Rheological evaluation of inter-grade and inter-batch variability of sodium alginate

Polymeric excipients are often the least well-characterized components of pharmaceutical formulations. The aim of this study was to facilitate the QbD approach to pharmaceutical manufacturing by evaluating the inter-grade and inter-batch variability of pharmaceutical-grade polymeric excipients. Sodium alginate, a widely used polymeric excipient, was selected for evaluation using appropriate rheological methods and test conditions. The materials used were six different grades of sodium alginate and an additional ten batches of one of the grades. To compare the six grades, steady shear measurements were conducted on solutions at 1%, 2%, and 3% w/w, consistent with their use as thickening agents. Small-amplitude oscillation (SAO) measurements were conducted on sodium alginate solutions at higher concentrations (4–12% w/w) corresponding to their use in controlled-release matrices. In order to compare the ten batches of one grade, steady shear and SAO measurements were performed on their solutions at 2% w/w and 8% w/w, respectively. Results show that the potential interchangeability of these different grades used as thickening agents could be established by comparing the apparent viscosities of their solutions as a function of both alginate concentration and shear conditions. For sodium alginate used in controlled-release formulations, both steady shear behavior of solutions at low concentrations and viscoelastic properties at higher concentrations should be considered. Furthermore, among batches of the same grade, significant differences in rheological properties were observed, especially at higher solution concentrations. In conclusion, inter-grade and inter-batch variability of sodium alginate can be determined using steady shear and small-amplitude oscillation methods.     

Inter-Grade and Inter-Batch Variability of Sodium Alginate Used in Alginate-Based Matrix Tablets

The purpose of this study is to characterize the inter-grade and inter-batch variability of sodium alginate used in the formulation of matrix tablets. Four different grades and three batches of one grade of sodium alginate were used to prepare matrix tablets. Swelling, erosion, and drug release tests of sodium alginate matrix tablets were conducted in a USP dissolution apparatus. Substantial differences in swelling and erosion behavior of sodium alginate matrix tablets were evident among different viscosity grades. Even different batches of the same grade exhibit substantial differences in the swelling and erosion behavior of their matrix tablets. The erosion behavior of sodium alginate matrix tablets can be partly explained by their rheological properties (both apparent viscosity and viscoelasticity) in solution. Sodium alginate with higher apparent viscosity and viscoelasticity in solution show slower erosion rate and higher swelling rate. Compacts prepared from grades or batches with higher viscosity and higher viscoelasticity show slower drug release. For grades or batches with similar apparent viscosities, apparent viscosities of sodium alginate solution at low concentration alone are not sufficient to predict the functionality of sodium alginate in matrix tablets. Viscoelastic properties of sodium alginate solutions at one high concentration corresponding to the polymer gel state, may be suitable indicia of the extended release behavior of sodium alginate matrix tablets.     

Alginate immobilization of recombinant Escherichia coli whole cells harboring l-arabinose isomerase for l-ribulose production

Recombinant Escherichia coli whole cells harboring Bacillus licheniformis l-arabinose isomerase (BLAI) were immobilized with alginate. The operational conditions for immobilization were optimized with response surface methodology. Optimal alginate concentration, Ca²⁺ concentration, and cell mass loading were 1.8% (w/v), 0.1 M, and 44.5 g L⁻¹, respectively. The interactions between Ca²⁺ concentration, alginate concentration, and initial cell mass were significant. After immobilization of BLAI, cross-linking with 0.1% glutaraldehyde significantly reduced cell leakage. The half-life of immobilized whole cells was 150 days, which was 50-fold longer than that of free cells. In seven repeated batches for l-ribulose production, the productivity was as high as 56.7 g L⁻¹ h⁻¹ at 400 g L⁻¹ substrate concentration. The immobilized cells retained 89% of the initial yield after 33 days of reaction. Immobilization of whole cells harboring BLAI, therefore, makes a suitable biocatalyst for the production of l-ribulose, particularly because of its high stability and low cost.     

