Characterization of calcium alginate and chitosan-treated calcium alginate gel beads entrapping allyl isothiocyanate

Calcium alginate (CA), chitosan-coated calcium alginate (CCA-I), and chitosan–calcium alginate complex (CCA-II) gel beads, in which an oil-in-water emulsion containing allyl isothiocyanate (AITC) was entrapped, were prepared and characterized for efficient oral delivery of AITC. The AITC entrapment efficiency was 81% for CA gel beads, whereas about 30% lower values were determined for the chitosan-treated gel beads. Swelling studies showed that all the gel beads suddenly shrunk in simulated gastric fluid (pH 1.2). In simulated intestinal fluid (pH 7.4), CA and CCA-I gel beads rapidly disintegrated, whereas CCA-II gel beads highly swelled without degradation probably due to the strong chitosan–alginate complexation. Release studies revealed that most entrapped AITC was released during the shrinkage, degradation, or swelling of the gel beads, and the chitosan treatments, especially the chitosan–alginate complexation, were effective in suppressing the release. CCA-II gel beads showed the highest bead stability and AITC retention under simulated gastrointestinal pH conditions.     

Phase separation in calcium alginate gels

Alginates are polysaccharides consisting of beta-D-mannuronate and alpha-L-guluronate units. In the presence of bivalent cations like calcium the guluronate blocks form physically cross-linked gels. The gelation properties of alginates play an important role in the stability of extracellular polymer substances and in the food industry. When stock solutions of Ca2+ ions and alginate are mixed, the gelation starts before the Ca2+ ions are evenly distributed, which leads to non-uniform gels. In this contribution, Ca alginate gels were prepared by in situ gelation using glucono-delta-lactone and CaCO3. In this way, uniform gels could be prepared directly in the measuring cell. Below a critical concentration, highly viscous solutions were obtained, which were below the critical point of gel formation. In these solutions at low rotational speeds a Schlieren peak arose, which became smaller and steeper with increasing time until a new meniscus could be detected. This behaviour is in contrast to the peak broadening due to diffusion after a synthetic boundary was formed. Evaluation of the data leads to negative diffusion coefficients. It has been shown by others that the mutual diffusion coefficient must be negative in the spinodal region. This phenomena is known as uphill diffusion and leads to phase separation of a binary system. The formation of the gel phase in this case is therefore discussed as uphill diffusion.     

Construction of bioengineered yeast platform for direct bioethanol production from alginate and mannitol

Brown macroalgae are a sustainable and promising source for bioethanol production because they are abundant in ocean ecosystems and contain negligible quantities of lignin. Brown macroalgae contain cellulose, hemicellulose, mannitol, laminarin, and alginate as major carbohydrates. Among these carbohydrates, brown macroalgae are characterized by high levels of alginate and mannitol. The direct bioconversion of alginate and mannitol into ethanol requires extensive bioengineering of assimilation processes in the standard industrial microbe Saccharomyces cerevisiae. Here, we constructed an alginate-assimilating S. cerevisiae recombinant strain by genome integration and overexpression of the genes encoding endo- and exo-type alginate lyases, DEH (4-deoxy-l-erythro-5-hexoseulose uronic acid) transporter, and components of the DEH metabolic pathway. Furthermore, the mannitol-metabolizing capacity of S. cerevisiae was enhanced by prolonged culture in a medium containing mannitol as the sole carbon source. When the constructed strain AM1 was anaerobically cultivated in a fermentation medium containing 6% (w/v) total sugars (approximately 1:2 ratio of alginate/mannitol), it directly produced ethanol from alginate and mannitol, giving 8.8 g/L ethanol and yields of up to 32% of the maximum theoretical yield from consumed sugars. These results indicate that all major carbohydrates of brown macroalgae can be directly converted into bioethanol by S. cerevisiae. This strain and system could provide a platform for the complete utilization of brown macroalgae.     

Growth and survival of phosphate-solubilizing bacteria in calcium alginate

Calcium alginate was superior to conventional charcoal-soil (3:1) carrier for phosphate-solubilizing bacteria (PSB). High populations of Pseudomonas striata (27) and Bacillus polymyxa (H-5) could be maintained in this polymer during storage. Incorporation of charcoal-soil (3:1) adversely affected the initial loading of these organisms in alginate gel. Alginate alone supported maximum survival of these organisms at elevated storage temperature (40 degrees C).     

