Review

Recent advances in
microencapsulation
of probiotics for
industrial
applications and
targeted delivery
Anil Kumar Anal
*
and
Harjinder Singh
Riddet Centre, Massey University, AgHort Building
Block C, Riddet Road, Private Bag 11 222, Palmerston
North 4442, New Zealand (Tel.: D64 6 3505356;
fax: D64 6 3505655; e-mail: a.anal@massey.ac.nz)
Because of their perceived health benets, probiotics have
been incorporated into a range of dairy products, including
yoghurts, soft-, semi-hard and hard cheeses, ice cream, milk
powders and frozen dairy desserts. However, there are still
several problems with respect to the low viability of probiotic
bacteria in dairy foods. This review focuses mainly on current
knowledge and techniques used in the microencapsulation
of probiotic microorganisms to enhance their viability during
fermentation, processing and utilization in commercial
products. Microencapsulation of probiotic bacteria can be
used to enhance the viability during processing, and also for
the targeted delivery in gastrointestinal tract.
Introduction
Probiotics have been dened in several ways, depending
on our understanding of the mechanisms of action of their
effects on the health and well-being of humans. The most
commonly used denition is that of Fuller (1989): probiot-
ics are live microbial feed supplements that benecially
affect the host by improving its intestinal microbial
balance. Recently Food and Agriculture Organization
(FAO) of the United Nations and the World Health Organi-
zation (WHO) dene probiotics as Live microorganisms
(bacteria or yeasts), which when ingested or locally applied
in sufcient numbers confer one or more specied demon-
strated health benets for the host (FAO/WHO, 2001).
The benecial effects of probiotics on the human gut ora
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 bodys natural defense mechanisms
(Dunne, 2001; Gismondo, Drago, & Lombardi, 1999). The
mechanism of anti-pathogenic effect may be through
decreasing the luminal pH by the production of short chain
fatty acids such as acetic acid, lactic acid or propionic acid,
rendering vital nutrients unavailable to pathogens, altering
the redox potential of the environment, producing hydrogen
peroxide or producing bacteriocins or other inhibitory
substances (Kailasapathy & Chin, 2000).
Probiotics may cause cell-mediated immune responses,
including activation of the reticulo-endothelial system,
augmentation of cytokine pathways and stimulation of
pro-inammatory pathways such as tumour necrosis factors
and interleukin regulation, without being a target of the
host immune system (Gill, Cross, Rutherfurd, & Gopal,
2001; Isolauri, 2000; Isolauri, Arvola, Sutas, Moilanen, &
Salminen, 2000). Probiotics may even activate macro-
phages directly (Tejada-Simon, Ustunol, & Pestka, 1999).
Recently, probiotics have been proposed for various treat-
ments of human intestinal barrier dysfunctions such as
lactose intolerance, acute gastroenteritis, food allergy, atopic
dermatitis, Crohns disease, rheumatoid arthritis, and colon
cancer (Kalliomaki, Salminen, Poussa, Arvilommi, &
Isolauri, 2003; Lee, Puong, Ouwehand, & Salminen, 2003;
Rinkinen, Jalava, Westermarck, Salminen, & Ouwehand,
2003; Salminen et al., 1998).
Lactic acid bacteria (LAB) are the most important pro-
biotic microorganisms typically associated with the human
gastrointestinal tract. These bacteria are Gram-positive,
rod-shaped, non-spore-forming, catalase-negative organ-
isms that are devoid of cytochromes and are of non-aerobic
habit but are aero-tolerant, fastidious, acid-tolerant and
strictly fermentative; lactic acid is the major end-product
of sugar fermentation (Axelsson, 1993). A few of the
known LAB that are used as probiotics are Lactobacillus
acidophilus, Lactobacillus amylovorous, Lactobacillus * Corresponding author.
0924-2244/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tifs.2007.01.004
Trends in Food Science & Technology 18 (2007) 240e251
casei, Lactobacillus crispatus, Lactobacillus delbrueckii,
Lactobacillus gasseri, Lactobacillus johnsonoo, Lacto-
bacillus paracasei, Lactobacillus plantarum, Lactobacillus
reuteri, Lactobacillus rhamnosus etc. (Makinen & Bigret,
1993).
Other common probiotic microorganisms are the bido-
bacteria. Bidobacteria are also Gram-positive and rod-
shaped but are strictly anaerobic. These bacteria can
grow at pH in the range 4.5e8.5. Bidobacteria actively
ferment carbohydrates, producing mainly acetic acid and
lactic acid in a molar ratio of 3:2 (v/v), but not carbon di-
oxide, butyric acid or propionic acid. The most recognized
species of bidobacteria that are used as probiotic organ-
isms are Bidobacterium adolescentis, Bidobacterium
animalis, Bidobacterium bidum, Bidobacterium breve,
Bidobacterium infantis, Bidobacterium lactis and Bido-
bacterium longum. Other than these bacteria, Bacillus
cereus var. toyoi, Escherichia coli strain nissle, Propionio-
bacterium freudenreichii, and some types of yeasts, e.g.
