Microencapsulation Technology For Probiotic

bacteria

Seminar On:-

Microencapsulation

Microencapsulation is defined as a technology of packaging solids, liquids or gaseous materials in miniature, sealed capsules that can release their contents at controlled rates under the influences of specific conditions (Anal & Stevens, 2005)

Purpose for microencapsulation

To make liquids behave like solids

Separate reactive materials

Reduce material toxicity

Provide environmental protection to compounds

Alter surface properties of the materials

Control release of materials

Reduce volatility or flammability of liquids

Mask the taste of bitter compounds

Principle of Encapsulation: Membrane barrier isolates cells from the host immune system while allowing transport of metabolites and extracellular nutrients. Membrane with size selective pores (30-70 kDa). Source: INOTECH Encapsulation.

Section of alginate microcapsules showing: A). the starch grains in cavities, B). L. acidophilus, and C). B. infantis located in thealginate matrix.

1. What functions must the encapsulated ingredients provide for the final product?

2. What kind of coating material should be selected ?

3. What processing conditions must the encapsulated ingredient survive before releasing its content ?

4. What is the optimum concentration of the active material in the microcapsule ?

5. By which mechanism will the ingredient be released from the microcapsule ?

6. What are the particle size, density, and stability requirements for the encapsulated ingredients ?

7. What are the cost constraints of the encapsulated ingredient ? [Shahidi and Han, 1993]

In designing the encapsulation process, the following questions should be asked:

The terms immobilization and encapsulation were used interchangeably in most reported literature

While encapsulation is the process of forming a continuous coating around an inner matrix that is wholly contained within the capsule wall as a core of encapsulated material, immobilization refers to the trapping of material within or throughout a matrix

A small percentage of immobilised material may be exposed at the surface, while this is not the case for encapsulated material

Difference between Immobilization and

Encapsulation

The microcapsule is composed of a semi permeable, spherical, thin and strong membranous wall

Therefore the bacterial cells are retained within the microcapsules

Moreover, compared to an entrapment matrix, there is no solid or gelled core in the microcapsule and its small diameter helps to reduce mass transfer limitations

Advantages of Micro-encapsulation Immobilization/

Entrapment

The nutrients and metabolites can diffuse through the semi permeable membrane easily

The membrane serves as a barrier to cell release and minimizes contamination

The encapsulated core material is released by several mechanisms such as mechanical rupture of the cell wall, dissolution of the wall, melting of the wall and diffusion through the wall

Encapsulation of probiotics in polymer systems

Advantages

Once entrapped/ encapsulated in matrix beads or in microcapsules, the cells are easier to handle than in a suspension or in slurry

The number of cells in beads or microparticles can be quantified, allowing the dosage to be readily controlled

Cryo and osmo-protective components can be incorporated into the matrix, enhancing the survival of cells during processing and storage

Finally, once the matrix beads/microcapsules have been dried, a further surface coating can be applied

This outer layer can be used to alter the aesthetic and sensory properties of the product and may also be functional, providing an extra level of protection to the cells

In addition, the coating layer can have desirable dissolution properties, which permit delayed release of the cells or release upon, for example, a change in pH

Culture Technique/Mechanism

Product Reference

B. bifidum, B. infantis Calcium alginate Mayonnaise Khalil and Mansour, 1998

L. paracasei Milk fat Cheddar cheese Stanton et al., 1998

Enterococcus faecium Milk fat Cheddar cheese Gardiner etal., 1998

B. bifidum, B. adolescentis

Cream White brined cheese

Ghoddusi and Robinson, 1998

B. bifidum, B. infantis, and B. longum

Calcium alginate gels Crescenza cheese Gobbeti et al., 1997

L. lactis subspp. lactis k-Carrageenan and locust bean gum

Fresh cheese Sodini et al., 1997

L. Casei Liquid core alginate capsule

Lactic acid Yoo et al., 1996

Lactobacilli Alginate Frozen dessert Sheu and Marshall, 1993

Immobilisation/ Encapsulation of cells for food/biotechnological application

Encapsulation of probiotics in k-carrageenan

Carrageenan is a natural polysaccharide that is extracted from marine macroalgae and is commonly used as a food additive

Gelation of k-carrageenan is generally dependent on a change in temperature.

The cell slurry is added to the heat-sterilized carrageenan solution at 40-450 C and gelation occurs by cooling to room temperature

The beads are formed after dropping the mixture of polymer and cells into a potassium chloride (KCl) solution

The conventional encapsulation method, with sodium alginate in calcium chloride (CaCl2), has been used to encapsulate L. acidophilus to protect this organism from the harsh acidic conditions in gastric fluid

Studies have shown that calcium-alginate immobilized cell cultures are better protected, shown by an increase in the survival of bacteria under different conditions, than the non-encapsulated state

Encapsulation of probiotics in alginate systems

Alginic acid, a natural polymer, is a polyuronic acid that is extracted from seaweeds and is composed of various proportions of 1-4 linked β-D-mannuronic (M) and α-L-guluronic (G) acids

These residues are present in various proportions depending on the source of the alginic acid

Alginic acid and its salts are block copolymers, containing both MM and GG homopolymer blocks and mixed blocks containing irregular sequences of M and G units

The binding of divalent cations and the subsequent gel formation are dependent on the composition and arrangement of the blocks of residues

The GG blocks have preferential binding sites for divalent counter-ions, such as Ca2+ , and the bound ions interact with other GG blocks to form linkages that lead to gel formation

On addition of sodium alginate solution to a calcium solution, interfacial polymerization is instantaneous, with precipitation of calcium alginate followed by a more gradual gelation of the interior as calcium ions permeate through the alginate systems

Encapsulation of probiotics in cellulose acetate phthalate (CAP)

Because of its ionizable phthalate groups, this cellulose derivative polymer is insoluble in acid media at pH 5 and lower but is soluble at pH higher than 6

In addition, CAP is physiologically inert when administered in vivo, and is, therefore, widely used as an enteric coating material for the release of core substances for intestinal targeted delivery systems

Rao, Shiwnarain, and Maharaj (1989) reported the encapsulation of B. pseudolongum in CAP using an emulsion technique

Microencapsulated bacteria survived in larger numbers (109 cfu/mL) in an acidic environment than non-encapsulated organisms, which did not retain any viability when exposed to a simulated gastric environment for 1 h.

Encapsulation of probiotics in proteins andpolysaccharide mixtures

Gelatin is useful as a thermally reversible gelling agent for encapsulation

Because of its amphoteric nature, it is also an excellent candidate for incorporating with anionic-gelforming polysaccharides, such as gellan gum

These hydrocolloids are miscible at pH >6, because they both carry net negative charges and repel one another

However, the net charge of gelatin becomes positive when the pH is adjusted below its isoelectric point and causes a strong interaction with the negatively charged gellan gum

In a recent study, Guerin, Vuillemard, and Subirade (2003) encapsulated Bifidobacterium cells in a mixed gel composed of alginate, pectin and whey proteins

They investigated the protective effects of gel beads without extra membrane and gel beads coated with extra membranes, formed by the conjugation of whey protein and pectin, in simulated gastric pH and bile salt solutions on the survival of free and encapsulated B. bifidum

Encapsulation of probiotics in chitosan

The biopolymer chitosan, the N-deacetylated product of the polysaccharide chitin, is gaining importance in the food and pharmaceutical field because of its unique polymeric cationic character, good biocompatibility, non-toxicity and biodegradability

Chitosan can be isolated from crustacean shells, insect cuticles and the membranes of fungi

The properties of chitosan vary with its source

The terms chitin and chitosan refer not to specific compounds but to two types of copolymers, containing the two monomer residues anhydro-N-acetyl-D-glucosamine and anhydro-D-glucosamine, respectively

Chitin is a polymer of b-(1-4)-2-acetamido-2- deoxy-D-glucopyranose and is one of the most abundant organic materials on earth and second to cellulose and murein, which is the main structural polymer of the bacterial cell wall

In order to achieve sufficient stability, chitosan gel beads and microspheres can be ionically cross-linked with Polyphosphates and sodium alginate

Encapsulation of probiotics in starch

Starch is a dietary component that has an important role in colonic physiology and functions and a potential protective role against colorectal cancer (Cassidy, Bingham, & Cummings, 1994)

Resistant starch is the starch that is not digested by pancreatic amylases in the small intestine and reaches the colon, where it can be fermented by human and animal gut microflora

The fermentation of carbohydrates by anaerobic bacteria produces short chain fatty acids and lowers the pH in the lumen

Resistant starch can be used to ensure the viability of probiotic populations from the food to the large intestine

Resistant starch also offers an ideal surface for adherence of the probiotics to the starch granule during processing, storage and transit through the upper gastrointestinal tract, providing robustness and resilience to environmental stresses.

Bacterial adhesion to starch may also provide advantages in new probiotic technologies to enhance delivery of viable and metabolically active probiotics to the intestinal tract

Talwalkar and Kailasapathy (2003) produced alginatee starch gel beads by dropping a mixture of alginatee starch-bacteria into a CaCl2 coagulation bath

The probiotic bacteria used for this study were L. acidophilus and B. lactis.

They found that encapsulation prevented cell death from oxygen toxicity

It is known that alginate gel beads restrict the diffusion of oxygen through the gel, creating anoxic regions in the centre of the beads

Benefit Product

Facilitates the production of oxygen-sensitive cultures Dried probiotic cultureFacilitates the recovery of centrifugation-sensitive cultures Dried probiotic cultureFacilitates the recovery of high EPS-producing cultures Dried probiotic cultureLess contamination problems Dried probiotic cultureCultures can be air-dried Dried probiotic cultureImproved survival on exposure to gastric solutions NutraceuticalImproved survival on exposure to bile solutions NutraceuticalImproved stability during storage in dried form NutraceuticalImproved acidification rate Dried sausagesImproved survival on heating Biscuits, powderImproved survival on freezing Ice cream, milk-based medium Improved retention in the finished product CheeseProtection against bacteriophages Fermented milksProtection against yeast contaminants Fermented milksImproved survival during storage Yoghurt, milk

Beneficial effects of probiotic microencapsulation

Microencapsulationtechniques

Types of materials for coating

Major steps in processes

Spray-drying Water-soluble polymers (i) Preparation of the solutions including microorganisms(ii) Atomization of the feed into spray(iii) Drying of spray (moisture evaporation)(iv) Separation of dried product form

Spray-congealing Waxes, fatty acids, water-soluble and water-insoluble polymers, monomers

(i) Preparation of the solutions containing core (e.g. probiotics)(ii) Solidification of coat by congealing the molten coating materials into non-solvent(iii) Removal of non-solvent materials by sorption, extraction or evaporation techniques

Fluidized-bed coating/air-suspension

Water-insoluble and water-soluble polymers, lipids, waxes

(i) Preparation of coating solutions(ii) Fluidization of core particles(iii) Coating of core particles with coating solutions

Techniques and processes used for encapsulating probiotic microorganisms

Microencapsulationtechniques

Types of materials for coating

Major steps in processes

Extrusion Water-soluble and water insoluble polymers

(i) Preparation of coating solution materials(ii) Dispersion of core materials(iii) Cooling or passing of core-coat mixtures through dehydrating liquid

Coacervation/phaseseparation technique

Water-soluble polymers (i) Core material is dispersed in a solution of coating polymer, the solvent for the polymer being the liquid manufacturing vehicle phase(ii) Deposition of the coating, accomplished by controlled, physical mixing of the coating and core materials in the vehicle phase(iii) Rigidifying the coating by thermal, cross-linking or desolvation techniques, to form self-sustaining microcapsules

Electrostatic method Oppositely charged polymers/ compounds

(i) Mixing of core and coating materials(ii) Extrusion of mixtures of core-coating materials in oppositely charged solutions(iii) Freeze-dry or oven-dry of microcapsules/microspheres/beads

Spray-coating methods for the microencapsulation of probiotics. The three technologies illustrated principally differ in the type of air fluidization employed and the site in the vessel where the coating material is sprayed.

Gel-particle technologies for the microencapsulation of probiotics. Three techniques used for the ME of probiotics in alginate gels. In the extrusion process (far left), the size of the particles can be adjusted by using vibrating nozzles or piezzo effects. With the emulsion processes (centre and right), agitation speed and conception of the mixers enable bead size adjustments. The emulsion processes are carried out by adding an alginate or carrageenan cell suspension to an oil phase. Solidification then occurs through the addition of either a CaCl2 solution or an acid solution. Co-encapsulation can be carried out by adding the second bioactive ingredient to the alginate solution, to the polymerising solution or to the coating solution.

Conclusions and Future Trends

Micro-encapsulation will assume importance in delivering viable strains of probiotic bacteria in large numbers to consumers

It will be used as a tool to co-encapsulate both prebiotic ingredients and probiotic bacteria within the same capsule to enhance growth and multiplication of these bacteria through symbiotic effects when they are released in the gastro-intestinal tract

In the future multiple-delivery may be developed, such as co-encapsulating prebiotics and probiotics as well as nutraceuticals, thus a new area of more complex nutritional matrices will need to be investigated

More in vivo studies should be conducted using human subjects to confirm the efficacy of micro or nano encapsulation in delivering probiotic bacteria and their controlled release in the gastro-intestinal system