This document discusses microencapsulation of probiotic bacteria. It begins by defining probiotics and their health benefits. Microencapsulation techniques can protect probiotic bacteria from harsh environments and target delivery to the gut. Common encapsulation methods include spray drying, extrusion and emulsion. Materials used for encapsulation include sodium alginate, cellulose acetate phthalate, chitosan and starch. Microencapsulation has applications in food and pharmaceutical products to enhance the viability of probiotic bacteria.

1. Presented by: Shalini
Department of Food science, Nutrition
& Technology
CSK HPKV, Palampur

2. Contents
Introduction
Microencapsulation
 Advantages of Microencapsulation
Materials used for Microencapsulation
Encapsulation techniques
Encapsulated probiotics in food products
Developments in the Food industry
Pharmaceutical Applications of Encapsulated probiotics
Conclusion

3. Introduction
• The World Health Organization (WHO) defines probiotics as“ live micro-
organisms, which, when administered in adequate amounts confer a health
benefit on the host.
• The concept of probiotics was first introduced in the 20th century by Noble prize
winner and father of modern immunology, Elie Metchnikoff (1845-1916), when
he noticed that the long, healthy lives of Bulgarian peasants resulted from their
consumption of fermented milk products.
• The term “Probiotics” was first introduced in 1965 by Lilly and Stillwell, when it
was described as growth promoting factors produced by microorganisms.
• The most commonly used probiotic microorganisms are Lactobacillus and
Bifidobacteria strains.

4. • Probiotic products are important functional foods as they represent about 65%
of the world functional food market.
• Probiotic bacteria have been incorporated into a wide range of foods, including
dairy products (such as yogurt, cheese, ice cream, dairy desserts) but also in
non-dairy dairy products (such as chocolate, cereals, juices) (Anal and Singh.
2007).
• The beneficial effects of probiotics on the human gut flora include antagonistic
effects and immune effects. The use of probiotic bacterial cultures stimulates the
growth of preferred microorganisms, crowds out potentially harmful bacteria
and reinforces the body’s natural defense mechanisms.
• Probiotics have been reported to play a therapeutic role by lowering cholesterol,
improving lactose tolerance and preventing some cancers (Kailasapathy and
Chin. 2000).

5. • Probiotic based products are associated with many health benefits. However, the
main problem is the low survival of these microorganisms in food products and
in gastrointestinal tract.
• To produce these beneficial effects in health, probiotics have to be able to survive
and multiply in the host.
• Probiotics should be metabolically stable and active in the product, survive
passage through the stomach and reach the intestine in large amounts.
• Providing probiotics with a physical barrier is an efficient approach to protect
microorganisms and to deliver them into the gut.
Microencapsulation of probiotic bacteria can be used to enhance the
viability during processing, and also for the targeted delivery in
gastrointestinal tract.

6. • Microencapsulation can be defined as the process
in which cells are retained within an encapsulating
membrane to reduce cell injury or cell lost, in a
way that result in appropriate microorganism
release in the gut (Sultana et al. 2000).
• 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).
A technology to Protect Probiotic Bacteria
A microcapsule consists of a semipermeable, spherical, thin, and strong membrane
surrounding a solid/liquid core, with a diameter varying from a few microns to 1
mm.
Microencapsulation

7. 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.

8. In designing the encapsulation process, the following
questions should be asked:
1) What function must be encapsulated ingredient 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 optimum concentration of the active material in the microcapsule ?
5) By which mechanism will the ingredient be released from the microcapsule ?
6) Which particle size, density, and stability requirements for the encapsulated
ingredients ?
7) What are the cost constraints of the encapsulated ingredient ?

9. Advantages of Micro-encapsulation
 The microcapsule is composed of a semipermeable, spherical, thin and strong
membranous wall.
 Therefore the bacterial cells are retained within the microcapsules (Jankowski
et al. 1997).
 More over, 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
semipermeable membrane easily.
 The membrane serves as a barrier to cell release and minimises 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 (Franjione and Vasishtha. 1995).

10. Materials used for Microencapsulation
• Various types of encapsulating
materials are used for the process.
• Namely:
– Alginate
– Gellan gum and xanthan gum
– k-Carrageenan
– Cellulose acetate phthalat
– Chitosan
– Starch
– Pectin
– Gelatin
– Whey proteins
– Chickpea protein
The selection of any material
depends on
1) its capsule forming capability,
2) its strength,
3) its enhancing viability of probiotics,
4) its cheapness,
5) its availability,
6) biocompatibility.

11. 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℃
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.

12. • The conventional encapsulation methods, 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 as increase in the survival of bacteria under different
conditions, than the non-encapsulated state.