Saccharomyces cerevisiae and Saccharomyces boulardii
have also been identied as having probiotic effects (Hol-
zapfel, Haberer, Geisen, Bjorkroth, & Schillinger, 2001).
Because of their perceived health benets, probiotic bac-
teria have been increasingly included in fermented dairy
products, including yoghurts, soft-, semi-hard and hard
cheeses, ice cream and frozen fermented dairy desserts
(Desmond et al., 2005; Dinakar & Mistry, 1994; Stanton,
Desmond, Fitzgerald, & Ross, 2003; Stanton et al., 2001;
Stanton, Ross, Fitzgerald, & Van Sinderen, 2005).
The ability of probiotic microorganisms to survive and
multiply in the host strongly inuences their probiotic ben-
ets. The bacteria should be metabolically stable and active
in the product, survive passage through the upper digestive
tract in large numbers and have benecial effects when in
the intestine of the host (Gilliland, 1989). The standard
for any food sold with health claims from the addition
of probiotics is that it must contain per gram at least
10
6
e10
7
cfu of viable probiotic bacteria (FAO/WHO,
2001). However, there are still several problems with re-
spect to the low viability of probiotic bacteria in dairy
foods. Several factors have been reported to affect the via-
bility of probiotics in fermented dairy products, including
titratable acidity, pH, hydrogen peroxide, dissolved oxygen
content, storage temperature, species and strains of associa-
tive fermented dairy product organisms, concentration of
lactic and acetic acids and even whey protein concentration
(Dave & Shah, 1997; Kailasapathy & Supriadi, 1996; Lan-
kaputhra, Shah, & Britz, 1996). Survival is, of course, es-
sential for organisms targeted to populate the human gut,
one of the most important issues in health benet provision
by probiotic bacteria.
Different approaches that increase the resistance of these
sensitive microorganisms against adverse conditions have
been proposed, including appropriate selection of acid- and
bile-resistant strains, use of oxygen-impermeable containers,
two-step fermentation, stress adaptation, incorporation of
micronutrients such as peptides and amino acids, and micro-
encapsulation (Gismondo et al., 1999).
Microencapsulation technology
Microencapsulation is dened as a technology of packag-
ing solids, liquids or gaseous materials in miniature, sealed
capsules that can release their contents at controlled rates
under the inuences of specic conditions (Anal & Stevens,
2005; Anal, Stevens, & Remunan-Lopez, 2006; Kailasapathy
& Masondole, 2005). A microcapsule consists of a semi-
permeable, spherical, thin, and strong membrane surrounding
a solid/liquid core, with a diameter varying from a few mi-
crons to 1 mm. A brief description of microencapsulation
techniques for encapsulation probiotic microorganisms is
given in Table 1. In a broad sense, encapsulation can be
used for many applications in the food industry, including
stabilizing the core material, controlling the oxidative reac-
tion, providing sustained or controlled release (both temporal
and time-controlled release), masking avours, colours or
odours, extending the shelf life and protecting components
against nutritional loss. Food-grade polymers such as algi-
nate, chitosan, carboxymethyl cellulose (CMC), carrageenan,
gelatin and pectin are mainly applied, using various micro-
encapsulation technologies (Table 2).
Microcapsules and microspheres can be engineered to
gradually release active ingredients. A microcapsule may
be opened by many different means, including fracture by
heat, solvation, diffusion, and pressure (Brannon-Peppas,
1997). A coating may also be designed to open in the
specic areas of the body. A microcapsule containing
acid-labile core materials that will be consumed by gastro-
intestinal uids must not be fractured until after it passes
through the stomach. A coating can therefore be used that
is able to withstand acidic conditions in the stomach acids
and allows those active ingredients to pass through the
stomach (Anal, Bhopatkar, Tokura, Tamura, & Stevens,
2003; Anal & Stevens, 2005). Fig. 1 illustrates the swelling,
erosion and disintegration of milk proteinepolysaccharides
microcapsules containing probiotics (Unpublished). These
microcapsules were rst incubated in simulated gastric
uid (SGF, pH 1.2) for 2 h and then transferred into simu-
lated intestinal uid (SIF, pH 7.4).
This review focuses on the current knowledge and tech-
niques used in the microencapsulation of probiotic micro-
organisms to enhance the performance of these organisms
during fermentation, downstream processing and utilization
in commercial products.