Rheological Behavior of Food Emulsions Mixed with Saliva: Effect of Oil Content, Salivary Protein Content, and Saliva Type

In this paper, we studied the effect of saliva on the rheological properties of β-lactoglobulin- and lysozyme-stabilized emulsions, prepared at pH = 6.7 in relation to variation of emulsions- and saliva-related parameters. The effect of oil–volume fraction (2.5% w/w to 10% w/w), salivary protein concentration (0.1 to 0.8 mg ml⁻¹), and the use of both stimulated and unstimulated saliva was investigated. Viscosity and storage modulus were measured before (η _emul and G′_emul, respectively) and after addition of saliva (η _mix and G′_mix). To better estimate the changes due to saliva-induced flocculation of the emulsions, the ratios η _mix/η _emul, G′_mix/G′_emul were calculated. In addition, tan δ (=the ratio of the loss and storage moduli) was investigated to evaluate the viscoelastic behavior of the emulsion/saliva mixtures. Increasing the oil–volume fraction and salivary protein concentration resulted in an increase in η _mix/η _emul and G′_mix/G′_emul, while a decrease in tan δ of the emulsion/saliva mixtures is occurring. When compared with unstimulated saliva, mixing β-lactoglobulin-stabilized emulsions with stimulated saliva led to a reduction in η _mix/η _emul and G′_mix/G′_emul, and an augment of tan δ at all measured deformations. In case of lysozyme-stabilized emulsions, the use of stimulated saliva increased G′_mix/G′_emul for γ < 3 when compared to unstimulated saliva. The effect of stimulated saliva on the η _mix/η _emul and tan δ in this mixture is similar to that of unstimulated saliva. These results indicate that the influence of stimulated saliva on the rheological parameters of emulsion/saliva mixtures largely depends on the type of emulsions. To conclude, our findings demonstrate that the rheological behavior of emulsions upon mixing with saliva is greatly affected by both saliva and emulsion properties.     

Alginate production by <Emphasis Type="Italic">Pseudomonas mendocina</Emphasis> in a stirred draft fermenter

In this study alginate production by Pseudomonas mendocina in a laboratory-scale fermenter was investigated. In the experiments the effect of temperature (25–31°C) and agitation (500–620 rev min⁻¹) at a constant air flow of 10 v/v/h were evaluated in relation to the rate of glucose bioconversion to alginate using response surface methodology (RSM). The fermenter configuration was also adapted to a system with a screw mixer and draft tube, due to the change in rheological characteristics of the fermentation broth. The adjusted model indicates a temperature of 29.1°C and agitation of 553 rev min⁻¹ for optimum alginate synthesis. In this fermentation system a Y _p/s of 44.8% was achieved. The alginate synthesized by P. mendocina showed a partially acetylated pattern as previously reported for alginates obtained from other Pseudomonas spp and Azotobacter vinelandii.     

<Emphasis Type="Italic">In Situ</Emphasis> Gelling Gelrite/Alginate Formulations as Vehicles for Ophthalmic Drug Delivery

The objective of this study was to develop an ion-activated in situ gelling vehicle for ophthalmic delivery of matrine. The rheological properties of polymer solutions, including Gelrite, alginate, and Gelrite/alginate solution, were evaluated. In addition, the effect of formulation characteristics on in vitro release and in vivo precorneal drug kinetic of matrine was investigated. It was found that the optimum concentration of Gelrite solution for the in situ gel-forming delivery systems was 0.3% (w/w) and that for alginate solution was 1.4% (w/w). The mixture of 0.2% Gelrite and 0.6% alginate solutions showed a significant enhancement in gel strength at physiological condition. On the basis of the in vitro results, the Gelrite formulations of matrine-containing alginate released the drug most slowly. For each tested polymer solution, the concentration of matrine in the precorneal area was higher than that of matrine-containing simulated tear fluid (STF) almost at each time point (p < 0.05). The area under the curve of formulation 16 (0.2%Gelrite/0.6%alginate) was 4.65 times greater than that of containing matrine STF. Both the in vitro release and in vivo pharmacological studies indicated that the Gelrite/alginate solution had the better ability to retain drug than the Gelrite or alginate solutions alone. The tested formulation was found to be almost non-irritant in the ocular irritancy test. The overall results of this study revealed that the Gelrite/alginate mixture can be used as an in situ gelling vehicle to enhance ocular retention.     

