Pullulan-sodium alginate based edible films: Rheological properties of film forming solutions

Rheological properties of pullulan, sodium alginate and blend solutions were studied at 20 °C, using steady shear and dynamic oscillatory measurements. The intrinsic viscosity of pure sodium alginate solution was 7.340 dl/g, which was much higher than that of pure pullulan (0.436 dl/g). Pure pullulan solution showed Newtonian behavior between 0.1 and 100 s⁻¹ shear rate range. However, increasing sodium alginate concentration in pullulan-alginate blend solution led to a shear-thinning behavior. The effect of temperature on viscosities of all solutions was well-described by Arrhenius equation. Results from dynamical frequency sweep showed that pure sodium alginate and blend solutions at 4% (w/w) polymer concentration were viscoelastic liquid, whereas the pure pullulan exhibited Newtonian behavior. The mechanical properties of pure sodium alginate and pullulan-alginate mixture were analyzed using the generalized Maxwell model and their relaxation spectra were determined. Correlation between dynamic and steady-shear viscosity was analyzed with the empirical Cox-Merz rule.     

Phase behavior of aqueous solutions of bovine serum albumin in the presence of dextran, at rest, and under shear

The demixing conditions for aqueous solutions of bovine serum albumin (BSA, fraction V) and for joint solutions of BSA plus dextran (DEX, M(w) = 2000 kg/mol) were determined by turbidimetric measurements as a function of composition, temperature, and shear rate. Aqueous solutions of BSA phase separate upon heating. Within the region of BSA concentrations between 0.05 and 32 wt %, the demixing temperature, T1, falls from ca. 65 degrees C to an almost constant value of 45 degrees C. Adding DEX to the BSA solutions reduces the homogeneous region of the mixture drastically where the amount of DEX required to lower T1 to 25 degrees C decreases rapidly as the concentration of BSA is raised. Experiments concerning the influences of shear have been performed for the ternary system up to 500 s(-1). They demonstrate that the content of dextran determines the sign of the effect. At low DEX concentrations, the mechanical field favors the homogeneous state (shear-induced mixing), whereas the opposite effect (shear-induced demixing) is observed at high DEX concentrations. Possible reasons for this observation are discussed.     

Molecular motions of polysaccharides in the solid state: dextran, pullulan and amylose.

Dextran, pullulan and amylose have been investigated by differential scanning calorimetry, thermogravimetric analysis, dynamic mechanical and dielectric spectroscopy over a wide range of temperatures and frequencies. No melting or glass transition is seen below the range of thermal degradation (about 300 degrees C) for either amylose or pullulan; only dextran shows a Tg at 223 degrees C (delta cp = 0.40 J/g deg). The viscoelastic spectrum of the 'dry' polysaccharides is characterized by a low temperature relaxation that occurs at -94, -73 and -59 degrees C, at 1 kHz, (activation energy 32, 39 and 52 kJ/mol) in dextran, pullulan and amylose respectively and is assigned to small entity local motions of the polysaccharide backbone. Absorbed water strongly modifies the relaxation spectrum, inducing a new relaxation below room temperature and dissipation regions associated with water loss above room temperature. The former appears at temperatures higher than the relaxation characteristic of the dry polymer and moves to lower temperature with increasing water content. In normal 'room humidity' conditions (about 10% absorbed water) the water-induced relaxation, attributed to the motion of complex polymer-water relaxing units, is the only observable feature in the dynamic mechanical and dielectric spectrum below room temperature.     