Spray- and freeze-dried probiotic products
Probiotic cultures for food applications are frequently
supplied in frozen or dried form, as either freeze-dried or
spray-dried powders (Holzapfel et al., 2001). The success-
ful spray drying of Lactobacilli and Bidobacteria has pre-
viously been reported for a number of different strains,
including L. paracasei (Desmond, Ross, OCallaghan,
241 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
Fitzgerald, & Stanton, 2002; Gardiner et al., 2000), Lacto-
bacillus curvatus (Mauriello, Aponte, Andol, Moschetti,
& Villani, 1999), L. acidophilus (Prajapati, Shah, &
Dave, 1987), L. rhamnosus (Corcoran, Ross, Fitzgerald,
& Stanton, 2004) and Bidobacterium ruminantium
(ORiordan, Andrews, Buckle, & Conway, 2001). However,
most probiotic bacteria do not survive well during the tem-
perature and osmotic extremes to which they are exposed
during the spray drying process (Selmer-Olsen, Sorhaug,
Birkeland, & Pehrson, 1999; Teixeira, 1979). When spray
drying is used for the preservation of potential probiotic
cultures, much of their activity is typically lost after
a few weeks of storage at room temperature. This is associ-
ated with stress that is induced by temperature changes,
phase changes and drying, a combination of which tends
to damage cell membranes and associated proteins.
One approach used by a number of researchers to im-
prove probiotic survival is the addition of protectants to
the media prior to drying. For example, the incorporation
of thermoprotectants, such as trehalose (Conrad, Miller,
Cielenski, & de Pablo, 2000), non-fat milk solids and/
or adnitol (Selmer-Olsen et al., 1999), growth promoting
factors including various probiotic/prebiotic combinations
(Desmond et al., 2002) and granular starch (Crittenden
et al., 2001) have been shown to improve culture viabil-
ity during drying and storage. Recently, incorporation of
the soluble ber, gum acacia, into a milk-based medium
prior to spray drying the probiotic L. paracasei was
found to increase its viability during storage, compared
with milk powder alone (Desmond et al., 2002). How-
ever, other soluble bers investigated, including inulin
and polydextrose, did not enhance probiotic viability
during spray drying or powder storage (Corcoran, Stanton,
Fitzgerald, & Ross, 2005).
Microencapsulation by spray drying is a well-established
process that can produce large amounts of material. Never-
theless, this economical and effective technology for protect-
ing materials is rarely considered for cell immobilization
because of the high mortality resulting from simultaneous
dehydration and thermal inactivation of microorganisms.
In response to these limitations, a low cost microencapsula-
tion method, which can be easily scaled up, to improve the
stability of probiotic lactic cultures has been proposed (Picot
&Lacroix, 2003a, 2003b). The technique consists of coating
milk fat droplets containing powder particles of freeze-dried
bacteria with whey protein polymers, using emulsication
and spray drying in a continuous two-step process. Success-
ful production of the resulting multiphase low diameter mi-
crocapsules requires rigorous control of the size distribution
of the different elements constituting the capsules. In partic-
ular, the material dispersed in the hydrophobic phase must be
larger than the bacterial cells and smaller than the fat glob-
ules. In order to decrease the powder particle size, in later
studies, Picot and Lacroix (2003c) micronized the powder
particles, produced by spray drying and emulsication
methods, using a spiral jet mill as a grinding system. The
Table 1. Techniques and processes used for encapsulating probiotic microorganisms
Microencapsulation
techniques
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) Solidication 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
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/phase
separation 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
242 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
Table 2. Encapsulation of probiotic bacteria in various polymer systems
Bacteria systems Polymers Microencapsulation technology Functionality References
Lactobacillus Carrageenan Gel beads Biomass production Klein and Vorlop (1985)
S. thermophilus Carrageenan Gel beads Biomass production Audet et al. (1988, 1990, 1991)
L. bulgaricus Carrageenan/locust bean gum Gel beads Biomass production Ouellette et al. (1994)
B. infantis
Lactobacillus
Carrageenan/locust bean gum
Gel beads Biomass production
Doleyres, Fliss et al. (2002),
Doleyres et al. (2004), Doleyres,
Paquin et al. (2002)
Bidobacterium
Bidobacterium Alginate/glycerol Gel beads Biomass production Kebary (1996)
Lactobacillus Alginate Gel beads Acid stable Chandramouli et al. (2004)
Bidobacterium
Bidobacterium pseudolongum Cellulose acetate phthalate Gel beads Acid and bile salt stable Rao et al. (1989)
L. delbrueckii Alginate/sodium lauryl sulphate Gel beads Biomass production Sheu et al. (1993)
L. casei Carrageenan/locust bean gum Emulsication Acid stable Chan and Zhang (2002)
L. lactis Gelatin/toluene-2-4-diisocyanate Gel beads Biomass production Hyndman et al. (1993)
Lactobacillus Alginate Gel beads Acid stable Chandramouli et al. (2004)
L. acidophillus Alginate Direct compression Acid stable Chan and Zhang (2002)
B. breve Alginate microspheres Emulsication Acid stable Hansen et al. (2002)
B. Longum
Pedicoccus acidilactei
Whey protein Micronization Acid stable Picot and Lacroix (2003a, 2003b, 2004)
Bidobacterium Alginate/chitosan Gel beads Acid stable/storage Lee et al. (2004)
Bidobacterium Alginate/pectin/whey protein Gel beads Acid stable/storage Guerin et al. (2003)
Bidobacterium Resistant starch Gel beads Acid stable/storage Crittenden et al. (2001)
Lactobacillus Starch Gel beads Acid stable/storage
Bidobacterium Waxy maize starch Gel beads/emulsication Acid stable/storage ORiordan et al. (2001)
Lactobacillus
Alginate/starch
Gel beads Acid stable
and stable during storage
Sultana et al. (2000), Kailasapathy
and Chin (2000) Bidobacterium
2
4
3
A
.
