Optimization and production of curdlan gum using Bacillus cereus PR3 isolated from rhizosphere of leguminous plant

Curdlan gum is a neutral water-insoluble bacterial exopolysaccharide composed primarily of linear β-(1,3) glycosidic linkages. Recently, there has been increasing interest in the applications of curdlan and its derivatives. Curdlan is found to inhibit tumors and its sulfated derivative possess anti-HIV activity. Curdlan is biodegradable, non-toxic towards human, environment and edible which makes it suitable as drug-delivery vehicles for sustained drug release. The increasing demand for the growing applications of curdlan requires an efficient high yield fermentation production process so as to satisfy the industrial needs. In this perspective, the present work is aimed to screen and isolate an efficient curdlan gum producing bacteria from rhizosphere of ground nut plant using aniline-blue agar. High yielding isolate was selected based on curdlan yield and identified as Bacillus cereus using gas-chromatography fatty acid methyl ester analysis. B. cereus PR3 curdlan gum was characterized using FT-IR spectroscopy, SEM, XRD and TGA. Fermentation time for curdlan production using B. cereus PR3 was optimized. Media constituents like carbon, nitrogen and mineral sources were screened using Plackett–Burman design. Subsequent statistical analysis revealed that Starch, NH₄NO₃, K₂HPO₄, Na₂SO₄, KH₂SO₄ and CaCl₂ were significant media constituents and these concentrations were optimized for enhancement of curdlan production up to 20.88?g/l.     

Optimization of uracil addition for curdlan (β-1→3-glucan) production by Agrobacterium sp.

Uracil, acting as a precursor of UDP-glucose, served as an activator on the production of curdlan with Agrobacterium sp. (ATCC 31750). The time of adding uracil was important to improve curdlan production. When uracil was added after ammonium depletion (at 26 h), it was used as a nitrogen source for cell growth. Although the cell concentration increased, the curdlan production was decreased. If uracil was added at 46 h, then uracil was degraded slowly but still activated curdlan production. With the addition of both sucrose (200 g) and uracil (1.5 g), the curdlan production was increased up to 93 g l^–1 after 160 h fermentation.     

Levan and levansucrases: Polymer,enzyme, micro-organisms and biomedical applications

Abstract

Nowadays, levan synthesis and its biocatalysis is a step claiming attention due to its promising uses in biomedical field. Levansucrase, as the enzyme responsible of levan biocatalysis is studied in this work concerning sequence homology, signal peptide, metal binding, etc. More than 650 different levansucrase sequences were studied and many interesting results were obtained such as phylogenetic trees and conserved residues that could be essential for biocatalysis machinery. Moreover, a review of levan applications in biomedicine and health field was made. It is showed the advances in applications such as a film agent, a carrier for drug delivery systems, an anti-inflammatory compound or its potential use for functional food as prebiotic.     

A new effective process for production of curdlan oligosaccharides based on alkali-neutralization treatment and acid hydrolysis of curdlan particles in water suspension

Biologically active β-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual β-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes.     

Production of extracellular water insoluble β-1,3-glucan (curdlan) fromBacillus sp. SNC07

β-1,3-Glucan (curdlan) is a water-insoluble polysaccharide composed exclusively of β-1,3 linked glucose residues. Extracellular curdlan was mostly synthesized byAgrobacterium species andAlcaligenes faecalis under nitrogen-limiting conditions. In this study, we screened the microorganisms capable of producing extracellular curdlan from soil samples. For the first time, we reported Gram-positive bacteriumBacillus sp. SNC 107 capable of producing extracellular curdlan in appreciable amounts. The effect of different carbon sources on curdlan production was studied and found that the yield of curdlan was more when glucose was used as carbon source. It was also found that maximum production was achieved when the initial concentration of ammonium and phosphate in the medium was 0.5 and 1.9 g/L respectively. In this study the curdlan production was increased from 3 to 7 g/L in shake flask cultures.     

Curdlan microspheres. Synthesis,characterization and interaction with proteins (enzymes,vaccines)

Microparticles of curdlan, synthesized through crosslinking with epichlorohydrin in organic suspension media, were chemically modified with the aim of introducing strongly and/or weakly acidic anionic and palmitoyl hydrophobic groups. Microparticles of both curdlan and curdlan derivatives were physico-chemically characterized. Study of the interaction with enzymes, such as lysozyme, and vaccines, such as tetanus anatoxin, showed a co-operative protein retention effect, induced by electrostatic and hydrophobic forces. The results of the in vitro release studies on support–protein complexes recommend them as potential controlled release systems.     

