20Micro-Encapsulation ofProbioticsJean-Antoine Meiners
20.1 Introduction
Micro-encapsulation is defined as the technology for packaging with the help of
protective membranes particles of finely ground solids, droplets of liquids or
gaseous materials in small capsules that release their contents at controlled rates
over prolonged periods of time under the influences of specific conditions (Boh,
2007). The material encapsulating the core is referred to as coating or shell.
The majority of microcapsules have spherical shapes and their diameter
varies from a few microns to 1 mm. However, some authors consider particles
of even more than one millimeter as microcapsules. For the purpose of this
chapter the term microcapsule refers to capsules whose aim is the protection
and controlled release of the active substance, and the term microsphere, a
commonly used term in the scientific literature, refers to granules, that do not
have a core-shell morphology, and can be simply defined as the embedding of an
active substance in a matrix (Watheley, 1996).
In general, encapsulation can be used to improve the stability of the active
substance during processing and storage, mask unpleasant flavors and odors,
control possible oxidative reactions, and provide controlled release at the right
place and the right time. As such, it has numerous applications in the food,
pharmaceutical, cosmetic, agricultural, textile, paper and paint industries. One of
the earliest published inventions was the carbonless copy paper in which tiny
microcapsules were fixed on the backside of a sheet of paper; these were crushed
by the pressure of writing, thus releasing their dye (Green, 1957). In the areas of
pharmaceuticals and chemicals, many products, e.g., enzymes, can cause health
and safety hazards when manipulated in a very fine powder form, due to excessive
dust formation; granulation into larger size particles and coating can be used to
alleviate such handling problems (Meesters, 2006). In terms of food applications,
the aims of encapsulation techniques are to improve the stability of bioactive
# Springer ScienceþBusiness Media, LLC 2009
ingredients during processing and storage and to prevent undesirable interactions
with the food matrix (Champagne and Fustier, 2008). An example is the case of
ascorbic acid, a highly reactive, water soluble and heat-labile compound, encap-
sulation of which can double its shelf life compared to the free form (Wilson and
Shah, 2007).
Various approaches can be used to open up the microcapsules to release the
active substance. These include (i) mechanical rupture of the membrane, e.g., in the
case of mastication of micro-encapsulated flavours in chewing gums, (ii) exposure
to high temperatures to make the coating material melt, a technology frequently
used for encapsulated chemical leavening agents in baked goods, (iii) dissolution of
the capsules when placed in solvents, (iv) exposure to specific pH, (v) biodegrada-
tion of the polymer coating by enzymes, (vi) diffusion of the active substance
through the polymer coating, (vii) high osmotic pressure inside the microcapsule
and (viii) combinations of the above (Pothakamury and Barnosa-Canovas, 1995).
In the area of microbial products, micro-encapsulation is used in order to
enhance the delivery of probiotic microorganisms into foods during processing
and storage, or to protect against the acidic conditions in the stomach and ensure
delivery into the intestine. In addition, microbial cells can also be immobilized
onto polymer matrices and used as biocatalysts in fermentation processes. The
main difference between encapsulation and immobilization is that in the latter
the polymer beads produced allow fast and easy diffusion of water and other
fluids, and thus the cells are biochemically active (Klein and Vorlop, 1985).
20.2 Micro-encapsulation Techniques and Processes
A variety of encapsulation techniques are available and include both chemical
processes, such as phase microseparation, coacervation, liposome encapsulation
molecular inclusion, as well as physical processes, such as spray drying, spray
chilling, prilling, spinning disk, fluidized bed coating, and extrusion. Certain
steps are common to many of these processes.
Basically the initial step is to introduce the active substance inside the shell;
this involves dispersion or atomization in order to position the membrane on the
outside of the microcapsule and the active substance in the core.
In the case of microcapsules containing a liquid core active substance an
emulsion is prepared, whose surface is polymerized in a subsequent step, the
interface condensed and/or the solvent evaporated. In the section below, the
different types of encapsulation methods are presented in more detail.
806 20 Micro-Encapsulation of Probiotics
20.2.1 Spray Drying
Spray drying is one of the most commonly used encapsulation method, as it is a
well established and cost-efficient technology; it is basically designed to evaporate
water from the dry matter. The solution is injected in a hot air stream in a closed
vessel and the solvent, whichmost of the times is water, is evaporated. The energy is
absorbed to evaporate the water and consequently the powder temperature can be
controlled. The residence time in the tower is one of the limiting factors; the
majority of the moisture has to evaporate during the fall time in the tower of the
particles (Adamiec and Marciniak, 2004). In order to be efficient, the total surface
area should be as large as possible and consequently the droplet sizes small, and if
possible, of the same size.
