The effect of degradation on κ-carrageenan/locust bean gum/konjac glucomannan gels at acidic pH

The feasibility of textural and rheological modification of gels containing κ-carrageenan (KC) and locust bean gum (LBG) by addition of konjac glucomannan (KGM) was investigated. Special attention was paid to the effect of polysaccharide degradation during heating at acidic pH. The general effect of polysaccharide degradation was to decrease the Young's modulus, while the fracture strain in extension was scarcely affected unless the degradation was very severe.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

Summary Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in -carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p _a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.     

Characterization and optimization of a new immobilized system of κ-carrageenan through interaction with carob bean gum and polyols

The optimum relationships of κ-carrageenan and carob bean gum were investigated in order to obtain an immobilization system with better compression resistance, trapping capacity, and storage stability, and less syneresis phenomenon, when compared to κ-carrageenan itself. With that objective, different concentrations of polyols (glycerol and propylene glycol) were added, because of their water-retention characterization in the containing system. In this way, an improved system with good compression resistance was obtained: 15 × 10⁻⁴ N/m² in modified κ-carrageenan gel without E. coli cells and 11 × 10⁻⁴ N/m² with Escherichia coli cells. In the modified κ-carrageenan gel, the syneresis phenomenon decreased. The enzymatic activity in the system was 18 U/g, which did not present a change over a storage period of six months.     

Diffusion coefficients of carbohydrates in modified κ-carrageenan gels with and without Escherichia coli immobilized

Effective diffusion coefficients (Dag) of carbohydrates in modified κ-carrageenan gels, with and without Escherichia coli immobilized cells were determined. A diffusion cell based on the phenomena of sorption from a “well-stirred fluid” was used. Dag values were smaller in the gel with cells than without them. The effect of temperature on Dag was also studied.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.     

Facile synthesis of fluorescent polysaccharides: Cytosine grafted agarose and κ-carrageenan

New fluorescent polymeric materials were synthesized by grafting the nucleobase cytosine on to the backbone of agarose and κ-carrageenan, employing a rapid water based method under microwave irradiation using potassium persulphate (KPS) as an initiator. The emission spectrum of the modified agarose and κ-carrageenan recorded in aqueous solution (5 × 10⁻⁵ M) exhibited emission maxima (λ_em,max) at 348 nm by excitation at 266 nm. The emission intensity was enhanced by ca. 104% and 60% compared to that of pure cytosine solution of the same concentration. When the concentration of the pure cytosine solution is made equivalent to the concentration of the cytosine molar component (3.09 × 10⁻⁵) and (3.5 × 10⁻⁵) present in 5 × 10⁻⁵ M solution of modified agarose and κ-carrageenan, respectively, then ca. 143% and 81% enhancement in emission intensity was observed. The remarkable fluorescent activity of the agarose-cytosine derivative may have potential uses as sensor in various applications.     

Preparation and characterization of konjac glucomannan–poly(acrylic acid) IPN hydrogels for controlled release

In this paper, we reported the synthesis and properties of interpenetrating polymer network (IPN) hydrogel systems designed for colon targeted drug delivery. The gels were composed of konjac glucomannan (KGM) and cross-linked poly(acrylic acid) (PAA) by N,N-methylene-bis-(acrylamide) (MBAAm). It was possible to modulate the swelling degree of the gels. And the swelling ratio has sensitive respondence to the environmental pH value variation. The degradation tests show that the hydrogels retain the enzymatic degradation character of KGM. In vitro release of model drug VB₁₂ was studied in the presence of Cellulase E0240 in pH 7.4 phosphate buffer at 37 °C. The accumulative release percent of the model drug reached 85.6% after 48 h and the drug release was controlled by the swelling and the degradation of the hydrogels. The results indicated that the IPN hydrogels can be exploited as potential carriers for colon-specific drug delivery.     

