International Journal of Food Microbiology 145 (2011) 162–168

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International Journal of Food Microbiology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Survivability of probiotics encapsulated in alginate gel microbeads using a novel

impinging aerosols method
Asma Sohail a, Mark S. Turner a, Allan Coombes b, Thor Bostrom c, Bhesh Bhandari a,⁎
a
School of Agriculture and Food Sciences, University of Queensland, St Lucia, 4072, Australia
b
School of Pharmacy, University of Queensland, Woolloongabba, 4102, Australia
c
Faculty of Science and Technology, Queensland University of Technology, 4001, Brisbane, Australia

a r t i c l e i n f o a b s t r a c t

Article history: Encapsulation of probiotic bacteria in cross-linked alginate beads is of major interest for improving the
Received 9 June 2010 survivability in harsh acid and bile environment and also in food matrices. Alginate micro beads (10–40 μm)
Received in revised form 28 October 2010 containing the probiotics Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were produced by a
Accepted 7 December 2010
novel technique based on dual aerosols of alginate solution and CaCl2 cross linking solution. Extruded macro
beads (approximately 2 mm diameter) produced by the conventional method and micro beads produced by
Keywords:
Microencapsulation
novel aerosols technique offered comparable protection to L. rhamnosus in high acid and bile environment.
Lactobacillus rhamnosus GG Chitosan coating of micro beads resulted in a significant increase in survival time of L. rhamnosus from 40 to
Lactobacillus acidophilus NCFM 120 min in acid condition and the reduction in cell numbers was confined to 0.94 log over this time. Alginate
macro beads are more effective than micro beads in protecting L. acidophilus against high acid and bile.
Chitosan coating of micro beads resulted in similar protection to L. acidophilus in macro beads in acid and
extended the survival time from 90 to at least 120 min. Viability of this organism in micro beads was 3.5 log
after 120 min. The continuous processing capability and scale-up potential of the dual aerosol technique offers
potential for an efficient encapsulation of probiotics in very small alginate micro beads below sensorial
detection limits while still being able to confer effective protection in acid and bile environment.
© 2010 Published by Elsevier B.V.

1. Introduction
Microencapsulation defines a process in which solid, liquid or
gaseous materials are retained within an encapsulating matrix or
membrane as small capsules and release their contents at controlled
rates over extended time periods (Champagne and Fustier, 2007).
Microencapsulation has been applied to add value to the food
products by stabilizing reactive, sensitive, or volatile ingredients and
also by protecting them against moisture, heat and other extreme
conditions (Anal and Singh, 2007; Pimentel-González et al., 2009).
Probiotics defined as “live micro organisms which, when admin-
istered in adequate amounts, confer a health benefit on the host”
(Araya et al., 2002) are common core materials that are often
encapsulated in alginate gel. It has been recommended that food
containing probiotic bacteria should contain at least 106 live micro
organisms per g or mL at the time of consumption in order to have
protective effects such as maintenance of normal intestinal micro
flora, enhancement of the immune system, reduction of lactose
intolerance and anti-cancer activity (Capela et al., 2006; Mokarram
et al., 2009; Picot and Lacroix, 2004; Manojlovic et al., 2010). In
⁎ Corresponding author. Tel.: + 61 7 33469192; fax: + 61 7 33651177.
E-mail address: b.bhandari@uq.edu.au (B. Bhandari).
addition to that probiotics should resist the natural barrier of high
acidity and bile content in the gastrointestinal tract (GIT) and show
their viability and activity at the site of action (Ding and Shah, 2007).
Since some Lactobacillus lack the ability to adequately survive GIT
conditions (Mandal et al., 2006) microencapsulation techniques have
been applied to improve acid, bile and heat tolerance of probiotics
(Ding and Shah, 2007) and also to inhibit unwanted reactions during
storage and processing which can result in loss of activity (Adhikari
et al., 2000; Chandramouli et al., 2004; Sultana et al., 2000). Probiotics
used for this study include Lactobacillus rhamnosus GG (ATCC 53130)
and Lactobacillus acidophilus NCFM.
In case of encapsulation of probiotics, many encapsulation
methods have been developed and generally involve spray drying
and entrapment in gel particles by emulsion or extrusion methods. In
the emulsion method, a hydrocolloid solution is emulsified in
vegetable oil and then added into an ionic solution to induce gelation.
Reaction between the hydrocolloid solution and ionic solution (CaCl2)
takes place in the oil phase when coalescence of the droplets
(hydrocolloid and ionic solution) takes place (Burey et al., 2008;
Lamprecht et al., 2001; Sugiura et al., 2005). However, there is a
difficulty in this method of removal of the oil for which chemicals may
be required and another separation step is added which can prove to
be messy and tedious. This method is also non-continuous and time
consuming (Burey et al., 2008).

0168-1605/$ – see front matter © 2010 Published by Elsevier B.V.

doi:10.1016/j.ijfoodmicro.2010.12.007
 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168
One widely reported extrusion method is dropping a millimetre
size alginate solution containing microorganism into the cross-linking
CaCl2 bath. Methods involve electrostatic atomization, spinning/
rotating disk atomization, vortex bowl disk atomization, and vibration
or jet cutter, that involve additional force to generate smaller beads.
Existing techniques are capable of producing macro particles (up to
5 mm) and micro particles (up to 200 μm) (Gibbs et al., 1999; Gouin,
2004; Shilpa et al., 2003; Zuidam and Shimoni, 2010) but have one or
more limitations such as scale-up restrictions. These limitations
include sensory detection of large beads on consumption of food
products, the requirement for solvents, and application of heat and
non-continuous production. Spray drying results in a high volume
output but produces water soluble capsules, which results in exposure
of core materials on rehydration. Acid resistant beads are necessary
for protection of acid-labile contents (unstable in acid environment)
in gastric fluids following consumption.
Alginate is the most common encapsulating matrix used for food
grade and non-food compounds because of its biocompatibility, safety
and low cost (Krasaekoopt et al., 2004; Martinsen and Smidsrød,
1989) Alginates are linear copolymer of 1, 4-linked β-D-mannuronic
acid (M) and α-L-guluronic acid (G) residues extracted from various
species of algae (Gombotz and Wee, 1998; Martinsen and Smidsrød,
1989). The polymer's bioadhesive properties facilitate coating with
other polymers (Gombotz and Wee, 1998; Mumper et al., 1994). The
mechanical properties and permeability of alginate capsules, for
example, can be modified using polycations such as chitosan (Peniche
et al., 2004) that are complex with the negatively charged alginate
chains and influence the release rate of encapsulated active
compounds.
Entrapment of compounds in alginate gels cross linked by metal
ions such as calcium can result in water insoluble capsules, but many
techniques are limited to batch production and are difficult to scale-
up. In this study a new continuous encapsulation process has been
developed (Bhandari, 2009) which uses separate impinging aerosols
of sodium alginate solution and calcium chloride cross-linking
solution to produce cross-linked alginate micro beads with an
average diameter of less than 40 μm (Fig. 1). Here we report on the
Fig. 1. Schematic of the double aerosol technique for encapsulation of probiotics in
alginate microbeads.
163
application of this technique for encapsulating the probiotics
L. rhamnosus GG and L. acidophilus NCFM and the survivability of
encapsulated bacteria in simulated gastric and intestinal fluids.
2. Materials and methods
2.1. Preparation of lactobacilli
Frozen cultures (in 40% v/v glycerol) of two strains of probiotic
bacteria L. rhamnosus GG (ATCC 53103) and L. acidophilus NCFM were
obtained from sample stocks at the University of Queensland. These
strains were inoculated into 100 mL of MRS (de Man, Rogosa, Sharpe
[Oxoid Ltd]) broth and incubated at 37 °C for 24 h for L. rhamnosus and
36 h for L. acidophilus, respectively. The cells were harvested by
centrifugation at 10,000 rpm (24,123 rcf) for 10 min and were
washed twice with sterile 0.1% peptone.
2.2. Encapsulation of lactobacilli in alginate microbeads
Bacterial cultures were suspended in 5 mL of sterile 0.1%
peptone and mixed with 45 mL of 2% (w/v) sodium alginate
solution (GRINSTED® Alginate FD 155, Danisco Textile Ingredients,
Australia) which had been sterilized at 121 °C for 15 min. An
aerosol of microbial suspension in alginate solution was injected
into the top of a Plexiglass cylinder through a two-fluid nozzle at a
pump speed of 12 mL/min using pressurized air at 65 psi. A counter
aerosol of sterile 0.1 M CaCl2 solution was injected from the base of
the vessel using a two-fluid nozzle at a speed of 9 mL/min using
compressed air at 50 psi. Alginate micro beads encapsulating the
probiotics were collected from the outlet at the base of the vessel
(Fig. 1).
Encapsulation of probiotics L. rhamnosus and L. acidophilus in
alginate macro beads was achieved by extrusion method (Krasaekoopt
et al., 2004). Cells were suspended in 5 mL of sterile 0.1% of peptone
and mixed with 45 mL of sterile 2% (w/v) sodium alginate solution.
The suspension was then injected through a sterile disposable, Idakko
22 G 1/2 needle (0.70 × 38 mm) into a bath of sterile 0.1 M CaCl2
solution. The macro beads were retained in the calcium chloride
solution for 30 min then rinsed with 0.1% peptone solution and stored
in the same medium at 4 °C prior to testing.
2.3. Coating of alginate microbeads with chitosan
Low molecular weight chitosan (20 mg) was dissolved in 2 mL of
10% glacial acetic acid to achieve a concentration of 0.1% (w/v). The pH
was adjusted to between 5.7 and 6.0 by adding 0.1 M NaOH. The
solution was filtered through a Whatman #4 filter paper and sterilized
by passage through a 0.45 μm Millipore filter. Alginate micro beads
encapsulating “L. rhamnosus and L. acidophilus” were immersed in the
chitosan solution and stirred at 100 rpm for 40 min using an orbital
shaker (IKA KS 260 basic, Labtek).
2.4. Determination of encapsulated cell numbers
Freshly prepared alginate microbeads encapsulating probiotics
(1 g) were broken down in 9 mL of 2% (w/v) sterile sodium citrate
solution at pH 6.0 by gently shaking at room temperature for 10 min
using an orbital shaker (IKA KS 260 basic, Labtek). Chitosan coated
alginate micro beads were blended in a stomacher (Colworth
stomacher 400) for 1 min prior to breakdown in sodium citrate
solution. Dilutions of 101 up to 108 were made in 0.1% peptone and
100 μL aliquots were plated on MRS agar. Colonies of L. rhamnosus
and L. acidophilus were enumerated following incubation at 37 °C for
24 and 36 h under anaerobic conditions.
164 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168

2.5. Acid tolerance of free and encapsulated probiotics from −160 °C to −85 °C over 2 h. The sample was held at −85 °C for
47 h before increasing the temperature to 20 °C over 15 h. Finally the
A low pH (acid tolerance) study of free and encapsulated sample was held at 20 °C for 1 h in Leica AFS. Samples were then
bacteria was carried out according to the method of Ding and Shah dehydrated with acetone and mounted in Epon by polymerisation at
(2007). MRS broth was adjusted to pH 2.0 with 5.0 M HCl and 60 °C for two days. Polymerised resin blocks were sectioned using a
sterilized by autoclaving at 121 °C for 15 min. One mL aliquots (109 Leica Ultracut UC6 ultra microtome and a diamond knife.
to 1010 CFU/mL) of free L. rhamnosus and L. acidophilus were used as Sections 60 nm in thickness were picked up on formvar coated
controls. Test samples consisted of freshly prepared alginate micro copper 200 mesh grids and stained with 5% uranyl acetate in 50%
beads (1 g) encapsulating probiotics produced by the double methanol for 3 min and in Reynolds lead citrate for 2 mins. Samples
aerosol method, chitosan-coated micro beads and extruded alginate were washed with water between and after staining. Sections were
macro beads encapsulating probiotics. Test and control samples examined in a JEOL 1010 (Japanese Electron Optical Limited)
were added to tubes containing 9 mL of modified MRS broth (pH transmission electron microscope operated at 80 kV and images
2.0) and incubated at 37 °C for 5, 20, 40, 60, 90 and 120 min. After were captured using an Olympus soft imaging mega view III digital
incubation for the specified time intervals, 1 mL aliquots of free camera.
cells and encapsulated cells were collected and neutralized by
adding 1 M NaOH. Media containing free cells were serially diluted
with 0.1% peptone and inoculated onto MRS agar plates. Encapsu- 2.8. Statistical analysis
lated bacteria were released from alginate micro or macrobeads and
enumerated by the method described in Section 2.4. All tests were Data was derived from the results of 3 independent experiments
repeated 3 times to estimate the standard error. and Analysis of variance was carried out using the ‘General Linear
Model’. Completely randomized design (CRD) with repeated mea-
2.6. Bile tolerance of free and encapsulated probiotics surement and split unit was used. Significant differences between the
means of cell counts were determined by using Tukey's simultaneous
Oxgall (Difco™, Bacto Lab Pty Ltd) was used as a bile salt to study test. All analyses were carried out using Minitab 15.
the bile tolerance of encapsulated and free cells. Prior to the
experiment the tolerance of both lactobacilli strains to oxgall was
assessed by inoculating them individually (109 CFU/mL) in sterile
MRS broth with 0.3, 0.5, 1.0, 1.5, 2.0, and 3.0% (w/v) added oxgall at
37 °C for 120 min. Bile tolerance was studied according to the method
described by Ding and Shah (2007), Kim et al. (2008) and Liong and
Shah (2005). Bacterial survival was evaluated by performing plate
counts with MRS agar. The oxgall concentration selected was the
lowest concentration that inhibited L. rhamnosus and L. acidophilus
survival. Suspensions of free bacteria (1 mL) containing 109 CFU/mL
and alginate encapsulated cells (1 g) were placed in a tube containing
9 mL of MRS broth modified with oxgall and incubated at 37 °C for 5,
60 and 120 min. Aliquots (1 mL) of free and encapsulated bacteria
were collected at each time interval. Free cells were serially diluted
with 0.1% peptone and inoculated on MRS agar plates. Enumeration of
the numbers of encapsulated bacteria was carried out as described
above. Following breakdown of the micro or macrobeads in sodium
citrate solution, bacterial survival was determined by serial dilution
and inoculation on MRS agar plates at 37 °C for 24 and 36 h
respectively as described in Section 2.4.

2.7. Size and morphology of probiotic loaded micro and macro beads

The size and shape of probiotic-loaded micro beads produced by

the double aerosol method and probiotic loaded macro beads
produced by extrusion were observed using an optical microscope.
Hydrated microbeads were studied using an environmental scanning
electron microscope (ESEM) (FEI Quanta 200 Environmental SEM (FEI
Company, USA)). Suspensions of alginate gel microbeads in distilled
water were mounted on a cooled peltier stage operating at a
temperature of 4 °C and examined using an accelerating voltage of
20.0 kV. Samples were rapidly dehydrated initially at a pressure of
4.00 Torr to remove excess water and reveal the microbeads. A
pressure of 5.00 Torr was then applied to prevent further dehydration
during observation of the microbeads. A further reduction of pressure
to 3.00 Torr was used to observe the final stages of dehydration of the
microbeads. Transmission electron microscopy (TEM) was used to
study the internal structure of the microbeads. For TEM analysis
hydrated samples were mixed with 2% agarose in a 100 μm membrane
carrier. Samples were cryo-fixed by cryo-substitution (1% osmium
tetroxide, 0.5% uranyl acetate, and 5% water in acetone) using Fig. 2. (A) Encapsulated beads (novel method) in optical microscope. (B) Encapsulated
EMPACT 2 HPF. The process was initiated by raising the temperature bead showing bacteria inside.
 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168 165

Fig. 3. (A) ESEM image of micro beads(carpet of beads) (B) ESEM image of hydrated encapsulated micro beads (arrows show the individual beads) (C) ESEM image of flattened
encapsulated beads (arrows show the impression of the flattened beads) and (D) ESEM image of fully dried beads showing salt deposits at the sides.

3. Results and discussion
3.1. Size and morphology of alginate micro beads and encapsulated cell
numbers
The cell suspensions used for encapsulation contained 9.3–9.7
(log CFU/mL). The number of cells encapsulated in alginate micro
beads using the double aerosol method, was in the range of 8.9–9.6
(log CFU/g) of beads. Extruded alginate macro beads were found to
contain 9.0–9.30 (log CFU/g), compared with the initial free cell load
of the starting suspension of 9.2–9.4 (log CFU/mL). Therefore a very
high cell loading was observed for both micro beads and macro
beads.
Alginate micro beads containing the probiotics L. rhamnosus and
L. acidophilus respectively with a mean size of 35 μm (10–40 μm
range) were readily produced using the double aerosol technique.
The average size of probiotic loaded alginate macro beads produced
by extrusion method was 1.9 mm (1.8–1.9 mm range). Both alginate
micro beads and macro beads were roughly spherical in shape and
there was no large variation in size or shape with the different
bacterial strains (Fig. 2).
ESEM was employed to investigate the size and morphology of
hydrated alginate gel microbeads. This technique has been widely
used to study the morphology of hydrated, delicate materials and
drug delivery systems such as polymeric microspheres and liposomes
(Elvira et al., 2004; Mohammed et al., 2004). The decided advantage of
ESEM compared to conventional SEM is that samples can be analysed
in the hydrated state without coating and investigations of material
behaviour can be made dynamically under controlled humidity
atmospheres. The sequence of ESEM images in Fig. 3 captures the
transition from fully hydrated alginate gel microbeads to dehydrated
film structures on lowering the vapour pressure in the sample
chamber. The ESEM image in Fig. 3A reveals a mat of hydrated
microbeads with sizes ranging from 10 to 15 μm. The reduced size
relative to that measured in optical micrographs (35 μm) can be
explained by a partial shrinkage of the microbeads during dehydra-
tion. Individual hydrated microbeads are shown in Fig. 3B. Gradual
dehydration of the alginate microbeads results in flattening and
coalescence of the gel structure to a film (Fig. 3C). The final stages of
drying gives rise to salt deposition on the film surface, originating
from the original CaCl2 cross linking solution (Fig. 3D).
The TEM images in Fig. 4 show the cross section of an alginate
micro bead (arrowed) approximately 35.26 μm in length, encapsu-
lating L. rhamnosus. A medial section of encapsulated rod shaped
bacterium approximately 3.8 × 0.8 μm is clearly visible within the
microbead (Fig. 4B).
166 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168
Fig. 4. (A) TEM image of the cross sectional area of the micro bead showing medial,
transverse and diagonal sections of the encapsulated bacteria inside the bead. (B) TEM
image of the medial section of the encapsulated bacteria inside the micro bead.
3.2. Acid tolerance of free and encapsulated probiotics
Fig. 5A and B shows the viability of free and encapsulated
L. rhamnosus and L. acidophilus bacteria after 120 min exposure to
the acid environment (pH 2.0). The viability of free L. rhamnosus
bacteria had decreased to undetectable levels after 20 min
(Fig. 5A). The survival of L. rhamnosus was extended to 40 min
(4.38 log CFU/mL) by encapsulation and protection was similar in
alginate micro beads and macro beads (P N 0.05). In the present
study, the survivability of L. rhamnosus was greatly enhanced by
chitosan coating of the micro beads in the high acid condition to
the extent that only 1.27 log CFU/mL reduction was observed in
120 min and the survival was 8.65 log CFU/mL at that time
whereas none other probiotics survived in any other treatment
after 120 min (P b 0.05). Other investigators have reported that
chitosan-coated macrobeads (1.89 mm) significantly improve the
survivability of L. acidophilus compared with free cells, as shown by
the D-value of 18.8 min for free cells compared with 50 min for
encapsulated bacteria (Krasaekoopt et al., 2004). Gbassi et al. (2009)
and Sultana et al. (2000) reported that probiotics encapsulation in
unmodified alginate beads (size 0.5–1.0 mm) was ineffective in
protecting probiotics in highly acidic environments. Truelstrup et al.
(2002) reported similar results in that bifidobacteria encapsulation
in Ca-alginate microcapsules with an average diameter of 20 μm and
70 μm did not significantly improve survival in SGF. However, other
studies have reported higher survival rates when lactobacilli
immobilized in alginate beads were incubated in SGF (Ding and
A10
9
Viable cells log cfu/mL
7
6 a
b
5 c
d
4
1
0 5 20 40 60 90 120
Incubation Time (min)
B 10
9
Viable cells log cfu/mL 7 a
6 b
c
5 d
4
1
0 5 20 40 60 90 120
Incubation Time (min)
Fig. 5. A. The viability of L. rhamnosus GG exposed to the high acid environment (pH 2.0).
(a) Free bacteria (b) bacteria encapsulated in alginate microbeads (c) bacteria
encapsulated in chitosan-coated alginate microbeads (d) bacteria encapsulated in
extruded alginate macrobeads. B. The viability of L. acidophilus NCFM to the high acid
environment (pH 2.0). (a) Free bacteria (b) bacteria encapsulated in alginate microbeads
(c) bacteria encapsulated in chitosan-coated alginate microbeads (d) bacteria encapsu-
lated in extruded alginate macrobeads.
Shah, 2007; Lee et al., 2004; Lee and Heo, 2000; Mandal et al., 2006;
Mortazavian et al., 2008; Picot and Lacroix, 2004). These conflicting
reports emphasise that a direct comparison of the results of
probiotic survivability experiments is complicated by factors such
as the encapsulation method used, culture preparation, bacteria
strain and assay procedures (Truelstrup et al., 2002). Acid and bile
resistance are well known to vary widely among strains within a
species and among species (Ding and Shah, 2007; Shah et al., 1995;
Truelstrup et al., 2002).
L. acidophilus (Fig. 5B) proved to be more acid-resistant than
L. rhamnosus with survival being measured at 90 min for L. acidophilus
in acid condition compared with 20 min for L. rhamnosus. Suspensions
of free L. acidophilus were reduced from an initial viable count of 9.28
to 3.83 log CFU/mL (5.45 log CFU/mL reduction) after 90 min. Over the
same time scale, L. acidophilus bacteria encapsulated in alginate micro
beads showed more survivability than free cells (P b 0.05) and the
survival was 4.26 log CFU/mL but the protection was inferior to
alginate macro beads (5.46 log CFU/mL (Fig. 5B)). Encapsulation of
L. acidophilus in uncoated micro beads or macro beads was ineffective
in protecting bacteria against high acid environment beyond 90 min.
In contrast, chitosan coated micro beads conferred similar protection
 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168 167

A10 study of acid tolerance, chitosan-coated micro beads resulted in

improved protection in the presence of bile salt (oxgall).
9 L. acidophilus was found to be more resistant than L. rhamnosus to
SIF and showed only a 1.70 log CFU/mL reduction after 2 h in a higher
8
Viable cells log cfu/mL

(3.0%) oxgall solution (Fig. 6B). Encapsulation of L. acidophilus in

7 extruded macro beads was more effective than alginate micro beads
6
(P b 0.05) in maintaining cell viability (Fig. 6B), but chitosan coating of
a
micro beads resulted in a similar protective effect to alginate macro
b
5 beads (P N 0.05). A reduction of 0.6 log CFU/mL was observed after
c 120 min where the survival was 9.53 log CFU/mL in chitosan coated
4
d micro beads.
3 Truelstrup et al. (2002) reported that probiotic bifidobacteria
2 encapsulated in micro beads with diameters below 100 μm did not
result in significant improvements in cell viability in SGF and macro
1
0 5 60 120 beads (1000 μm) caused coarseness in food texture. Mokarram et al.
Incubation Time (min) (2009) considered that bacteria should be encapsulated within
micro beads of a particular size range (50–75 μm) in order to
achieve protection in gastro-intestinal conditions. The results
B10 presented here support and highlight previous findings that the
survivability of encapsulated bacteria is highly dependent on the
9
strain encapsulated. In addition, chitosan coating of the porous
8 alginate gel matrix provides major improvements in protection in
Viable cells log cfu/mL

low pH environments by restricting diffusion of the acid medium.

7
Our results further demonstrate that chitosan coating of micron-
6 size, alginate gel beads below 40 μm can confer equivalent probiotic
a
protection to millimetre size macro beads.
5 b
c
4 4. Conclusion
d
3
Alginate micro beads, 10–40 μm in size, encapsulating the
2 probiotics L. rhamnosus GG and L. acidophilus NCFM were produced
by a novel technique involving dual aerosols of alginate solution and
1
0 5 60 120 CaCl2 cross linking solution. Micro beads and extruded macro beads
Incubation Time (min) (approximately 2 mm diameter) offered similar protection to
L. rhamnosus GG in the acid and bile tolerance study. Chitosan coating
Fig. 6. A. The viability of L. rhamnosus GG in the presence of oxgall 0.5%. (a) Free of micro beads resulted in a significant increase in survival time of the
bacteria (b) bacteria encapsulated in alginate microbeads (c) bacteria encapsulated in
probiotic from 40 to 120 min and the cell numbers were reduced by
chitosan-coated alginate microbeads (d) bacteria encapsulated in extruded alginate
macrobeads. B. The viability of L. acidophilus NCFM in the presence of oxgall 3.0%. 1.27 log only. Alginate macro beads were more effective than micro
(a) Free bacteria (b) bacteria encapsulated in alginate microbeads (c) bacteria beads in protecting L. acidophilus NCFM against acid and bile
encapsulated in chitosan-coated alginate microbeads (d) bacteria encapsulated in tolerance. However chitosan coating of micro beads resulted in
extruded alginate macrobeads. similar protection to macro beads in high acid environment and
extended the survival time from 90 to at least 120 min. The cell
numbers were reduced by 6 log over 2 h. These findings demonstrate
to macro beads and, moreover, extended survival times to 120 min the potential of the dual aerosol technique for continuous encapsu-
where the survival was 3.49 log CFU/mL. A reduction of cell counts of lation process of probiotics in alginate micro beads which is as
6.03 log CFU/mL was measured after 120 min. effective as other alginate gel encapsulation technique to protect
Kim et al. (2008) reported that free L. acidophilus was completely probiotics in the presence of high acid and bile salts respectively.
inactivated after 60 min incubation at pH 1.2, while bacteria
encapsulated in calcium alginate beads retained viability above
Acknowledgement
104 CFU/mL at pH 1.5 after 3 h. Mokarram et al. (2009) have also
also reported that encapsulation of L.acidophilus and L.rhamnosus in
The authors express their appreciation to Mr. Rick Webb in the
calcium alginate microcapsules with alginate mono and double layer
centre for microscopy and microanalysis, University of Queensland,
coating with a size of 23 μm (uncoated beads), 47 μm (mono layer
Australia and Mr. Malik Adil Nawaz for their assistance in the
coated beads) and 75 μm (double layer coated beads) protects the
transmission electron microscopy.
probiotics in SGF.

3.3. Bile tolerance of free and encapsulated probiotics References

The effect of oxgall (0.5% and 3.0%) on the viability of L. rhamnosus
and L. acidophilus respectively is shown in Fig. 6A and B. The viability
of free cells of L. rhamnosus (Fig. 6A) dropped by 3.35 log CFU/mL
within the first five minutes and then remained stable over 2 h
(6.0 log CFU/mL). L. rhamnosus encapsulated in either alginate micro
beads produced by the double aerosol method or extruded alginate
macro beads showed similar survivability after 120 min. Thus the
effect of size of the beads was not significant (P N 0.05). Similar to the
Adhikari, K., Mustapha, A., Grun, I.U., Fernando, L., 2000. Viability of microencapsulated
bifidobacteria in set yogurt during refrigerated storage. Journal of Dairy Science 83,
1946–1951.
Anal, A.K., Singh, H., 2007. Recent advances in microencapsulation of probiotics for
industrial applications and targeted delivery. Trends in Food Science and
Technology 18, 240–251.
Araya, M., Morelli, L., Reid, G., Sanders, M.E., Stanton, C., Pineiro, M., 2002. Guidelines for
the evaluation of probiotics in food. Joint FAO/WHO working group report on
drafting guidelines for the evaluation of probiotics in food, London, ON, Canada.
Bhandari, B., 2009. Device and method for preparing microparticles. University of
Queensland, Australia.
168 A. Sohail et al. / International Journal of Food Microbiology 145 (2011) 162–168
Burey, P., Bhandari, B.R., Howes, T., Gidley, M.J., 2008. Hydrocolloid gel particles:
formation, characterization, and application. Critical Reviews in Food Science and
Nutrition 48, 361–377.
Capela, P., Hay, T.K.C., Shah, N.P., 2006. Effect of cryoprotectants, prebiotics and
microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried
yoghurt. Food Research International 39, 203–211.
Champagne, C.P., Fustier, P., 2007. Microencapsulation for the improved delivery of
bioactive compounds into foods. Current Opinion in Biotechnology 18, 184–190.
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. Journal of Microbiological Methods 56, 27–35.
Ding, W.K., Shah, N.P., 2007. Acid, bile, and heat tolerance of free and microencapsu-
lated probiotic bacteria. Journal of Food Science 72, M446–M450.
Elvira, C., Fanovich, A., Fernández, M., Fraile, J., San Román, J., Domingo, C., 2004.
Evaluation of drug delivery characteristics of microspheres of PMMA-PCL-
cholesterol obtained by supercritical-CO2 impregnation and by dissolution–
evaporation techniques. Journal of Controlled Release 99, 231–240.
Gbassi, G.K., Vandamme, T., Ennahar, S., Marchioni, E., 2009. Microencapsulation of
Lactobacillus plantarum spp in an alginate matrix coated with whey proteins.
International Journal of Food Microbiology 129, 103–105.
Gibbs, B.F., Kermasha, S., Alli, I., Mulligan, C.N., 1999. Encapsulation in the food industry:
a review. International Journal of Food Sciences and Nutrition 50, 213–224.
Gombotz, W.R., Wee, S.F., 1998. Protein release from alginate matrices. Advanced Drug
Delivery Reviews 31, 267–285.
Gouin, S., 2004. Microencapsulation: industrial appraisal of existing technologies and
trends. Trends in Food Science and Technology 15, 330–347.
Kim, S.-J., Cho, S.Y., Kim, S.H., Song, O.-J., Shin, I.I.S., Cha, D.S., Park, H.J., 2008. Effect of
microencapsulation on viability and other characteristics in Lactobacillus acidophi-
lus ATCC 43121. LWT Food Science and Technology 41, 493–500.
Krasaekoopt, W., Bhandari, B., Deeth, H., 2004. The influence of coating materials on
some properties of alginate beads and survivability of microencapsulated probiotic
bacteria. International Dairy Journal 14, 737–743.
Lamprecht, A., Schafer, U., Lehr, C.M., 2001. Influences of process parameters on
preparation of microparticle used as a carrier system for Omega-3 unsaturated fatty
acid ethyl esters used in supplementary nutrition. Journal of Microencapsulation
18, 347–357.
Lee, K.Y., Heo, T.R., 2000. Survival of Bifidobacterium longum immobilized in calcium
alginate beads in simulated gastric juices and bile salt solution. