Seminar On:-

Microencapsulation Technology
For Probiotic bacteria
 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 the
alginate matrix.
 In designing the encapsulation process, the following
questions should be asked:

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]
 Difference between
Immobilization and Encapsulation

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
 Advantages of Micro-encapsulation
Immobilization/ Entrapment

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
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
 Immobilisation/ Encapsulation of cells for
food/biotechnological application

Culture Technique/Mechanis Product Reference

m

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. Cream White brined Ghoddusi and

adolescentis cheese Robinson, 1998
B. bifidum, B. infantis, Calcium alginate gels Crescenza cheese Gobbeti et al., 1997
and B. longum
L. lactis subspp. lactis k-Carrageenan and locust Fresh cheese Sodini et al., 1997
bean gum
L. Casei Liquid core alginate Lactic acid Yoo et al., 1996
capsule
Lactobacilli Alginate Frozen dessert Sheu and Marshall,
1993
 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 40-45 0

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 and
polysaccharide 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
 Beneficial effects of probiotic
microencapsulation

Benefit Product

Facilitates the production of oxygen-sensitive cultures Dried probiotic culture

Facilitates the recovery of centrifugation-sensitive cultures Dried probiotic culture
Facilitates the recovery of high EPS-producing cultures Dried probiotic culture
Less contamination problems Dried probiotic culture
Cultures can be air-dried Dried probiotic culture
Improved survival on exposure to gastric solutions Nutraceutical
Improved survival on exposure to bile solutions Nutraceutical
Improved stability during storage in dried form Nutraceutical
Improved acidification rate Dried sausages
Improved survival on heating Biscuits, powder
Improved survival on freezing Ice cream, milk-based medium
Improved retention in the finished product Cheese
Protection against bacteriophages Fermented milks
Protection against yeast contaminants Fermented milks
Improved survival during storage Yoghurt, milk
 Techniques and processes used for encapsulating
probiotic microorganisms

Microencapsulation Types of materials for Major steps in processes

techniques coating
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- (i) Preparation of the solutions containing core
soluble and water-insoluble (e.g. probiotics)
polymers, monomers (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 Water-insoluble and water- (i) Preparation of coating solutions
coating/ soluble polymers, lipids, (ii) Fluidization of core particles
air-suspension waxes (iii) Coating of core particles with coating solutions
Microencapsulation Types of materials for Major steps in processes
techniques coating
Extrusion Water-soluble and water (i) Preparation of coating solution materials
insoluble polymers (ii) Dispersion of core materials
(iii) Cooling or passing of core-coat mixtures
through dehydrating liquid
Coacervation/phase Water-soluble polymers (i) Core material is dispersed in a solution of
separation coating polymer, the solvent for the polymer
technique 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 (i) Mixing of core and coating materials
polymers/ compounds (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