Incorporation of nisin in micro-particles of calcium alginate

Nisin was successfully incorporated into a matrix of calcium alginate and ground into micro-particles smaller than 150 μm. Formation of micro-particles and incorporation of nisin was verified by scanning electron microscopy and by the reduction in the inactivation of nisin activity with proteolytic enzymes. Incorporation efficiency was 87–93% and the nisin in the alginate-incorporated form was 100% active against an indicator culture of Lactobacillus curvatus both in MRS broth and reconstituted skim milk.     

Metabolism and calcium antagonism of sodium alginate oligosaccharides

Sodium alginate oligosaccharides (NaAOs) consisting of a mixture of eight oligosaccharides have previously been reported to lower blood pressure. We investigated in this study the excretion of NaAOs into the urine or feces, and attempted to elucidate the mechanism for lowering blood pressure by using isolated mesenteric arteries from the rabbit. The recovery rate of P8, which is the main component of NaAOs, was 5.2% and 58.9% over 48 hours in the urine and feces, respectively. The mechanism for lowering blood pressure appeared to be NaAOs having calcium antagonist activity, especially voltage-operated calcium channels. Our results suggest that NaAOs are substantially excreted into the feces, although some of them may be absorbed internally, exerting antagonist activity towards the calcium channels, especially voltage-operated calcium channels.     

Diffusion of proteases in calcium alginate beads.

Diffusion of proteases from Bacillus subtilis and Serratia marcescens within calcium alginate beads has been assayed, and the experimental data fitted into a mathematical model for diffusion into a finite volume liquid medium. Values of effective diffusivity were calculated for the studied proteases and compared with the available data in the literature for molecules of lower molecular weight.     

Characterization of nitrifying bacteria immobilized in calcium alginate

Activated sludge has been fed with a medium containing ammonium ions as the sole nitrogen source. Biomass collected from this continuous culture was immobilized in calcium alginate. The influence of pH, temperature, and the size and cell load of the biocatalyst beads on the nitrifying activity was determined, as well as the storage and operational stability of the system. The results are compared with those obtained with Nitrosomonas europaea. It has been concluded that the mixed culture is more difficult to work with than the pure strain and that the reproducibility of the results is lower. The trends found, however, were largely similar, except for the operational stability which was poorer in the case of the immobilized mixed culture.     

Characterization of Nitrosomonas europaea immobilized in calcium alginate

Nitrosomonas europaea cells have been immobilized in calcium alginate and the resulting preparation was used as a biocatalyst for the oxidation of NH⁺₄ to NO^?₂. Characterization of this immobilized biocatalyst was done according to the guidelines recommended by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The most important indications obtained from the results are: (a) at low concentrations of substrate, either ammonium ions or oxygen, diffusion limitation will play a role; (b) inhibition by nitrite ions accumulating in the support is not rapidly controlling the efficiency of the immobilized cells; (c) accumulation of hydrogen ions is a rate-limiting factor, especially in unbuffered solutions; (d) the activity of immobilized N. europaea can increase as a result of growth in the support under conditions which would cause washout of free cells. This last result shows the potential of immobilized N. europaea for nitrification of wastewater. The development of a system applying a cheaper and more stable support is, however, a prerequisite for this application.     

Optimization of alpha-amylase immobilization in calcium alginate beads

alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.     

Characterization of Gluconobacter oxydans immobilized in calcium alginate

Summary Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.     

Diffusion characteristics of calcium alginate gels.

The diffusivity of a protein solute (bovine serum albumin) within calcium alginate gels made from sodium alginate of different guluronic acid content was determined. It was found that protein diffusion within alginate gels, prepared to be isotropic in structure, was greatest for gels prepared from sodium alginate of low guluronic acid content as opposed to those prepared from sodium alginate of high guluronic acid content. This finding was explained in terms of the difference in flexibility of the polymer backbone of the two alginates. The greater the polymer backbone flexibility, the greater the solute diffusivity within the gel.     

Culture of neural stem cells in calcium alginate beads

Neural stem cells (NSCs) with the capacity of extensive self-renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse-derived NSCs were cultured in three-dimensional calcium alginate beads (Ca-Alg-Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca-Alg-Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, beta-tubulin, and GFAP. The results show that the 2-mm diameter Ca-Alg-Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 x 10(5) cells x mL-1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca-Alg-Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca-Alg-Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca-Alg-Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.     