Photobiochemistry of folates: A photochemical reduction of folic acid

Exposure of deaerated folic acid solutions containing an electron donor to UV radiation (310–390 nm, I = 0.4 W m⁻²) induced formation of dihydrofolic acid (DHFA), a photoexcitation which gave tetrahydrofolic acid (THFA). Only DHFA was formed in the presence of EDTA (E′_o = +0.40 V), while the presence of stronger reductants—NADH (E′_o = −0.32 V) and boron hydride (E′_o = −0.48 V)—induced photoreduction to THFA. It was demonstrated that UV radiation had no effect on the THFA formylation, giving the coenzyme 5,10-methenyltetrahydrofolic acid and its transformation into another coenzyme, 5-formyltetrahydrofolic acid.     

The Surface Display of the Alginate Lyase on the Cells of Yarrowia lipolytica for Hydrolysis of Alginate

The alginate lyase structural gene (AlyVI gene) was amplified from plasmid pET24-ALYVI carrying the alginate lyase gene from the marine bacterium Vibrio sp. QY101 which is a pathogen of Laminaria sp. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying the alginate lyase could form clear zone on the plate containing sodium alginate, indicating that they had high alginate lyase activity. The cells displaying alginate lyase can be used to hydrolyze poly-β-d-mannuronate (M) and poly-α-l-guluronate (G) and sodium alginate to produce different lengths of oligosaccharides (more than pentasaccharides). This is the first report that the yeast cells displaying alginate lyase were used to produce different lengths of oligosaccharides from alginate.     

Effect of Sodium Alginate Addition on Physical Properties of Rennet Milk Gels

Effect of addition of sodium alginate (alginate) to milk on the storage modulus (G′), water holding capacity (WHC) and hardness of rennet gels was evaluated as a function of alginate (0–0.25 g/100 g) and fat (0.5–3.0 g/100 g) concentrations. There was a significant effect of alginate addition on ionic calcium in milk and whey (Ca²⁺), and particle size distribution in alginate added milk. Results showed a positive correlation of alginate with WHC; negative correlation of alginate and positive correlation of fat with G′; and negative correlation of interaction of fat and alginate with gel hardness of rennet gels. Hence, the rennet gels with lower fat content and higher added alginate tended to be softer due to the high water holding capacity of the alginate particles.     

Investigation of the Effects of Hydroalcoholic Solutions on Textural and Rheological Properties of Various Controlled Release Grades of Hypromellose

Hypromellose (hydroxypropyl methylcellulose, HPMC) matrices are widely used in the formulation of sustained release dosage forms. The integrity and performance of an HPMC matrix formulation depends on rapid hydration and gel formation upon ingestion. Due to the recent alert issued by the Food and Drug Administration regarding the potential negative influence of alcoholic beverages on extended release (ER) formulations, several researchers have evaluated the potential influence of hydroalcoholic media on drug release from ER dosage forms. It has been reported that HPMC matrix formulations do not show “dose dumping” in hydroalcoholic media. The purpose of this study was a fundamental investigation on the effect of hydroalcoholic solutions (0–40% v/v ethanol) on textural and rheological properties of different viscosity grades of neat HPMC, as the functional ingredient within a hydrophilic matrix. In general, hydroalcoholic solutions had little effect on gel formation and mechanical properties of hydrated compacts, while the rheological behavior of HPMC showed dependency on the ethanol content of such solutions.     

Extraction and characterization of an alginate from the brown seaweed <Emphasis Type="Italic">Sargassum turbinarioides</Emphasis> Grunow

Matrix polysaccharide from the brown algae Sargassum turbinarioides collected in the coastal waters of Nosy Be (Madagascar) in the Indian Ocean was isolated and its structure was studied by ¹H-NMR spectroscopy, FT-IR, SEC-MALLS and HPAEC. An alginate with a molecular weight of 5.528 × 10⁵ g mol⁻¹ was identified as sole polysaccharide. Values of the M/G ratio, F _GG, F _MM and F _GM (or F _GM) blocks were measured at respectively 0.94, 0.39, 0.36 and 0.25 and compared with those of alginates from other Sargassum species. This sodium alginate appeared similar to some of the other Sargassum alginates with M/G < 1, high values of homopolymeric blocks (η < 1) and significant polyguluronic block content.     

