International Dairy Journal 13 (2003) 313

Review
Evaluation of encapsulation techniques of probiotics for yoghurt
Wunwisa Krasaekoopt, Bhesh Bhandari*, Hilton Deeth
School of Land and Food Sciences, The University of Queensland, St. Lucia, Qld. 4072, Australia
Received 19 June 2001; accepted 10 October 2002

Abstract

The health benets provided by probiotic bacteria have led to their increasing use in fermented and other dairy products.
However, their viability in these products is low. Encapsulation has been investigated to protect the bacteria in the products
environment and improve their survival. There are two common encapsulation techniques, namely extrusion and emulsion, to
encapsulate the probiotics for their use in the fermented and other dairy products. This review evaluates the merits and limitations of
these two techniques, and also discusses the supporting materials and special treatments used in encapsulation processes.
r 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Probiotics; Microencapsulation; Survival; Extrusion; Emulsion

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2. Techniques for microencapsulation of bacterial cells in hydrocolloid beads . . . . . . . . . . . . . . . . . . 5
2.1. Extrusion technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.1.1. Supporting material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2. Emulsion technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.2.1. Continuous phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
2.2.2. Supporting material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3. Special treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.1. Cross-linking with cationic polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.2. Coating with other polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.3. Mixing with starch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.4. Incorporation of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4. Advantages and limitations of extrusion and emulsion encapsulation techniques . . . . . . . . . . . . . . 10
5. Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

1. Introduction microbial balance (Fuller, 1992). Claimed benets

Probiotics are live microbial supplements, which
benecially affect the host by improving its intestinal
*Corresponding author. Tel.: +617-33469192; fax: +617-33651177.
E-mail address: b.bhandari@mailbox.uq.edu.au (B. Bhandari).
include controlling intestinal infection, controlling
serum cholesterol levels, improving lactose utilization
in persons who are lactose maldigestors, and possessing
anticarcinogenic activity.
Probiotic bacteria, which are commensals of the
human gut, have been reported to inhibit the growth

0958-6946/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 5 8 - 6 9 4 6 ( 0 2 ) 0 0 1 5 5 - 3
4 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313
of undesirable microorganisms and food poisoning
bacteria, such as Salmonella, that can be encountered
in the gastrointestinal tract (Huges & Hoover, 1991;
Lim, Huh, & Baek, 1993). The probiotic effect has been
attributed to production of acid (Rasic & Kurmann,
1983), production of bacteriocins (Tagg, Dajani, &
Wannamaker, 1976), competition with pathogens (Gurr,
1987), and enhancement of the immune system (Fuller,
1992). Probiotic bacteria produce b-galactosidase which
is benecial for people with lactose intolerance. The
normal yoghurt cultures, Lactobacillus delbrueckii ssp.
bulgaricus and Streptococcus thermophilus, produce b-
galactosidase in yoghurt, but these bacteria cannot
survive and grow in the intestinal tract due to their low
bile salt tolerance. In contrast, probiotic bacteria such as
Lb. acidophilus and Bifidobacterium bifidum can survive
and grow in the intestinal tract and produce b-
galactosidase in the presence of bile (Noh & Gilliland,
1993). Kim and Gilliland (1983) concluded that
improvement in lactose digestion resulted from this
intestinal enzyme production and not from hydrolysis
of lactose before consumption of milk containing
Lb. acidophilus. Probiotics have also been claimed to
have anticarcinogenic or antimutagenic activities. This
may result from one or more factors, such as inhibition
of the carcinogen and/or procarcinogen, inhibition of
bacteria that convert procarcinogens to carcinogens,
activation of the hosts immune system, and reduction
of the intestinal pH to reduce microbial activity (Gilli-
land, 1989; Rasic & Kurmann, 1983). In addition,
hypocholesterolemic effects (lowering of blood choles-
terol) have been reported, especially for Lb. acidophilus
strains (Buck & Gilliland, 1994; Gilliland, 1989).
Many different microorganisms are added to dairy
products for their probiotic potential (Fuller, 1997;
Gibson & Fuller, 1998). These include Lactobacilli such
as Lb. acidophilus, Lb. casei, Lb. delbrueckii ssp.
bulgaricus, Lb. reuteri, Lb. brevis, Lb. cellobiosus, Lb.
curvatus, Lb. fermentum, and Lb. plantarum; Gram-
positive cocci such as Lactococcus lactis ssp. cremoris,
Str. thermophilus, Enterococcus faecium, Str. diacetylac-
tis, and Str. intermedius; and Bidobacteria such as
B. bifidum, B. adolescentis, B. animalis, B. infantis,
B. longum, and B. thermophilum.
In many countries standards have been developed for
the numbers of the probiotic bacteria in fermented
products. For example, a minimal content (106 cfu g 1)
was established for bidobacteria added to fermented
milks by regulation recently approved by the countries
of MERCOSUR (Argentina, Paraguay, Brazil, and
Uruguay) (Pagano, 1998). In Japan, a standard has
been developed by the Fermented Milks and Lactic Acid
Bacteria Beverages Association, which requires a mini-
mum of 107 viable probiotic bacteria cells per millilitre
to be present in fresh dairy products (Robinson, 1987).
A suggested minimum level for probiotic bacteria in
yoghurt is 106 (Robinson, 1987; Kurman & Rasic, 1991)
or daily intake should be about 108 (Anonymous, 1992).
The Australian Food Standards Code does not specify
any requirements regarding the number of probiotic
bacteria in fermented products.
The ability of microorganisms to survive and multiply
in the host strongly inuences their probiotic benets.
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 host (Gilliland, 1989). Many studies
have shown low viability of probiotics in yoghurt and
fermented milk (Gilliland & Speck, 1977; Anonymous,
1992; Iwana, Masuda, Fujisawa, Suzuki, & Mitsuoka,
1993; Shah, Lankaputhra, Britz, & Kyle, 1995; Dave &
Shah, 1997; Shah & Lankaputhra, 1997; Gardini,
Lanciotti, Guerzoni, & Torriani, 1999; Schillinger,
1999; Vinderola, Bailo, & Reinheimer, 2000). Ravula
and Shah (1998) also reported that very high levels of
probiotic bacteria do not survive in fermented frozen
dairy desserts. They reported that the count declined by
56 log cycles within 812 weeks of storage at 181C.
Other studies of survival of probiotics have produced
similar results (Holcomb, Frank, & McGregor, 1991;
Laroia & Martin, 1991; Hekmat & McMahon, 1992). In
addition, Lankaputhra and Shah (1995) found that
survival of Lb. acidophilus and Bifidobacterium spp. is
low in the presence of acid and bile salts.
Protection of probiotics by microencapsulation in
hydrocolloid beads has been investigated for improving
their viability in food products and the intestinal tract
(Rao, Shiwnarain, & Maharaj, 1989). This has been
proposed for various dairy fermentations (Champagne,
Gaudy, Poncelet, & Neufeld, 1992a; Champagne
et al., 1992b; Champagne, Girard, & Rodrigue, 1993;
Champagne, Lacroix, & Sodini-Gallot, 1994) such as
fermentation of whey (Audet, Paquin, & Lacroix, 1989)
and continuous inoculation of milk for yoghurt
manufacture (Prevost & Divies, 1988). Additional
benets of microencapsulation of cells include: protec-
tion of cells inside the beads from bacteriophages
(Steenson, Klaenhammer, & Swaisgood, 1987); in-
creased survival during freeze drying and freezing
(Kearney, Upton, & Loughlin, 1990; Sheu & Marshall,
1993; Sung, 1997; Kim & Yoon, 1995); and greater
stability during storage (Kim, Kamara, Good, &
Enders, 1988; Kebary, Hussein, & Badawi, 1998;
Reuter, 1990).
Microencpasulation of various bacterial cultures
including probiotics has been a common practice for
extending their storage life and converting them into a