The principle of the technique is based on a spray, which is created by forcing
the fluid through an orifice. The energy required to overcome the pressure drop
upon exiting the orifice is supplied by the spray dryer feed pump. Pressure nozzles
coupled to high pressure feed pumps, which produce pressures of as much as 3000
psig, have the advantage of producing a narrow particle size distribution, but can
have severe damaging effects on a microorganism’s cell structure. Despite the fact
that they are less energy efficient and produce narrower particle size distribution,
the two-fluid nozzle is very popular for laboratory equipment, probably since it
allows working with small flow rates. The most popular nozzle type for industrial
spray drying is the centrifugal atomizer, where a spray is created by passing the fluid
across or through a rotating wheel or disk. The benefit for micro-encapsulation
purposes is that rather large particles, up to 100 mm, can be produced this way.
20.2.2 Spray Chilling and Cooling
Spray chilling and spray cooling (or congealing), the second being operated at
lower temperatures, involve mixing thoroughly the core and amolten shell material
with a melting temperature well above the operating temperature (usually lipids),
and atomizing through a two media nozzle into a cooling chamber in order to
solidify the droplet instantaneously. One of the limitations is the speed in heat
transfer of the energy freed during the re-crystallization. The use of cooling media
has allowed high volume and high speed production. Another limiting factor is the
difference in surface tension between the matrix and the core material. Conse-
quently, the core is not always placed in the center of the microcapsule, which may
affect the protective properties of the microcapsule (Meiners, 2004).
Micro-Encapsulation of Probiotics 20 807
20.2.3 Prilling
Hawley’s Condensed Chemical Dictionary says that prills are ‘small round or
acicular aggregates of a material’. Prilling can be a useful technology, when solidifi-
cation of the shell material is an instantaneous reaction. The core and the shell
material are mixed thoroughly and the dispersion is pumped to flow over a sonically
vibrating dispersion head. Gravity allows the droplets to fall into the collection
device for cooling or polymerization (Wu et al., 2007). More advanced and sophis-
ticated equipment have been developed that limit considerably the residence time
of the dispersion and the need for large cooling towers (Meiners, 2004).
20.2.4 Spinning Disk
A comparable technology to prilling is the spinning disk technology, which uses
centrifugal forces for droplet separation.
The droplet volumes and their mutual spacing are governed by the channel
geometry and the frequency of rotation. Devices exist that combine spinning disk
forces with high frequency droplet separation (Chesnokov, 2001).
20.2.5 Fluidized Bed
Fluidized bed technology is based on the separation of individual particles in a
gas stream and the fixation of the membrane substance by polymerization, drying
or crystallization around the core. The solvation and drying steps can be avoided,
which for thermo-sensitive materials may represent a major advantage. Agglom-
eration and retention in the filter system cause the use of the fluidized bed system
to be difficult with products having strong adhesive properties (Guignon, 2002).
20.2.6 Extrusion
Micro-encapsulation by extrusion involves projecting an emulsion core and
coating material through a nozzle at high pressure. It involves preparing a
hydrocolloidal solution, adding the active substance and extrusing the suspension
through a nozzle in the form of droplets into a hardening solution or setting bath
(Krasaekoopt et al., 2003). Carbohydrate matrices in the glassy state have very
808 20 Micro-Encapsulation of Probiotics
good barrier properties and extrusion is a convenient process enabling the
encapsulation of active substances in such matrices (Gouin, 2004).
20.2.7 Coacervation
Coacervation consists of the separation of colloid particles from a solution, which
then agglomerate into a separate liquid phase called coarcevate. A number of hydro-
colloid systems have been evaluated for coacervation micro-encapsulation including
among others the gelatine/gum acacia, heparin/gelatine, carageenan, chitosan, soy
protein, lactogloboulin/gum acacia and the guar/dextran system (Gouin, 2004).
20.2.8 Liposomes
Liposomes are artificially made microscopic membrane vesicles consisting of one
or more concentric layers of lipids. They are formed by dispersing the lipid
formulation in a solvent system, decreasing the solvent volume and then re-
dispersing the film of lipid/solvent in an aqueous phase (Bangham, 1995).
20.2.9 Inclusion Complexation
Cyclodextrins are cyclic oligomers, who have the ability to form inclusion com-
plexes with the active substances. They are typically used for the protection of
unstable and high value chemicals, such as flavours. Oil-in-water emulsions using
cyclodextrins can also be subsequently spray dried (Astray et al., 2009).
20.3 Technologies used for the Immobilization andMicro-encapsulation of Microganisms
Immobilization of living cells was the first form of micro-encapsulation and
was pioneered about 50 years ago for medical purposes, and (Chang, 1964).
The first patent demonstrating the biocompatibility of polymers and the resis-
tance of the encapsulated material to sterilization conditions was granted in 1965
(Mauvernay, 1965).
Immobilization of microorganisms for bioprocessing purposes became im-
portant for the development of the continuous fermentation process. Much of the
Micro-Encapsulation of Probiotics 20 809
research work was done by the brewing industry using immobilized yeast cells
and the dairy industry using immobilized lactic acid producing bacteria (Prevost
and Divies, 1988).
The pore size of the microcapsules produced has an important effect on the
cellular biochemical activities, as it controls the rate of retention/passage of
undesirable metabolites (Klein et al., 1983). Different methods have been used
to immobilize microorganisms, including, physical entrapment in a polymeric
network, attachment or adsorption to a carrier, and membrane entrapment.
