Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Inhomogeneous alginate gel spheres: an assessment of the polymer gradients by synchrotron radiation-induced X-ray emission, magnetic resonance microimaging, and mathematical modeling

It has been previously demonstrated that calcium alginate gels prepared by dialysis often exhibit a concentration inhomogeneity being the polymer concentration considerably lower in the center of the gel than at the edges. Inhomogeneity may be a preferred structure in microcapsules due to low porosity and higher stability so that it is interesting to evaluate the polymer gradient in spherically symmetrical small alginate beads (1.0-0.7 mm diameter) obtained in different conditions. In this paper, two complementary techniques have been used to investigate this aspect. The concentration gradient of alginate has been analyzed by measuring both the spatial distribution of calcium ions in sections of alginate gel spheres, by means of x-ray fluorescence spectroscopy, and the T2 relaxation behavior on intact gel beads using magnetic resonance microimaging. The experimentally determined gradients from three-dimensional gels provide data to reevaluate the parameter estimates in the recently reported mathematical model for alginate gel formation (A. Mikkaelsen and A. Elgsaeter, Biopolymers, 1995, Vol. 36, pp. 17-41). The model may account for the gels being less inhomogeneous when nongelling sodium or magnesium ions are added during gelation.     

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.     

Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation

Recently, interest has focused on hepatocytes’ implantation to provide end stage liver failure patients with a temporary support until spontaneous recovery or a suitable donor becomes available. To avoid cell damage and use of an immunosuppressive treatment, hepatic cells could be implanted after encapsulation in a porous biomaterial of bead or capsule shape. The aim of this study was to compare the production and the physical properties of the beads, together with some hepatic cell functions, resulting from the use of different material combinations for cell microencapsulation: alginate alone or combined with type I collagen with or without poly-L-lysine and alginate coatings. Collagen and poly-L-lysine increased the bead mechanical resistance but lowered the mass transfer kinetics of vitamin B12. Proliferation of encapsulated HepG2/C3A cells was shown to be improved in alginate-collagen beads. Finally, when the beads were subcutaneously implanted in mice, the inflammatory response was reduced in the case of alginate mixed with collagen. This in vitro and in vivo study clearly outlines, based on a systematic comparison, the necessity of compromising between material physical properties (mechanical stability and porosity) and cell behavior (viability, proliferation, functionalities) to define optima hepatic cell microencapsulation conditions before implantation.     

Enhanced production of bioethanol and ultrastructural characteristics of reused Saccharomyces cerevisiae immobilized calcium alginate beads

Yeast immobilized on alginate beads produced a higher ethanol yield more rapidly than did free yeast cells under the same batch-fermentation conditions. The optimal fermentation conditions were 30 °C, pH 5.0, and 10% initial glucose concentration with 2% sodium alginate beads. The fermentation time using reused alginate beads was 10-14 h, whereas fresh beads took 24 h, and free cells took 36 h. All bead samples resulted in nearly a 100% ethanol yield, whereas the free cells resulted in an 88% yield. Transmission electron microscopy (TEM) showed that the shortened time and higher yield with the reused beads was due to a higher yeast population per bead as well as a higher porosity. The ultrastructure of calcium alginate beads and the alginate matrix structure known as the “egg-box” model were observed using TEM.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Immobilization of recombinant nanobiofiber CS3 fimbriae onto alginate beads for improvement of cadmium biosorption

