from pear tree exudate in China. They were selected for growth at 37°C, screened for levan production and then characterized as Gram-negative, facultatively anaerobic rods. Nucleotide sequences from 16S rRNA genes, the housekeeping genes dnaJ, gyrB, recA and rpoB alignments, BLAST searches and phenotypic data confirmed by MALDI-TOF analysis showed that these bacteria belong to the genus Gibbsiella and resembled strains isolated from diseased oaks in Britain and Spain. Gibbsiella-specific PCR primers were designed from the proline isomerase and the levansucrase genes. Acid secretion was investigated by screening for halo formation on calcium carbonate agar and the compound identified by NMR as acetic acid. Its production by Gibbsiella spp. strains was also determined in culture supernatants by GC/MS analysis after derivatization with pentafluorobenzyl bromide. Some strains were differentiated by the PFGE patterns of SpeI digests and by sequence analyses of the lsc and the ppiD genes, and the Chinese Gibbsiella strain was most divergent. The newly investigated bacteria as well as Gibbsiella querinecans, Gibbsiella dentisursi and Gibbsiella papilionis, isolated in Britain, Spain, Korea and Japan, are taxonomically related Enterobacteriaceae, tolerate and secrete acetic acid. We therefore propose to unify them in the species Gibbsiella acetica sp. nov.

Construction of a Bacillus amyloliquefaciens strain for high purity levan production

Bacillus amyloliquefaciens NK-1 has the potential to produce levan and poly-gamma-glutamic acid (γ-PGA) simultaneously. However, it is not possible to purify each single product from the same strain because the extraction process is identical. We deleted the pgs cluster (for γ-PGA synthesis) from the NK-1 strain and constructed a γ-PGA-deficient NK-ΔLP strain. Nuclear magnetic results showed that the NK-ΔLP strain could produce high purity levan product. However, its levan titer was only 1.96 g L(-1) in the basal medium. Single-factor experimental and response surface methodology was used to optimize the culture condition, leading to levan titer of 13.9 and 22.6 g L(-1) in flask culture and in a 5-L bioreactor, respectively. The levan purity can reach to 92.7% after 48 h cultivation. Furthermore, the relationship between levanase (LevB) and levan molecular weight was studied. The results showed that LevB resulted in the production of low molecular weight levan and its expression level determined the ratio of high and low molecular weight levan. We also deleted the sac cluster (for levan synthesis) from the NK-1 strain and constructed a levan-deficient NK-L strain. The NK-L strain exhibited increased purity of γ-PGA product from 79.5 to 91.2%.

Antioxidant and anti-inflammatory levan produced from Acetobacter xylinum NCIM2526 and its statistical optimization

Levan is a homopolymer of fructose naturally obtained from both the plants and microorganisms. Along with the general properties of a biopolymer like bio-compatibility, bio-degradability, renewability, flexibility, and eco-friendliness, levan also offers some important biomedical properties such as anti-oxidant, anti-inflammatory, anti-carcinogenic, anti-AIDS and hyperglycaemic inhibitor. In this study, we have demonstrated the microbial production of therapeutically potential levan by batch fermentation process in sucrose rich medium using Acetobacter xylinum NCIM 2526. The produced Levan was characterized using various physicochemical techniques such as FTIR, 1H NMR, 13C NMR spectroscopy, TGA and HPLC. The biomedical potential of the isolated A. xylinum levan for its anti-oxidant and anti-inflammatory activities was exploited in vitro. Further the present study also focused on the optimization of levan production using one factor at a time approach followed by a statistical method, central composite design (CCD) with selected variables. The yield of levan was increased significantly from 0.54 to 13.25g/L with the optimized variables.

High Levan Production by Bacillus licheniformis NS032 Using Ammonium Chloride as the Sole Nitrogen Source

In this study, levan production by Bacillus licheniformis NS032 isolated from a petroleum sludge sample was investigated. High levan yield was obtained in a wide range of sucrose concentrations (up to 400g/L) and, contrary to most levan-producing strains, using ammonium chloride as the sole N source. Interaction between sucrose, ammonium chloride, and initial pH of the medium in a low sucrose (60-200g/L) and a high sucrose (300-400g/L) system was analyzed by response surface methodology. According to the calculated model in the low sucrose system, maximum predicted levan yield was 47.8g/L (sucrose 196.8g/L, ammonium chloride 2.4g/L, pH 7.0), while in the high sucrose system, levan yield was 99.2g/L (sucrose 397.6g/L, ammonium chloride 4.6g/L, pH 7.4). In addition, protective effect of microbial levan against copper toxicity to Daphnia magna is observed for the first time. The acute toxicity (48h EC50) of copper decreased from 0.14 to 0.44mg/L by levan in concentration of 50ppm.

