Production of prebiotic gellan oligosaccharides based on the irradiation treatment and acid hydrolysis of gellan gum.

Biologically active gellan oligosaccharides (GOSs), newly found plant elicitors and biostimulants, are produced from the hydrolysis of gellan gum. Traditional hydrolysis with concentrated acid suffers from the problems of high pollution and low functional oligosaccharide yield because the process is difficult to control. Irradiation (60Co γ-ray) with a dosage ranging from 0 kGy to 175 kGy was used to degrade gellan gum efficiently and cleanly into low molecular weight (Mw) gellan with an average Mw ranging from 449,119 Da to 72,903 Da. The low Mw gellan irradiated at 70 kGy was further hydrolyzed with low concentration acid (0.5 mol/L HCl) to produce GOSs with DPs mainly 4 and 8, indicating that the Rha-ß-(1 â†’ 3)-Glc bonds in gellan gum were easily cut to produce residues with tetrasaccharide repeat subunits. Besides antioxidant activity, GOSs were also proved with prebiotic activity by in vitro fecal fermentation in a self-designed bionic intestinal reactor.

Physicochemical and biological characterization of a xanthan gum-polyvinylpyrrolidone hydrogel obtained by gamma irradiation.

Xanthan gum (XG) and polyvinylpyrrolidone (PVP) are two polymers with low toxicity, high biocompatibility, biodegradability, and high hydrophilicity, making them promising candidates for multiple medical aspects. The present work aimed to synthesize a hydrogel from a mixture of XG and PVP and crosslinked by gamma irradiation. We assessed the hydrogel through a series of physicochemical (FT-IR, TGA, SEM, and percentage of swelling) and biological (stability of the hydrogel in cell culture medium) methods that allowed to determine its applicability. The structural evaluation by infrared spectrum demonstrated that a crosslinked hydrogel was obtained from the combination of polymers. The calorimetric test and swelling percentage confirmed the formation of the bonds responsible for the crosslinked structure. The calorimetric test evidenced that the hydrogel was resistant to decomposition in contrast to non- irradiated material. The determination of the swelling degree showed constant behavior over time, indicating a structure resistant to hydrolysis. This phenomenon also occurred during the test of stability in a cell culture medium. Additionally, microscopic analysis of the sample revealed an amorphous matrix with the presence of porosity. Thus, the findings reveal the synthesis of a novel material that has desirable attributes for its potential application in pharmaceutical and biomedical areas.

A review of the enzymatic, physical, and chemical modification techniques of xanthan gum.

Xanthan gum (XG), a bacterial polysaccharide has numerous valuable characteristics in the food, biomedical, pharmaceuticals, and agriculture sector. However, XG has also its particular limitations such as its vulnerability to microbial contamination, inadequate mechanical and thermal stability, unusable viscosity, and poor water solubility. Therefore, XG's structure and conformation need to be modified enzymatically, chemically, or physically to improve its optimistic features and decrease the formation of crystals, increase antioxidant ability, and radical scavenging activity. We have found out different means to modify XG and elaborate the importance and significance of the modified structure of XG. In this review, different enzymes are reviewed for XG degradation, which modifies their structure from different points (main chain or side chain). This article also reviews various physical methods (ultrasound, shear, pressure, sonication, annealing, and heat treatments) based on prevailing publications to alter XG conformation and produce low molecular weight (LMW) and less viscous end-product. Moreover, some chemical means are also discussed that result in modified XG through crosslinking, grafting, acetylation, pyruvation, as well as by applying different chemical agents. Overall, the current progress on XG degradation is very auspicious to develop a new molecule with considerable uses, in various industries with future assessments.

Effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides produced by yoghurt starter cultures.

In this study, the effects of GSM 1800 band radiation on composition, structure and bioactivity of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus were determined. For this, GSM 1800 band radiation was applied to both cultures and characteristics of EPSs extracted from the control groups (K) and the radiation stressed groups (R) were determined. An alteration in the chemical composition of the EPSs was observed and EPS production levels and molecular weights of the EPSs increased following the GSM 1800 band radiation application. Alterations in the functional groups, thermal and morphological characteristics of EPSs following the GSM 1800 band radiation application were confirmed by FTIR, TGA and SEM analysis, respectively. Importantly no alterations in the antioxidant and antibacterial activity of the EPSs were observed following the radiation application. These results suggested the effects of the GSM radiation on final characteristics of EPSs from yogurt starter cultures.