Characterization of a denitrifying bacterium immobilized in κ-carrageenan

Summary Cells of a pure strain of a denitrifying bacterium have been immobilized in -carrageenan. The influence of pH and temperature on the immobilized cells was determined, as well as the operational stability in a continuous gas-lift loop reactor. Several steady states at different nitrate loadings were reached with respect to activity and dry cell weight. The results show that the immobilized cells form a stable system which rapidly reacts to changes in substrate (nitrate) supply. The maximum conversion rate of the immobilized cells is higher than the rate usually observed with immobilized denitrifying cells but lower than in fluidized-bed systems with attached biomass. It is shown that with increasing immobilized concentration the K _m value apparently increases to such an extent that for growing cells no zero-order kinetics may be assumed. Offprint requests to: R. H. Wijffels     

Production of a high activity of an extremely thermostable β-mannanase by the thermophilic eubacterium Rhodothermus marinus, grown on locust bean gum

The production of a highly thermostable mannanase by Rhodothermus marinus was increased 16.5-fold by optimising the concentrations of locust bean gum and yeast extract using central composite designs. The optimised medium and culture conditions yielded mannanase activity at 495 nkat ml–1 (248 nkat mg–1 protein). In addition, -L-arabinofuranosidase, -xylanase, -xylosidase, -glucosidase, -mannosidase, -galactosidase, -galactosidase and endoglucanase activities were detected at 32 nkat ml–1, 30 nkat ml–1, 16 nkat ml–1, 15 nkat ml–1, 0.1 nkat ml–1, 1 nkat ml–1, 0.5 nkat ml–1 and 8 nkat ml–1, respectively. No filter paper cellulase activity could be detected. The optimum pH of the mannanase was 5.0–6.5 and it showed high stability from pH 5 to 10 after 16 h incubation at 50 °C. The enzyme activity was maximum at 85 °C, with half lives of 45.3 h at 85 °C and 4.2 h at 90 °C. This is the first report on the production of such a high activity of extremely thermostable mannanase by an extreme thermophilic bacterium. © Rapid Science Ltd. 1998     

Impact of magnetic nanofillers in the swelling and release properties of κ-carrageenan hydrogel nanocomposites

Natural polysaccharides such as κ-carrageenan are an important class of biomaterials for drug delivery. The incorporation of magnetic nanoparticles in polysaccharide hydrogels is currently being explored as a strategy to confer to the hydrogels novel functionalities valuable for specific bio-applications. Within this context, κ-carrageenan magnetic hydrogel nanocomposites have been prepared and the effect of magnetic (Fe₃O₄) nanofillers in the swelling of the hydrogels and in the release kinetics and mechanism of a model drug (methylene blue) has been investigated. In vitro release studies demonstrated the applicability of the composites in sustained drug release. The mechanism controlling the release seems to be determined by the strength of the gel network and the extent of gel swelling, both being affected by the incorporation of nanofillers. Furthermore, it was demonstrated that the release rate and profile could be tailored using variable Fe₃O₄ nanoparticles load. Thus, this seems to be a promising strategy for the development of drug delivery systems with tailored released behavior.     

Production of 3,6-anhydro-D-galactose from κ-carrageenan using acid catalysts

This study describes acid-catalyzed production of 3,6-anhydro-D-galactose (D-AnG) from κ-carrageenan, a sulfated polysaccharide with an alternating backbone consisting of D-AnG and D-galactose (D-Gal). We analyzed four hydrolysis products (D-AnG, 5-hydroxymethylfurfural (HMF), levulinic acid (LA), and D-Gal) and reducing sugar contents during acid hydrolysis. Acid screening was carried out using seven acid catalysts which have different acidity. The catalysts showing high D-AnG production and high selectivity were chosen for subsequent experiments. We selected four acid catalysts (HCOOH, CH₃COOH, HNO₃, and HCl), and studied the effects of catalyst acidity, hydrolysis temperature T, and reaction time t on the production of D-AnG and other hydrolysis products. The optimal condition for maximum production of D-AnG by κ-carrageenan hydrolysis was T = 100°C and t = 30 min using 0.2 M HCl. Under this condition, 2.81 g/L D-AnG (33.5% of theoretical maximum) could be obtained from 2% (w/v) κ-carrageenan. In general, the maximum values of D-AnG, D-Gal, and the sum of two by-products (HMF and LA) increased with the acidity of catalysts. However, HNO3 was an exception in that the maximum production levels of HMF and LA were unusually low compared with other acid catalysts. D-AnG was successfully purified from acid hydrolysates using silica gel chromatography and the product was nearly 100% pure. This effective D-AnG production could facilitate future studies on the conversion of D-AnG to biofuels and biochemicals.     

Performance of growing Nitrosomonas europaea cells immobilized in κ-carrageenan

Summary Nitrosomonas europaea cells were immobilized in -carrageenan. The performance of the immobilized cells was investigated in an airlift loop reactor under wash-out conditions with respect to freely suspended cells. When fed with solutions of ammonia up to 16 mM, high substrate conversions were accomplished. Sudden increases in ammonium-ion concentration hardly influenced the conversion. Observations made by scanning electron microscopy showed that the immobilized cells were initially growing homogeneously across the beads, but as growth proceeded, a biomass density gradient developed, eventually resulting in a kind of biofilm.Offprint requests to: R. H. Wijffels     

Effect of potassium chloride and cationic drug on swelling,erosion and release from κ-carrageenan matrices

The basic objective of this work was to study the effect of model cationic drug metformin HCl on swelling and erosion and, in turn, the release of KCl and drug itself, from the κ-carrageenan matrices. Water uptake by the matrix up to 2 hours was found to increase with KCl concentration from the plain matrix. Erosion was not affected by concentration of KCl. Incorporation of drug favors water uptake, but in presence of KCl it was found to be reduced. Drugcontaining matrices have shown higher release of KCl as compared with plain batches. Drug release was retarded as KCl concentration increased up to 5%, above which the reduced cohesivity of the matrix caused increase in drug release.     

The immune regulation of κ-carrageenan oligosaccharide and its desulfated derivatives on LPS-activated microglial cells

Neurodegenerative disease involves an inflammatory response in the central nervous system characterized by an increase in inflammatory cytokines and activation of microglial cell. To reveal the immune regulation activity of κ-carrageenan oligosaccharides (KOS) on microglia cell activated by LPS and the relationship between the sulfate group content of KOS and its immune regulation activity, KOS was prepared by enzymatic hydrolysis. The degradation products of κ-carrageenan were analyzed by high performance liquid chromatography (HPLC), ESI-TOF-MS and (13)C NMR spectroscopy, and the results indicated that the hydrolyzed products of the κ-carrageenase were κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate, respectively. Then desulfated derivatives of KOS (DSK) were obtained with DMSO-methanol-pyridine method. The effect of KOS and DSK on the viability of microglia cell activated by LPS was determined with MTT method. Griess assay and ELISA method were used to determine the contents of NO/NO(2-), TNF-α and IL-10 released by activated microglia cell, respectively. The results showed that KOS could inhibit the viability and content of NO, TNF-α and IL-10 released by LPS-activated microglia cell dose dependently. Compared with that of KOS, the inhibiting activity of DSK is weaker. So it could be concluded that KOS could protect microglial cell from being activated by LPS, and there was a positive relationship between the sulfate group content of KOS and its protection function.     

Interactions between κ-carrageenan and chitosan in nanolayered coatings—Structural and transport properties

The interactions between κ-carrageenan and chitosan, two oppositely charged polysaccharides, have been investigated through microcalorimetric and quartz crystal microbalance measurements. Microcalorimetric measurements show that κ-carrageenan/chitosan interaction is an exothermic process and that the alternate deposition of κ-carrageenan and chitosan results in the formation of a nanolayered coating mainly due to the electrostatic interactions existing between the two polyelectrolytes (though other types of interactions may also be involved). Quartz crystal microbalance measurements confirmed that the alternating deposition of κ-carrageenan and chitosan resulted in the formation of a stable multilayer structure. The κ-carrageenan/chitosan nanolayered coating, assembled on a polyethylene terephthalate (PET) support, was characterized in terms of its surface (contact angle measurements) and gas barrier properties (water vapor and O₂ permeabilities) and analyzed by scanning electron microscopy (SEM). The water vapor permeability (WVP) and the oxygen permeability (O₂P) of the κ-carrageenan/chitosan nanolayers were found to be 0.020 ± 0.002 × 10⁻¹¹ and 0.043 ± 0.027 × 10⁻¹⁴ g m⁻¹ s⁻¹ Pa⁻¹, respectively. These results contribute to a better understanding of the type of interactions that play role during the construction of this type of nanostructures. This knowledge can be used in the establishment of an approach to produce edible, biodegradable multilayered nanostructures with improved mechanical and barrier properties for application in, e.g. food and biomedical industries.     