The Effect of Shearing in the Thermo-mechanical Properties of Candelilla Wax and Candelilla Wax�CTripalmitin Organogels

We investigated the shearing effect in the thermo-mechanical properties of candelilla wax (CW) and candelilla wax–tripalmitin (CW-TP) organogels developed using safflower oil as the liquid phase. A shear rate of 600 s⁻¹ was applied during cooling (1 °C/min) of 3% CW and 3% CW-1% TP solutions, until reaching nucleation (i.e., 47 °C) or metastable conditions (i.e., 52 °C) then allowing the development of the organogel under quiescent conditions at 15 °C. The thermal and rheological properties of these organogels were compared with the ones of organogels developed under static conditions. The X-ray results showed that under static conditions the CW crystallized in an orthorhombic ( O _^ ) \left( {{{\hbox{O}}_{ \bot }}} \right) subcell packing with a lateral stacking with a length (L) equivalent to the hydrocarbon chain’s length. During organogelation of the 3% CW-1% TP solution, CW and TP co-crystallized in a O _^ {{\hbox{O}}_{ \bot }} subcell packing with TP in a 2L stacking organization. The application of shearing did not modify the molecular packing of the crystals or the melting properties of the organogels. However, the CW and CW-TP organogels developed with shearing showed the formation of larger crystals (i.e., fiber-like or plate-like) in comparison with the ones developed under static conditions. The presence of 1% TP increased the solid fat content, and thus the CW-TP organogels showed higher elasticity (G′) than CW organogels. The organogels developed with the application of shearing until reaching metastable conditions (i.e., CW 52 °C and CW-TP 52 °C) had the highest G′ and yield stress of all organogels investigated. In the same way, creep and recovery analyses showed that CW 52 °C and CW-TP 52 °C organogels had the highest resistance to deformation, lowest instantaneous recovery, and highest extended recovery of all organogels studied. We concluded that application of shear and the extent of its application as a function of supercooling determine crystal size and the proportion of transient to junction zones throughout the three-dimensional crystal network and, therefore, the organogels' rheological properties.     

Phosphomannose-isomerase (<Emphasis Type="Italic">pmi</Emphasis>) gene as a selectable marker for <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of rapeseed

Nodal segments (4 ± 1 mm long) of Hibiscus moscheutos (hardy hibiscus) were excised from in vitro proliferating microshoots and utilized to evaluate initial factors involved in bulk alginate encapsulation. The factors evaluated were; storage vessel type, volume and multiple rinse effects of CaCl₂ solutions, and sodium alginate concentrations (2.5, 2.75, 3.0 or 3.25%) for bulk alginate encapsulation. Results indicate that vessels utilized for bulk alginate encapsulation should have a lower base area (L × W) to height ratio to reduce the amount of alginate matrix shrinkage resulting in exposure of nodal segments. Increased volumes and multiple 50 mM CaCl₂ solution rinses did not have an effect on alginate solidification. Shoot length, root number, and root length significantly decreased in a linear manner from nodal explants stored for 4 weeks with increasing concentrations of sodium alginate. This research suggests an innovative technique for alginate encapsulation of H. moscheutos utilizing bulk methods as an alternative to single bead alginate encapsulation.     

In vitro investigation and biomechanical modeling of the effects of PLF-68 on osteoarthritis in a three-dimensional model

