O-maleoyl derivative of low-molecular-weight κ-carrageenan: Synthesis and characterization

Low-molecular-weight (LMW) κ-carrageenan was achieved through mild hydrochloric acid hydrolysis of κ-carrageenan. The acylation of low-molecular-weight κ-carrageenan was performed by use of tetrabutylammonium (TBA) salt of the anionic polysaccharide fragments, maleic anhydride, 4-dimethylaminopyridine and tributylamine under homogeneous conditions in N,N-dimethylformamide at 60 °C. Analysis of FT-IR spectrum of O-maleoyl κ-carrageenan showed that a monoester derivative with maleoyl group was formed when LMW κ-carrageenan reacted with maleic anhydride. Investigation of ¹H NMR spectroscopy revealed that the maleoylation took place regioselectively at C-2 of the κ-carrageenan 3-linked unit.     

Mixtures of pregelatinised maize starch and κ-carrageenan: Compatibility, rheology and gelation

Compatibility, flow and visco-elastic properties of a pregelatinised maize starch mixed with κ-carrageenan were investigated. After cooking of the pregelatinised starch, some undissolved granules remained in solution. Aqueous mixtures of κ-carrageenan and starch were studied at 60 °C and 20 °C by combining rheological measurements and microscopic observations under conditions allowing gelation of carrageenan and non-gelation of starch. The viscometric study of mixed dilute solutions of amylose from pregelatinised starch and carrageenan showed that the components are slightly incompatible. Mixture viscosity and elastic modulus were studied at 60 °C in details as a function of mixture composition for a total polymer concentration of 3%; both were found to be significantly higher than the corresponding theoretical additive values. This finding was interpreted by starch granules excluded volume effect. At 20 °C, no noticeable increase of mixture elastic modulus was found as compared with the additive value. The absence of the synergistic effect is supposed to be due to the formation of highly inhomogeneous gels with agglomerates of undissolved granules.     

Rheological and calorimetric study of the sol–gel transition of κ-carrageenan

Rheological and DSC techniques were used to study the effect of κ-carrageenan and KCl concentrations, 0–300 mM, on the sol–gel transition as well as on the linear viscoelasticity, at 25 °C, of the resulting gels. In heating and cooling DSC tests, the peak temperature was taken as the sol–gel transition point. In rheological tests, sol–gel transitions were determined from the variation of dynamic moduli with frequency and temperature, the independence of the phase angle on frequency and the evolution with temperature of dynamic moduli on cooling and heating at constant frequency and strain. Transition temperatures from DSC and rheology were in good agreement among them and with those previously reported. The three procedures yielded similar results, but the transition temperatures were more easily determined through the independence of the phase angle on frequency. Frequency sweeps showed gel behavior with stiffness increasing with polysaccharide and salt concentration. Below 100 mM KCl, G′ increased notably, whereas higher concentrations produced only marginal increases.     

Conformation and association of κ-carrageenan in the presence of locust bean gum in mixed NaI/CsI solutions from rheology and cryo-TEM

Mixtures of locust bean gum (LBG) with κ-carrageenan (KC) in 0.1 M aqueous solutions of the mixed salts NaI/CsI were investigated by cryo-transmission electron microscopy (cryo-TEM) and dynamic viscoelastic measurements. Previous studies have shown that as the cesium content is increased in such mixed salt solutions, a transition occurs from molecularly dispersed helices to ‘superhelical rods’ of KC. We now found that LBG stabilises the superhelical rods, shifting the transition to a lower content of Cs for the mixtures than for KC alone. The formation of superhelical rods was evidenced both by cryo-TEM images and by an onset of thermal hysteresis in the coil–helix transition of KC. In the mixtures, the transition temperatures on cooling and heating were insensitive to the proportions of LBG and KC present at all cesium contents. Under conditions where no helix aggregation occurred (no hysteresis) the mixtures showed high tan δ values and low storage moduli. Under aggregated conditions, gels formed, and gels with added LBG had enhanced moduli compared to gels with KC alone. On the basis of these results we propose that LBG associates to the super-helical rods of KC.     