powder form for ease of their use. There are several
techniques such as spray drying, freeze drying, uidized
bed drying for encapsulating the cultures and converting
them into a concentrated powdered form. However,
the bacteria encapsulated by these techniques are
 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313
completely released in the product. In this case, the
cultures are not protected from the product environment
or during the passage through the stomach or intestinal
tract. Encapsulation in hydrocolloid beads entraps or
immobilizes the cells within the bead matrix, which in
turn provides protection in such an environment. This
paper reviews the techniques for encapsulation of
probiotic bacteria in hydrocolloid beads, particularly
for the use in yoghurt. Application of immobilized cells
in hydrocolloids for probiotic biomass production is
been described.
2. Techniques for microencapsulation of bacterial cells in
hydrocolloid beads
Microencapsulation is a process in which the cells are
retained within an encapsulating membrane to reduce
cell injury or cell loss. The encapsulation techniques
applied to probiotics for the use in fermented milk
products or biomass production can be classied into 2
groups, depending on the method used to form the
beads: extrusion (droplet method) and emulsion or two-
phase system. Both extrusion and emulsion techniques
increase the survival of probiotic bacteria by up to
8095% (Audet et al., 1988; Rao et al., 1989; Sheu &
Marshall, 1991; Sheu & Marshall, 1993; Sheu, Marshall,
& Heymann, 1993; Jankowski, Zielinska, & Wysakows-
ka, 1997; Kebary et al., 1998).
2.1. Extrusion technique
Extrusion is the oldest and most common approach to
making capsules with hydrocolloids (King, 1995). It
simply involves preparing a hydrocolloid solution,
adding microorganisms to it, and extruding the cell
suspension through a syringe needle in the form of
droplets to free-fall into a hardening solution or setting
bath (Fig. 1). The size and shape of the beads depend on
the diameter of the needle and the distance of free-fall,
respectively (Table 1). This method is the most popular
due to its ease, simplicity, low cost, and gentle
formulation conditions ensuring high retention of cell
viability.
2.1.1. Supporting material
The supporting material used for extrusion is alginate,
which is a linear heteropolysaccharide of d-mannuronic
and l-guluronic acid extracted from various species of
algae (Smidsrod, Haug, & Lian, 1972). Depending on
the source, the composition and the sequence in l-
guluronic acid and d-mannuronic acid vary widely. The
functional properties of alginate as supporting material
correlate strongly with the composition and sequence of
l-guluronic acid and d-mannuronic acid. Divalent
cations such as Ca2+ bind preferentially to the polymer
5
of l-guluronic acid. The length of the polymer of d-
mannuronic acid is, therefore, the main structural
feature contributing to gel formation (Smidsrod et al.,
1972; Skjak-Braek, Larsen, & Smidsrod, 1986).
To form beads, a cell suspension is mixed with a
sodium alginate solution, and the mixture dripped into a
solution containing a multivalent cation (usually Ca2+
in the form of CaCl2). The droplets form gel spheres
instantaneously, entrapping the cells in a three-dimen-
sional lattice of ionically cross-linked alginate. The
success of the alginate gel encapsulation technique is due
to the gentle environment it provides for the entrapped
material, cheapness, simplicity, and its biocompatibility
(Klein, Stock, & Vorlop, 1983; Tanaka, Masatose, &
Veleky, 1984; Martinsen, Skjak-Braek, & Smidsrod,
1989).
The concentrations of alginate used to form the gel
vary. Jankowski et al. (1997) used a very low concentra-
tion of 0.6% to form a gel with 0.3 m CaCl2. Others have
used 12% alginate and 0.051.5 m CaCl2 (Table 1). The
size of the beads is approximately 23 mm in diameter.
Moreover, the size and sphericity of the bead depend
mainly on the viscosity of the sodium alginate solution
and the distance between the syringe and the calcium
chloride collecting solution (Smidsrod & Skjak-Braek,
1990). As the concentration, and hence viscosity, of
sodium alginate increases, the size of the beads
decreases. The extruder orice diameter is another
important factor, which regulates droplet size. Using a
0.27-mm syringe, Smidsrod and Skjak-Braek (1990)
obtained a bead size of 23 mm. The composition of the
alginate also inuences bead size; small beads result
from low guluronic alginates (Martinsen et al., 1989).
2.2. Emulsion technique
In this technique, a small volume of the cell-polymer
suspension (discontinuous phase) is added to a large
volume of a vegetable oil (continuous phase) such as
soybean oil, sunower oil, canola oil or corn oil. The
mixture is homogenized to form a water-in-oil emulsion.
Once the water-in-oil emulsion is formed, the water-
soluble polymer must be insolubilized (cross-linked) to
form tiny gel particles within the oil phase (Fig. 1). The
smaller the internal phase particle size of the emulsion,
the smaller the nal microparticles will be. The
insolubilization method of choice depends on the type
of supporting material used. The beads are harvested
later by ltration. The size of the beads is controlled by
the speed of agitation, and can vary between 25 mm and
2 mm. This technique has been used successfully to
encapsulate lactic acid bacteria for batch (Lacroix,
Paquin, & Arnaud, 1990) and continuous fermentation
(Audet, Lacroix, & Paquin, 1992) (Table 2).
6 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