Use of gamma irradiation technology for modification of bacterial cellulose nanocrystals/chitosan nanocomposite film.

The objective of this work was to investigate the influence of different gamma ray dosages (5, 10, and 10 kGy) on the structural, mechanical, surface and barrier properties of chitosan (Ch) based nanocomposite film. The results showed gamma irradiation caused an increase in the surface hydrophobicity, water vapor permeability and sensitivity of films to water and also, yellowness and opacity of films increased, simultaneously. By increasing the irradiation doses up to 10 kGy, the mechanical properties of Ch/BCNC film was significantly enhanced. As observed by FTIR spectra, no change occurred in the chemical functional groups of the films during irradiation. XRD studies confirmed that crystallinity of films was increased after irradiation. The nanocomposite film irradiated by 10 kGy had the highest thermal stability. In conclusion, gamma radiation can be considered as a safe method for sterilization of foods and modification of Ch/BCNC film properties.

Scytonemin Plays a Potential Role in Stabilizing the Exopolysaccharidic Matrix in Terrestrial Cyanobacteria.

Cyanobacteria are photosynthetic oxygen-evolving prokaryotes that are distributed in diverse habitats. They synthesize the ultraviolet (UV)-screening pigments, scytonemin (SCY) and mycosporine-like amino acids (MAAs), located in the exopolysaccharide (EPS) matrix. Multiple roles for both pigments have gradually been recognized, such as sunscreen ability, antioxidant activity, and heat dissipation from absorbed UV radiation. In this study, a filamentous terrestrial cyanobacterium Nostoc flagelliforme was used to evaluate the potential stabilizing role of SCY on the EPS matrix. SCY (∼3.7 %) was partially removed from N. flagelliforme filaments by rinsing with 100 % acetone for 5 s. The physiological damage to cells resulting from this treatment, in terms of photosystem II activity parameter Fv/Fm, was repaired after culturing the sample for 40 h. The physiologically recovered sample was further desiccated by natural or rapid drying and then allowed to recovery for 24 h. Compared with the normal sample, a relatively slower Fv/Fm recovery was observed in the SCY-partially removed sample, suggesting that the decreased SCY concentration in the EPS matrix caused cells to suffer further damage upon desiccation. In addition, the SCY-partially removed sample could allow the release of MAAs (∼25 %) from the EPS matrix, while the normal sample did not. Therefore, damage caused by drying of the former resulted from at least the reduction of structural stability of the EPS matrix as well as the loss of partial antioxidant compounds. Considering that an approximately 4 % loss of SCY led to this significant effect, the structurally stabilizing potential of SCY on the EPS matrix is crucial for terrestrial cyanobacteria survival in complex environments.

Protective effects of reactive functional groups on chondrocytes in photocrosslinkable hydrogel systems.

Photocrosslinkable hydrogels are frequently used in cartilage tissue engineering, with crosslinking systems relying on cytotoxic photoinitiators and ultraviolet (UV) light to form permanent hydrogels. These systems are rarely assessed in terms of optimization of photoinitiator or UV dosage, with non-cytotoxic concentrations from literature deemed sufficient. We hypothesized that the number of reactive functional groups present within a hydrogel polymer is highly relevant when crosslinking, affording cytoprotection to chondrocytes by preferentially interacting with the highly reactive radicals that are formed during UV-mediated activation of a photoinitiator. This was tested using two photocrosslinkable hydrogel systems: gelatin methacrylamide (GelMA) and gellan gum methacrylate (GGMA). We further assessed the effects of two different UV dosages on chondrocyte differentiation while subject to a single photoinitiator dosage in the GGMA system. Most notably, we found that a higher ratio of reactive groups to photoinitiator molecules offers cytoprotective effects, and future developments in photocrosslinkable hydrogel technology may involve assessment of such ratios. In contrast, we found there to be no effect of UV on chondrocyte differentiation at the two chosen dosages. Overall the optimization of photocrosslinkable systems is of great value in cartilage tissue engineering and these data provide a groundwork for such concepts to be developed further. STATEMENT OF SIGNIFICANCE: Photocrosslinkable hydrogels, which use photoinitiators and predominantly ultraviolet light to form stable matrices for cell encapsulation and tissue development, are promising for cartilage tissue engineering. While both photoinitiators and ultraviolet light can damage cells, these systems have generally not been optimized. We propose that the ratio of reactive functional groups within a polymer to photoinitiator molecules is a critical parameter for optimization of photocrosslinkable hydrogels. Using photocrosslinkable gelatin and gellan gum, we found that a higher ratio of reactive groups to photoinitiator molecules protected chondrocytes, but did not affect chondrocyte differentiation. The principle of cytoprotection by functional groups developed in this work will be of great value in optimizing photocrosslinkable hydrogel systems for cartilage and other tissue engineering applications.