K
.
A
n
a
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,
H
.
S
i
n
g
h
/
T
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e
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i
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o
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i
e
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&
T
e
c
h
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o
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y
1
8
(
2
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7
)
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2
5
1
effect of micronization on cell viability was governed mainly
by the nal particle size of the processed culture. They re-
vealed that micronization is an effective means of reducing
the powder particle size of freeze-dried cultures with an ac-
ceptable mortality rate before producing microcapsules with
low diameters for the protection of sensitive probiotic bacte-
ria. However, the use of micronized cultures in cell encapsu-
lation technologies that require heat treatment, such as spray
drying, may reduce cell viability.
Direct dispersion of fresh cells in a heat-treated whey
protein suspension followed by spray drying was found to
be an alternative and less destructive microencapsulation
method, with survival rates after spray drying of 26% for
B. breve and 1.4% for the more heat-sensitive B. longum
(Picot & Lacroix, 2004). Even though the viability of the
bacteria after spray drying remained low, these microparti-
cles showed cell protection in gastric juice and controlled
release of probiotic bacteria under simulated intestinal
conditions.
Furthermore, the addition of cryoprotectants during the
freeze drying of lactobacilli has been used to help overcome
inactivation during drying and stabilization during storage.
In a recent study, freeze-dried Lactobacillus bulgaricus sur-
vived better during storage at 20

C over 10 months when
cells had been grown in the presence of fructose, lactose or
mannose or when glucose, fructose, monosodium glutamate
or sorbitol was added to the drying medium (Carvalho et al.,
2002, 2003, 2004a, 2004b). In particular, trehalose,
Fig. 1. Photographs showing the disintegration mechanism (swelling and burst effect) of whey protein isolateealginateechitosanecalcium chloride
microcapsules incubated in simulated gastrointestinal uids; simulated gastric uid (SGF, pH 1.2), and simulated intestinal uid (SIF, pH 7.4) at
37

C: (a) dried microcapsules; (b) microcapsules incubated in SGF for 2 h; (c) microcapsules incubated in SIF for 2 h; (d) microcapsules incubated
in SIF for 8 h (Unpublished).
244 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
a disaccharide of glucose, has been found to be effective at
protecting the bacterial cells during freezing and drying
(Garcia De Castro, Bredholt, Strom, & Tunnaclife, 2000).
Encapsulation of probiotics in polymer systems
Encapsulation of probiotics in a biodegradable polymer
matrix has a number of 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 quanti-
ed, allowing the dosage to be readily controlled. Cryo-
and osmo-protective components can be incorporated into
the matrix, enhancing the survival of cells during process-
ing and storage. Finally, once the matrix beads/microcap-
sules 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 func-
tional, 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.
Various polymer systems have been used to encapsulate
probiotic microorganisms to protect against low pH and
high bile concentrations and to enhance physical stability
during downstream processing. Microcapsule or bead sys-
tems using various biopolymers are very easy to prepare
on a lab-scale, and any ingredients can be encapsulated,
whether it is hydrophilic, hydrophobic, liquid, or a viscous
oil, a solid etc. However, the scaling up of the process is
very difcult and processing costs are very high. Moreover,
most of the conventionally produced microcapsules (e.g.
calcium alginate beads/microcapsules), tend to be very po-
rous which allows fast and easy diffusion of water and other
uids in and out of the matrix.