Use of holographic laser interferometry to study the diffusion of polymers in gels

The aim of this study was to demonstrate the potential for holographic interferometry to be used for diffusion studies of large molecules in gels. The diffusion and partitioning of BSA (67,000 g/mol) and pullulans (5,900-112,000 g/mol) in agarose gel were investigated. The gel diffusion coefficients obtained for BSA were higher when distilled water was used as a solvent compared to those obtained with 0.1 M NaCl as the solvent. Furthermore, the gel diffusion coefficient increased with increasing BSA concentration. The same trend was found for liquid BSA diffusion coefficients obtained by DLS. BSA partition coefficients obtained at different agarose gel concentrations (2-6%, w/w) decreased slightly with increasing gel concentration. However, all BSA gel diffusion coefficients measured were significantly lower than those in pure solvent and they decreased with increasing agarose concentration. The gel diffusion coefficients obtained for pullulans decreased with increasing pullulan molecular weight. The same effect from increased molecular weight was seen in the liquid diffusion coefficients measured by DLS. The pullulan partition coefficients obtained decreased with increasing molecular weight. However, pullulans with a larger Stokes' radius than BSA had partition coefficients that were higher or approximately the same as BSA. This implied that the pullulan molecules were more flexible than the BSA molecules. The results obtained for BSA in this study agreed well with other experimental studies. In addition, the magnitude of the relative standard deviation was acceptable and in the same range as for many other methods. The results thereby obtained showed that holographic interferometry is a suitable method for studying diffusion of macromolecules in gels.     

Flow birefringence of xanthan and other polysaccharide solutions

A xanthan sample with molecular weight M = 2.2 x 10(6) was investigated in three solvents: bidistilled water, 0.2 M aqueous NaCl and cadoxen by flow birefringence and viscometry methods in dilute solutions. It was shown that the optical shear rate coefficients of xanthan in aqueous and cadoxen media differ by two orders of magnitude. An estimation of xanthan optical anisotropy in different conformational states has been made and compared with values for other polysaccharides: dextran, pullulan, cellulose and chitosan. The process of denaturation and the flow birefringence of renaturated xanthan in aqueous solutions (after heat treatment at 121 degrees C) have also been studied.     

A rapid purification of rat alpha 1-foetoprotein by phase partition

The phase partition system dextran/polyethylene glycol (2 : 1, w/w) was chosen to separate rat alpha 1-foetoprotein from rat serum albumin, which is its main contaminant due to their having close physicochemical properties. The optimization of this method necessitated a systematic study of the behaviour of rat serum albumin in the system under consideration. This article describes the optimum conditions, in terms of pH, ionic strength and the concentration of polymer solutions, for the purification and recovery of alpha 1-foetoprotein. After a prepurification of rat foetal serum by CM-cellulose chromatography, a single partition step permitted the recovery of 15% of the total alpha 1-foetoprotein present in the rat serum. The purity of this alpha 1-foetoprotein was demonstrated by its binding parameters and by analytical gel electrophoresis.     

DWS microrheology of a linear polysaccharide

Diffusing wave spectroscopy has been used to measure the rheological behavior of pullulan (M(w) = 1 x 10(5)) aqueous solutions up to concentration of 40 g/dL. It was found that these solutions were mainly viscous, with the loss modulus G' higher than the elastic modulus G'. The plot of the specific viscosity eta(sp) as a function of pullulan concentration showed two critical concentrations c = 4 g/dL and c = 15 g/dL. For c < c, eta( sp) approximately c(1.25+/-0.05); for c < c < c, eta( sp) approximately c(2+/-0.05); and for c > c, eta( sp) approximately c(4.5+/-0.5). These results are in very good agreement with those reported in the literature.     

Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Synthesis, physicochemical and spectroscopic characterization of copper(II)-polysaccharide pullulan complexes by UV-vis, ATR-FTIR, and EPR

