Ronald J. Neufeld

Production of alginate beads by emulsification/internal gelation. I. Methodology

SummarySmall diameter alginate beads (microspheres) were formed via internal gelation of alginate solution emulsified within vegetable oil. Gelation was initiated by addition of an oil-soluble acid thereby reducing the pH of the alginate solution and releasing soluble Ca2+ from the citrate complex. Smooth, spherical, micron-sized beads were formed. The mean diameter ranged from 200 to 1000 μm, controlled by the reactor impeller design and rotational speed. The technique has potential for large-scale and continuous applications in immobilization.

Immobilization of Cells for Application in the Food Industry

Immobilization of cells offers advantages to the food process industries, including enhanced fermentation productivity and cell stability and reduced downstream processing costs due to facilitated cell recovery and recycle. This article summarizes the varied immobilization methodologies, including adsorption, entrapment, covalent binding, and microencapsulation. Examples of interest to the food industry are provided, together with a review of the physiological effects of immobilization. Topics in process engineering include immobilized cell bioreactor configurations and the scale-up potential of the various immobilization techniques.

Microencapsulation of lipophilic drugs in chitosan-coated alginate microspheres

Chitosan-coated alginate microspheres containing a lipophilic marker dissolved in an edible oil, were prepared by emulsification/internal gelation and the potential use as an oral controlled release system investigated. Microsphere formation involved dispersing a lipophilic marker dissolved in soybean oil into an alginate solution containing insoluble calcium carbonate microcrystals. The dispersion was then emulsified in silicone oil to form an O/W/O multiple phase emulsion. Addition of an oil soluble acid released calcium from carbonate complex for gelation of the alginate. Chitosan was then applied as a membrane coat to increase the mechanical strength and stabilize the microspheres in simulated intestinal media. Parameters studied included encapsulation yield, alginate concentration, chitosan molecular weight and membrane formation time. Mean diameters ranging from 500 to 800 micron and encapsulation yields ranging from 60 to 80% were obtained. Minimal marker release was observed under simulated gastric conditions, and rapid release was triggered by transfer into simulated intestinal fluid. Higher overall levels of release were obtained with uncoated microspheres, possibly due to binding of marker to the chitosan membrane coat. However the slower rate of release from coated microspheres was felt better suited as a delivery vehicle for oil soluble drugs.

Microencapsulation of DNA within alginate microspheres and crosslinked chitosan membranes for in vivo application.

Calf thymus DNA was microencapsulated within crosslinked chitosan membranes, or immobilized within chitosan-coated alginate microspheres. Microcapsules were prepared by interfacial polymerization of chitosan, and alginate microspheres formed by emulsification/ internal gelation. Diameters ranged from 20 to 500 Μm, depending on the formulation conditions. Encapsulated DNA was quantifiedin situ by direct spectrophotometry (260 nm) and ethidium bromide fluorimetry, and compared to DNA measurements on the fractions following disruption and dissolution of the microspheres. Approximately 84% of the DNA was released upon core dissolution and membrane disruption, with 12% membrane bound. The yield of encapsulation was 96%. Leakage of DNA from intact microspheres/capsules was not observed. DNA microcapsules and microspheres were recovered intact from rat feces following gavage and gastrointestinal transit. Higher recoveries (60%) and reduced shrinkage during transit were obtained with the alginate microspheres. DNA was recovered and purified from the microcapsules and microspheres by chromatography and differential precipitation with ethanol. This is the first report of microcapsules or microspheres containing biologically active material (DNA) being passed through the gastrointestinal tract, with the potential for substantial recovery.

External versus internal source of calcium during the gelation of alginate beads for DNA encapsulation

Alginate gels produced by an external or internal gelation technique were studied so as to determine the optimal bead matrix within which DNA can be immobilized for in vivo application. Alginates were characterized for guluronic/mannuronic acid (G/M) content and average molecular weight using 1H-NMR and LALLS analysis, respectively. Nonhomogeneous calcium, alginate, and DNA distributions were found within gels made by the external gelation method because of the external calcium source used. In contrast, the internal gelation method produces more uniform gels. Sodium was determined to exchange for calcium ions at a ratio of 2:1 and the levels of calcium complexation with alginate appears related to bead strength and integrity. The encapsulation yield of double-stranded DNA was over 97% and 80%, respectively, for beads formed using external and internal calcium gelation methods, regardless of the composition of alginate. Homogeneous gels formed by internal gelation absorbed half as much DNAse as compared with heterogeneous gels formed by external gelation. Testing of bead weight changes during formation, storage, and simulated gastrointestinal (GI) conditions (pH 1.2 and 7.0) showed that high alginate concentration, high G content, and homogeneous gels (internal gelation) result in the lowest bead shrinkage and alginate leakage. These characteristics appear best suited for stabilizing DNA during GI transit.