Spherical polymer beads with diameters ranging from
0.3 to 3.0 mm and immobilizing active biomass are pro-
duced using extrusion or emulsication techniques, by ther-
mal (k-carrageenan, gellan, agarose, gelatin) or ionotropic
(alginate, chitosan) gelation of the droplets. Some of these
systems are discussed in more detail in the following
sections.
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. Elevated temperatures (60e80

C) are needed to
dissolve the polymer at concentrations ranging from 2 to
5% (Klein & Vorlop, 1985). Gelation of k-carrageenan is
generally dependent on a change in temperature. The cell
slurry is added to the heat-sterilized carrageenan solution
at 40e45

C and gelation occurs by cooling to room temper-
ature. The beads are formed after dropping the mixture of
polymer and cells into a potassium chloride (KCl) solution.
Audet, Paquin, and Lacroix (1988) reported the inhibitory
effect of KCl on some bacteria such as Streptococcus ther-
mophilus and L. bulgaricus. Later they used a combination
of k-carrageenan and locust bean gum to encapsulate LAB
to enhance their stability during biomass production in dairy
products (Audet, Paquin, & Lacroix, 1990, 1991). The gel
bead strength can be enhanced using another polymer,
such as locust bean gum. A ratio of carrageenan to locust
bean gum of 2:1, through specic interaction of the galacto-
mannan chains of locust bean gum with carrageenan, has
been found to give the synergistic effects and to form the
strong gel beads. Ouellette, Chevalier, and Lacroix (1994)
immobilized a pure culture of B. infantis in k-carrageenan/
locust bean gum beads and also used this system to contin-
uously ferment skimmed milk supplemented with 1% yeast
extract. Immobilized cell growth and cell release from the
gel beads into the circulating milk allowed for a steady in-
oculation of the feed for a maximal cell volumetric produc-
tivity of approximately 1 10
12
cfu/mL.
Recently, Doleyres, Fliss, and Lacroix (2002, 2004) and
Doleyres, Paquin, LeRoy, and Lacroix (2002) immobilized
probiotic cells in carrageenan and locust bean gum gel
beads by ionotropic gelation method to produce a mixed
lactic culture containing a non-competitive strain of bido-
bacteria and a competitive LAB strain, during repeated
batches and continuous cultures. The two-stage continuous
fermentation system, composed of a rst reactor containing
cells of the two strains separately immobilized in carra-
geenan/locust bean gum gel beads and a second reactor op-
erated with free cells released from the rst reactor, allowed
the continuous production of a concentrated mixed culture
with a strain ratio of a composition that depended on
temperature. Cells produced by this technology exhibited
important physiological changes and increased stress toler-
ance. The tolerance of these cells to stresses, such as freeze
drying, hydrogen peroxide and simulated gastrointestinal
conditions, increased markedly with culture time and after
15 days was higher than the tolerance of cells produced by
a conventional method.
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 1e4 linked b-D-mannuronic (M) and a-L-
guluronic (G) acids. These residues are present in various
proportions depending on the source of the alginic acid. Al-
ginic 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 bind-
ing of divalent cations and the subsequent gel formation are
dependent on the composition and arrangement of the
blocks of residues (Gemeiner, Rexova-Benkova, S

vec, &
Norrlow, 1994). The GG blocks have preferential binding
sites for divalent counter-ions, such as Ca
2
, and the bound
ions interact with other GG blocks to form linkages that
lead to gel formation. On addition of sodium alginate solu-
tion to a calcium solution, interfacial polymerization is in-
stantaneous, with precipitation of calcium alginate followed
by a more gradual gelation of the interior as calcium ions
245 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
permeate through the alginate systems. The size of the
beads is generally dependent on the viscosity of the poly-
mer solution, the diameter of the orice and the distance
between the outlet and the coagulation solution (Anal
et al., 2003; Anal & Stevens, 2005).
Various researchers (Chandramouli, Kailasapathy,
Peiris, & Jones, 2004; Lee, Cha, & Park, 2004; Sheu,
Marshall, & Heymann, 1993) have studied factors affecting
bead preparation, such as concentrations of alginate and
CaCl
2
, timing of hardening of the beads and cell concentra-
tions on encapsulation of probiotics. The conventional
encapsulation method, with sodium alginate in calcium
chloride (CaCl
2
), has been used to encapsulate L. acidophi-
lus to protect this organism from the harsh acidic conditions
in gastric uid. Studies have shown that calcium-alginatee
immobilized cell cultures are better protected, shown by an
increase in the survival of bacteria under different condi-
tions, than the non-encapsulated state. The results from
these studies indicate that the viability of encapsulated
bacteria in simulated gastric uid increases with an increase
in capsule size.