In order to entrap the biomass in microcapsules simultaneous with the
membrane formation, it is important for the that droplets to be generated simul-
taneously with the membrane. For this purpose, specific equipment has been
developed and polymers have been selected. The polymers generally form a gel
under the influence of ionization or thermosetting.
For the purpose of droplet separation with a narrow size distribution, a
pumping system under gravity provides a constant uninterrupted flow of the
liquid. In the early droplet generators, the liquid consisted of a solution of the
biomass and the dissolved polymer. Upon falling into the collection bath filled
with a solution containing the ionic solution, the polymer membrane was cross-
linked to form self sustaining microcapsules, encapsulating the biomass droplet
within the membrane (Sheu and Marshall, 1993). The more recent versions use a
nozzle designed for co-extrusion, creating a polymer network around the droplet
during its fall into the collection bath. The different dripping systems can be
identified according to the principles below. > Figures 20.1–20.4 depict the
various types of nozzles used.
� Dripping without assistance by gravity into the collection bath
� Dripping assisted by an air stream
� Dripping assisted by an electrostatic force
� Laminar jet break up assisted by vibration
� Jet break up assisted by rotation and vibration
� Co-extrusion assisted by laminar jet break up.
The use of static mixers has also appeared to be very promising, since it allows
large volume and cost efficient production. The device consists of mixer elements
contained in a cylindrical (tube) or squared housing. These can vary from 6 mm
to 6 meters diameter. Static mixer elements consist of a series of baffles. As the
streams move through the mixer, the non-moving elements continuously blend the
materials (Maa and Hsu, 1996).
810 20 Micro-Encapsulation of Probiotics
20.4 Objectives for the Micro-encapsulation ofProbiotics
A major function of micro-encapsulation is to provide protection against the high
acidity of the gastric fluids. A microcapsule containing the probiotic must not be
fractured until it passes through the stomach. Since the biological release mecha-
nism is triggered by the higher pH in the upper intestine, a coating can be used that
. Figure 20.1Dripping assisted by an electrostatic force (courtesy of Nisco Engineering AG).
. Figure 20.2Dripping assisted by aerodynamically jetting (courtesy of Nisco Engineering AG).
Micro-Encapsulation of Probiotics 20 811
withstands the low pH and can release its content at a pH similar to that of the large
intestine (e.g., pH 5.5 to 7). The survival of commercial probiotics in conditions
of very low pH was recently investigated; the researchers assessed in vitro the
survival of 32 probiotic strains, all isolated from commercially available products
. Figure 20.3Dripping assisted by co-axial air (courtesy of Nisco Engineering AG).
. Figure 20.4Jet cutter (courtesy of Genialab AG).
812 20 Micro-Encapsulation of Probiotics
in simulated gastric contents. Approximately 50% of them were viable after
20 minutes exposure; eighteen strains were further tested in respect to their bile
tolerance; five of them showed poor tolerance, whereas seven of them showed
moderate tolerance and the rest high tolerance (Gibson et al., 2005). The above
highlight the importance of developing effective micro-encapsulation methods,
especially for strains that are less robust than others. Another function of micro-
encapsulation of probiotics is to enhance the viability of the cells during processing
and storage. To this end, it is important to highlight that the final application of
the product may dictate the use of totally different technologies, or different shell
or carrier materials. For example, in the case of dried products (e.g., foods or
neutraceuticals), the water content, and in particular the water activity, is one of
the most important factors influencing cell survival, as it has a direct effect on
the metabolic activity of microorganisms. Water activity (aw) describes the
(equilibrium) amount of water available for the hydration of materials. Water
activity is the effective mole fraction of water, defined as aw¼lwXw ¼ p/p0,
where lw is the activity coefficient of water, Xw is the mole fraction of water in
the aqueous fraction, p is the partial pressure of water above the material and p0 is
the partial pressure of pure water at the same temperature. Based on the above, a
non-water permeable capsule material would be the first type of material to
evaluate for using in a dried product. If the capsule material is chosen from
non-ionisable polymers, the protection against the gastric acidity would likely
improve too.
In addition to the above, it must be noted that the suitability of a particular
encapsulation technology or material depends on the properties of the specific
probiotic strain (e.g., acid tolerance, oxygen tolerance, bile tolerance). It is generally
difficult to simply transfer the results from one strain to another; significant amount
of experimental work is needed to identify and optimise suitable encapsulation
methods. Below is a list of applications of encapsulated probiotics in the food and
non-food sectors.
� Cell immobilisation (e.g., industrial fermentations)
� Micro-encapsulation of probiotics in dairy and beverage applications
� Micro-encapsulation of probiotics in dry food applications
� Micro-encapsulation of probiotics in nutritional or medical supplements
>Table 20.1 lists the encapsulation technologies and the types of shell
materials commonly used for the micro-encapsulation of probiotics.