Bioactive copper(II) complexes with polysaccharides, like pullulan and dextran, are important in both veterinary and human medicine for the treatment of hypochromic microcitary anemia and hypocupremia. In aqueous alkaline solutions, Cu(II) ion forms complexes with the exopolysaccharide pullulan and its reduced low-molecular derivative. The metal content and the solution composition depend on pH, temperature, and time of the reaction. The complexing process begins in a weak alkali solution (pH >7) and involves OH groups of pullulan monomer (glucopyranose) units. Complexes of Cu(II) ion with reduced low-molecular pullulan (RLMP, M_w 6000 g mol⁻¹) were synthesized in water solutions, at the boiling temperature and at different pH values ranging from 7.5 to 12. The Cu(II) complex formation with RLMP was analyzed by UV–vis spectrophotometry and other physicochemical methods. Spectroscopic characterizations (ATR-FTIR, FT-IRIS, and EPR) and spectra–structure correlation of Cu(II)–RLMP complexes were also carried out.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Optimization of high molecular weight pullulan production by Aureobasidium pullulans in batch fermentations

Of five strains of Aureobasidium pullulans studied, NRRL Y-2311-1 yielded the highest titer (26.2 g/L) of pullulan and formed the lowest amount of melanin-like pigment. Sucrose was superior to glucose as the carbon and energy source on the basis of yield and titer of pullulan produced. Pullulan titer was higher (26.2 vs 5.1 g/L), biomass concentration was lower (6.9 vs 12.7 g/L), and DO was lower (0 vs 60% of saturation) when the fermenter was agitated by a marine propeller compared to Rushton impellers. Pullulan produced by strain NRRL Y-2311-1 ranged in weight-average molar mass (M(w)) from 486 KDa and number-average molar mass (M(n)) from 220 Da on day 1 of growth to 390 KDa and 690 Da on day 6; M(w) declined by about 35% from day 1 to day 3, the day of maximum pullulan titer. For the other strains, the ranges of molar mass on the day of maximum pullulan titer were 338-614 KDa (M(w)) and 100-6820 Da (M(n)).     

Synthesis and characterisation of dextran and pullulan sulphate

Dextrans and pullulans of different molar masses in the range of 10(4)-10(5) g/mol were sulphated via a SO3-pyridine complex. The degree of substitution achieved was DS = 2.4 and DS = 1.4 for dextran sulphate and DS = 2.0 and DS = 1.4 for pullulan sulphate, respectively. Confirmation of sulphation was given by FTIR spectroscopy. Asymmetrical S=O and symmetrical C-O-S stretching vibrations were detected at 1260 and 820 cm(-1). Reactivity of the polysaccharide C-atoms was determined by 13C NMR spectroscopy: For dextran this was C-3 > C-2 > C-4, while for pullulan it was C-6 > C-3 > C-2 > C-4.     

Alternating bioactivity of polymeric layer-by-layer assemblies: anticoagulation vs procoagulation of human blood

The layer-by-layer assembly between cationic chitosan and anionic dextran sulfate was analyzed quantitatively by a quartz crystal microbalance technique in the absence and presence of 0.2, 0.5, and 1 M NaCl in the polymer solution. The apparent film thickness increased upon increasing the NaCl concentration. The anti- versus procoagulant activity of these films against whole human blood was studied by the immersion of a substrate into blood for 30 min incubation time at 37 degrees C. The substrate was coated with films of varying NaCl concentrations and assembly step numbers. There was a critical concentration for the alternating activity; above a concentration of 0.5 M NaCl, both anti- and procoagulation could be observed on the dextran sulfate and chitosan surfaces, respectively. The underlying layer of the assembly was necessary for this alternating activity; after a five-step assembly, the activity was realized. The adsorption of a cationic dye (methylene blue) onto the films revealed that the anionic-charge density derived from dextran sulfate on the film surface was linearly increased with increased NaCl concentration. There was a critical charge density of the dextran sulfate for the anticoagulant activity. An assembly was also constructed from a combination of chitosan and heparin, but the activity was different from that of the former system; strong anticoagulant activity was observed even on the chitosan surface. We suggest that the polymer species and/or the assembly conditions are key factors for realizing the alternating bioactivities of films prepared by the layer-by-layer assembly.     