However, Hansen, Allan-Wojtas, Jin, and Paulson (2002)
reported that very large calcium alginate beads (>1 mm)
cause a coarseness of texture in live microbial feed supple-
ments and that small beads of size less than 100 mm do not
signicantly protect the bacteria in simulated gastric uid,
compared with free cells. These studies indicate that these
bacteria should be encapsulated within a particular size
range. They tested nine different strains of Bidobacterium
spp. for their tolerance to simulated gastrointestinal condi-
tions, and observed some variations among the strains for
resistance to gastric uid (pH 2e3) and bile salts (5 and
10 g/L). Among these strains, only a strain B. lactis
Bb-12 was found to be resistant to low pH and bile salts.
They also encapsulated some of the strains in alginate
microspheres to evaluate their resistance properties in gas-
tric uid and to bile salts. They obtained alginate micro-
spheres (20e70 mm) by emulsifying the mixture of cells
and sodium alginate in vegetable oil and subsequently
cross-linking with CaCl
2
. Cryo-scanning electron micro-
scopy revealed that these microparticles were densely
loaded with probiotic bacteria and were porous. The loaded
alginate microparticles remained stable during storage at
4

C in 0.05 M CaCl
2
and in milk (2% fat), sour cream
and yoghurt for up to 16 days and in simulated gastric uid
(pH 2.0) for 1 h at 37

C. However, the microparticles ex-
posed to low pH did not improve the survival of acid-
sensitive bidobacteria. Kebary (1996) also showed that B.
bidum survived in higher numbers in frozen milk in beads
made from alginate than in beads made from k-carrageenan.
Recently, Chen, Chen, Liu, Lin, and Chiu (2005) used
prebiotics (fructooligosaccharides or isomaltooligosacchar-
ides), a growth promoter (peptide) and sodium alginate as
coating materials to microencapsulate different probiotics
such as L. acidophilus, L. casei, B. bidum 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.
Chan and Zhang (2002) developed an encapsulation
technique of compression coating, which permits the stabi-
lization of lyophilized cells during storage. This technique
involves compressing the lyophilized cell powder into
a core tablet and then compressing coating materials around
this core to form the nal compact. The objective of this
work was to investigate the use of methacrylic acid copoly-
mer as an enteric coating material for the compression
coating of an industrially sourced strain of L. acidophilus.
It was hoped that this enteric coating material would pro-
tect the cells as they passed through the stomach, and,
when used together with pectin, could be used to target
the release of the probiotics to the terminal ileum and the
beginning of the colon in the human gastrointestinal tract.
The release prole and the viability of the probiotic cells
during passage through the simulated gastrointestinal tract
were investigated. The coating material used was a mixture
of sodium alginate and hydroxypropyl cellulose in the
weight ratio 9:1. The encapsulated cells showed a 10
4
e10
5
-
fold increase in cell survival compared with free cells under
acidic conditions. The formation of a hydrogel barrier by
the compacted sodium alginate layer was shown to retard
the permeation of the acidic uid into the cells. In vitro
tests further revealed that the release of encapsulated cells
in the human digestive tract could occur near the end of
the ileum and the beginning of the colon. The mechanism
of cell release was considered to be primarily due to erosion
of the alginate gel layer.
Cui, Goh, Kim, Choi, and Lee (2001) prepared poly-L-
lysineecross-linked alginate microparticles loaded with
bidobacteria. They used an air atomization method to
spray the alginateebacteria culture in a coagulation bath
containing CaCl
2
. The microparticles were further cross-
linked with poly-L-lysine. The survival of bidobacteria
from the alginateepoly-L-lysine microparticles was much
higher, even in the lower pH media. Due to stability of
poly-L-lysineecross-linked alginate microparticles in gas-
tric uid, bidobacteria can be protected without losing
their survivability. The survival of bidobacteria loaded in
the particles remained highest (2.67 10
9
cfu/g) at pH 6.8
while the number is reduced at lower pH (1.5, exposure
time, 2 h) to 5.0 10
7
cfu/g. However, only 1e3% of the
unencapsulated bidobacteria can survive in lower pH.
The stability of the free-owing bidobacteriaealginatee
poly-L-lysine microparticles was also improved during
storage at 4

C in a refrigerator, compared with free
cultures.