Isolation and Characterization of Levan from Moderate Halophilic Bacteria Bacillus licheniformis BK AG21

Levan is fructose polymer as the result of biosynthesis by levansucrase. This study is aimed to explore the potential of moderate halophilic bacteria Bacillus licheniformis BK AG21 isolated from Bledug Kuwu, Purwodadi Central Java, in producing levan. This bacteria displayed positive potential in producing levan based on the appearance of slime mucoid on a sucrose medium. The optimum production of levan was attained when the culture medium containing sucrose and peptone as respective carbon and nitrogen sources was shaking incubated with speed of 150rpm for 24hours at 37 oC. Thermal gravimetric analysis revealed that levan produced by B. licheniformis BK AG21 decomposed at 214 oC. The structure of the isolated levan was elucidated with FTIR, 1H and 13C NMR spectroscopies. Based on the obtained spectroscopic data, the isolated levan was composed of β-(2,6)-linkages of fructose residues.

Heterologous Expression and Characterization of Thermostable Levansucrase (BsSacB) from Bacillus subtilis BB03

Thermostable levansucrase encoding gene (sacB) with its promoter was isolated from Bacillus subtilis BB03. BsSacB is composed of 1419 bp, encoding 473 amino acid residues and with promoter regions TTGCAA (-35) and TAGAAT (-10). The amino acid sequence analysis of BsSacB showed conserved motifs of microbial fructosyltransferase belonging to Glycoside Hydrolase family 68 (GH68). Comparative analysis of the protein structure was carried out with 3D model of BsSacB constructed using PyMol software. BsSacB gene was expressed in Escherichia coli and the purified enzyme demonstrated thermo stability up to 50°C with a high hydrolytic activity at pH 6.0 and presence of Ca2+ promoted the activity by 15%. An attempt was made to further characterize the thermostable recombinant levansucrase from B.subtilis BB03 using crude sucrose rich substrates. Optimal levan production was seen with molasses (1.94±0.04 g/L) at 15 h as compared to cane juice (1.0±0.01 g/L) at 12 h. An increase in levan formation with gradual decrease in hydrolytic activity was distinctly evident in both the molasses and juice media.

Screening and Characterization of Levan Secreted by Halophilic Bacterium of Halomonas and Chromohalobacter Genuses Originated from Bledug Kuwu Mud Crater

Five bacterial isolates from the genus of Halomonas and Chromohalobacter isolated from Bledug Kuwu mud crater, located in mainland Purwodadi-Grobogan, Central Java, Indonesia, have been assayed for levan production. Initial screening was conducted on modified Belgith medium using sucrose as the major carbon source, in which the colonies of levan-producing bacteria will have a slimy mucoid appearance when grown on this medium. The screening results showed only one positive bacterial species, which was Chromohalobacter japonicus BK AB18 that identified as potential levan producer. Thermal stability of the isolated levan has been charachterized by TGA, which gave approximate decomposition temperature about 211 oC. The structure of the levan has been elucidated by FTIR and NMR spectroscopies. FTIR spectrum of the isolated levan displayed high similarity to that of levan isolated from Bacillus methylotrophicus. The chemical shifts of carbon and proton NMR spectra of the isolated levan also exhibited high similiarity to those of levan isolated from Pseudomonas fluorescens and Zymonas mobils.

Review on production, characterization and applications of microbial levan

Levan is a homopolymer of fructose naturally obtained from both plants and microorganisms. Microbial levans are more advantageous, economical and industrially feasible with numerous applications. Bacterial levans are much larger than those produced by plants with multiple branches and molecular weights ranging from 2 to 100 million Da. However levans from plants generally have molecular weights ranging from about 2000 to 33,000 Da. Microbial levans have wide range of applications in food, medicine, pharmaceutical, cosmetic and commercial industrial sectors. With excellent polymeric medicinal properties and ease of production, microbial levan appear as a valuable and versatile biopolymer of the future. The present article summarizes and discusses the most essential properties of bioactive microbial levan and recent developments in its production, characterization and the emerging applications in health and industry.