Micro-Encapsulation of Probiotics 20 813
. Table 20.1
Technologies used for the micro-encapsulation of probiotics (Adapted from Anal and Singh,
2007)
Technology Shell material Micro-encapsulation steps
Spray drying Water soluble polymers (a) Incorporate the microorganisms intothe solution
(b) Atomization into spray
(c) Evaporation of solvent
(d) Separation of powder
Spray chilling Lipids, waxes (a) Incorporate the micro organisms intothe melt
(b) Atomizing into spray
(c) Solidification of the coating by chillingbelow the melting temperature
Air suspensioncoating
Water insoluble and watersoluble polymers, lipids, waxes
(a) Preparation of the coating melt orsolution
(b) Fluidizing the core particles
(c) Atomizing small droplets of thecoating material around the core particles
(d) Drying, solidifying, crystallizing thecoating with core in the center
Extrusion; Jetcutter; Staticmixer
Water soluble polymers (a) Active substance is dissolved into thepolymer solution
(b) Dripping the solution into thecollection bath
(C1) Cross linking the polymer withdivalent ions
(C2) Gelling the polymer bythermosetting
(C3) Complexing with a polyelectrolyte
Co-extrusion Water soluble polymers (A1) Active substance is pumped to theinner nozzle port
(A2) Polymer is pumped to the outernozzle port
(b) Dripping the active substance andpolymer into the collection bath
(C1) Cross linking the polymer withdivalent ions
(C2) Gelling the polymer bythermosetting
814 20 Micro-Encapsulation of Probiotics
20.5 Biopolymers
It is obvious that the biopolymers applied must be biocompatible and non toxic
to the cells. The most popular biopolymers for micro-encapsulation of micro-
organisms are discussed below:
Alginate Alginate is one of the most used polymers for the encapsulation of
microorganisms. It can be found in the cell walls and intercellular spaces of brown
algae. It is a linear co-polymer with homo-polymeric blocs, covalently linked in
different sequences, depending to the source of algae. Alginic acid, the free acid from
alginate is the intermediate product in the commercial alginates and has limited
stability. In order to make stable water soluble products alginic acid is transformed
into a range of salts i.e., Ca-alginate; Na-alginate; K-alginate; Mg-alginate; NH4-
alginate.The ratio of mannuronic acid to gluconic acid and the structure of the
polymer determine the properties of alginate in solution. Alginates may be
prepared with a wide range of molecular weights. Alginate capsules are formed
by dripping an aqueous alginate solution into a solution containing a multi-
valemt cation, usually a calcium salt. The calcium ion attaches to two polymer
strands by replacing the salt bond, and can thus form a very fine network.
Carrageenan Carrageenan is family of a naturally occurring sulphated poly-
saccharide which fill the voids of the cellulosic structure of red seaweed. Carragee-
nans are made up of repeating galactose units and 3,6 anhydrogalactose, joined by
alternating glycosidic linkages. They are divided into three classes, k-, i- and
l- carrageenan, which have different properties. The plant source and the extraction
method determine to a large extent the molecular structures and properties; e.g.,
carrageenans with different amounts of sulphate ester groups will associate differ-
ently with metal ions. All types are all soluble in hot water, but only the k- type is
soluble in cold water. Gelation of carrageenan involves helix formation, and is
induced by cooling down to room temperature from a hot solution. Potassium and
calcium ions are also essential for gelling, as they stabilize the gel, prevent swelling,
or induce gelation (Krasaekoopt et al., 2003).
The strength of the carrageenan capsules can be improved using locust bean
gum (LBG) at a ratio of carrageenan to LBG of 2:1. LBG is part of the galacto-
mannan family. It is extracted from the kernels of the carob tree; it forms a food
reserve for the seeds and helps to retainwater under arid conditions. LBG consists of
a backbone of b-(1,4)-D-mannose units; approximately every fourthmannose unit
there is a substitution by a a-D-galactose side chain. The level of substitution is
important for the properties of the gum, as the galactomannan can associate and
self cross-link. LBG requires heating to dissolve in water and is rarely used as a
Micro-Encapsulation of Probiotics 20 815
single shell material (Rees, 1972). The increased strength of carrageenan/LBG
capsules is due to synergistic effects, which are attributed to the interaction
between the double stranded helix of k- carrageenan and the un-branched
segments of the D- mannose backbone of the LBG molecule.
Chitosan Chitosan is a weak anionic polysaccharide consisting mainly of
b(1,4) linked glucosamine units together with some N-acetylglucosamine units.
It is produced by de-acetylation of chitin extracted from crustacean shells. It is
positively charged and water soluble at pH below 6.5, and this enables it to form
polyelectrolyte complexes with negatively charged materials, such as polypho-
sphates, [Fe(CN)6]4- and [Fe(CN)6]3-, and citrate (Peniche et al., 2003).