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,
246 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
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) re-
ported the encapsulation of B. pseudolongum in CAP using
an emulsion technique. Microencapsulated bacteria sur-
vived in larger numbers (10
9
cfu/mL) in an acidic environ-
ment than non-encapsulated organisms, which did not
retain any viability when exposed to a simulated gastric
environment for 1 h. Favaro-Trindale and Grosso (2002)
encapsulated B. lactis and L. acidophilus in CAP polymer
using a spray drying method. This study evaluated the resis-
tance of microencapsulated microorganisms in acid and
high bile salt concentrations. Spray-dried microcapsules
of CAP containing B. lactis and L. acidophilus were effec-
tive in protecting both these microorganisms when inocu-
lated into media with pH values similar to those in the
human stomach. Microencapsulated L. acidophilus suffered
a reduction of only 1 log at pH 1 after 2 h of incubation,
and the population of B. lactis was reduced by only 1 log
immediately after inoculation into a pH 1 medium and be-
tween 1 and 2 h after inoculation into a pH 2 medium. After
inoculation of the CAP microcapsules loaded with bacteria
into bile solution (pH 7), complete dissolution of the pow-
der indicated that both the wall material and the process
used in the preparation of the microcapsules were adequate
in protecting the bacteria, to pass undamaged through the
acidic conditions of the stomach, followed by their rapid
liberation in the pH of the intestine.
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-gel-
forming 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). Hyndman, Groboillot, Poncelet, Champagne, and
Neufeld (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, Vuillemard, and Subirade
(2003) encapsulated Bidobacterium cells in a mixed gel
composed of alginate, pectin and whey proteins. They in-
vestigated 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. bidum. After 1 h of incubation
in acidic solution (pH 2.5), the free cell counts decreased
by 4.75 log, compared with a decrease of <1 log for entrap-
ped cells. The free cells did not survive after 2 h of
incubation at pH 2.5, whereas the immobilized cells de-
creased by about only 2 log. After incubation (1 or 3 h)
in 2 and 4% bile salt solutions, the mortality for B. bidum
cells in membrane-free gel beads (4e7 log) was greater
than that for free cells (2e3 log). However, the counts of
cells immobilized in membrane-coated gel beads decreased
by <2 log. The double membrane coating enhanced the re-
sistance of the cells to acidic conditions and higher bile salt
concentrations.
Encapsulation of probiotics in chitosan
The biopolymer chitosan, the N-deacetylated product of
the polysaccharide chitin, is gaining importance in the food
and pharmaceutical eld 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 specic 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 or-
ganic materials on earth and second to cellulose and murein,
which is the main structural polymer of the bacterial cell
wall. In order to achieve sufcient stability, chitosan gel
beads and microspheres can be ionically cross-linked with
polyphosphates (Anal & Stevens, 2005) and sodium algi-
nate (Anal et al., 2003).
Krasaekoopt, Bhandari, and Deeth (2003, 2004) evaluated
the survival of probiotics encapsulated in chitosan-coated
alginate beads in yoghurt and in UHT- and conventionally
treated milk during storage. They used L. acidophilus 547,
L. casei 01 and B. bidum 1994 as model organisms for their
study. The survival of the encapsulated bacteria was higher
than that of the free cells by approximately 1 log. The number
of probiotic bacteria was maintained above the recommen-
ded therapeutic minimum (10
7
cfu/g) throughout storage
for the lactobacilli but not for the bidobacteria. Lee et al.
(2004) carried out a similar study and compared various chi-
tosans (different molecular weights) for coating conventional
alginate beads. They investigated the effects of chitosane
alginate microparticles on the survival of L. bulgaricus
KFRI763 in simulated gastric juices and simulated intestinal
uid and on their stability during storage at 4 and 22

C. The
probiotic loaded in alginate microparticles was prepared by
spraying a mixture of sodium alginate and cell culture into
a CaCl
2
echitosan solution using an air-atomizing device.
When the microorganism was exposed to gastric uid (pH
2.0) for 1 h, none survived. In contrast, an impressive and
high survival rate was obtained when the sprayed particles
were coated with chitosan. They concluded that the microen-
capsulation of LAB with alginate and a chitosan coating of-
fers an effective means of delivering viable bacterial cells to
the colon and maintaining their survival during refrigerated
storage.
247 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
Encapsulation of probiotics in starch
Starch is a dietary component that has an important role
in colonic physiology and functions and a potential protec-
tive 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 microora. The fermentation of carbohy-
drates by anaerobic bacteria produces short chain fatty
acids and lowers the pH in the lumen (Kleessen et al.,
1997; Le Blay, Michel, Blottie`re, & Cherbut, 1999). Resis-
tant 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 pro-
biotics to the starch granule during processing, storage and
transit through the upper gastrointestinal tract, providing
robustness and resilience to environmental stresses. Bacte-
rial adhesion to starch may also provide advantages in new
probiotic technologies to enhance delivery of viable and
metabolically active probiotics to the intestinal tract
(Crittenden et al., 2001).