A cell surface display system with metalbinding properties was previously developed using CS3 fimbriae, which are hollow tubes 20 nm-thick and 2 nm in diameter. In this study, hybrid CS3 pili were separated from recombinant Escherichia coli and entrapped in calcium alginate gel beads in order to improve their stabilization and also adsorption of heavy metals. The surface morphology of the gel beads containing pili was investigated by scanning electron microscopy (SEM). Immunofluorescence microscopy was employed to confirm the attachment of nanobiofibers to the alginate beads. The effects of three variables (sodium alginate concentration, protein to alginate mass ratio, and bead size) at two levels each on Cd²⁺ biosorption efficiency were investigated by full factorial experimental design. A second-order polynomial equation modeled the design space for the process response of cadmium removal capacity. The optimal values of the factors were obtained as follows: 1% sodium alginate concentration, 0.25 protein to alginate mass ratio, and a 6 mm bead size. Under these conditions, Cd²⁺ was adsorbed at 45.45 mg/g to the nanobiofiber. The results indicate that the immobilized recombinant hybrid CS3 pili may be an appropriate biosorbent for removal of heavy metals from polluted aquatic environments.     

Enhancing the Viability of Lactobacillus plantarum Inoculum by Immobilizing the Cells in Calcium-Alginate Beads Incorporating Cryoprotectants

Many literature reports have cited the importance of the rehydration conditions of lyophilized cultures in determining viability. The rate of rehydration and the volume of fluid used have been identified as two important factors. One possible means of controlling these is by immobilizing the cells before lyophilization within a gel matrix in which the subsequent rehydration rate and fluid volume would be controlled by the properties of the gel. In this study Lactobacillus plantarum was immobilized and lyophilized in Ca-alginate beads in which 1 M glycerol or 0.75 M adonitol with skim milk were incorporated as a cryoprotectant. The properties of these Ca-alginate beads were examined before and after lyophilization and rehydration. The beads incorporating glycerol were smaller and stronger than those with adonitol. After lyophilization, size decreased and strength increased but to a greater extent in the beads with glycerol, indicating that the microenvironment within the two bead types was probably different. The protective effect of the bead microenvironment on immobilized L. plantarum was also examined. Lyophilization and rehydration within the alginate beads with either polyol yielded higher survival rates than that attained with free cell cultures during rehydration in optimal or suboptimal conditions. During rehydration under suboptimal conditions, the immobilized cell survival was greatest when 0.75 M adonitol was the incorporated cryoprotectant.     

Encapsulation of astaxanthin-rich Xanthophyllomyces dendrorhous for antioxidant delivery

Calcium alginate gel (CAG) beads were used to entrap the antioxidant astaxanthin-rich Xanthophyllomyces dendrorhous (ASX) by ionic gelation. ASX-CAG bead entrapment efficiency and release behavior, as influenced by alginate and CaCl₂ concentration and hardening time, were investigated. The optimized bead preparation conditions that gave rise to an efficient ASX release pattern were 1.5% alginate, 50 mM CaCl₂, and a 5 min hardening time. The antioxidant activity of non-encapsulated ASX was maintained for 4 days and then sharply decreased, whereas encapsulated ASX was maintained for 6 days. These results revealed that physical entrapment of ASX within CAG beads could be an effective technique for protecting the antioxidant activity of ASX from lipid peroxidation.     

Effects of sterilization treatments on some properties of alginate solutions and gels.

Aqueous sodium alginate solutions were subjected to various heat sterilization treatments. Sodium alginate powder was also treated by both gamma-irradiation and ethylene oxide sterilization. The effects of these treatments on the viscosities of sodium alginate solutions and both the diameter and strength of the beads formed in 0.1 M CaCl2 solutions were determined quantitatively. The viscosity of sodium alginate solutions and the gel strength of the calcium alginate beads decreased with increasing sterilization temperature while the bead diameters were found to increase. All these effects can be attributable to a reduction in the degree of polymerization of the alginate molecules as a result of the heat treatments. Ethylene oxide and gamma-irradiation treatments caused similar effects. Standard conditions for sterilization are necessary for comparative studies with alginate beads.     