Starch Starch is a polysaccharide consisting of a large number of glucose
monosaccharide units joined together by glycoside bonds. Starch consists of two
types of molecules, amylose (normally 20 – 30%) and amylopectin (normally
70 – 80%). Both consist of polymers of a-D-glucose units. Of the two components
of starch, amylose has the most useful functions as a hydrocolloid. Chemical
modification of starches, such as cross-linking, oxidization, acetylation, and hydro-
xypropylation may confer interesting changes in functionality. For example, octe-
nylsuccinic acid anhydride (OSAN)-modified starches are popular for their
emulsifying properties, as they contain both hydrophobic and hydrophillic groups.
In addition, resistant starch, i.e., the indigestible form of starch, offers an ideal
surface for adherence for the probiotic microorganisms during processing, storage
and their passage through the gastrointestinal tract (GIT) (Anal and Singh, 2007).
Mixing starch with k-carrageenan, alginate, xanthan gum and low molecular
weight sugars is a popular practice in micro-encapsulation as they reduce starch
retrogradation. Starch derivatives mostly hydrolyzed forms such as dextrins and
maltodextrins, are also frequently used as carrier material for spray and freeze
drying (Anal and Singh, 2007).
Gum arabic Gumarabic is a hydrocolloid produced by the natural exudation
of acacia trees. Because it is a complex mixture of molecules and the fact that the
material varies significantly with the source, the investigation of the exact molecu-
lar structures still attracts considerable research interest. It is generally composed
of a highly branched polysaccharide fraction consisting of galactose, arabinose,
rhamnose and glucuronic acids. It also contains an arabinogalactan-protein
complex in which arabinogalactan chains are covalently linked to a protein chain
through serine and hydroxyproline groups (Dror et al., 2006). The protein plays an
important role in the functionality of the gum. The simultaneous presence of
hydrophilic carbohydrates and hydrophobic protein gives the molecule its emul-
sification and stabilization properties (Randall et al., 1988).
816 20 Micro-Encapsulation of Probiotics
Gum arabic is used to produce gum arabic / gelatine coacervates, which have
been used as encapsulation materials. The formation of coacervates is possible
only at pH values below the isoelectric point of gelatine, which is approximately
at pH 8. It is at these values that gelatine becomes positively charged, whereas
gum arabic continues to be negatively charged. The formation of stable micro-
capsules using mixtures of gum arabic and whey protein has also been reported
(Weinbreck et al., 2004).
Pectin Pectin is a major cell wall component in plants, playing a role in the
control of cell growth and the defence against the invasions of microorganisms.
Pectins are composed of a a-D-galacturonic acid, which are interrupted by single
a-L-rhamnose residues. A major difference between pectins is their content in
methyl esters. The degree of esterification (DE) is defined by the numberof esterified
D-galacturonic acid residues. High methoxyl pectin forms gels due to hydropho-
bic interactions and hydrogen bonding between pectin molecules. Low methoxyl
pectin forms gels in the presence of di- and polyvalent cations, which cross-link
and neutralise the negative charges of the pectin molecule (Wher et al., 2004).
Gelatin Gelatin is a high molecular mass polypeptide derived from con-
nective animal tissue, such as bone and skin. The protein chain unfolds upon
melting and upon cooling it forms a coil-helix structure and entraps water,
forming a reversible gel. Gelatin is extracted after acid pre-treatment and has
an isoelectric point (IP) of 7–9.4; gelatin after lime pre-treatment has an IP of
4.5–5.3. The possibility to vary the IP, and thus the charge by adjusting the
pH makes gelatin a favourite candidate for micro-encapsulation. However, in
order for the capsules to be self sustainable cross-linking is required; and the
cytotoxity of the traditionally applied organic solvents makes the process less
suitable for microorganisms (Hyndman et al., 1993). However, as mentioned
previously, gelatin is used due to its amphoteric nature in co-operation with
anionic carbohydrates to form gum/gelatine coacervates.
Whey protein Whey protein is the protein obtained from whey during
cheese making. Whey proteins are obtained by ultrafiltration, during which,
the low molecular weight compounds such as lactose, minerals, vitamins and
non-protein nitrogen are removed in the permeate while the proteins become
concentrated in the retentate. After ultrafiltration, the retentate is pasteurized,
sometimes evaporated, and then dried usually by spray drying at low tempera-
tures in order to avoid significant protein denaturation.
Whey protein is very popular for its film forming characteristics and is used
as a protective material in spray drying, resulting in a water soluble microcapsule
system (Picot and Lacroix, 2004).
Micro-Encapsulation of Probiotics 20 817
Other polymers There is a large choice of acid resistant polymers with
designed release at intestinal pH values. Most of them have been approved for
pharmaceutical purposes, while only few of those have food approval: hydro-
xypropyl methylcellulose; methylcellulose; ethylcellulose; hydroxypropyl methyl-
cellulose phthalate; hydroxypropyl methylcellulose acetate succinate; poly(methyl
methacrylate); carboxymethyl cellulose; polyvinyl acetate phthalate; methylcellu-
lose phthalate; cellulose acetate phthalate; polyvinyl acetate phthalate; polyvinyl
pyrrolidone; carboxypolymethylene. Most of these products are only soluble
in alcohol and cannot be brought into direct contact with the culture; therefore
they can be applied as an outer top coating only. Finished products are in general
found in the pharmaceutical or the nutritional supplement markets.