A group of researchers (Mattila-Sandholm et al., 2002)
worked on stabilization of LAB and to formulate new types
of foods fortied with encapsulated health-promoting bac-
teria in the starch system. In their study, large potato starch
granules (50e100 mm), which were enzymatically treated
to obtain a porous structure, were used as a carrier. Sub-
sequently, amylose, the linear polymer of starch, was solu-
bilized, cooled and precipitated over the bacteria-lled
starch granules. Finally, the whole product, together with
the growth medium, was freeze dried to a powder form.
They found the encapsulated LAB can survive at least
6 months at room temperature under normal atmospheric
humidity, and at least 18 months when frozen.
Talwalkar and Kailasapathy (2003) produced alginatee
starch gel beads by dropping a mixture of alginatee
starchebacteria into a CaCl
2
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 re-
gions in the centre of the beads. In another report, they used
a modied method to encapsulate probiotic bacteria in an
alginateestarch system. The incorporation of Hi-Maize
starch improved the encapsulation of viable bacteria
compared with the bacteria encapsulated without starch
(Iyer & Kailasapathy, 2005; Sultana et al., 2000).
Conclusions and future directions
Sophisticated shell materials and technologies have been
developed and an extremely wide variety of functionalities
can now be achieved through microencapsulation. Any type
of triggers can be used to prompt the release of the encap-
sulated ingredients, such as pH changes, mechanical stress,
temperature, enzymatic activity, time, osmotic force, etc.
Encapsulated probiotic bacteria can be used in many
fermented dairy products, such as yoghurt, cheese, cultured
cream and frozen dairy desserts, and for biomass pro-
duction. In the encapsulated form, the probiotics are pro-
tected from bacteriophage and harsh environments, such
as freezing and gastric solutions. Thus, encapsulation facil-
itates the manufacture of fermented dairy products in which
the bacteria have consistent characteristics and higher sta-
bility during storage and higher productivity than non-
encapsulated bacteria. With the encapsulated products, the
residence time, acidity and continuous inoculation of milk
with a constant bacilli/cocci ratio can be controlled at a
desired pH.
The use of microencapsulated probiotics for controlled-
release applications is a promising alternative to solving the
major problems of these organisms that are faced by food
industries. Even so, the challenges are to select the appro-
priate microencapsulation technique and encapsulating
materials. To date, the research on the encapsulation of pro-
biotics has focused mainly on maintaining the viability of
the probiotic bacterial cells at low pH and high bile
concentrations.
One important challenge for cell encapsulation is the
large size of microbial cells (typically 1e4 mm) or particles
of freeze-dried culture (more than 100 mm). This character-
istic limits cell loading for small capsules or, when large
size capsules are produced, can negatively affect the tex-
tural and sensorial properties of food products in which
they are added. In almost all cases, gel entrapment using
natural biopolymers, such as calcium alginate, carrageenan,
gellan gum, and chitosan are favored by researchers. How-
ever, although promising on a laboratory scale, the devel-
oped technologies for producing gel beads still present
serious difculties for large-scale production of food-grade
microencapsulated microorganisms.
Another major challenge is to improve the viability of
probiotics during the manufacturing processes, particularly
heat processing. Consequently, there appears to be no com-
mercial probiotic products available that are stable at high
temperatures. Keeping in view the importance of producing
thermoresistant probiotic microorganisms, as well as the
interests of food and pharmaceutical companies, new
approaches are needed in further research. There are at least
two options: (1) discovering new strains of probiotic bacte-
ria that are naturally heat stable or that have been geneti-
cally modied and (2) developing an encapsulation
system that effectively acts like an insulation material.
Our group is currently exploring approaches to tackle this
challenging area and is focusing on developing novel en-
capsulation systems. This is based on an understanding of
the thermal conductivity properties of several food-grade
biopolymers and lipids that are used as encapsulating shell
materials, individually and in combination. These microen-
capsulation systems may also control the diffusion of oxy-
gen across the wall and may ensure a smaller log reduction
in the viability of cells in foods. Coating of capsules with
some lipids, with high melting points, may also provide
248 A.K. Anal, H. Singh / Trends in Food Science & Technology 18 (2007) 240e251
low moisture conditions and an anaerobic environment
for probiotic bacteria and may possibly improve thermal
stability.
Application of this research could be particularly
important for the production of functional dairy products
containing high concentrations of viable bacteria and
bioingredients from LAB. Immobilization can efciently
protect cells, making this approach potentially useful for
delivery of viable bacteria to the lower gastrointestinal tract
of humans via fermented, beverages and other functional
food products.
Acknowledgement
The authors would like to thank Fonterra Co-operative
Group Limited, New Zealand for nancial support of this
work.
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