Microencapsulation of the probiotic cultures
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.