20.6 Applications of Micro-encapsulation ofProbiotics
In this section, some examples from the various studies on micro-encapsulation
of probiotics are presented. The area of cell immobilization for use in fermenta-
tion systems has attracted considerable interest recently for both probiotic and
non probiotic lactic acid bacteria (LAB). The advantages of immobilized cells
over free cells include enhanced biological stability, high biomass concentration,
increased product yields, increased product stability, and the ability to separate
and re-use cells (Dervakos and Webb, 1991; Lacroix and Yidirim, 2007). As such,
immobilisation techniques have the potential for enhancing the performance of
probiotics and produce strains with specific physiological and functional char-
acteristics, as well as improved technological properties.
The alginate system has been studied extensively for the encapsulation of
probiotics. The research suggests that the size of the beads affects considerably cell
survival under simulated gastrointestinal conditions (Lee et al., 2000; Chandramouli
et al., 2004). Beads of less than 100 mm in size did not significantly protect
Bifidobacterium cells; the protection was increased as the beads became bigger,
especially for very large beads with sizes higher than 1 mm. However, such large
beads cause a coarseness of texture in food systems (Hansen et al., 2002).
Starch has also been investigated as a potential encapsulation material for
probiotics. Researchers have developed a micro-encapsulation technology that
involves encasing bifidobacteria in the hollow core of partially hydrolyzed
granular starch, which is then encapsulated with an outer coating of amylose.
818 20 Micro-Encapsulation of Probiotics
This technology is designed to protect the probiotic bacteria from adverse
environmental conditions during processing, in products during storage, and
during passage through the upper GIT for delivery in the large intestine (Mattila-
Sandholm et al., 2002).
Mixtures of various polysaccharides and in some cases proteins have also been
investigated as a potential encapsulation materials for probiotics. For example,
alginate was combined with pectin and whey proteins in order to protect Bifido-
bacterium bifidum during transit through the GIT. A clear improvement in cell
survival was observed compared to free cells, although still a significant percentage
of the cells were dying (Guerin et al., 2003). In another study alginate-starch gel
beads were produced to protect Lactobacillus acidophilus and Bifidobacterium
lactis; it was found that encapsulation prevented cell death from oxygen toxicity
(Talwalkar and Kailasapathy, 2003). It was also found that chitosan-coated alginate
beads were offering protection to various LAB strains during storage in milk
(Krasaekoopt et al., 2004), as well as in simulated gastrointestinal fluids (Lee
et al., 2004). In another study, a polysaccharide from kiwifruit was combined with
alginate and chitosan and shown to improve the survival of Lactobacillus rham-
nosus at low pH (Ying et al., 2007). A low-cost micro-encapsulation technique has
been recently proposed, which consists of coating milk fat droplets containing
powder particles of freeze dried cultures with whey protein and polymers, using
emulsification and spray drying in a continuous two step process. Rigorous control
of the size distribution of the different elements constituting the microcapsule is
required. In particular, the size of the material dispersed in the hydrophobic phase
must be larger than that of the globule (Picot and Lacroix, 2004).
The use of polymers as immobilisation matrices for probiotics during freeze
and spray drying has also been investigated. It was shown that immobilization of
Lactobacillus acidophilus with k-carrageenan could enhance the temperature
tolerance of the freeze dried cells and improve their storage stability (Tsen et al.,
2002). The results from other studies regarding the role of specific polymers on
cell survival during storage were less conclusive, and were highly dependent on
the species (Champagne et al., 1996a, 1996b).
In the case of spray drying, most of the probiotic strains do not survive
well the high temperatures and the osmotic stress to which they are exposed to
in the process. The temperature and phase changes, and drying, stress the cells
and damage their membranes. As a result their activity is typically lost after a
few weeks of storage at room temperature (Anal and Singh, 2007). One of the
methods that has been proposed to circumvent this is the incorporation of
Micro-Encapsulation of Probiotics 20 819
acacia gum into the drying medium; it was shown that this increased significanlty
the survival rate of Lactobacillus paracasei during storage at a variety of tempera-
tures (Desmond et al., 2002).
The size of the market for probiotics in animal feed applications has increased
significantly after the ban on antibiotics as growth promoters. Since feed additives
are generally stored under ‘barn conditions’, i.e., ambient temperature, oxygen and
moisture, micro-encapsulation can help to improve the survival of probiotics.
In this regard, and taking into account the relationship between high moisture
content and death, a watertight shell is required to achieve high protection. Fat
encapsulated probiotics have shown to be ‘barn stable’ for a period of over 2 years
without refrigeration (Meiners, 2004). In this process, also called ‘hot melt’
process, very thin layers of molten lipids are applied to the surface of freeze
dried cultures in fluidized bed equipment. After re-crystallization, very tight
microcapsules are formed. The properties of the microcapsules depend on the
choice of lipids, the moisture residue in the fluidizing air stream and the process
parameters, such as the droplet size of the molten lipid and the temperature of the
atomizing air.