Doctors that Harm,Doctors that Heal: Reimagining Medicine in Post-Conflict Aceh,Indonesia

ABSTRACT

This article explores the fears and desires that conflict survivors invest in biomedicine in post-conflict Aceh, Indonesia. After examining common criticisms of Acehnese doctors, I go on to discuss the practise of Acehnese medical travel to Malaysia and the desirable depictions of biomedicine that are generated through cross-border clinical encounters. Building on Pinto [2004. Development Without Institutions: Ersatz Medicine and the Politics of Everyday Life in Rural North India. Cultural Anthropology, 19(3):337–364], I argue that in the post-conflict period, biomedicine acts as both a site of terror and ‘point of imagination and longing’ that guides my interlocutors' understandings of the changing world around them. The article brings debates about medicine and violence into dialogue with a body of post-conflict ethnography, showing how popular imaginaries of biomedicine can shape the process of post-conflict world building.     

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l⁻¹. As the cell concentration increased from 0.42 to 1.68 g l⁻¹ in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l⁻¹. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell⁻¹ h⁻¹ irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l⁻¹ under nitrogen-limiting conditions, as high as 65 g l⁻¹ of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183. Received 25 October 1999/ Accepted in revised form 21 July 2000     

Improved curdlan fermentation process based on optimization of dissolved oxygen combined with pH control and metabolic characterization of <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45–60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD⁺, NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20–50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.     

Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).     

Optimal production of curdlan by Agrobacterium sp. with feedback inferential control of optimal pH profile

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l^–1 in 120 h) was achieved with feedback optimal control.     

Pullulan: biosynthesis,production, and applications

Pullulan is a linear glucosic polysaccharide produced by the polymorphic fungus Aureobasidium pullulans, which has long been applied for various applications from food additives to environmental remediation agents. This review article presents an overview of pullulan’s chemistry, biosynthesis, applications, state-of-the-art advances in the enhancement of pullulan production through the investigations of enzyme regulations, molecular properties, cultivation parameters, and bioreactor design. The enzyme regulations are intended to illustrate the influences of metabolic pathway on pullulan production and its structural composition. Molecular properties, such as molecular weight distribution and pure pullulan content, of pullulan are crucial for pullulan applications and vary with different fermentation parameters. Studies on the effects of environmental parameters and new bioreactor design for enhancing pullulan production are getting attention. Finally, the potential applications of pullulan through chemical modification as a novel biologically active derivative are also discussed.     

Purified curdlan and its hydroxyalkyl derivatives: preparation, properties and applications

Curdlan, a β-1,3-glucan produced by fermentation of Alkaligenes faecalis, is a non-ionic gel-forming polysaccharide that is, along with its hydroxalkyl derivatives, a potentially important matrix for life science applications. The commercially available material contains residual nucleic acids, cellular debris and other contaminants that can interfere with electrophoretic separations and visualization procedures. Simple procedures have been developed for purification of the curdlan and coherent gel formation. Curdlan gels can be formed in a variety of chaotrope-containing, or chaotropic, solvents, including 40% formamide, 7 M urea. The chaotropes can be retained or subsequently removed by leaching. Heat treatment before or after leaching enables thermostable gel formation. Hydroxyethyl and glyceryl derivatives of curdlan have been prepared. Depending on the degree of substitution (DS), a whole spectrum of derivatives with a range of unique properties can be obtained. Lower DS hydroxyethyl derivatives form clear, elastic gels in 6 M urea, while the higher DS hydroxyethyl derivatives are soluble in hot water and gel on cooling to form clear, elastic, thermoreversible gels. Partial depolymerization of curdlan by γ-irradiation reduces the viscosity of subsequent preparations, enabling the preparation of higher concentration, more sieving gels. Use of selected preparations to form unique matrices for electrophoretic separations has been demonstrated.     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

Biotechnological production and applications of Cordyceps militaris,a valued traditional Chinese medicine

Cordyceps militaris is a potential harborer of biometabolites for herbal drugs. For a long time, C. militaris has gained considerable significance in several clinical and biotechnological applications. Much knowledge has been gathered with regard to the C. militaris's importance in the genetic resources, nutritional and environmental requirements, mating behavior and biochemical pharmacological properties. The complete genome of C. militaris has recently been sequenced. This fungus has been the subject of many reviews, but few have focused on its biotechnological production of bioactive constituents. This mini-review focuses on the recent advances in the biotechnological production of bioactive compositions of C. militaris and the latest advances on novel applications from this laboratory and many others.     