Effects of light wavelengths on extracellular and capsular polysaccharide production by Nostoc flagelliforme.

The influences of different wavelengths of light (red 660nm, yellow 590nm, green 520nm, blue 460nm, purple 400nm) and white light on extracellular polysaccharide (EPS) and capsular polysaccharide (CPS) production by Nostoc flagelliforme in liquid culture were demonstrated in this study. The results showed that, compared with white light, red and blue lights significantly increased both EPS and CPS production while yellow light reduced their production; purple and green lights stimulated EPS production but inhibited CPS formation. Nine constituent monosaccharides and one uronic acid were detected in both EPS and CPS, and their ratios showed significant differences among treatment with different light wavelengths. However, the advanced structure of EPS and CPS from various light conditions did not present obvious difference through Fourier transform infrared spectroscopy and X-ray diffraction characterization. These findings establish a basis for development of high-yielding polysaccharide production process and understanding their regulation.

Effects of light intensity on components and topographical structures of extracellular polysaccharides from the cyanobacteria Nostoc sp.

A study on the effects of light intensity (40 and 80 µE/m(2)/sec) on the components and topographical structures of extracellular polysaccharides (EPS) was carried out in cyanobacteria Nostoc sp.. EPS yield increased with light intensity. However, light intensity did not significantly affect the EPS fractions and monosaccharide composition. Higher light intensity generally resulted in higher protein content of EPS in similar fractions. The topographical structure of EPS, investigated by atomic force microscopy, appeared as spherical lumps, chains and networks. The long chains were observed at higher light intensity. Thus, light intensity affected the yield and nature of EPS.

The mechanical properties and cytotoxicity of cell-laden double-network hydrogels based on photocrosslinkable gelatin and gellan gum biomacromolecules.

A major goal in the application of hydrogels for tissue engineering scaffolds, especially for load-bearing tissues such as cartilage, is to develop hydrogels with high mechanical strength. In this study, a double-network (DN) strategy was used to engineer strong hydrogels that can encapsulate cells. We improved upon previously studied double-network (DN) hydrogels by using a processing condition compatible with cell survival. The DN hydrogels were created by a two-step photocrosslinking using gellan gum methacrylate (GGMA) for the rigid and brittle first network, and gelatin methacrylamide (GelMA) for the soft and ductile second network. We controlled the degree of methacrylation of each polymer so that they obtain relevant mechanical properties as each network. The DN was formed by photocrosslinking the GGMA, diffusing GelMA into the first network, and photocrosslinking the GelMA to form the second network. The formation of the DN was examined by diffusion tests of the large GelMA molecules into the GGMA network, the resulting enhancement in the mechanical properties, and the difference in mechanical properties between GGMA/GelMA single networks (SN) and DNs. The resulting DN hydrogels exhibited the compressive failure stress of up to 6.9 MPa, which approaches the strength of cartilage. It was found that there is an optimal range of the crosslink density of the second network for high strength of DN hydrogels. DN hydrogels with a higher mass ratio of GelMA to GGMA exhibited higher strength, which shows promise in developing even stronger DN hydrogels in the future. Three dimensional (3D) encapsulation of NIH-3T3 fibroblasts and the following viability test showed the cell-compatibility of the DN formation process. Given the high strength and the ability to encapsulate cells, the DN hydrogels made from photocrosslinkable macromolecules could be useful for the regeneration of load-bearing tissues.