Sodium alginate Microbial cell suspension

Mix

Extrusion Technique Emulsion Technique

Emulsification in vegetable oil

Cell suspension

CaCl2

Drop in Addition of calcium chloride to

Calcium chloride solution Break emulsion and form gel

Microbial cell

Liquid core

Alginate
Calcium alginate bead
Fig. 1. Flow diagram of encapsulation of bacteria by the extrusion and emulsion techniques.

2.2.1. Continuous phase 2.2.2. Supporting material

For food applications, vegetable oils are used as
the continuous phase. Some studies have used
white light parafn oil (Rao et al., 1989) and mineral
oil (Groboillot et al., 1993). In some cases emulsiers
are added to form a better emulsion, because the
emulsiers lower the surface tension, resulting in
smaller spheres (Adamson, 1982). The most com-
mon emulsier used is Tween 80 at 0.2% (Sheu &
Marshall, 1993; Sheu et al., 1993; Kebary et al., 1998).
Sheu et al. (1993) used Tween 80 together with 0.5%
sodium lauryl sulphate, which produced a bead size of
2535 mm.
There are many supporting materials used with the
emulsion technique. These include a mixture of k-
carageenan and locust bean gum (Audet et al., 1988,
1989; Arnaud et al., 1992), cellulose acetate phthalate
(Rao et al., 1989), alginate (Sheu et al., 1991, 1993; Sheu
& Marshall, 1993; Larisch et al., 1994; Kebary et al.,
1998), chitosan (Groboillot et al., 1993), and gelatin
(Hyndman, Groboillot, & Poncelet, 1993).
2.2.2.1. Carrageenan. k-Carageenan is a natural poly-
saccharide extracted from marine macroalgae, com-
monly used as a food additive. Elevated temperatures
Table 1
Encapsulation of lactic acid and probiotic bacteria by the extrusion technique

Bacteria Supports used Conc. of alginate Conc. of CaCl2 Special treatment Diameter of bead Application Reference
(%) (m) (mm)

Lactobacillus delbrueckii Alginate 1.875 1.5 Na 2.5 Yoghurt Prevost, Divies, and
ssp. bulgaricus Rousseau (1985)
Streptococcus thermophilus

Str. lactis ssp. diacetylactis Alginate 1.875 1.5 N 2.6 Cheese Prevost and Divies (1987)
Str. cremoris

Str. cremoris Alginate 1 0.1 N b Phage protection Steenson et al. (1987)

Lb. delbrueckii ssp. Alginate 1.875 1 N 2.5 Yoghurt Prevost and Divies (1988)
bulgaricus
Str. Thermophillus

Lactococcus lactis ssp. Alginate 2 0.05 Coated with low 2 Biomass Zhou, Martins, Groboillot,
cremoris MWc chitosan production Champagne, and Neufeld
(1998)

Lb. plantarum Alginate 2 0.1 Added glycerol 2 Biomass Kearney et al. (1990)
production

