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.     

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.     

The production of ethanol by Saccharomyces cerevisiae immobilized in polycation-stabilized calcium alginate gels

Summary Polycation treatment of preformed calcium alginate beads produced a matrix with higher resistance to phosphate ions. The treatment of immobilized Saccharomyces cerevisiae in the calcium alginate beads inhibited respiration of the entrapped cells but did not reduce ethanol production.     

Biological control of phytophagous ladybird beetles Epilachna vigintioctopunctata (Col., Coccinellidae) by chitinolytic phylloplane bacteria Alcaligenes paradoxus entrapped in alginate beads

The chitinase secreting strain KPM‐012A of Alcaligenes paradoxus was isolated from tomato leaves and vitally entrapped in sodium alginate gel beads to provide a new method for biocontrol of phytophagous ladybird beetles Epilachna vigintioctopunctata. First, the peritrophic membrane was dissected from the adult ladybird beetles that ingested the suspension of KPM‐012A after starvation to observe degradation of the midgut surface by the bacteria under electron microscopy. The peritrophic membrane around the bacteria was degraded, suggesting the release of chitinase from the ingested bacteria. Large amounts of chitinase were successfully released from KPM‐012A‐entrapped calcium alginate beads. This chitinase release from the microbial beads was sustained for 1 week and was sufficient to digest the peritrophic membrane. Daily supply of tomato leaves treated with the microbial beads caused considerable suppression of leaf feeding and oviposition by the adult ladybird beetles, indicating that this method is effective for decreasing population of insect pests in the subsequent generation. Thus, the present study provided an experimental basis for the biocontrol measures of herbivorous insect pests by the chitinolytic bacteria entrapped in alginate beads.     

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.     

Chromium (VI) uptake by grape stalks wastes encapsulated in calcium alginate beads: equilibrium and kinetics studies

Abstract

The removal of hexavalent chromium from aqueous solution using grape stalks wastes encapsulated in calcium alginate (GS–CA) beads was investigated. Cr(VI) sorption kinetics were evaluated as a function of chromium initial concentration and grape stalks (GS) content in the calcium alginate (CA) beads. The process follows pseudo second-order kinetics. Transport properties of hexavalent chromium on GS–CA beads was characterised by calculating chromium diffusion coefficient using the Linear Absorption Model (LAM). Langmuir isotherms, at pH 3.0 were used to describe sorption equilibrium data as a function of GS percentage in the CAbeads. Maximum uptake obtained was 86.42 mmol of Cr(VI) per L of wet sorbent volume. Results indicated that both kinetic and equilibrium models describe adequately the adsorption process.     

Viability of ectomycorrhizal fungus mycelium entrapped in calcium alginate gel

 We studied the viability of fragmented mycelium of Pisolithus tinctorius and Paxillus involutus entrapped in calcium alginate gel to determine the efficacy of this method of producing ectomycorrhizal fungus inoculum. Fungi were grown in MMN solution at 28  °C before being fragmented in a blender and subsequently entrapped in calcium alginate. We tested different ratios of alginate and mycelium suspension to 0.7 M CaCl₂. The ratio 8 : 10 resulted in well-formed beads of the highest viability for Paxillus involutus (99%) and for Pisolithus tinctorius (75%). Paxillus involutus mycelium was more than 90% viable when entrapped mycelium was 10 to 50 days old, and Pisolithus tinctorius attained its highest viability (55%) for 20- to 40-day-old mycelium. Gel entrapped Paxillus involutus mycelium grew well at all temperatures after 30 days of storage, but viability significantly decreased after 60 days storage at 6  °C on dry filter paper. For gel-entrapped Pisolithus tinctorius mycelium, viability was highest when stored at 25  °C in 0.7 M CaCl₂. Entrapment of Paxillus involutus fragmented mycelium in calcium alginate beads under the conditions that we propose can be used successfully to produce inoculum. Accepted: 11 October 1998     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Lactococcus lactis release from calcium alginate beads.

Cell release during milk fermentation by Lactococcus lactis immobilized in calcium alginate beads was examined. Numbers of free cells in the milk gradually increased from 1 x 10(6) to 3 x 10(7) CFU/ml upon successive reutilization of the beads. Rinsing the beads between fermentations did not influence the numbers of free cells in the milk. Cell release was not affected by initial cell density within the beads or by alginate concentration, although higher acidification rates were achieved with increased cell loading. Coating alginate beads with poly-L-lysine (PLL) did not significantly reduce the release of cells during five consecutive fermentations. A double coating of PLL and alginate reduced cell release by a factor of approximately 50. However, acidification of milk with beads having the PLL-alginate coating was slower than that with uncoated beads. Immersing the beads in ethanol to kill cells on the periphery reduced cell release, but acidification activity was maintained. Dipping the beads in aluminum nitrate or a hot CaCl2 solution was not as effective as dipping them in ethanol. Ethanol treatment or heating of the beads appears to be a promising method for maintaining acidification activity while minimizing viable cell release due to loosely entrapped cells near the surface of the alginate beads.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

Removable colored coatings based on calcium alginate hydrogels

This article describes the creation of a nontoxic, biodegradable coating using calcium alginate and FD&C approved dyes. The coating is robust but is rapidly removed upon treatment with disodium ethylenediamine tetraacetate (EDTA). Dye leaching from calcium alginate films was studied, and it was determined that the efficiency of dye retention is proportional to the degree of cross-linking. Degradation rates were studied on calcium alginate beads serving as a model for a coating. We determined that degradation rates depend on the gel's cross-linking and on the amount of EDTA used. Bead size also influenced the degradation rates; smaller beads degraded faster than larger beads. We show that the coating can be used as an easily removable and environmentally friendly logotype on an artificial turf surface. Applications of these coatings can be extended to food, cosmetic, medicinal, and textile uses and to wherever nontoxic, easily removable colored coating is desired.     