Entrapment of concanavalin A-glycoenzyme complexes in calcium alginate gels

Glucose oxidase, invertase, and amyloglucosidase were entrapped in calcium alginate gels as concanavalin A complexes in order to prevent the leaching out of the enzymes from the porous matrix. The free as well as the gel-entrapped concanavalin A-glycoenzyme complexes exhibited a relatively high effectiveness factor, eta, indicating good accessibility to the substrates. Concanavalin A-invertase complex exhibited marked broadening of pH-activity and temperature-activity profiles and was highly resistant to temperature inactivation even after entrapment in the alginate beads. It was possible to entrap considerable quantities of invertase as concanavalin A complex in the beads without a marked decrease in eta. A column containing crosslinked concanavalin A-invertase complex entrapped in alginate beads retained the ability to completely hydrolyze 1M sucrose even after continuous operation for over four months.     

Microencapsulation of yeast cells in the calcium alginate membrane

Summary Cells of Saccharomyces cerevisiae (ATCC 24858) were encapsulated in the calcium alginate membrane and cultured. Swelling of the capsule was prevented by adding 0.2 g CaCl₂ to 1 L growth medium. The dry cell concentration based on the inner volume of the capsule reached 309 g/L, which was much higher than could be obtained by cell entrapment. All the cells remained inside the capsule during the cultivation. The flux of CO₂ through the capsule membrane increased approximately twice by adding a nonionic surfactant to the CaCl₂ solution during the step of capsule formation.     

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase

The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.     

Steroid transformation by living cells immobilized in calcium alginate

Summary Whole cells of Arthrobacter simplex were immobilized in a living state in calcium alginate gel. The bacteria showed steroid-¹-dehydrogenase activity and the production of prednisolone from cortisol was investigated. The ¹-dehydrogenase activity of the immobilized cells could be increased about ten-fold by incubation in nutrient media (e.g., containing 0.5% peptone abd 0.2% glucose). The reason for this activation was examined and it was found that the immobilized cells were capable of multiplying when supplied with nutrients. Furthermore, provided that an inducer, cortisol, was present, the steroid-¹-dehydrogenase activity increased in proportion to the increase in the number of cells and it was thus concluded that microbial growth was the cause of activation.Experiments on repeated, batch-wise pseudocrystallofermentation with immobilized A. simplex cells also showed that immobilized cells could be advantageously used for pseudocrystallofermentation of steroids.     

Diffusivity of Cu2+ in calcium alginate gel beads

The diffusivity of Cu(2+) in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. (c) 1994 John Wiley & Sons, Inc.     

Immobilization of goat brain aminopeptidase B in calcium alginate beads

Aminopeptidase B, an arginyl aminopeptidase, was purified from goat brain with a purification factor of ～280 and a yield of 2.7%. It was entrapped in calcium alginate together with bovine serum albumin. The optimal conditions for immobilization for maximum activity yield were 1% CaCl₂ and 2.5% alginate. The immobilized enzyme retained ～62% of its initial activity and could be used for five successive batch reactions with retention of 30% of the initial activity. The pH and temperature optima of the free and immobilized enzyme were pH 7.4, 45°C and pH 7.8, 50°C respectively, while the pH and thermal stability as well as the stability of the enzyme in organic solvents were improved significantly after entrapment. The K_m value for the immobilized enzyme was about twofold higher than that of the soluble enzyme. Because of this increased stability, the immobilized enzyme may be useful in the meat processing industry.     

Gelation conditions and transport properties of hollow calcium alginate capsules

The diameter, membrane thickness, and compression intensity of hollow Ca-alginate capsules were measured at different gelation conditions, such as the reactant concentration, dropping velocity, and gelation time. The optimum operation conditions for preparing capsules were determined at 100 g/L CaCl(2), 10 g/L sodium alginate (Na-alginate), a dropping velocity of 150 droplets/min, and a gelation time of 10 min. Diffusion of some saccharide and amino acid from bulk solution into capsules was investigated, and the diffusion coefficients were calculated by the developed mathematical model. All the tested substances can diffuse easily into the capsules. The combined diffusion coefficients of the capsule D(m) are 92-99% as large as their diffusion coefficients in pure water, while the diffusion coefficients in the capsule membrane D(1) are 60-95% as large as those. By employing polyethylene glycol (PEG) and bovine serum albumin (fraction V) (BSA(V)), the molecular weight cut-off of the capsule was determined. For linear macromolecules, hollow Ca-alginate capsules have a molecular weight cut-off of 4000. No diffusion of BSA(V) into the capsules was observed.