金纳米粒在药物传递系统中的应用

金纳米粒是一种新型纳米载体,具有独特的理化、光学和生物学性质,且具有低毒性、低免疫原性、生物相容性好、体表面积大、易制备、粒径和形态可控、表面易修饰等优点,在生物医学领域和药物传递系统中具有广阔的应用前景。综述金纳米粒在小分子药物和基因药物传递系统中的应用研究新进展。     

Effect of metabolic structures and energy requirements on curdlan production by<Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

A comprehensive metabolic network was proposed forAlcaligenes faecalis and employed in a stoichiometrically based flux balance model for curdlan production optimization. The maximal yield of curdlan was evaluated for curdlan batch production. Various metabolic structures and metabolic pathway distributions related with the curdlan maximal yield was evaluated. The results showed that the energy efficiency rather than the substrate supply was the major constraint for the enhancement of curdlan production. The increase in specific rate of glucose uptake could enhance curdlan production yield due to the decrease of the ratio of metabolic maintenance to substrate consumption. However, some of the energy loss and nutrient limitation associated with the increase of metabolic maintenance would adversely affect the conversion efficiency of the substrate.     

Comparison of curdlan and its carboxymethylated derivative by means of Rheology, DSC, and AFM

Curdlan was carboxymethylated in an aqueous alkaline medium using monochloroacetic acid as the etherifying agent. The structure of carboxymethylated curdlan (CMc) was analyzed by FT-IR and NMR spectroscopy, which revealed that the carboxymethyl group was introduced mainly at the C-6 position as well as at the C-2 and C-4 positions. Furthermore, CMc was compared with the native curdlan by using rheology and DSC methods. It was found that in water, both polysaccharides behaved as pseudoplastic fluids and fit the power law and Herschel-Bulkley rheological models well. Both the storage shear modulus G' and the loss shear modulus G' of CMc aqueous solutions decreased and became more frequency dependent with decreasing concentration in comparison with the curdlan aqueous suspensions. The modulus-temperature curve also suggested that the gel characteristic of curdlan has been lost after chemical modification, which is consistent with the DSC results. AFM images revealed differences in the conformation of native and carboxymethylated curdlan, which changed from the aggregation of macromolecules to triple helices. All the experimental results suggest that the hydrogen bonds that bind curdlan with interstitial water to form the micelles have been destroyed completely and that the hydrophobic interactions related to the methylene groups at C-6 formed above 55 degrees C disappeared due to the introduction of the hydrophilic carboxymethyl group.     

Curdlan intake changes gut microbial composition,short-chain fatty acid production,and bile acid transformation in mice

Indigestible polysaccharides, such as dietary fibers, benefit the host by improving the intestinal environment. Short-chain fatty acids (SCFAs) produced by gut microbial fermentation from dietary fibers exert various physiological effects. The bacterial polysaccharide curdlan benefits the host intestinal environment, although its effect on energy metabolism and SCFA production remains unclear. Hence, this study aimed to elucidate the effect of curdlan intake on gut microbial profiles, SCFA production, and energy metabolism in a high-fat diet (HFD)-induced obese mouse model. Gut microbial composition of fecal samples from curdlan-supplemented HFD-fed mice indicated an elevated abundance of Bacteroidetes, whereas a reduced abundance of Firmicutes was noted at the phylum level compared with that in cellulose-supplemented HFD-fed mice. Moreover, curdlan supplementation resulted in an abundance of the family Bacteroidales S24-7 and Erysipelotrichaceae, and a reduction in Deferribacteres in the feces. Furthermore, curdlan supplementation elevated fecal SCFA levels, particularly butyrate. Although body weight and fat mass were not affected by curdlan supplementation in HFD-induced obese mice, HFD-induced hyperglycemia was significantly suppressed with an increase in plasma insulin and incretin GLP-1 levels. Curdlan supplementation elevated fecal bile acid and SCFA production, improved host metabolic functions by altering the gut microbial composition in mice.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.