In this study, it was hypothesized that Pluronic F-68 (PLF-68) increases matrix synthesis of osteoarthritis (OA) chondrocytes in addition to its well-documented cell survival effect. To test this hypothesis, rat articular chondrocytes were embedded in agarose discs and were exposed to 5-azacytidine (Aza-C) to induce OA-like alterations. Chondrocytes were then treated with PLF-68 (8 and 12 mg/ml) for 10 days. Aza-C-exposed and PLF-68-untreated chondrocytes and Aza-C-unexposed and PLF-68-untreated chondrocytes were used as negative and positive control groups, respectively. Dynamic hydrostatic pressure (max 0.2 MPa, 0.1 Hz) was applied to discs for 30 min/day (5 days/week). Cell viability, collagen and proteoglycan deposition in discs were determined. Unconfined compression stress relaxation tests were performed to determine peak stress and material parameters of discs—namely spring constants (k ₁ and k ₂), damping coefficient (η), instantaneous modulus (E ₀) and relaxed modulus (E _∞) using Kelvin model to evaluate the functional coherence of the matrix. PLF-68 treatment significantly increased the collagen deposition in discs and viability of OA-like chondrocytes. A dose-dependent increase was also observed for elastic stiffness parameters (k ₁, k ₂, E ₀ and E _∞). Same positive effect of PLF-68 was not observed for proteoglycan deposition. However, dose-dependent increase in η suggests that PLF-68 treatment resulted with the deposition of functional matrix. This is the first study which reports that PLF-68 has also positive effect on collagen synthesis of OA cells. As a conclusion, our results suggest that PLF-68 has a potential for recovery from OA-like alterations, which should be further analyzed.     

Alginate molecular mass produced by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> in response to changes of the O<Subscript>2</Subscript> transfer rate in chemostat cultures

Alginate production by Azotobacter vinelandii growing in chemostat cultures was evaluated under different O₂ transfer rates (OTR). As a result of modifying the culture’s agitation rate from 300 to 500 rpm, the OTR increased from 9 to 15.1 mmol l⁻¹ h⁻¹ and a slight variation in the alginate production (1.7–2.2 g l⁻¹) was observed. At a constant growth rate (0.1 h⁻¹), the mean molecular mass of the alginate was strongly influenced by changes in the OTR, varying from 860 to 1,690 kDa. These results support a possible relationship between alginate polymerization-depolymerization process and the O₂ uptake rate.     

Alginate production by Azotobacter vinelandii DSM576 in batch fermentation

The kinetics of growth and alginate production from glucose in a nitrogen and phosphate-rich medium by Azotobacter vinelandii DSM576 were studied in a laboratory fermenter at pH 7 and 35°C. Batch fermentations were carried out both without control of dissolved oxygen concentration (DO) and at 1, 2, 5 and 10% DO. Although growth was faster at higher DO, maximum biomass concentration was lower. No alginate was produced at 10% DO. Alginate production was faster at 5 and 2% DO but higher alginate concentrations and yields were obtained without DO control. Alginate production was growth-associated at 5% DO, but significant amounts of alginate were produced after growth had stopped at lower DO values. In fermentations without DO control the molecular weight of the polymer reached a maximum (11–17.6 × 10⁴) when specific growth rate was between 0.02 and 0.04 h⁻¹ and residual concentration of ammoniacal nitrogen was between 0.01 and 0.02 g L⁻¹ and then sharply decreased. Received 15 August 1997/ Accepted in revised form 08 January 1998     

Propagation of three orchid genera using encapsulated protocorm-like bodies

Summary Protocorm-like bodies (plbs) of the orchid Dendrobium ‘Sonia’ were obtained from fractionated plb explants on Murashiga and Skoog (1962) medium (MS) supplemented with 4.44 μM 6-benzylaminopurine (BA). The developmental stage of plbs to be encapsulated, concentrations of sodium alginate (2–5%) and calcium chloride (25–100 mM), and concentration of MS salts in the matrix were assessed. Fractionated plbs 13–15 d after culture were at the leaf primordia stage and found suitable for encapsulation studies. The best encapsulation response was observed with 3% sodium alginate upon complexation with 75 mM CaCl₂.2H₂O. An encapsulation matrix prepared with MS medium (three-quarter strength) supplemented with 0.44 μM BA and 0.54 μM α-naphthaleneacetic acid gave 100% conversion of encapsulated plbs to plants after storage. A viability of >85% of encapsulated plbs of Dendrobium ‘Sonia’ was observed for 75 d at 4°C. Encapsulated plbs of orchids Dendrobium, Oncidium, and Cattleya were stored at 4°C for 75, 60 and 30 d, respectively, with >88% germination. All plants survived potting in media having either wood charcoal pieces alone or in combination with brick pieces.     

