Microencapsulation of the probiotic cultures

/Microencapsulation of the probiotic cultures
Microencapsulation of the probiotic cultures 2018-05-12T05:36:23+00:00

Microencapsulation of the probiotic cultures

Introduction

Alginate, commercially available as alginic acid, sodium salt, commonly called sodium alginate, is a linear polysaccharide normally isolated from many strains of marine brown seaweed and algae, thus the name alginate. The copolymer consists of two uronic acids: D-mannuronic acid (M) and L-guluronic acid (G). Because it is the skeletal component of the algae it has the nice property of being strong and yet flexible.
Alginic acid can be either water soluble or insoluble depending on the type of the associated salt. The salts of sodium, other alkali metals, and ammonia are soluble, whereas the salts of polyvalent cations, e.g., calcium, are water insoluble, with the exception of magnesium. The alginate polymer itself is anionic (i.e., negatively charged) overall. Polyvalent cations bind to the polymer whenever there are two neighboring guluronic acid residues. Thus, polyvalent cations are responsible for the cross-linking of both different polymer molecules and different parts of the same polymer chain. The process of gelation, simply the exchange of calcium ions for sodium ions, is carried out under relatively mild conditions. Because the method is based on the availability of guluronic acid residues, which will not vary once given a batch of the alginate, the molecular permeability does not depend on the immobilization conditions. Rather, the pore size is controlled by the choice of the starting material.
2 Na(Alginate) + Ca++ ——-> Ca(Alginate)2 + 2 Na+
The ionically linked gel structure is thermostable over the range of 0-100ºC; therefore heating will not liquefy the gel. However, the gel can be easily redissolved by immersing the alginate gel in a solution containing a high concentration of sodium, potassium, or magnesium. Maintaining sodium:calcium <= 25:1 will help avoid gel destabilization. Citrate or phosphate pH buffers cannot be effectively used without destabilizing the alginate gel.
Alginate is currently widely used in food, pharmaceutical, textile, and paper products. The properties of alginate utilized in these products are thickening, stabilizing, gel-forming, and film-forming. Alginate polymers isolated from different alginate sources vary in properties. Different algae, or for that matter different part of the same algae, yield alginate of different monomer composition and arrangement. There may be sections of homopolymeric blocks of only one type of monomer (-M-M-M-) (-G-G-G-), or there may be sections of alternating monomers (-M-G-M-G-M-). Different types of alginate are selected for each application on the basis of the molecular weight and the relative composition of mannuronic and guluronic acids. For example, the thickening function (viscosity property) depends mainly on the molecular weight of the polymer; whereas, gelation (affinity for cation) is closely related to the guluronic acid content. Thus, high guluronic acid content results in a stronger gel.
Cell immobilization is done through: covalent bonding, affinity bonding, physical adsorption, and entrapment in synthetic and natural polymer matrices.
One of the problems for immobilisation is the mass transfer resistance imposed by the fact that the substrate has to diffuse to the reaction site and inhibitory or toxic products must be removed to the environment. Oxygen transfer is often the rate limiting step in a suspended cell culture, and it is more so in an immobilized cell culture. Oxygenation in an immobilized cell culture is one of the major technical problems that remain to be solved. In light of the oxygenation problems, immobilization techniques have been mainly confined to anaerobic processes in which either obligate (strict) anaerobes are employed or only the anaerobic components of the facultative metabolic mechanisms are selectively utilized.
An immobilized cell bioreactor is well suited for those cells whose growth phases and product formation phases are uncoupled. Cell biomass and primary metabolites are growth associated products, but secondary metabolites such as antibiotics and various enzymes are produced during the stationary phase. The uncoupling of the phases means that productive cells cannot compete with the non-productive cells in a continuously operated suspension fermentor because the productive cells spend the nutritional and energy resources producing chemicals in quantities far above the amount necessary for their survival, instead of reproducing themselves to propagate further. On the contrary, cell growth in an immobilized cell reactor must be severely limited if gel swelling or breakage is to be avoided. However, once the cells are immobilized, the cell viability must be concomitantly sustained over a long period of time. Thus, immobilization is advantageous for sustaining slowly growing cells.

Requirements

  1.  4 % sodium alginate solution, 25 mL
  2. 1.5 % calcium chloride solution, 100 mL
  3. Bakers’ yeast, dried, 2.5 g
  4. 8 % sucrose solution, 150 mL
  5. Universal indicator solution, 1 mL, diluted with
  6. 1 mL of distilled or deionised water

Procedure

  1. Mix the dried yeast with 25 mL of distilled water in a small beaker.
  2. Cover and leave to rehydrate for 10 minutes at room temperature.
  3. Dissolve 30g of sodium alginate in 1 liter to make a 3% solution. Sodium alginate solution is best prepared by adding the powder to agitated water, rather than vice versa, to avoid the formation of clumps. Prolonged stirring may be necessary to achieve the complete dissolution of sodium alginate. After sodium alginate is completely dissolved, leave the solution undisturbed for 30 minutes to eliminate the air bubbles that can later be entrapped and cause the beads to float.
  4. Add 25 ml of sodium alginate solution to the yeast suspension. Stir well.
  5. Draw up some of the yeast/alginate mixture into a syringe.
  6. The beads are formed by dripping the polymer solution from a height of approximately 20 cm into an excess (100 ml) of stirred 0.2M CaCl2 solution with a syringe and a needle at room temperature. A diameter of 0.5-2 mm can be readily achieved with a syringe and a needle.
  7. Leave the immobilised yeast cell beads to harden in the calcium chloride solution for 1-2 hrs. The alginate will be ionically cross-linked by the calcium ions. Wash the beads with a fresh calcium crosslinking solution.
  8. Separate the beads from the solution using a strainer.
  9. Place the beads in a sugar solution in a conical flask. Stoppera flask with a bung that has been fitted with a fermentation lock. If universal indicator is added to the fermentation lock, the indicator will change colour as carbon dioxide is produced.
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