Lc. lactis ssp. lactis bv. Alginate 1.5 0.2 Coated with Cream Prevost and Divies (1992)
diacetylactis alginate
Lc. lactis ssp. cremoris Alginate 2 0.1 N Biomass Morin, Bernier-Cardou,
production and Champagne (1992)

Str. thermophilus Alginate 2 0.1 N Biomass Champagne et al. (1993)

production
Lb. delbrueckii ssp.
W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

bulgaricus
Lc. lactis ssp. lactis bv. Alginate 1.8 1.5 N Cachon and Divies (1993)
diacetylactis
Lb. acidophillus Alginate 0.6 0.3 Mixed with starch 5 Biomass Jankowski et al. (1997)
production
a
N, no treatment.
b
, no record.
c
MW, molecular weight.
7
Table 2 8
Encapsulation of probiotic bacteria by the emulsion technique

Bacteria Support used Continuous phase Special treatment Diameter of bead Application Reference
a
Streptococcus thermophilus 3% k-carrageenan Soy oil N 0.52 mm Yoghurt Audet et al. (1988)
and locust bean gum
(2:1)
Lactobacillus delbrueckii ssp.
bulgaricus

Str. Thermophilus 3% k-carrageenan Soy oil N 0.51 mm b Audet et al. (1989)

and locust bean gum
(2:1)
Lactococcus lactis ssp. lactis Lb.
delbrueckii ssp. bulgaricus

Bifidobacterium pseudolongum 10% Cellulose acetate White light parafn N Rao et al. (1989)
phthalate oil

Lb. delbrueckii ssp. bulgaricus 3% Alginate Vegetable oil with 2% 2% emulsier added Ice cream Sheu and Marshall
emulsier (1991)

Lb. casei ssp. casei 3% k-carrageenan Vegetable oil N 12 mm Biomass Arnaud, Lacroix, and
and locust bean gum production Choplin (1992)
(11:1)

Lc. lactis ssp. cremoris Chitosan (4%) Mineral oil Cross-linked with 150 mm Biomass Groboillot,
hexamethylene production Champagne, Darling,
diisocyanate or and Poncelet (1993)
glutaldehyde

Lb. delbrueckii ssp. bulgaricus 3.6% Alginate Vegetable oil 6% glycerol or 30 mm Frozen dessert Sheu et al. (1993)
containing 0.2% mannitol added
Tween 80
Lb. delbrueckii ssp. bulgaricus 3% Alginate Vegetable oil 0.5% sodium lauryl 2535 mm Frozen ice milk Sheu and Marshall
containing 0.2% sulphate added (1993)
W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313

Tween 80

Lc. lactis ssp. cremoris 24% Gelatin Sunower seed oil Cross-linked with 271+168 mm Biomass Hyndman,
toluene-2,4- production Groboillot, and
diisocyanate Poncelet (1993)

Lc. lactis ssp. cremoris 2% Alginate Canola oil Coated with poly-l- 50 mm1 mm Biomass Larisch, Poncelet, and
lysine production Champagne (1994)