Sterilization of exopolysaccharides produced by deep-sea bacteria: impact on their stability and degradation.

Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry.

Ultrasonic depolymerization of an exopolysaccharide produced by a bacterium isolated from a deep-sea hydrothermal vent polychaete annelid.

Low frequency ultrasound was used to depolymerize a high-molecular-weight exopolysaccharide (EPS) produced by a deep-sea hydrothermal bacterium Alteromonas macleodii subsp. fijiensis biovar deepsane. The influence of several parameters was examined including the duration of ultrasonic irradiation, EPS concentration, reaction temperature and volume of the sonicated solution. With the aim of optimizing the depolymerization, the native EPS was simultaneously treated with hydrogen peroxide and ultrasound. This study identified the sonication conditions that produce low-molecular-weight derivatives from the native EPS (>10(6)Da) with good reproducibility.

Gamma-irradiation of lyophilised wound healing wafers.

Lyophilised wafers are being developed as drug delivery systems that can be applied directly to the surface of suppurating wounds. They are produced by the freeze-drying of polymer solutions and gels. This study investigates the possibility of sterilising these glassy, solid dosage forms with gamma-irradiation and determining the rheological properties of rehydrated wafers post-irradiation. One series of wafers was formulated using sodium alginate (SA) modified with increasing amounts of methylcellulose (MC), the other being composed of xanthan gum (XG) and MC. Batches were divided into three lots, two of which were exposed to 25 and 40 kGrays (kGy) of Cobalt-60 gamma-irradiation, respectively, the third being retained as a non-irradiated control. Apparent viscosities of solutions/gels resulting from the volumetric addition of distilled water to individual wafers were determined using continuous shear, flow-rheometry. Flow behaviour on proprietary suppurating surfaces was also determined. Large reductions in viscosity were apparent for irradiated SA samples while those of XG appeared to be largely unaffected. In addition, an increase in the yield stress of xanthan formulations was observed. Xanthan wafers appeared to withstand large doses of irradiation with no detrimental effect on the rheology of reconstituted gels. This offers the possibility of manufacturing sterilisable delivery systems for wounds.

Dynamic light scattering investigation of sodium caseinate and xanthan mixtures.

Aqueous solutions of sodium caseinate and xanthan at pH 7 and containing 0.1 M NaCl, and their mixtures were investigated using dynamic light scattering. Sodium caseinate solutions showed a bimodal distribution of relaxation rates; with the aggregate peak distribution predominating. Xanthan solutions showed a single distribution at low concentrations (< or =0.06 wt.%) and a bimodal distribution at higher concentrations. The sodium caseinate-xanthan mixture modes were independent of the total biopolymer concentration, and behaved as a superposition of sodium caseinate solution alone and xanthan solution alone. This indicates that there is no interaction between xanthan and sodium caseinate in the range of concentrations considered in this study.

Electron beam fragmentation of bacterial polysaccharides as a method of producing oligosaccharides for the preparation of conjugate vaccines.

End-group mediated conjugation of bacterial polysaccharides (PSs) to carrier proteins containing T-helper cell epitopes renders such polysaccharides immunogenic also in young infants. Optimal construction of such conjugate vaccines requires fragmentation of the PS prior to the coupling reaction. In the present study a general simple and inexpensive method for the fragmentation of PSs is presented. It is based on the irradiation of isolated PSs in an electron beam accelerator. Exposure of isolated pneumococcal capsular polysaccharides (PnPSs) to ionizing radiation resulted in their partial depolymerization in a radiation dose-dependent manner. Radiation, unlike sonication, generated PnPS fragments of molecular size lower than 50 kDa and as small as 1.5 kDa when high radiation doses were used. These PnPS fragments have terminal reducing groups that can be easily used for chemical activation and subsequent coupling to any chosen carrier protein. The radiation-produced PnPS fragments retained their antigenic epitopes, when compared to native, full-size PnPSs as determined by enzyme-linked immunoassay.

Response of humans to gamma-irradiated reference Escherichia coli endotoxin.