Calcium-alginate beads coated with polycationic polymers: Comparison of chitosan and DEAE-dextran

Abstract The retention capacity of alginate beads coated by two polycationic polymers, chitosan or diethylaminoethyl-dextran (DEAE-dextran) was studied. When beads were stored in water or in a 0·9% NaCl solution, the chitosan coating was stable for more than 5 months or less than 2 months, respectively. The greater the concentration of the polycationic polysaccharides in the formation solution of the beads, the lower the haemoglobin release. Haemoglobin release depended on the pH conditions of bead formation. With chitosan, the lowest release was observed at pH 5·4 (4·4% of released haemoglobin) and at pH 2 (1·5%), respectively, during bead formation and storage. With DEAE-dextran the lowest release was observed at pH 4 (10%).

Investigation of the mechanism of metal uptake by denatured Rhizopus arrhizus biomass

Chemical treatments to denatured Rhizopus arrhizus biomass were found to cause reductions in metal uptake capacities as high as 60%. Phosphate and carboxyl groups were identified as important moeities involved in metal binding. Scatchard analyses of uptake isotherms of native biomass confirm the existence of more than one type of binding site. An uptake model involving binding to a multiplicity of nonequivalent sites is proposed. The primary interactions are due to a complexation mechanism involving sites in the biomass containing carboxylate, phosphate, and other functional groups. Uptake may be enhanced by electrostatic attraction to negatively charged functional groups, but this is a secondary mechanism.

DNA protection from extracapsular nucleases, within chitosan‐ or poly‐L‐lysine‐coated alginate beads

DNA was immobilized within alginate matrix using an external or an internal calcium source, and then membrane coated with chitosan or poly-L-lysine. Membrane thickness increased with decreasing polymer molecular weight and increasing degree of deacetylation (chitosan). Beads were exposed to a 31,000 molecular weight nuclease to determine the levels of DNA protection offered by different membrane and matrix combinations. Almost total hydrolysis of DNA was observed in alginate beads following nuclease exposure. Less than 1% of total double-stranded DNA remained unhydrolyzed within chitosan- or poly-L-lysine-coated beads, corresponding with an increase in DNA residuals (i.e. double- and single-stranded DNA, polynucleotides, bases). Chitosan membranes did not offer sufficient DNA protection from DNase diffusion since all of the double-stranded DNA was hydrolyzed after 40 min of exposure. Both chitosan and poly-L-lysine membranes reduced the permeability of alginate beads, shown by enhanced retention of DNA residuals after DNase exposure. The highest level of DNA protection within freshly prepared beads was obtained with high molecular weight (197,100) poly-L-lysine membranes coated on beads formed using an external calcium source, where over 80% of the double-stranded DNA remained after 40 min of DNase exposure. Lyophilization and rehydration of DNA beads also reduced permeability to nucleases, resulted in DS-DNA recoveries of 60% for chitosan-coated, 90% for poly-L-lysine-coated, and 95% for uncoated alginate beads.

Production of alginate beads by emulsification/internal gelation. II. Physicochemistry

Alginate microspheres were produced by emulsification/internal gelation of alginate sol dispersed within vegetable oil. Gelification was initiated within the alginate sol by a reduction in pH (7.5 to 6.5), releasing calcium from an insoluble complex. Smooth, spherical beads with the narrowest size dispersion were obtained when using low-guluronic-acid and low-viscosity alginate and a carbonate complex as the calcium vector. A more finely dispersed form of the complexed calcium within the alginate sol promotes a more homogeneous gelification. Microsphere mean diameters ranging from 50 μm to 1000 μm were obtained with standard deviations ranging from 35% to 45% of the mean.

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca‐alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell‐free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from105 cfu ml−1 to 103 cfu ml−1 after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non‐immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.