B. bifidum B. infantis 3% Alginate orn oil containing Added glycerol Ice milk Kebary et al. (1998)
0.2% Tween 80
a
N, no treatment.
b
, no record.
 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313
(60801C) are needed to dissolve the polymer at
concentrations ranging from 2% to 5% (Klein &
Vorlop, 1985). Gelation of carrageenan is induced by
temperature changes. The cell slurry is added to the
heat-sterilized polymer solution at 40451C and gelation
occurs by cooling to room temperature. After the beads
are formed, K ions (in the form of KCl) are used to
stabilize the gel and to prevent swelling, or to induce
gelation. Audet et al. (1988) reported that KCl has an
inhibitory effect on some lactic acid bacteria such as Str.
thermophilus and Lb. delbrueckii ssp. bulgaricus. Mono-
valent ions such as Rb+, Cs+ and NH+ 4 result in
stronger gels (Tosa et al., 1979). Locust bean gum, at a
ratio of carrageenan to locust bean gum of 2:1, increases
the strength of gels through specic interaction of its
galactomannan chains with carrageenan (Takata, Tosa,
& Chibata, 1977; Miles, Morris, & Carroll, 1984).
2.2.2.2. Cellulose acetate phthalate (CAP). Due to the
presence of ionisable phthalate groups, this polymer is
insoluble in acid media at a pH of 5 or lower but is
soluble when the pH is increased to 6 or higher (Malm,
Emerson, & Hiatt, 1951). In addition, CAP is physio-
logically inert when administered in vivo and is, there-
fore, widely used as an enteric coating material for the
release of drugs and other pharmaceutical substances in
the intestine. Rao et al. (1989) studied a procedure for
microencapsulation of B. pseudolongum with CAP, using
the emulsion technique. Microencapsulated B. pseudo-
longum survived the simulated gastric environment in
larger numbers (109 cfu mL 1) than non-encapsulated
organisms, which did not retain any viability when
exposed to a simulated gastric environment for 1 h.
2.2.2.3. Chitosan. Chitosan is a positively charged
linear polysaccharide formed by deacetylation of chitin
extracted from crustacean shells. It is water soluble
below pH 6, and, like alginate, forms a gel by ionotropic
gelation. Chitosan, a polycation with amine groups, can
be cross-linked by anions or polyanions, such as poly
phosphates, [Fe(CN)6]4 , [Fe(CN)6]3 , polyaldehydro-
carbonic acid (Klein & Vorlop, 1983). Chitosan
exhibited inhibitory effects on different types of lactic
acid bacteria (Groboillot et al., 1993). To overcome
viability problems with chitosan, Zhou, Martins, Gro-
boillot, Champagne, and Neufeld (1998) used this
polymer for cell encapsulation to coat alginate beads
by soaking the beads in chitosan solution (0.4%) with
gentle shaking for 40 min. Cell loading in beads ranged
from 108 to 1010 cfu g 1.
2.2.2.4. Gelatin. Gelatin, a protein, is useful as a
thermally reversible gelling agent for encapsulation.
Because of its amphoteric nature, it also is an excellent
candidate for cooperation with anionic polysaccharides
like gellan gum. These hydrocolloids are miscible at pH
9
> 6, since they both carry net negative charges and repel
one another. However, when the pH is adjusted below
gelatins isoelectric point, the net charge on the gelatin
becomes positive, causing an interaction with the
negatively charged gellan gum (King, 1995). Hyndman
et al. (1993) used high concentration gelatin (24%) to
encapsulate Lc. lactis ssp. cremoris by cross-linking with
toluene-2, 4-diisocyanate for biomass production.
2.2.2.5. Alginate. In the emulsion technique, alginate is
added prior to emulsion formation. The addition of an
oil-soluble acid, such as acetic acid, reduces the alginate
pH from 7.5 to approximately 6.5 and initiates gel
formation with Ca2+ (Poncelet, Poncelet, Beaulieu, &
Neufeld, 1993). Following gelation, the beads are
partitioned into water and washed to remove residual
oil.
3. Special treatments
Despite the suitability of alginate as the entrapment
matrix material, gel entrapment in alginate has some
limitation due to low stability in the presence of
chelating agents such as phosphate, lactate, and citrate.
The chelating agents share afnity for calcium and
destabilize the gel (Smidsrod & Skjak-Braek, 1990).
Thus, stability problems are encountered during lactic
acid fermentation (Roy, Goulet, & Duy, 1987) and
cause cell release from the beads. In the case of other
matrix material, such as chitosan, the entrapped cells
can be released from the beads during fermentation and
cause low initial loading for the next fermentation.
Therefore, special treatments, such as coating the beads,
are applied in order to improve the properties of
