Screening for synergistic interactions in dilute polysaccharide solutions

A simple viscometric approach has been used to screen for binding interactions between different polysaccharides in very dilute solution where exclusion effects should be negligible. The method involves preparing stock solutions to approximately the same, low, viscosity (η_sp≈1), dialysing to identical ionic conditions, mixing in various proportions, and looking for departures from the initial common viscosity.

Mixtures of xanthan or de-acetylated xanthan with locust bean gum (LBG) or konjac glucomannan (KM) show massive enhancement of viscosity, as anticipated from the formation of synergistic gels at higher concentrations. However, no viscosity changes on mixing with LBG or KM were observed for other conformationally ordered bacterial polysaccharides (welan and rhamsan) or for alginate and pectin with sufficient Ca²⁺ to induce almost complete conversion to the dimeric ‘egg box’ form, demonstrating that conformational rigidity is not, in itself, sufficient for other polysaccharides to form heterotypic junctions with mannan or glucomannan chains.

Interactions of carrageenans with LBG appear to depend on both conformation and the extent of aggregation. Mixtures of LBG with K⁺ kappa carrageenan in 100mM KCl (which is known to promote extensive aggregation of double helices) gave erratic values for rotational viscosity and showed typical gel-like mechanical spectra under low-amplitude oscillation. Disordered carrageenans (K⁺ kappa in water and lambda in 100mM KCl) showed no evidence of interaction with LBG. Negative results were also obtained for iota carrageenan under ionic conditions believed to promote ordering without significant aggregation (100mM KCl). However, under conditions where limited aggregation might be expected (iota carrageenan in 90 mM CaCl₂; Me₄N⁺ kappa carrageenan in 150 mM Me₄NI), significant reductions in viscosity were observed on mixing with LBG, which may indicate some intermolecular association but without the formation of an extended network structure.     

The influence of carrageenan on molecular mobility in low moisture amorphous sugars

The influence of less than 1% of κ-carrageenan on the mobility of glucose syrup was studied in the context of the glass–rubber transition using proton NMR relaxometry. Glass-transition temperatures, (T_g) were measured by differential scanning calorimetry (DSC) on glucose syrup samples containing 0 or 0.9% κ-carrageenan, between 0 and 1.4% KCl, and at water contents from 3.5 to 16% (wwb). Potassium chloride was added to vary the extent of gelation of the carrageenan in order to assess the effect of the biopolymer network on molecular mobility.

Contrary to the reported increase of the rheologically determined glass-transition temperature, in the presence of gelling agents, the addition of 0.9% κ-carrageenan to glucose syrup with and without KCl, had no effect on the DSC measured T_g. In addition, there was no effect on molecular mobility in the glassy region. The presence of carrageenan only significantly affected the mobile part of the NMR free induction decay at relatively high temperatures.     

The influence of locust bean gum and dextran on the gelation of κ-carrageenan

The interaction of κ-carrageenan with locust bean gum and dextran has been studied by rheology, differential scanning calorimetry (DSC), and electron spin resonance spectroscopy (ESR). Rheological measurements show that the carrageenan gel characteristics are greatly enhanced in the presence of locust bean gum but not in the presence of dextran. Carrageenan/locust bean gum mixtures show two peaks in the dsc cooling curves. The higher temperature peak corresponds to the temperature of gelation and its intensity increases at the expense of the lower temperature peak as the proportion of locust bean gum in the mixture increases. Furthermore, the DSC heating curves show enhanced broadening when locust bean gum is present, indicating increased aggregation. These results are taken as evidence of carrageenan/locust bean gum association. The gelation process has also been followed by ESR using spin-labeled carrageenan. On cooling carrageenan solutions, an immobile component appears in the ESR spectra signifying a loss of segmental mobility consistent with chain stiffening due to the coil → helix conformational transition and helix aggregation. For carrageenan/locust bean gum mixtures, carrageenan ordering occurs at temperatures corresponding to the higher temperature DSC setting peak and the temperature of gelation. Similar studies using spin-labeled locust bean gum show that its mobility remains virtually unaffected during the gelation process. It is evident, therefore, that carrageenan and locust bean gum interact only weakly. It is proposed that at low carrageenan concentrations the gel network consists of carrageenan helices cross-linked by locust bean gum chains. At high carrageenan concentrations the network is enhanced by the additional self-aggregation of the “excess” carrageenan molecules. For carrageenan/dextran mixtures, only one peak is observed in the dsc cooling curves. The onset of gelation shifts to higher temperatures only at very high (20%) dextran concentrations and this is attributed to volume exclusion effects. Furthermore, there is no enhanced broadening of the peaks in the DSC heating curves as for the carrageenan/locust bean gum systems. It is therefore concluded that carrageenan/dextran association does not occur. The difference in behavior between locust bean gum and dextran is attributed to the greater flexibility of the dextran chains. © 1996 John Wiley & Sons, Inc.     