13. Encapsulation of probiotics in
alginate systems
• Alginate is a naturally derived polysaccharide extracted from various species of
algae and composed of β-D-mannuronic and α- L-guluronic acids.
• The composition of the polymer chain varies in amount and in sequential
distribution according to the source of the alginate and this influences
functional properties of alginate as supporting material.
• Alginate hydrogels are extensively used in cell encapsulation and calcium
alginate is preferred for encapsulating probiotics because of its simplicity, non-
toxicity, biocompatibility and low cost.
• Sodium alginate is also used for encapsulating probiotics

14. • The success of the use of alginate in microencapsulation of probiotics is due to
the basic protection against acidity it provides to the cells
• Alginates with a high content of guluronic acid blocks (G blocks) are preferable
for capsule formation because of their high mechanical stability, high porosity
and tolerance to salts and chelating agents (Nicetic et al. 1999).
Chen et al. 2005 used prebiotics (fructooligosaccharides or
isomaltooligosaccharides), a growth promoter (peptide) and sodium alginate as
coating materials to microencapsulate different probiotics such as L. acidophilus, L.
casei, B. bifidum and B. longum. A mixture containing sodium alginate (1% w/v)
mixed with peptide (1% w/w) and fructooligosaccharides (3% w/w) as coating
materials produced the highest survival in terms of probiotic count.

15. 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 et al. 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.

16. 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 (King. 1995).

17. • Hyndman et al. 1993 used high concentrations of gelatin (24% w/v) to
encapsulate Lactobacillus lactis by cross-linking with toluene-2,4-
diisocyanate for biomass production.
• In a recent study, Guerin et al. 2003 encapsulated Bifidobacterium cells in
a mixed gel composed of alginate, pectin and whey proteins.

18. 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.
 In order to achieve sufficient stability, chitosan gel beads and microspheres can
be ionically cross-linked with polyphosphates and sodium alginate.

19. 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 et al.
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.

20. • 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 alginate 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.

22. Encapsulation systems
(a) reservoir type, (b) matrix type, and (c) coated
matrix type.
Different types of encapsulates can be
found, the reservoir type and the
matrix type.
 The reservoir type has a shell around
the core material and this is why it
can also be called a capsule.
 In the case of matrix type, the active
agent is dispersed over the carrier
material and can also be found on
the surface.
 A combination of these two types gives a third type of capsule: the matrix where
the active agent is recovered by a coating

23. Size range provided by each technique

24. General plan describing steps to produce
microcapsules
Encapsulation technology is usually held in three stages.

25. Spray Drying
The most commonly used microencapsulation method in the food industry, is
economical and flexible, and produces a good quality product
The process involves:
 the dispersion of the core material into a polymer solution,
 forming an emulsion or dispersion,
 followed by homogenisation of the liquid,
 then atomisation of the mixture into the drying chamber.
This leads to evaporation of the solvent (water) and hence the formation of matrix
type micro capsules.
Advantage Disadvantage
It can be
operated on a
continuous basis
the high temperature used in
the process may not be
suitable for encapsulating
probiotic bacterial cultures
Proper adjustment and control of the
processing conditions such as the inlet
and the outlet temperatures can achieve
viable encapsulated cultures of desired
particle size distribution.

26. • At an inlet temperature of 100℃ and low outlet temperature of 45℃,
Bifidobacterium cells were encapsulated satisfactorily to produce micro spheres
with gelatinised modified starch as a coating material (O’Riordan et al. 2001).
• A previous report indicated that survival of probiotic bacteria during spray
drying decreased with increasing inlet temperatures (Mauriello et al. 1999)
• Inlet temperatures of above 60℃ resulted in poor drying and the sticky product
often accumulated in the cyclone and sometimes in the receiving flask.
• Higher inlet temperatures (>120℃ resulted in higher outlet temperatures
(>60℃) and significantly reduced the viability of encapsulated bifidobacteria
(O’Riordan et al. 2001).

27. Spray-drying Procedure
 The solution is pressured and
then atomized to form a
‘‘mist’’ into the drying
chamber.
 The hot gas (air or nitrogen)
is blown in the drying
chamber too.
 This hot gas allows the
evaporation of the solvent.
 The capsules are then
transported to a cyclone
separator for recovery.

28. Extrusion technique
• Extrusion is a physical technique to encapsulate probiotic living cells and uses
hydrocolloids (alginate and carrageenan) as encapsulating materials.
• The Micro-encapsulation of probiotic cells by extrusion involves projecting the
solution containing the cells through a nozzle at high pressure.
• Extrusion of polymer solutions through nozzles to produce capsules is mainly
reported on a laboratory scale, where simple devices such as syringes are
applied. If the droplet formation occurs in a controlled manner (contrary to
spraying) the technique is known as prilling.
• This is preferably done by the pulsation or vibration of the jet nozzle.

29. Principle of the technique
 Simple needle droplet-generator
that usually is air driven (a) and
pinning disk device (b).
 The probiotic cells are added to
the hydrocolloid solution and
dripped through a syringe
needle or a nozzle spray
machine in the form of droplets
which are allowed to free-fall
into a hardening solution such
as calcium chloride.
Extrusion technologies

30. Emulsion technique
• The discontinuous phase (small volume of the cell polymer suspension) is added
to a large volume of oil (continuous phase).
• The mixture is homogenized to form water-in-oil emulsion.
• Once the water-in-oil emulsion is formed, the water soluble polymer is
insolubilized (cross-linked) to form the particles within the oil phase.
• The beads are harvested later by filtration.

31. • The size of the beads is controlled by the speed of agitation, and can
vary between 25 μm and 2 mm.
• For food applications, vegetable oils are used as the continuous
phase.
• Some studies have used white light paraffin oil and mineral oil.
• Emulsifiers are also added to form a better emulsion, because the
emulsifiers lower the surface tension, resulting in smaller particles
(Krasaekoopt et al. 2003).

32. Spray coating technology
A liquid coating
material is sprayed over
the core material and
solidifies to form a layer
at the surface. The
liquid coating material
can be injected from
many angles over the
core material: fluid-bed
top spray coating (a),
fluid-bed bottom spray
coating with the
Wurster device (b), and
fluid bed tangential
spray coating (c)

33. Encapsulated
Probiotics in Food
Products

34. Cheese
• Many studies have reported the use of encapsulated probiotic cells and
particularly in Cheddar cheese.

35. Yogurt
• The incorporation of probiotic living cells in yogurt
enhances its therapeutic value. However, there is poor
level of probiotic viability in yogurt because of the low
pH (from 4.2 to 4.6).
• Studies have shown that the use of encapsulated
probiotic bacteria was better for their survival.
• The incorporation of probiotic cells into yogurts could
be carried out without making many modifications
from the traditional process (Kailasapathy. 2009).

36. • Kailasapathy 2005 studied the survival and effect of free and calcium-induced
alginate–starch encapsulated probiotic bacteria (Lactobacillus acidophilus and
Bifidobacterium lactis) pH, exopolysaccharide productionand their influence
on the sensory properties of yoghurt. The results showed that Addition of
probiotic bacteria (free or encapsulated) reduced acid development in yogurt
during storage. There was an increased survival of 2 and 1 log cell numbers of
L. acidophilus and B. lactis, respectively due to protection of cells by
microencapsulation.
• This study has shown that incorporation of free and encapsulated probiotic
bacteria do not substantially alter the overall sensory characteristics of
yogurts and microencapsulation helps to enhance the survival of probiotic
bacteria in yogurts during storage.

38. Frozen dairy desserts
 It is not easy to incorporate probiotic microorganisms into frozen desserts because
of high acidity in the product, high osmotic pressure, freeze injury and exposure
to the incorporated air during freezing.
 The introduction of probiotic bacteria in an encapsulated form into frozen
desserts may overcome these difficulties and could produce useful markets and
health benefits (Chen and Chen. 2007).
Godward and Kailasapathy (2003) studied the incorporation of probiotic cells in
ice cream in different states.The results have shown that free cells survive better
than encapsulated cells. Freshly encapsulated probiotic cells had greater survival
than those which were freeze-dried after encapsulation and co-encapsulation of L.
acidophilus and B. bifidum enhances the survival of both strains.
Finally, addition of probiotics does not affect air incorporation into ice cream.

40. Other food products
• Most of the products containing probiotic cells are dairy products and it is
necessary to develop other food carrier for probiotics owing to lactose
intolerance in certain populations.
• Efforts have been made to identify new food carriers.
• For example, good quality mayonnaise was obtained when incorporating
encapsulated bifidobacteria.
• Calcium alginate provides protection for bifidobacteria against the bactericidal
effects of vinegar.

42. Developments in the Food industry
 During the past few years, food products containing encapsulated probiotic cells
have been introduced on the market.
 In 2007, Barry Callebaut developed a process to produce chocolate containing
encapsulated probiotic cells with the Probiocap technology in partnership with
Lal’food. According to Barry Callebaut, the addition of encapsulated probiotic
cells has no influence on chocolate taste, texture and mouth feel. A consumption
of 13.5 g per day of probiotic chocolate seems to be sufficient to ensure the
balance of the intestinal microflora.
 In Korea, yogurts containing encapsulated LAB are available on the market
under the brand name Doctor-Capsule

43. Pharmaceutical Applications of
Encapsulated probiotics
• Probiotic supplements are available in different forms.
• The two most popular forms are:
1. Capsules and
2. Freeze dried powders.
• Probiotic strains of L. acidophilus 50 ME are sold as micro-encapsulated by
Institut Rosell/ Lallemand The Americas, Montreal, Canada.
• Probiocap (micro encapsulated L. acidophilus 50 ME in an hydrophobic matrix)
marketed by this company claims to have increased tolerance to gastric juices,
improved survival during tableting, enhanced temperature resistance during
food processing and extended shelf life at room temperatures.

44. Conclusion
 Microencapsulation has been proven to be one of the most efficient
methods for maintaining viability and stability of probiotics, as it protects
probiotics during food processing and storage, as well as in gastric
conditions.
 Microencapsulation can achieve a wide variety of functionalities
according to the development of the technology and nowadays,
encapsulated probiotic cells can be incorporated in many types of food
products.

45. • Probiotics can be found not only in dairy products, but also in chocolate
or cereals too.
• In the future multiple-devivery 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.