encapsulated beads. Coated beads not only prevent cell
release but also increase mechanical and chemical
stability. Cross-linking with cationic polymers, coating
with other polymers, mixing with starch, and incorpor-
ating additives can improve stability of beads.
3.1. Cross-linking with cationic polymers
Alginate beads have been stabilized by cross-linking
with cationic polymers such as polyethyleneimine and
polypropyleneimine, or polyethyleneimine and glutar-
aldehyde (Marx, 1989). Formation of a membrane
around the beads and the spraying of the beads with
glutaraldehyde have been proposed as stabilizing
techniques to minimize cell release (Kolot, 1988).
Groboillot et al. (1993) showed that the membrane
formed with 4% chitosan cross-linked with hexamethy-
lene diisocyanate or glutaraldehyde resulted in stronger
microcapsules. The reaction of the bifunctional reagent
with chitosan resulted in bridge formation linking the
chitosan molecules. The length of the bridge depends on
10 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313
the type of cross-linking agent. Hyndman et al. (1993)
obtained a similar result when toluene diisocyanate was
used to cross-link the gelatin. The microcapsules were
more resistant to breakage at the higher concentration.
3.2. Coating with other polymers
Overgaard, Scharer, Moo-Young, and Bols (1991)
produced alginate beads coated with a chitosan lm.
The beads were obtained by dropping an alginate
solution into a mixture of calcium chloride and chitosan
solution. McKnight, Ku, and Goosen (1988) reported
the coating of alginate beads with chitosan while Zhou
et al. (1998) found that suspending alginate beads in a
low molecular weight chitosan solution formed a
membrane, which reduced cell release by 40%. Chit-
osan, a positively charged polyamine, forms a semi-
permeable membrane around a negatively charged
polymer such as alginate. This membrane does not
dissolve in the presence of Ca2+ chelators or antigelling
agents and thus enhances stability of the gel (Smidsrod
& Skjak-Braek, 1990), and provides a barrier to cell
release. It was observed that low-molecular-weight
chitosan diffuses more readily into the alginate gel
matrix, resulting in a denser membrane than with high-
molecular-weight chitosan (McKnight et al., 1988).
Tanaka, Irie, and Ochi (1989) reported the coating of
gel beads by a cell-free alginate gel layer. Furthermore,
coating of beads with a poly amino acid such as poly l-
lysine is of interest due to its better biocompatibility
(Larisch et al., 1994), and potential for application in the
food industry, such as yoghurt production (Champagne
et al., 1992a). Champagne et al. (1992a) showed that
coating alginate beads with a single layer of poly l-
lysine did not signicantly reduce the release of cells but
double coating with poly l-lysine and alginate reduced
cell release by a factor of approximately 50.
3.3. Mixing with starch
Alginate/starch liquid core capsules offer the ability to
encapsulate Lb. acidophilus without loss of viability and
fermentation ability (Jankowski et al., 1997). Capsule
membranes allow sufcient diffusion of nutrients and
metabolites to maintain growth of encapsulated cells.
3.4. Incorporation of additives
With incorporation of cryoprotectants such as glycer-
ol, survival of encapsulated cells after lyophilisation and
rehydration is enhanced because these agents confer
additional protection (Kearney et al., 1990). The
survival of bidobacteria increased signicantly to
88.5% (Kebary et al., 1998), and Lb. delbrueckii ssp.
bulgaricus to 90% (Sheu et al., 1993) because these
agents reduced ice crystal formation by binding with
Table 3
Positive and negative features of extrusion and emulsion techniques
Extrusion Emulsion
Technological feasibility Difcult to scale up Easy to scale up
Cost Low High
Simplicity High Low
Survival of microorganism 8095% 8095%
Size of bead 25 mm 25 mm2 mm
water. The beads with glycerol also exhibited a 43%
decrease in size due to the higher alginate concentration
per unit volume in the beads with glycerol binding with
water.
4. Advantages and limitations of extrusion and emulsion
encapsulation techniques
For encapsulation of probiotics, both extrusion and
emulsion techniques can be applied. Advantages and
disadvantages of these techniques are shown in Table 3.
Extrusion is a relatively simple technique. It usually
produces entrapped, rather than encapsulated core
material, although encapsulation can be achieved
through co-extrusion devices or dropping into a bath
of coating material which react at the droplet surface.