The effect of immobilization on recombinant protein production in plant cell culture

The effects of encapsulation on the production of recombinant human proteins by Nicotiana tabacum cells were investigated using alginate, carrageenan, and agar as immobilization matrices. Experiments showed that cell encapsulation in alginate increased the production of human granulocyte-macrophage colony-stimulating factor (GM-CSF) in tobacco cells by approximately 50%. Alginate also yielded the highest quality beads and the most reproducible growth results. The most likely cause for this increased protein production is the altered growth conditions within the alginate beads resulting in a prolonged exponential growth phase. To characterize these effects, we compared growth performance and protein production for various gel geometries, bead sizes, and volume fractions of beads.     

Determination of the radial distribution of Saccharomyces cerevisiae immobilised in calcium alginate gel beads

Summary The dissolution of alginate gel beads in 20 g sodium citrate /l produces a linear decrease in bead diameter. The rate of dissolution is dependent on the concentration of CaCl₂ within the gel beads. This method allows the controlled release of Saccharomyces cerevisiae from alginate gel beads and permits the simple and rapid determination of the radial distribution of cell concentration.     

Effect of skim milk-alginate beads on survival rate of bifidobacteria

In this study, an attempt was made to increase the survival rate of bifidobacteria entrapped in alginate in the gastrointestinal tract, and to investigate the potential industrial applications, for example lyophilized capsules and yogurt. First, the protective effect of various food additives on bifidobacterial survivability was determined after exposure to simulated gastric juices and bile salts. The additives used in this study were skim milk (SM), poly dextrose (PD), soy fiber (SF), yeast extract (YE), chitosan (CS), κ-carageenan (κ-C) and whey, which were added at 0.6% concentration (w/v) to 3% alginate-bifidobacterial solution. In the simulated gastric juices and bile salts, the protective effect of 0.6% skim milk-3% alginate (SM-A) beads on the survival rate of bifidobacteria proved to be higher than the other additives. Second, the hydrogen ion permeation was detected through SM-A vessel without bifidobacterial cells at different SM concentrations (0.2%, 0.4%, 0.6%, 0.8%, and 1.0%). There were no differences in terms of the pH decrease in SM-A vessels at 0.6%, 0.8%, and 1.0% (w/v) SM concentrations. The survival rate of bifidobacteria in SM-A beads would appear to be related to the SM buffering capacity against hydrogen ions and its tendency to reduce the pore size of bead. In this experiment, the survival rate of bifidobacteria entrapped in beads containing 0.6% SM showed the highest viability after exposure to simulated gastric juices for 3 h, thereby indicating that 0.6% SM is the optimum concentration for 3% alginate bead preparation. Third, the effect of SM-A beads on the freeze-drying and yogurt storage for 10 days was investigated. SM-A beads were found to be more efficient for freeze drying and yogurt storage than untrapped cells and the alginate bead. Consequently, the survival rate of bifidobacteria entrapped in SM-A beads was increased in simulated gastric juices, bile salts and probiotic products such as lyophilized capsules and yogurt, SM-A beads can be expected to produce high value probiotic products.     

Transport limitation of chlorine disinfection of Pseudomonas aeruginosa entrapped in alginate beads