Conjugal transfer of plasmid DNA between streptococci immobilized in calcium alginate gel beads.

A rapid and simple technique was developed for conjugation between group N and group D streptococci by using cells entrapped within calcium alginate gel beads. With this method, the frequencies of transfer of lactose metabolism from Streptococcus lactis ME2 to S. lactis LM2302 were comparable to those achieved with agar surface matings. Conjugal transfer of the chloramphenicol and erythromycin resistance plasmid pVA797::Tn917 from S. faecalis V1229 to S. faecalis V1102 in alginate beads occurred at frequencies comparable to those achieved with filter matings. The results demonstrated efficient conjugal transfer of plasmid DNA among alginate-immobilized streptococcal cells and suggested that this method could be used as an alternative to conventional solid-surface and filter matings with these organisms.     

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.     

Biosorption of no-carrier-added radionuclides by calcium alginate beads using 'tracer packet' technique

The aim of this study was to determine the adsorption behaviour of various micronutrient elements e.g., (61)Cu, (62,63)Zn, (66,67,68)Ga, (66,67,69)Ge, (71,72)As present in no-carrier-added state, with calcium alginate (CA) using 'tracer packet' technique. High Ge and Ga and moderate Cu removal were achieved at pH 7 and pH 5, respectively. Results on the studies to recover all the three radionuclides from the calcium alginate beads using desorbing reagents, HCl, thiourea, ammonium oxalate and sodium nitrite showed that 0.1 M HCl and 0.1 M ammonium oxalate removed Cu and Ge moderately. The amount of Ga desorbed by all the washing liquids was almost negligible, except sodium nitrite.     

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.     

Influence of immobilization parameters on growth and lactic acid production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> and <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis> co-immobilized in calcium alginate gel beads

Streptococcus thermophilusand Lactobacillus bulgaricus were co-immobilized in different systems with varying calcium (0.1–1.5M) and alginate (1–2<><>, w/v) concentrations. Highest lactic acid production was 35 g l₁ when both bacteria were in high viscosity beads (1<><>, w/v alginate) hardened in 0.1 M CaCl₂ .The gel bead composition affected size and distribution of entrapped lactic acid bacteria.     

Feasibility study on the use of calcium alginate-entrapped hybridoma cells for IgM production

In order to test the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production, HO-22-1 hybridoma cells entrapped into calcium alginate beads with varying alginate concentrations were cultivated in spinner flasks. It was observed that the IgM produced by the entrapped cells could diffuse out of the calcium alginate beads regardless of alginate concentrations tested (0.8–2.5%). Since the increase in alginate concentrations showed an adverse effect on cell growth and maximum cell concentration, the use of lower alginate concentration was desirable for higher volumetric monoclonal antibody (MAb) productivity. When the entrapped cells in 0.8% alginate beads were cultivated in repeated-fed batch mode, the reduction of serum concentration in the medium from 10% to 1% did not decrease the volumetric IgM production. Taken together, the data obtained here showed the feasibility of using calcium alginate-entrapped hybridoma cells for IgM production.Alginate was generously provided by the Kelco company. This work was supported by the Ministry of Science and Technology, Korea.     

Elucidation of optimum conditions for immobilization of viable cells by using calcium alginate

Different factors which affect the stability of calcium alginate gel beads entrapping viable cells during fermentation were investigated. It was found that among others, the initial population of cells per ml of gel beads, the length of period of incubation in CaCl₂ solution, and the concentration of sodium alginate used for the immobilization were the most important factors affecting the stability of the gel beads during fermentation. By using an initial cell population of about 10⁵ cells per ml of 2.0% sodium alginate, and incubating the beads for at least 22 h in a CaCl₂ solution after immobilization, the percentage of beads which developed cracks during fermentation was highly reduced. Also, without the addition of CaCl₂ into the fermenting broth, the gel beads were stable for nine consecutive batch fermentations.     

Immobilization of cellulase from newly isolated strain Bacillus subtilis TD6 using calcium alginate as a support material

Bacillus subtilis TD6 was isolated from Takifugu rubripes, also known as puffer fish. Cellulase from this strain was partially purified by ammonium sulphate precipitation up to 80% saturation, entrapped in calcium alginate beads, and finally characterized using CMC as the substrate. For optimization, various parameters were observed, including pH maximum, temperature maximum, sodium alginate, and calcium chloride concentration. pH maximum of the enzyme showed no changes before and after immobilization and remained stable at 6.0. The temperature maximum showed a slight increase to 60 °C. Two percent sodium alginate and a 0.15 M calcium chloride solution were the optimum conditions for acquisition of enzyme with greater stability. K (m) and V (max) values for the immobilized enzyme were slightly increased, compared with those of free enzyme, 2.9 mg/ml and 32.1 μmol/min/mL, respectively. As the purpose of immobilization, reusability and storage stability of the enzyme were also observed. Immobilized enzyme retained its activity for a longer period of time and can be reused up to four times. The storage stability of entrapped cellulase at 4 °C was found to be up to 12 days, while at 30 °C, the enzyme lost its activity within 3 days.