20.7 Conclusion
The micro-encapsulation of probiotics has received a lot of attention, as it can help
to improve probiotic survival during processing and storage, as well as during
passage through the GI tract. A number of encapsulation techniques, such as spray
drying, spray coating, phase separation and extrusion, a well as natural biopoly-
mers, such as alginates, carrageenan, chitosan and pectin have been studied.
Although promising at a laboratory scale many of these technologies are difficult
to scale up. Further research is needed for the development of scalable and effective
technologies as well as for the design of controlled release delivery systems.
List of Abbreviations
DE Degree of Esterification
GIT Gastrointestinal Tract
IP Isoelectric Point
LAB Lactic Acid Bacteria
LBG Locust Bean Gum
820 20 Micro-Encapsulation of Probiotics
References
Adamiec J, Marciniak E (2004) Microencapsu-
lation of Oil/Matrix/Water system during
spray drying process. Proceedings of
the 14th International Drying Sympo-
sium, Sao Paulo, Brazil, 22–25 Vol. C,
2043–2050
Anal AK, Singh H (2007) Recent advances
in microencapsulation of probiotics for
industrial applications and targeted deliv-
ery. Trends in Food Science & Technology
18: 240–251
Astray G, Gonzalez-Barreiro C, Mejuto JC,
Rial-Otero R, Simal-Gandara J (2009)
A review on the use of cyclodextrins in
food. Food Hydrocolloids (In presss)
Bangham AD (1995) Surrogate cells or trojan
horses - the discovery of liposomes. Bio-
essays 17: 1081–1088
Boh B (2007) Developements et applications
industrielles des microcapsules. In:
Vandamme, Thierry F. (ed.). Microencap-
sulation: des sciences aux technologies.
Paris: Lavoisier, pp. 9–22
Champagne CP, Fustier P (2007) Microencap-
sulation for the improved delivery of bio-
active compounds into foods. Current
Opinion in Biotechnol 18: 184–190
Champagne CP, Mondou F, Raymond Y,
Brochu E (1996a) Effect of immobi-
lization in alginate on the stability of
freeze-dried Bifidobacterium longum,
Bioscience Microflora 15: 9–15
Champagne CP, Mondou F, Raymond Y, Roy D
(1996b) Effect of polymers and storage
temperature on the stability of freeze-
dried lactic acid bacteria. Food Research
International 29: 555–562
Chandramouli V, Kailasapathy K, Peiris P,
Jones M (2004) An improved method of
microencapsulation and its evaluation to
protect Lactobacillus spp. in simulated
gastric conditions. J Microbiol Methods
56: 27–35
Chang TMS (1964) Semipermeable microcap-
sules. Science 146: 524–525
Chesnokov YJ (2001) Short capillary waves on
the surface of a stretching cylindrical jet of
a viscous liquid. J Applied Mechanics and
Technical Physics 42: 431–436
Dervakos GA, Webb C (1991) On the merits of
viable-cell immobilization. Biotechnol
Advances 9: 559–612
Desmond C, Ross RP, O’Callaghan E, Fitzgerald
G, Stanton C (2002) Improved survival
of Lactobacillus paracasei NFBC 338 in
spray-dried powders containing gum aca-
cia. J Appl Microbiol 93: 1003–1011
Dror Y, Cohen Y, Yerushalmi-Rozen R (2006)
Structure of gum arabic in aqueous solu-
tion. J Polymer Science Part B-Polymer
Physics 44: 3265–3271
Gibson GR, Rouzaud, G, Brostoff J, Rayment N
(2005) An evaluation of probiotic effects
in the human gut: microbial aspects,
Final Technical report for FSA project ref
G01022
Gouin S (2004) Microencapsulation: industrial
appraisal of existing technologies and
trends. Trends in Food Science & Techno-
logy 15: 330–347
Green BK (1957) U.S. Patent 2800457
Guerin D, Vuillemard JC, Subirade M (2003)
Protection of bifidobacteria encapsulated
in polysaccharide-protein gel beads against
gastric juice and bile. J Food Protection 66:
2076–2084
Guignon B, Duquenoy A, Dumoulin, ED
(2002) Fluid bed encapsulation of parti-
cles: Principles and practice. Drying Tech-
nology 20: 419–447
Hansen LT, Allan-Wojtas PM, Jin YL, Paulson
AT (2002) Survival of Ca-alginate micro-
encapsulated Bifidobacterium spp. in milk
and simulated gastrointestinal conditions.
Food Microbiol 19: 35–45
Hyndman CL, Groboillot AF, Poncelet D,
Champagne CP, Neufeld RJ (1993)
Microencapsulation of lactococcus-lactis
within cross-linked gelatin membranes.