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.     

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     

Rheology and synergy of κ-carrageenan/locust bean gum/konjac glucomannan gels

The rheology and melting of mixed polysaccharide gels containing konjac glucomannan (KGM), locust bean gum (LBG) and κ-carrageenan (KC) were studied. Synergy-type peaks in the Young's modulus at optimal mixing ratios were found for both KC/LBG and KC/KGM binary gels at a fixed total polysaccharide content (1:5.5 for LBG:KC and 1:7 for KGM:KC). The Young's modulus peak for KC/KGM was higher than for KC/LBG gels. The same stoichiometric mixing ratios were found when either LBG or KGM was added to KC at a fixed KC concentration, where the Young's modulus increased up to additions at the stoichiometric ratio, but leveled off at higher LBG or KGM additions. Addition of KGM or LBG to the 2-component gels beyond the stoichiometric (optimal) mixing ratio at a fixed total polysaccharide content led to a decrease in the Young's modulus and an increase in the rupture strain and stress in extension, and both trends were stronger for KGM than for LBG.     

The influence of LBG addition to carrageenan on the mechanical stability of the gel and the fermentative activity of immobilized propionic acid bacteria

In the first part of the experiments, the mechanical properties of 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/locust bean gum (LBG) gels stored in various concentrations of propionic and acetic acids and their mixtures were examined. The stability of these materials was measured by uniaxial compression between two parallel plates using the Instron Universal Testing Machine. A mathematical model explaining the dependence of the destroying force on the storage time was chosen for data analysis. Using this model, the average rate of gel deterioration was calculated. The structural properties of the examined gels were most influenced by the highest concentration of propionic and acetic acids and their mixtures (1% acetic acid and 2% propionic acid). The addition of LBG to carrageenan decreased the gel destroying force and increased its resistance to acids. In the second part of the experiments, the Propionibacterium freudenreichii subsp. shermanii NCFB 1081 and NCFB 566 were immobilized in a living state in 1%, 2% and 3% carrageenan and 1%, 2% and 3% carrageenan/LBG gels. The ammonia consumption, glucose utilization, production of propionic and acetic acids and the biosynthesis of vitamin B₁₂ were examined. An increase in the productivity of propionic acid and a significant decrease in the vitamin B₁₂ produced in the biosynthesis were observed when immobilized cells were used. The immobilization of cells enhanced the productivity of propionic acid by up to 40% compared to free cells. The best results were obtained for the second and third applications of immobilized cells in all concentrations of carrageenan gels and 2% and 3% carrageenan/LBG gels The results showed that carrageenan/LBG is a better support material for the immobilization of propionic acid bacteria than the pure carrageenan.     

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.     

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.     

Spatial and temporal variations of Chondrus crispus (Gigartinaceae, Rhodophyta) carrageenan content in natural populations from Galicia (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes and tetrasporophytes of Chondrus crispus were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν- and μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 50% of the total carrageenans). In tetrasporophytes, the presence of λ-carrageenan was confirmed. Carrageenan content in gametophytes (37.4?±?1.68% DW) was higher than in tetrasporophytes (29.13?±?0.76% DW). Spatial and temporal variation in carrageenan content in both life cycle phases appears to be related mainly to seawater and air temperatures, insolation, water movement and desiccation. The highest values of carrageenan content were recorded in those localities where higher values of precipitation, wind speed or water movement occurred. A bimodal temporal pattern on carrageenan content was observed. Fronds showed a high carrageenan content in spring and autumn. During these seasons, the content was over 40% in gametophytes and 30% in the tetrasporophytes. In summer and winter, these values down in both life cycle phases below 30%. In general the highest carrageenan contents were related to highest seawater temperatures. On the contrary, high air temperature and high insolation appeared to be unfavourable for carrageenan production. GLM models were obtained to predict carrageenan production from natural C. crispus populations, along Galician coast.     

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.     

Effect of epiphyte infection on physical and chemical properties of carrageenan produced by Kappaphycus alvarezii Doty (Soliericeae,Gigartinales, Rhodophyta)

Epiphytism of filamentous red algae is a serious problem in Kappaphycus farms in the Philippines, Indonesia, Malaysia, and Tanzania. The causative organism of epiphyte outbreak has been identified as Neosiphonia apiculata (Hollenberg) Masuda and Kogame, but its actual effect on carrageenan quality has not yet been established. Therefore, yield and quality of carrageenan from healthy and infected specimens were examined. Infected specimens showed 20.5?±?2.5 % DW lower carrageenan yield compared with the healthy seaweed (65.5?±?4.2 % DW). Infected specimens also had a higher phenolic and fatty acid content, compared with healthy specimens. The carrageenan from the infected seaweed showed 74.5?±?2.8 % lower viscosity, 52.6?±?3.6 % lower gel strength, 22.9?±?1.5 % higher syneresis, and 5 °C higher melting temperature as compared with carrageenan from healthy specimens. FTIR and ¹³C-NMR analysis of carrageenan from infected seaweed did not show any differences in their functionality or carbon atom chemical shift as compared with healthy and standard k-carrageenan. However, size exclusion chromatography showed the infected carrageenan molecular size to be 80 kDa as compared with 800 kDa for the healthy and standard k-carrageenan. These findings prove that infection of Kappaphycus by the filamentous red algae epiphyte, N. apiculata, reduces carrageenan molecular size and affects the physical properties of the carrageenan.     

Characterization of Carrageenan Extracted from <Emphasis Type="Italic">Hypnea bryoides</Emphasis> in Oman

Carrageenophyte red seaweed from Oman, Hypnea bryoides, extracted using three different processes: an aqueous, a mild alkaline, and a more vigorous alkaline extraction was investigated. The resulting extract precipitated by alcohol was subject to chemical and rheological measurements. The total carbohydrate [ranged from 36.78 to 41.65 g/100 g], and ash [39.04 to 43.11 g/100 g] were the most abundant components in H. bryoides and contrary to the two, lipid content was found at a minimum [ranging from 2.95 to 3.38 g/100 g]. Alkali treatment with NaOH allowed complete conversion of kappa (κ) carrageenan form as detected by FTIR analysis. Total yield by alkali treatments gave higher yields (33%) compared with aqueous treatments (12%). However, subsequent aqueous treatment produced mixed carrageenan (μ and κ) with higher molecular weight compared with the alkali treatments which produced single carrageenan form (κ) with molecular weight of 4.1 × 10⁵ Da. The effects of thermal history on gel–sol and sol–gel transition were investigated by differential scanning calorimeter (DSC) and rheology on a pure sample and 1.5% κ-carrageenan mixture added with 30 mM KCl. Transition temperatures from DSC and rheology showed comparable results and were in good agreement with those previously reported.     

Changes in the polyelectrolyte-amphiphile interaction due to helix-coil transition induced by specific counterions or variations in temperature

For the system κ-carrageenan/amitriptyline it is shown that the degree of binding of amitriptyline is closely related to the carrageenan conformation as regulated by the counterions (Na⁺ or K⁺). The adsorption becomes much more pronounced when the carrageenan molecule is in the helix form (counterion K⁺) than when it has a coil conformation (counterion Na⁺). Furthermore, for the helical state the adsorption becomes strongly cooperative. It is also shown experimentally that the release from the adsorbed state has a conversion temperature at about 42°C (helix-coil transition). The effect is also related to the linear charge density. For κ-carrageenan with a higher charge density the adsorption is strong and cooperative both in the presence of Na⁺ and K⁺ ions. © 1996 John Wiley & Sons, Inc.     

Qualitative and quantitative analysis of carrageenan content in gametophytes of Mastocarpus stellatus (Stackhouse) Guiry along Galician coast (NW Spain)

Qualitative and quantitative differences in carrageenan composition of gametophytes of the rhodophyte Mastocarpus stellatus (Gigartinales) were observed in this study. Carrageenans in gametophytes belong to the kappa family (κ-, ι-, ν-, μ-carrageenan). The dominant fractions were κ- and ι-carrageenan (more than 80 % of the total carrageenans). Mean total carrageenan content in gametophytes was of 37.32?±?1.21 % DW. Spatial and seasonal variations were observed, mainly related to changes on environmental and oceanographic factors and the role of carrageenans in adapting the fronds to these changes. Maximum values in carrageenan content were observed for San Román (Biscay Bay) in May and for Laxe and Mougás (Atlantic coast) in June. The results of this study indicated that spatial differences in carrageenan content were due to interactions of different factors, rather than the effect of a single factor. Fronds from San Román had higher carrageenan content (43.23?±?1.87 % DW) than those collected at two sites of the Atlantic coast, Mougás and Laxe (32.20?±?1.14 % DW). San Román is exposed to the open sea, windy and oriented to the north, and the water temperature is higher in summer than in the Atlantic coast. However, seasonal variations in carrageenan content resulted to be more related to other factors directly correlated with the input of energy in the ecosystems (irradiance, sunshine hours and insolation). Thus, carrageenan content began to increase in early spring when the number of sunlight hours increased. Maximum values were reached in late spring or early summer, just before maximum values of irradiance and air temperature were achieved.     