An artificial biofilm system consisting of Pseudomonas aeruginosa entrapped in alginate and agarose beads was used to demonstrate transport limitation of the rate of disinfection of entrapped bacteria by chlorine. Alginate gel beads with or without entrapped bacteria consumed chlorine. The specific rate of chlorine consumption increased with increasing cell loading in the gel beads and decreased with increasing bead radius. The value of an observable modulus comparing the rates of reaction and diffusion ranged from less than 0.1 to 8 depending on the bead radius and cell density. The observable modulus was largest for large (3-mm-diameter) beads with high cell loading (1.8 x 10(9) cfu/cm(3)) and smallest for small beads (0.5 mm diameter) with no cells added. A chlorine microelectrode was used to measure chlorine concentration profiles in agarose beads (3.0 mm diameter). Chlorine fully penetrated cell-free agarose beads rapidly; the concentration of chlorine at the bead center reached 50% of the bulk concentration within approximately 10 min after immersion in chlorine solution. When alginate and bacteria were incorporated into an agarose bead, pronounced chlorine concentration gradients persisted within the gel bead. Chlorine did gradually penetrate the bead, but at a greatly retarded rate; the time to reach 50% of the bulk concentration at the bead center was approximately 46 h. The overall rate of disinfection of entrapped bacteria was strongly dependent on cell density and bead radius. Small beads with low initial cell loading (0.5 mm diameter, 1.1 x 10(7) cfu/cm(3)) experienced rapid killing; viable cells could not be detected (<1.6 x 10(5) cfu/cm(3)) after 15 min of treatment in 2.5 mg/L chlorine. In contrast, the number of viable cells in larger beads with a higher initial cell density (3.0 mm diameter, 2.2 x 10(9) cfu/cm(3)) decreased only about 20% after 6 h of treatment in the same solution. Spatially nonuniform killing of bacteria within the beads was demonstrated by measuring the transient release of viable cells during dissolution of the beads. Bacteria were killed preferentially near the bead surface. Experimental results were consistent with transport limitation of the penetration of chlorine into the artificial biofilm arising from a reaction-diffusion interaction. The methods reported here provide tools for diagnosing the mechanism of biofilm resistance to reactive antimicrobial agents in such applications as the treatment of drinking and cooling waters. (c) 1996 John Wiley & Sons, Inc.     

Stability of alginate-immobilized algal cells

Investigations were carried out using immobilized Chlorella cells to determine the diameter, compressibility, tolerance to phosphate chelation, and ability to retain algal cells during incubation of various alginate beads. These physical bead characteristics were found to be affected by a variety of interactive factors, including multivalent cation type (hardening agent) and cell, cation, and alginate concentration, the latter exhibiting a predominant influence. The susceptibility of alginate beads to phosphate chelation was found to involve a complex interaction of cation type, concentration, and pH of phosphate solution. A scale of response ranging from gel swelling to gel shrinking was observed for a range of conditions. However, stable calcium alginate beads were maintained in incubation media with a pH of 5.5 and a phosphate concentration of 5muM. A preliminary investigation into cell leakage from the beads illustrated the importance of maintaining a stable gel structure and limiting cell growth to reduce leakage.     

Tuning structural durability of yeast-encapsulating alginate gel beads with interpenetrating networks for sustained bioethanol production

Microorganisms have become key components in many biotechnological processes to produce various chemicals and biofuels. The encapsulation of microbial cells in calcium cross-linked alginate gel beads has been extensively studied due to several advantages over using free cells. However, industrial use of alginate gel beads has been hampered by the low structural stability of the beads. In this study, we demonstrate that the incorporation of interpenetrating covalent cross-links in an ionically cross-linked alginate gel bead significantly enhances the bead's structural durability. The interpenetrating network (IPN) was prepared by first cross-linking alginate chemically modified with methacrylic groups, termed methacrylic alginate (MA), with calcium ions and subsequently conducting a photo cross-linking reaction. The resulting methacrylic alginate gel beads (IPN-MA) exhibited higher stiffness, ultimate strength and ultimate strain and also remained more stable in media either subjected to high shear or supplemented with chelating agents than calcium cross-linked alginate gel beads. Furthermore, yeast cells encapsulated in IPN-MA gel beads remained more metabolically active in ethanol production than those in calcium cross-linked alginate gel beads. Overall, the results of this study will be highly useful in designing encapsulation devices with improved structural durability for a broad array of prokaryotic and eukaryotic cells used in biochemical and industrial processes.     