J Chemical Technol Biotechnol 56: 259–263
Micro-Encapsulation of Probiotics 20 821
Klein J, Stock J, Vorlop KD (1983) Pore-size
and properties of spherical ca-alginate
biocatalysts. European J Appl Microbiol
Biotechnol 18: 86–91
Klein J, Vorlop KD (1985) Immobilization
Techniques – Cells. In: Moo-Young, M
(ed.). Comprehensive Biotechnology,
vol. 2, Pergamon Press, Oxford, UK,
203–224
Krasaekoopt W, Bhandari B, Deeth H (2003)
Evaluation of encapsulation techniques of
probiotics for yoghurt. International
Dairy J 13: PII S0958–6946(0902)00155-
00153
Lacroix C, Yidirim S (2007) Fermentation
technologies for the production of pro-
biotics with high viability and function-
ality. Current Opinion in Biotechnol 18:
176–183
Lee JS, Cha DS, Park HJ (2004) Survival of
freeze-dried Lactobacillus bulgaricus KFRI
673 in chitosan-coated calcium alginate
microparticles. J Agricultural and Food
Chemistry 52: 7300–7305
Lee KY, Heo TR (2000) Survival of
Bifidobacterium longum immobilized in
calcium alginate beads in simulated gas-
tric juices and bile salt solution. Appl En-
viron Microbiol 66: 869–873
Maa YF, Hsu C (1996) Liquid-liquid emulsifi-
cation by static mixers for use in micro-
encapsulation. J Microencapsulation 13:
419–433
Mattila-Sandholm T, Myllarinen P, Crittenden
R, Mogensen G, Fonden R, Saarela M
(2002) Technological challenges for
future probiotic foods. International
Dairy Journal 12: PII S0958–6946(0901)
00099-00091
Mauvernay RY (1965) Brevet d’Invention, BE
66701
Meesters GMH (2006) Agglomeration of
enzymes, microorganisms and flavours.
In: Salman, Agba; Hounslow, Michael;
Seville, Jonathan P.K. (ed.). Granulation,
Volume 11 (Handbook of Powder Tech-
nology), 555-591
Meiners JA (2004) Some like it hot. Glatt Inter-
national Times, no. 18
Peniche C, Arguelles-Monal W, Peniche H,
Acosta N (2003) Chitosan: An attractive
biocompatible polymer for microencap-
sulation. Macromolecular Bioscience 3:
511–520
Picot A, Lacroix C (2004) Encapsulation of
bifidobacteria in whey protein-based
microcapsules and survival in simulated
gastrointestinal conditions and in yoghurt.
International Dairy Journal 14: 505–515
Pothakamury UR, BarbosaCanovas GV (1995)
Fundamental aspects of controlled release
in foods. Trends in Food Science & Tech-
nology 6: 397–406
Prevost H, Divies C (1988) Continuous pre-
fermentation of milk by entrapped yogurt
bacteria .1. development of the process.
Milchwissenschaft-Milk Science Interna-
tional 43: 621–625
Randall RC, Phillips GO, Williams PA (1988)
The role of the proteinaceous component
on the emulsifying properties of gum ara-
bic. Food Hydrocolloids 2: 131–140
Rees DA (1972) Shapely polysaccharides -
eighth colworth medal lecture. Biochemi-
cal Journal 126: 257–&
Sheu TY, Marshall RT (1993) Microentrapment
of lactobacilli in calcium alginate gels.
J Food Science 58: 557–561
Talwalkar A, Kailasapathy K (2004) Compari-
son of selective and differential media for
the accurate enumeration of strains of
Lactobacillus acidophilus, Bifidobacter-
ium spp. and Lactobacillus casei complex
from commercial yoghurts. International
Dairy Journal 14: 143–149
Tsen JH, Chen HH, King VA (2002) Survival of
freeze-dried Lactobacillus acidophilus
immobilized in kappa-carrageenan gel.
J General and Appl Microbiol 48: 237–241
Watheley TL (1996) Microcapsules: Preparation
by interfacial polymerization and interfacial
complexation and their applications. In:
Benita, Simon (ed.). Micro-capsules prepa-
ration, Micro-encapsulation methods and
822 20 Micro-Encapsulation of Probiotics
industrial applications. Informa Health
Care: 349–366
Wehr JB, Menzies NW, Blamey FPC (2004)
Alkali hydroxide-induced gelation of
pectin. Food Hydrocolloids 18: 375–378
Weinbreck F, Minor M, De Kruif CG (2004)
Microencapsulation of oils using whey
protein/gum arabic coacervates. J Micro-
encapsulation 21: 667–679
Wilson N, Shah NP (2007) Microencapsulation
of vitamins. Asean Food Journal 14: 1–14
Wu Y, Bao C, Zhou Y (2007) An innovated
tower-fluidized bed prilling process. Chi-
nese Journal of Chemical Engineering 15:
424–428
Ying DY, Parkar S, Luo XX, Seelye R, Sharpe JC,
Barker D, Saunders J, Pereira R, Schroder
R (2007) Microencapsulation of probio-
tics using kiwifruit polysaccharide and al-
ginate chitosan. Proceedings of the 6th
International Symposium on Kiwifruit,
Vols 1 and 2: 801–808
Micro-Encapsulation of Probiotics 20 823