Properties of pellets manufactured by wet extrusion/spheronization process using κ-carrageenan: Effect of process parameters

The aim of this study was to systematically evaluate the pelletization process parameters of κ-carrageenan-containing formulations. The study dealt with the effect of 4 process parameters—screw speed, number of die holes, friction plate speed, and spheronizer temperature—on the pellet properties of shape, size, size distribution, tensile strength, and drug release. These parameters were varied systematically in a 2⁴ full factorial design. In addition, 4 drugs—phenacetin, chloramphenicol, dimenhydrinate, and lidocaine hydrochloride—were investigated under constant process conditions. The most spherical pellets were achieved in a high yield by using a large number of die holes and a high spheronizer speed. There was no relevant influence of the investigated process parameters on the size distribution, mechanical stability, and drug release. The poorly soluble drugs, phenacetin and chloramphenicol, resulted in pellets with adequate shape, size, and tensile strength and a fast drug release. The salts of dimenhydrinate and lidocaine affected pellet shape, mechanical stability, and the drug release properties using an aqueous solution of pH 3 as a granulation liquid. In the case of dimenhydrinate, this was attributed to the ionic interactions with κ-carrageenan, resulting in a stable matrix during dissolution that did not disintegrate. The effect of lidocaine is comparable to the effect of sodium ions, which suppress the gelling of carrageenan, resulting in pellets with fast disintegration and drug release characteristics. The pellet properties are affected by the process parameters and the active pharmaceutical ingredient used.     

Repeated Enzymatic Hydrolysis of Polygalacturonic Acid,Chitosan and Chitin Using a Novel Reversibly-soluble Pectinase with the Aid of κ-carrageenan

Aspergillus niger pectinase, together with κ-carrageenan, could be precipitated in the presence of 0.2% KCl and re-dissolved by ten-fold dilution of the salt. The free as well as this reversibly-soluble (rs) enzyme were evaluated for hydrolysis of polygalacturonic acid, chitosan and chitin. The rs-enzyme showed 92%, 80% and 74% activity (as compared to the corresponding amount of enzyme when present as a free enzyme) towards the three substrates, respectively. There was no significant change in the pH and temperature optima of the rs-enzyme. This preparation could be reused six times without loss of any detectable polygalacturonase activity. This biocatalyst design was found to be efficient for the hydrolysis of polygalacturonic acid, chitosan and chitin.     

NMR study on hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides: experimental evidence of hydrogen bonding and chemical exchange interactions in κ/μ oligosaccharides

The hydroxy protons of κ- and κ/μ-hybrid carrageenan oligosaccharides have been studied by NMR spectroscopy in 85% H(2)O/15% acetone-d(6). Hydration and hydrogen bonding interactions in di- (κ), tetra- (κκ), hexa (κκκ), and octa- (κκκκ) κ-oligosaccharides and hexa- (κμκ), octa- (κμμκ), and deca- (κμμμκ) κ/μ-oligosaccharides have been investigated by measuring the chemical shifts, temperature coefficients, and chemical exchange of the hydroxy protons. These NMR parameters indicate that no strong and persistent intramolecular hydrogen bonds involving hydroxy protons stabilize the structure of κ-carrageenan oligosaccharides in aqueous solution. In the κ/μ-oligosaccharides, the presence of chemical exchange between OH3 of α-d-Gal-6-sulfate (D6S) and OH2 of β-d-Gal-4-sulfate (G4S) across the β-d-Gal-4-S-(1→4)-α-d-Gal-6-S linkage reveals the existence of a weak hydrogen bond interaction between the two hydroxyl groups. The smaller temperature coefficients of OH2_D6S and OH3_D6S indicate reduced hydration, interpreted as spatial proximity to the 4-sulfate group and O5 ring oxygen of the neighboring G4S residues, respectively. These first experimental results on the conformation of κ/μ-carrageenan oligosaccharides shine light on the potential role of "kinks" in the properties of the three-dimensional carrageenan gel network.