This method can be difcult for large-scale production
because of slow formation of beads compared with the
emulsion technique.
On the other hand, the emulsion technique is
relatively new to the food industry and easy to scale
up for large-scale production. It provides both encapsu-
lated and entrapped core materials. The size of the beads
formed by this method is smaller (25 mm to 2 mm) than
that of beads produced by the extrusion method
(25 mm). The size of beads from the extrusion method
depends mainly on the size of the needle used, while the
size of beads from the emulsion method depends on the
speed of agitation and the type of emulsier used. Due
to the need for a vegetable oil, the operating cost of the
emulsion technique may be higher than that of the
extrusion technique.
5. Applications
Because of the many benets offered by encapsula-
tion, entrapped microorganisms can be used to advan-
tage for producing dairy products such as yoghurt,
cheese and frozen milk products, as well as for biomass
production. Prevost, Divies and Rousseau (1985)
reported that the continuous manufacture of yoghurt
with entrapped microorganisms (Lb. delbrueckii ssp.
bulgaricus and Str. thermophilus) is more complicated
than the traditional batch method, but presents many
advantages. It is possible to obtain a product with
 W. Krasaekoopt et al. / International Dairy Journal 13 (2003) 313
constant characteristics because the residence time,
acidity and continuous inoculation of milk with a
constant bacilli/cocci ratio can be controlled at a desired
pH. Prevost and Divies (1988) also obtained a similar
result. Audet et al. (1988) used encapsulated Str.
thermophilus and Lb. delbrueckii ssp. bulgaricus to
produce yoghurt. The viability of these bacteria
remained very high throughout the entrapment steps
and subsequent storage. The bead diameter inuenced
the fermentation rate; smaller beads (0.51.0 mm) had
higher cell release rates, lactose utilization and acid
production by the entrapped cells, reaching values found
for free cells.
The use of encapsulated microorganisms reduced the
incubation time by 50% and 60% for fresh fermented
cheese production (Prevost & Divies, 1987) and cream
fermentation (Prevost & Divies, 1992), respectively.
Sheu and Marshall (1993) reported that about 40%
more lactobacilli survived freezing of ice cream when
they were entrapped in calcium alginate than when they
were not entrapped. Moreover, encapsulation protected
the microorganisms in batch-frozen and continuously
frozen ice milk mixes (Sheu et al., 1993). Kebary et al.
(1998) reported that encapsulation of bidobacteria
signicantly improved their survival throughout storage
from 4344% to about 5060% while Rao et al. (1989)
showed that encapsulated B. pseudolongum could
survive in a simulated gastric environment in larger
numbers than non-encapsulated microorganisms.
Encapsulation can also be applied for biomass
production since the culture is protected from attack
by bacteriophage due to the exclusion of phage particles
from the gel matrix (Steenson et al., 1987). Moreover,
encapsulated culture provides high stability of cells and
high productivity for metabolite production with high
agitation rates (Arnaud et al., 1992). Champagne et al.
(1993) reported that it was possible to use encapsulated
microorganisms to produce bacterial densities
(123.1  108 mL 1) 6 times higher than with classical
cell free suspensions (18.6  108 mL 1). Morin, Bernier-
Cardou and Champagne (1992) suggested that addition
of CaCO3 to the fermentation medium for biomass
production improved bead stability. On the other hand,
the encapsulated culture may increase the processing
time. For example, encapsulated Lc. lactis ssp. cremoris
took 17% longer than free cells to reduce the pH of milk
to 4.5 (Larisch et al., 1994). Groboillot et al. (1993)
reported that loss of acidication activity during
encapsulation was recovered in subsequent fermentation
to levels similar to that of free-cell fermentation.
6. Conclusion
Encapsulated probiotic bacteria can be used in many
fermented dairy products, such as yoghurt, cheese,
11
cultured cream and frozen dairy desserts, and for
biomass production. In the encapsulated form, the
probiotics are protected from bacteriophage and harsh
environments, such as freezing and gastric solutions.
Thus, encapsulation facilitates the manufacture of
fermented dairy products in which the bacteria have
constant characteristics, higher stability during storage,
and higher productivity than non-encapsulated bacteria.
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