Physicochemical and rheological properties of a novel emulsifier,EPS-R,produced by the marine bacterium<Emphasis Type="Italic">Hahella chejuensis</Emphasis>

The rheological properties of an exopolysaccharide, EPS-R, produced by the marine bacteriumHahella chejuensis strain 96CJ10356 were investigated. The E₂₄ of 0.5% EPS-R was 89.2%, which was higher than that observed in commercial polysaccharides such as xanthan gum (67.8%), gellan gum (2.01%) or sodium alginate (1.02%). Glucose and galactose are the main sugars in EPS-R, with a molar ratio of ∼1∶6.8, xylose and ribose are minor sugar components. The average molecular mass, as determined by gel filtration chromatography, was 2.2×10³ KDa. The intrinsic viscosities of EPS-R were calculated to be 16.5 and 15.9 dL/g using the Huggins and Kraemer equations, respectively, with a 2.3 dL/g overlap. In terms of rigidity, the conformation of EPS-R was similar to that of caboxymethyl cellulose (5.0×10⁻²). The rheological behavior of EPS-R dispersion indicated that the formation of a structure intermediate between that of a random-coil polysaccharide and a weak gel. The aqueous dispersion of EPS-R at concentrations ranging from 0.25 to 1.0% (w/w) showed a marked shear-thinning property in accordance with Power-law behavior. In aqueous dispersions of 1.0% EPS-R, the consistency index (K) and flow behavior index (n) were 1,410 and 0.73, respectively. EPS-R was stable to pH and salts.     

Surface Rheology Parameters of Source-Specific Surfactant Films as Indicators of Organic Matter Dynamics

The paper contains the results of natural film experiments carried out on inland and coastal waters in the Dead Vistula catchment area and mouth during 2000–2002, using the integrated Langmuir trough–Wilhelmy plate system. The static film parameters result from the generalized scaling procedures applied to the surface pressure–area isotherms. They appear to reflect in a quantitative and sensitive way the film composition (A_lim, M_w, E_isoth), film solubility and the miscibility of its components (via R, ΔS_c and y factors), and surface concentration (π_eq, Γ_eq). The adsorption kinetics parameters: effective diffusion coefficient D_eff/D and activation energy barrier E_a/RT are derived from dynamic surface pressure. There is a reason to suggest that certain classes of film-forming components or ‘end-members’ may dominate the static and dynamic surface properties. Variation in the surface rheological parameters of source-specific biosurfactants is postulated to reflect organic matter dynamics in natural waters and were measured for the Dead Vistula river, its tributaries and the adjacent coastal area.     

Transient changes in transpiration, and stem and soil CO2 efflux in longleaf pine (Pinus palustris Mill.) following fire-induced leaf area reduction

In 20-year-old longleaf pine, we examined short-term effects of reduced live leaf area (A _L) via canopy scorching on sap flow (Q; kg H₂O h⁻¹), transpiration per unit leaf area (E _L; mm day⁻¹), stem CO₂ efflux (R _stem; μmol m⁻² s⁻¹) and soil CO₂ efflux (R _soil; μmol m⁻² s⁻¹) over a 2-week period during early summer. R _stem and Q were measured at two positions (1.3-m or BH, and base of live crown—BLC), and R _soil was measured using 15 open-system chambers on each plot. E _L before and after treatment was estimated using Q measured at BLC with estimates of A _L before and after scorching. We expected Q to decrease in scorched trees compared with controls resulting from reduced A _L. We expected R _stem at BLC and BH and R _soil to decrease following scorching due to reduced leaf area, which would decrease carbon supply to the stem and roots. Scorching reduced A _L by 77%. Prior to scorching, Q at BH was similar between scorch and control trees. Following scorching, Q was not different between control and scorch trees; however, E _L increased immediately following scorching by 3.5-fold compared to control trees. Changes in E _L in scorched trees corresponded well with changes in VPD (D), whereas control trees appeared more decoupled over the 5-day period following treatment. By the end of the study, R _stem decreased to 15–25% in scorched trees at both stem positions compared to control trees. Last, we found that scorching resulted in a delayed and temporary increase in R _soil rather than a decrease. No change in Q and increased E _L following scorching indicates a substantial adjustment in stomatal conductance in scorched trees. Divergence in R _stem between scorch and control trees suggests a gradual decline in stem carbohydrates following scorching. The absence of a strong R _soil response is likely due to non-limiting supplies of root starch during early summer.