Rheological properties of mixtures of κ-carrageenan from Hypnea musciformis and galactomannan from Cassia javanica

Mixed gels of κ-carrageenan (κ-car) from Hypnea musciformis and galactomannans (Gal) from Cassia javanica (CJ) and locust bean gum (LBG) were compared using dynamic viscoelastic measurements and compression tests. Mixed gels at 5 g/l of total polymer concentration in 0.1 M KCl showed a synergistic maximum in viscoelastic measurements for κ-car/CJ and κ-car/LBG at 2:1 and 4:1 ratios, respectively. The synergistic maximum obtained from compression tests carried out for mixed gels at 10 g/l of total polymer concentration in 0.25 M KCl was the same for both κ-car/CJ and κ-car/LBG gels. An enhancement in the storage modulus (G′) and the loss modulus (G″) was observed in the mechanical spectra for the mixtures in relation to κ-car. The proportionally higher increase in G″ compared with G′, as indicated by the values of the loss tangent (tan δ), suggests that the Gal adhere non-specifically to the κ-car network.     

Behavior of κ-carrageenan in glycerol and sorbitol solutions

The solubility of κ-carrageenan in low water-content solvents is important in food applications where complete solubilization is required for proper development of structure and rheology. The effect of glycerol and sorbitol on the gelation and conformational helix transition of κ-carrageenan was studied using rheology and optical rotation. Glycerol/water solutions from 0–100 wt% glycerol and sorbitol solutions from 0–100% saturation were studied over the temperature range 0–90°C. The results were analyzed in terms of solvent solubility parameters, water chemical potential, and solvent dielectric constant. Effective cohesive energy density parameters could not be inferred for the carrageenan, but the gelation temperature could be correlated with solvent dielectric constant. Hydrogen bonding interactions control the carrageenan helix formation. The cohesive energy density as a measure of solvent quality accounts for hydrogen bonding but not Coulombic interactions, and the Coulombic interactions scale on dielectric constant. This indicates the dominant role of electrostatics on the gelation process.     

Responsiveness of κ-carrageenan microgels to cationic surfactants and neutral salts

The objective of this study was to examine the responsiveness of chemically cross-linked κ-carrageenan microspheres to different types of neutral salt electrolytes as well as to surfactants of varying chain lengths. In the presence of increasing salt concentration microsphere size changed radically from D[4,3] values of 320 μm to approximately 160 μm. The level of salt concentration needed to bring about this change varied depending on electrolyte type. This common behaviour was attributed to the difference in free cationic counter-ions concentration between the inside and outside of the microsphere and can be explained due to the effect of the Donnan equilibrium. The rheological properties of these microgels in their swollen and collapsed states were also explored with results showing that the collapsed microspheres had a greater impact on the viscosity of the system probably as a result of some aggregation of the collapsed microgels at rest due to surface charge screening at these high salt concentrations. The effect of surfactant on microsphere size showed a dramatic drop in D[4,3] values from 320 μm to approximately 120 μm for BAC, DoTAB, MTAB and CTAB at specific critical concentrations. This critical aggregation concentration was found to increase linearly on a log–log scale with the critical micelle concentration of these surfactants in water, indicating that the alkyl chain length of the surfactants had an effect on the critical aggregation concentration.     

Physical and stability characteristics of Burkholderia cepacia lipase encapsulated in κ-carrageenan

In this work, a novel approach for lipase immobilization was exploited. Lipase from Burkholderia cepacia was encapsulated into κ-carrageenan by co-extrusion method to form a liquid core capsule. The diameter of the encapsulated lipase was found to be in the range of 1.3–1.8 mm with an average membrane thickness of 200 μm and 5% coefficient of variance. The encapsulation efficiency was 42.6% and 97% moisture content respectively. The encapsulated lipase was stable between pH 6 and 9 and temperature until 50 °C. The encapsulated lipase was stable until disintegration of the carrier when stored at 27 °C and retained 72.3% of its original activity after 6 cycles of hydrolysis of p-NPP. The encapsulated lipase was stable in various organic solvents including methanol, ethanol, iso-propanol, n-hexane and n-heptane. Kinetic parameters K_m and V_max were found to be 0.22 mM and 0.06 μmol/min for free lipase and 0.25 mM and 0.05 μmol/min for encapsulated lipase respectively.     

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.     

Studies on κ-carrageenan/locust bean gum mixtures in the presence of sodium chloride and sodium iodide

The solution properties of κ-carrageenan and κ-carrageenan/locust bean gum mixtures have been studied by small deformation oscillation measurements and differential scanning calorimetry (DSC) in the presence of sodium chloride and sodium iodide. Both salts induced the κ-carrageenan to undergo a coil-helix conformational change as noted by an increase in the storage and loss moduli (G′, G′) and by an exothermic peak in the DSC cooling curves. The enthalpy ΔH_c-h and temperature of the conformational transition T_c-h were higher in Nal compared to NaCl and T_c-h increased with increasing the concentration of both electrolytes. Gelation was not observed for carrageenan or carrageenan/locust bean gum mixtures in the presence of up to 200 mM Nal. Although carrageenan alone did not gel in the presence of 100 mM NaCl, a weak gel was obtained for a mixture containing 0.9%/0.1% carrageenan/locust bean gum. Furthermore, the mixture showed hysteresis in both the rheological and DSC cooling and heating curves. A strong gel was produced for carrageenan alone in the presence of 200 mM NaCl and the gel strength increased on adding a small proportion of locust bean gum (0.9%/0.1%). © 1997 John Wiley & Sons, Inc. Biopoly 41: 657–671, 1997     

Conformational change and aggregation of κ-carrageenan studied by flow field-flow fractionation and multiangle light scattering

The relatively novel combination of flow field-flow fractionation (FFF) and multiangle light scattering (MALS) was employed to study a nondegraded κ-carrageenan in different 0.1M salt solutions. The applicability of the technique was tested, and the effects of salt type and salt composition on the molar mass and radius of gyration were studied. A conformational ordering was induced at room temperature by switching the solvent from 0.1M NaCl (coil form) to 0.1M NaI (helix form). An approximate doubling of the average molar mass and an increase in radius of gyration was then observed, in agreement with results obtained previously using size exclusion chromatography–MALS. This increase in size was attributed to conformational ordering and to the formation of double helices. Severe aggregation was observed above 40% CsI in the 0.1M mixed salt solution of CsI and NaI. This was ascribed to the association of helices into large aggregates. For these large associates, having molar masses of several millions, a reversal of the elution order in flow FFF was detected. © 1998 John Wiley & Sons, Inc. Biopoly 45: 85–96 1998     

Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A

The alcohols methanol, ethanol, propan-1-ol and 2,2,2-trifluoroethanol were added to aqueous solution of κ-casein A. Far u.v. circular dichroism spectroscopy revealed a greater proportion of -helix structure in the presence of alcohol; the effectiveness of the alcohol in causing this structural change increased in the order methanol to 2,2,2-trifluoroethanol. No alterations in secondary or tertiary structure were found in κ-casein A on reduction and S-carboxymethylation using near and far u.v.c.d. spectroscopy. The isolated macropeptide section of κ-casein was less sensitive to the addition of alcohols; it appeared that the linkage of macropeptide to the para-κ-casein section affected the case with which solvent induced conformational changes occurred. The conformational changes found in the presence of alcohols could not be correlated simply with the changes in the dielectric constant or with the alcohol-induced collapse of the hairy macropeptide layer of casein micelles or ther precipitation.     

NMR investigations of the 4-ethyl guaicol self-diffusion in iota (ι)-carrageenan gels

Self-diffusion coefficient of an aroma molecule (4-ethyl guaicol) was measured using the pulsed field gradient spin echo NMR (PGSE-NMR) method in order to investigate the influence of a macromolecular matrix on its diffusion and release processes. Iota (ι)-carrageenan was used for its ability to form thermoreversible gels in aqueous salt solutions. Variations of the ι-carrageenan and the salt concentrations permitted various gels with different thermal and rheological properties to be obtained. These latter were modified by an isotope effect obtained by preparing gels in D₂O. The NMR self-diffusion measurements realised for water and the aroma molecules indicated neither chemical interactions with ι-carrageenan, nor obstruction effects from the polysaccharide chains. In ι-carrageenan gels, the diffusional phenomenon was highly dependent on the heterogeneous gel structure and controlled by hydrodynamic interactions due to frictional drag between each molecule of the system and water microviscosity changes.     

Physical characteristics of calcium induced κ- carrageenan networks

The presence of an optimum counter-ion concentration in calcium-induced κ-carrageenan gels at low polymer concentrations of 5 and 10 g/l is observed. At approximately the stoicheometric molar ratio of 1 calcium per carrageenan sulphate, a gel with high elastic modulus, high optical clarity and fine network structure is observed. On further increase of counter-ion concentration beyond this optimum, elastic modulus decreases significantly associated with sharp increase in the gels turbidity together with a network characterised with coarse and large-pore mesh.

The quite complete characterisation of the various gel networks both mechanically by ways of oscillatory and static rheology and optically by turbidimetry and cryo-SEM shows that the extensive structural charge neutralisation of the polysaccharide by divalent calcium ions is responsible for a marked aggregation of the polymer strands reminiscent of precipitation. At lower counter-ion to polymer ratios, onset of gelation might prevent such phase separation.     

Unusual intramolecular N→O acyl group migration occurring during conjugation of (−)-DHMEQ with cysteine

Previously we found that (−)-DHMEQ, a specific NF-κB inhibitor, covalently bound to a specific cysteine of NF-κB component proteins. In the course of formation of the (−)-DHMEQ and protected cysteine conjugate, we observed an unusual intramolecular N→O acyl group migration.     

Chondroprotective and anti‐inflammatory effects of amurensin H by regulating TLR4/Syk/NF‐κB signals

The low‐grade, chronic inflammation initiated by TLR4‐triggered innate immune responses has a central role on early osteoarthritis. Amurensin H is a resveratrol dimer with anti‐inflammatory and anti‐apoptotic effects, while its effects on TLR‐4 signals to inhibit osteoarthritis are still unclear. In the present study, treatment with amurensin H for 2 weeks in monosodium iodoacetate‐induced mice significantly slows down cartilage degeneration and inflammation using macroscopic evaluation, haematoxylin and eosin (HE) staining and micro‐magnetic resonance imaging. In IL‐1β‐stimulated rat chondrocytes, amurensin H suppresses the production of inflammatory mediators including nitric oxide, IL‐6, IL‐17, PGE2 and TNF‐α using Greiss and ELISA assay. Amurensin H inhibits matrix degradation via decreasing levels of MMP‐9 and MMP‐13 using Western blot assay, promotes synthesis of type II collagen and glycosaminoglycan using immunostaining and safranin O staining, respectively. Amurensin H inhibits intracellular and mitochondrial reactive oxygen species (ROS) generation, and mitochondrial membrane depolarization using DCFH‐DA, MitoSOX Red and JC‐1 assay as well. IL‐1β stimulates TLR4 activation and Syk phosphorylation in chondrocytes, while amurensin H inhibits TLR4/Syk signals and downstream p65 phosphorylation and translocation in a time and dose‐dependent manner. Together, these results suggest that amurensin H exerts chondroprotective effects by attenuating oxidative stress, inflammation and matrix degradation via the TLR4/Syk/NF‐κB pathway.     

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1β (IL-1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL-1β to its cellular receptors or could interfere with its signaling pathway at a post-receptor level. Addition of glucosamine to rat chondrocytes treated with IL-1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein-1. After treatment with IL-1β, glucosamine increased the expression of mRNA encoding the type II IL-1β receptor. These results emphasize the potential role of two regulating proteins of the IL-1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.     

NF‐κB regulates a cassette of immune/inflammatory genes in human pregnant myometrium at term

The onset of human labour resembles inflammation with increased synthesis of prostaglandins and cytokines. There is evidence from rodent models for an important role for nuclear factor‐κB (NF‐κB) activity in myometrium which both up‐regulates contraction‐associated proteins and antagonizes the relaxatory effects of progesterone. Here we show that in the human, although there are no differences in expression of NF‐κB p65, or IκB‐α between upper‐ or lower‐segment myometrium or before or after labour, there is nuclear localization of serine‐256‐phospho‐p65 and serine‐536‐phospho‐p65 in both upper‐ and lower‐segment myometrium both before and after the onset of labour at term. This shows that NF‐κB is active in both upper and lower segment prior to the onset of labour at term. To identify the range of genes regulated by NF‐κB we overexpressed p65 in myocytes in culture. This led to NF‐κB activation identical to that seen following interleukin (IL)‐1β stimulation, including phosphorylation and nuclear translocation of p65 and p50. cDNA microarray analysis showed that NF‐κB increased expression of 38 genes principally related to immunity and inflammation. IL‐1β stimulation also resulted in an increase in the expression of the same genes. Transfection with siRNA against p65 abolished the response to IL‐1β proving a central role for NF‐κB. We conclude that NF‐κB is active in myocytes in both the upper and lower segment of the uterus prior to the onset of labour at term and principally regulates a group of immune/inflammation associated genes, demonstrating that myocytes can act as immune as well as contractile cells.