Immobilization of Saccharomyces uvarum cells in porous beads of polyacrylamide gel for ethanolic fermentation

Summary A procedure which does not involve the use of an immiscible organic solvent phase is described for the entrapment of yeast cells in porous beads of polyacrylamide gel. The cells are rapidly dispersed at 4° C in an aqueous solution containing sodium alginate and acrylamide-N,Nmethylene-bis-acrylamide monomer, and the suspension is immediately dropped into a solution of calcium formate to give calcium alginate coated beads. Polyacrylamide gel forms within the bead. The calcium alginate is subsequently leached out of the composite bead with either sodium citrate or potassium phosphate buffer solution. Cells of Saccharomyces uvarum ATCC 26 602 entrapped in such polyacrylamide beads ferment cane molasses in batch mode at higher specific ethanol productivity than a free cell suspension. Their volumetric productivity in continuous fermentation is higher than that of Ca²⁺-alginate immobilized cells.NCL Communication No. 4383     

Culture of chondrocytes in alginate gel: Variations in conditions of Gelation influence the structure of the alginate gel, and the arrangement and morphology of proliferating chondrocytes

Summary Sodium alginate, which gels in the presence of calcium ions, is commonly used for culture of anchorage-independent cells, such as chondrocytes. Normally, the gel appears microscopically homogeneous but, depending on the conditions of gelation, it may contain a varying number of small channels that extend inward from the surface. We have examined the influence of these channels on the morphology of cultured chondrocytes entrapped in alginate beads. Growth-plate or articular chondrocytes cultured in alginate normally proliferate and form rounded cell clusters but, in alginate beads containing numerous channels, many chondrocytes become aligned and form columns similar to those in the growth plate in vivo. As the pattern of cellular growth and morphology in alginate is profoundly influenced by the presence of channels in the gel, further studies were conducted to determine what specific conditions of gelation affect their formation. The channels are especially numerous when both the alginate and the gelling solutions lack sodium ions or other monovalent cations. The channels are cavities in the gel formed by particulate blocking of the rapid diffusion of calcium ions from the gelling solution into the boundary of the calcium alginate solution, and hence they extend inward from cells at the surface of the alginate gel. An understanding of the conditions under which these channels develop makes it possible either to avoid their formation or, alternatively, to enhance the number of channels in order to encourage proliferating cells to grow in radial columns, rather than in a less organized pattern characteristic of most culture systems.     

Immobilization of the marine microalga Phaeodactylum tricornutum in alginate for in situ experiments: Bead stability and suitability

Microalgae immobilization in alginate matrixes has been recently used to perform in situ experiments. However, the susceptibility of alginate matrixes to cation chelating agents and to antigelling cations, which can cause bead disruption or dissolution, is a major limitation for in situ exposures in estuarine and marine systems. The ultimate goal of this study was to produce alginate beads stable in seawater and suited for Phaeodactylum tricornutum growth. For this, different concentrations of alginate isolated from Macrocystis pyrifera (1.5, 1.9 and 2.3% [w/v]) and Laminaria hyperborea (4.0, 4.9 and 5.8% [w/v]), two concentrations of the hardening cations calcium and strontium (2.0 and 4.0% [w/v]), and the use of the polycation chitosan were investigated. Only beads found to be more stable after 16 days of exposure in seawater were inoculated with the microalga. P. tricornutum immobilized in beads prepared from 5.8% L. hyperborea alginate and in all beads in which a chitosan hardening treatment was applied showed a weak microalgal growth. Beads prepared using 4.9% of L. hyperborea alginate and a 4% (w/v) strontium solution were found to be the most stable and the most suitable for microalgal growth, and were exposed in the field, under natural fluctuating conditions of light and temperature. In situ growth rates of immobilized P. tricornutum cells demonstrated the potential of these beads for future use in in situ experiments in estuarine and marine systems.     

Growth of an adherent continuous cell line entrapped in a peg/alginate matrix

Summary CHO-K1 cells, an anchorage-dependent line, were entrapped in beads prepared from a Na alginate/polyethylene glycol mixture and grown, through successive passages, to an average maximum density of 4.5×10⁷ viable cells/g of bead. Cell growth and viability was unaffected by repeated alginate re-solubilization and reformation of the gel beads through five passages.