Alginate/PVA Beads for Levan Production by Zymomonas mobilis

AbstractThe levan is a biopolymer of great importance to the food industry since it is capable of defining and modifying the structure of one food, acting as stabilizer, thickener, gelling agent and being largely responsible for the texture of processed foods. The levan production by bacterial cell immobilization may potentialize the results of these studies, having advantages such as: high cell concentrations inside the reactor, increase the substrate absorption rate, improve the performance and reduce the risk of microbial contamination. Thus, this study aims to evaluate the levan production by immobilized Zymomonas mobilis in hybrid system of alginate/polyvinyl alcohol (PVA) when submitted to different sucrose concentrations (5, 10, 25 and 30%), pH (5.7 and 7.0) and incubation temperature of 30C for 12, 18 and 24 h. The results showed that the best levan production rate was 18.66 g/L at 30% sucrose concentration, with productivity 1.55 g/L/h at pH 7.0.Practical ApplicationsThe biopolymer called levan is a nontoxic, nonmutagenic, odorless, tasteless and soluble dietary fiber; it has physiological effects that depend on its size and linkage type, and its fermentability. It can be produced by bacterial fermentation like Zymomonas mobilis. The administration of levan in human diet (5.5–6 g, twice a day) for 2–3 months reduced body weight and body fat mass. The input of levan showed in some studies an increase of divalent cation (Zn, Mg and Fe) and calcium absorption in rats. In the food industry, levan improves the rheological properties and functional characteristics of dairy products, such as yogurt. Its application includes use as a stabilizer, emulsifier, surface agent, encapsulating agent and flavor and fragrances carrier.

Effective stimulating factors for microbial levan production by Halomonas smyrnensis AAD6T

Levan is a bioactive fructan polymer that is mainly associated with high-value applications where exceptionally high purity requirements call for well-defined cultivation conditions. In this study, microbial levan production by the halophilic extremophile Halomonas smyrnensis AAD6(T) was investigated systematically. For this, different feeding strategies in fed-batch cultures were employed and fermentation profiles of both shaking and bioreactor cultures were analyzed. Initial carbon and nitrogen source concentrations, production pH, NaCl and nitrogen pulses, nitrogen and phosphorous limitations, trace elements and thiamine contents of the basal production medium were found to affect the levan yields at different extends. Boric acid was found to be the most effective stimulator of levan production by increasing the sucrose utilization three-fold and levan production up to five-fold. This significant improvement implied the important role of quorum sensing phenomenon and its regulatory impact on levan production mechanism. Levan produced by bioreactor cultures under conditions optimized within this study was found to retain its chemical structure. Moreover, its biocompatibility was assessed for a broad concentration range. Hence H.smyrnensis AAD6(T) has been firmly established as an industrially important resource microorganism for high-quality levan production.

High production of plant type levan in sugar beet transformed with timothy (Phleum pratense) 6-SFT genes

Levan, a type of fructan, is an oligomer or polymer with mainly a β(2,6)-linked fructose chain attached to sucrose. We introduced two timothy genes, PpFT1 and PpFT2, coding for two homologous sucrose:fructan 6-fructosyltransferases into sugar beet. Sugar beet produces a high concentration of sucrose, a starting substrate in fructan synthesis, in the root. Among transgenic T1 lines, we obtained sugar beet transformants that accumulated large amounts of β(2,6)-linked levans (about 20 to 75mgg−1 FW) in the roots. The transformed sugar beet plants possessing PpFT1 or PpFT2 produced linear levans with different degrees of polymerization (DP). Namely, the PpFT1 transformants accumulated mainly high DP levans including those with DP>40, while the PpFT2 transformants accumulated levans with DP between 3 and 40. Chromatograms showed that PpFT2 produces pure β(2,6)-linked linear levans compared with fructans synthesized by PpFT1. These levans belong to the high DP class of plant fructans, but have much shorter DP than that of levans generally produced by microorganisms.

Green synthesis of silver and gold nanoparticles employing levan, a biopolymer from Acetobacter xylinum NCIM 2526, as a reducing agent and capping agent

With a vision of finding greener materials to synthesize nanoparticles, we report the production and isolation of levan, a polysaccharide with repeating units of fructose, from Acetobacter xylinum NCIM2526. The isolated levan were characterized using potassium ferricyanide reducing power assay, Fourier transform infra-red (FTIR) spectroscopy and 1H nuclear magnetic resonance spectroscopy (1H NMR). To exploit levan in nanotechnology, we present a simple and greener method to synthesize silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) using biopolymer, levan as both reducing and stabilizing agents. The morphology and stability of the AgNPs and AuNPs were examined by transmission electron microscopy (TEM) and UV–vis absorption (UV–vis) spectroscopy. The possible capping mechanism of the nanoparticles was postulated using FTIR studies. As synthesized biogenic nanoparticles showed excellent catalytic activity as evidenced from sodium borohydride mediated reduction of 4-nitro phenol and methylene blue.