This study assesses the effect of gamma-irradiation of endotoxin given intravenously to healthy human volunteers. The national reference standard endotoxin derived from E. coli was placed in aqueous medium in sterile-sealed ampoules and divided into four groups. One group received endotoxin with no radiation while the other three received endotoxin with gamma-irradiation at doses of 0.18, 0.36, or 1.08 Mrad. These doses of radiation cause characteristic alterations to the endotoxin molecule, primarily to the O-polysaccharide moiety. Each of the four different preparations of endotoxin was given intravenously to four volunteers at a concentration of 4 ng/kg. The responses for clinical symptoms, cortisol, and growth hormone were significantly and progressively reduced by increasing the irradiation to the endotoxin. Most strikingly, no clinical symptoms were noted with the endotoxin exposed to the highest dose of radiation (1.08 Mrad). Fever, vital signs, white blood cell count, and differential exhibited no statistically significant differences among the groups, but the kinetics of change were altered by increasing doses of gamma-irradiation. Irradiated endotoxin was significantly more effective in decreasing the platelet count than untreated endotoxin. The fever index correlated significantly with maximum temperature, change in temperature, white blood cell count index, mature neutrophil count index, and the cortisol index. Thus, there is dissociation of biological activities for endotoxin in humans due to molecular changes primarily in the O-polysaccharide moiety from exposure to gamma-irradiation.

Ultrasonic irradiation of bacterial polysaccharides. Characterization of the depolymerized products and some applications of the process.

Ultrasonic irradiation (u.i.) has been used to depolymerize biopolymers including DNA, dextran, and the Vi capsular polysaccharide from Citrobacter freundii. Representative bacterial polysaccharides were subjected to u.i. and the effect of this energy upon their molecular weight and chemical structure was characterized. U.i. depolymerized a neutral polysaccharide (dextran) and acidic polysaccharides containing either a phosphoric diester linkage [Haemophilus influenzae type b (Hib) and pneumococcus types 6A and 6B] or a uronic acid moiety (pneumococcus type 9N). Prolonged u.i. depolymerized all the polysaccharides to a finite and similar molecular mass (approximately 50 000 daltons). The rate of depolymerization induced by u.i. depended on the viscosity of the solvent and the concentration of the polysaccharide. 13C-N.m.r. data of the native Hib polysaccharide and its depolymerized products indicated that u.i. did not alter the chemical structure of the repeating units. Determination of the monophosphate terminal residues by 31P-n.m.r. spectroscopy and of the reducing end groups by the Park-Johnson reaction indicated that both the phosphoric diester and the glycosidic linkages were cleaved. The Vi polysaccharide, prepared as an investigational vaccine, could not be analyzed for its chemical structure by 13C-n.m.r. spectroscopy owing to its high viscosity but depolymerization by u.i. permitted this analysis. The finite molecular weight of the products observed after prolonged u.i. is best explained by the postulation that the mechanical torque necessary to rupture the linkages is dependent upon the length of the polysaccharide. The method of u.i. for depolymerization is useful for the preparation of homogeneous, low-molecular-weight polysaccharides without alteration of the chemical structure of the repeating units.

Effect of ionizing radiation on chemical and biological properties of Salmonella minnesota R595 lipopolysaccharide.

Lipopolysaccharide of the Salmonella minnesota Re mutant R595 was irradiated with 60Co gamma doses of 50, 100, 150 and 200 kGy. The irradiated preparations were less toxic, less active in the Shwartzman reaction and as activators of the complement system, but they had retained the protection activity against the lethal action of endotoxin. The irradiation resulted in a dose-dependent decrease in the amounts of constituents (glucosamine, KDO, fatty acids) of the original lipopolysaccharide. With increasing irradiation doses increasing amounts of the irradiated material became dialysable (up to 21% in the 200 kGy sample). Only 50% of total fatty acids were present in the 200 kGy preparation compared to the parent lipopolysaccharide. The degradation products formed during irradiation have not been identified.

Radiation induced alterations in the endotoxin of S. typhimurium.

The lipopolysaccharide (LPS) of S. typhimurium has been shown to be significantly detoxified after in vivo irradiation at 500 krad. Radiation is thus a useful method for converting endotoxin into toxoid. The structural alterations in the detoxified LPS are shown to be mainly in the lipid A molecule, resulting in the loss of beta-hydroxymyristic acid.