Determination of the molecular weight of microbial polysaccharides using high performance exclusion chromatography

An automatic method of determining the molecular weight parameters (Mw, Mn) of microbial polysaccharides such as dextran, pullulan was developed based on the use of high performance size-exclusion chromatography on the two types of columns: Zorbax PSM 60 + 300 + 1000 and SynChropack GPC 100 + 500 + 1000. The Mw and Mn values were determined for a number of domestic and foreign dextran preparations. Changes in the molecular weight of pullulan and hydroxyethylstarch resulted from acid and enzymatic hydrolysis were estimated.     

Swelling of Skinned Muscle Fibers of the Frog: Experimental Observations

Frog skeletal muscle fibers, mechanically skinned under water-saturated silicone oil, swell upon transfer to aqueous relaxing medium (60 mM KCl; 3 mM MgCl₂; 3 mM ATP; 4 mM EGTA; 20 mM Tris maleate; pH = 7.0; ionic strength 0.15 M). Their cross-sectional areas, estimated with an elliptical approximation, increase 2.32-fold (±0.54 SD). Sarcomere spacing is unaffected by this swelling. Addition of 200 mM sucrose to relaxing medium had no effect on fiber dimensions, whereas decreasing pH to 5.0 caused fibers to shrink nearly to their original (oil) size. Decreasing MgCl₂ to 0.3 mM caused fibers to swell 10%, and increasing MgCl₂ to 9 mM led to an 8% shrinkage. Increasing ionic strength to 0.29 M with KCl caused a 26% increase in cross-sectional area; decreasing ionic strength to 0.09 M had no effect. Swelling pressure was estimated with long-chain polymers, which are probably excluded from the myofilament lattice. Shrinkage in dextran T10 (number average mol wt 6,200) was transient, indicating that this polymer may penetrate into the fibers. Shrinkage in dextran T40 (number average mol wt 28,000), polyvinylpyrrolidone (PVP) K30 (number average mol wt 40,000) and dextran T70 (number average mol wt 40,300) was not transient, indicating exclusion. Maximal calcium-activated tension is decreased by 21% in PVP solutions and by 31% in dextran T40 solutions. Fibers were shrunk to their original size with 8 × 10^-2 g/cm³ PVP K30, a concentration which, from osmometric data, corresponds to an osmotic pressure (II/RT) of 10.5 mM. As discussed in the text, we consider this our best estimate of the swelling pressure. We find that increasing ionic strength to 0.39 M with KCl decreases swelling pressure slightly, whereas decreasing ionic strength to 0.09 M has no effect. We feel these data are consistent with the idea that swelling arises from the negatively charged nature of the myofilaments, from either mutual filamentary repulsion or a Donnan-osmotic mechanism.     

Effects of the high-molecular-weight cryoprotectant dextran on fluid secretion by Calliphora salivary glands

The rate of fluid secretion by isolated salivary glands of Calliphora was inhibited as a linear function of dextran and poly vinyl pyrrolidone concentrations in the range 15–35% w/w. This inhibition was not overcome by supramaximal concentrations of 5-hydroxytryptamine, nor was it caused by a decreased availability of K⁺ from the medium. Although the polymers caused large decreases of freezing point (and vapor pressure) of the incubation medium, the glands did not respond to this by secreting a more K ⁺-rich saliva. When dextran and polyvinyl pyrrolidone were added as powders to salt solutions, the total freezing-point depression of the mixture was equal to the sum of that exerted by the pure salt solution and that expected for the polymer concentration. The activities of K⁺ and Cl^?, as measured by ion-selective electrodes, were not increased in solutions by the addition of dextran. Dextran was demonstrated by electron microscopy to penetrate into the basal clefts and intercellular spaces of the isolated glands. These results demonstrate that addition of dextran (and probably of polyvinyl pyrrolidone) does not decrease the solvent activity of water in physiological salt solutions. The inhibition of fluid secretion by isolated salivary glands of Calliphora seems therefore due only to the altered physical characteristics of the medium.