Effects of improved post-harvest handling on the chemical constituents and quality of carrageenan in red alga,Kappaphycus alvarezii Doty

Red alga Kappaphycus alvarezii Doty is an important commercial species widely cultivated in southeast Asian countries for its polysaccharide, kappa-carrageenan. Common post-harvest handling technique involves sun-drying of harvested seaweed on platforms at the farms. Quantity and quality of carrageenan varies depending on the duration and care taken during the post-harvest handling of the raw seaweed. In this study, dynamics of moisture content, water activity index (aw), carrageenan yield, and carrageenan quality were investigated by subjecting the seaweed to three post-harvest methods: (1) freeze-drying (FD), (2) shade-drying (SD), and (3) direct sun-drying (DSD). Seaweed dried under FD and SD produced high yield (56–58 %), superior gel strength (1,454–1,424 g cm^?2), high viscosity (57–58 cPs), and low syneresis (15–17 %). But, carrageenan extracted from DSD seaweed gave 28 % lower yield, 38 % lower gel strength, 27 % lower viscosity, and 8–9 % higher syneresis. In addition, gelling temperature and melting temperature of the DSD carrageenan were lower by 4 and 9 °C, respectively. Molecular size analyses of carrageenan extracted from seaweed dried under FD and SD contained carrageenan of 700 KDa (80 %) and 200 KDa (4–10 %). However, carrageenan extracted from DSD seaweed contained smaller carrageenan molecules, 460 KDa (55 %), 210 KDa (25 %), and <100 KDa (20 %). Further, scanning electron microscope images illustrated the severe effects of DSD on the morphology of seaweed cells. Therefore, SD technique was found to be the best post-harvest processing technique that gave quality carrageenan in a high quantity. Due to its simplicity and low cost, it is a practical approach to be practiced in southeast Asian countries.     

On the interchain associations in aqueous solutions of a succinoglycan polysaccharide

Formation of interchain associations between succinoglycan chains have been studied by comparing weight average molecular weight, intrinsic viscosity of succinoglycan as a function of the conditions to prepare the solutions (polymer concentration, the heating temperature adopted compared with T_m). The different solutions obtained were characterized by their Newtonian viscosity, the storage and loss moduli and their sensitivity to the temperature. It was found that interchain associations, first stabilized mainly during the disorder-order transition convert to more stable associations by aging at temperatures below but not too far from T_m. These associations appear from succinoglycan solutions characterized by an overlap parameter higher than about 8 and modify only very slightly the conformational transition parameters obtained from microcalorimetry measurements.     

Steady-state and pulsed NMR studies of gelation in aqueous agarose

Steady-state and pulsed NMR techniques have been used to investigate molecular motion in sols and gels of agarose. In passing through the sol–gel transition, the molecular mobility of water molecules is reduced only by a small amount, whereas motion of the polymer chains is greatly attenuated. The results are discused in terms of the network theory of gelation, with references to the role of water in the process and the nature of the “junction zones” between polymer chains. T₂ and line-width measurements are dominated by exchange broadening. The effects of exchange rate and differences in relaxation time between the exchanging sites are discussed. The temperature hysteresis behavior of agarose gels has been investigated and the effects of “ageing” correlated with changes in nuclear relaxation times. The synergistic increase in gel strength obtained on adding locust bean gum (LBG) to agarose has been investigated. The results indicate that LBG does not form double-helix junctions and may decrease rates of gelation by steric effects. At high agarose concentration, the LBG remains mainly in solution in interstitial water, but at low agarose concentration, it is suggested that the LBG can link gel aggregates together into a self-supporting structure, producing a synergistic increase in gel strength. Comparisons have been made between the nature of the agarose–LBG interaction and agarose–cellulose interactions in biological systems.     

Structure and thermal stability of pyruvated carrageenans from the red alga Coccotylus truncatus

The composition, structure, and thermal stability of carrageenans from unattached Coccotylus truncatus (the Baltic Sea, Estonia) were investigated. The complex polysaccharide was characterized by ¹³C NMR and FTIR spectroscopy, ICP-OES and gel permeation chromatography methods. The main components of C. truncatus galactan are 3,6-anhydro-α-d-galactose-2-sulfate (30 ± 1.5%) and β-d-galactose-4-sulfate (45.3%), indicating a ι-carrageenan backbone. As the minor components, α-d-galactose-2,6-disulfate (12 ± 2%) from ν-carrageenan and 4′,6′-pyruvated β-d-galactopyranosyl residues (1.4%) from pyruvated α-carrageenan are found to be present, latter being responsible for the undersulfated nature of the galactan. The native polysaccharide with the average molecular weight of about 1500 kDa is highly susceptible to thermal degradation. The high-temperature treatment of this galactan gives products with 3,6-anhydro-α-d-galactose units predominantly at the reducing end. The carrageenan extraction from C. truncatus gives characteristically low yields (12-17%); weak gelling ability of the polysaccharides from this seaweed species (gel strength 30-40 g/cm²) does not depend significantly on extraction conditions.     

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.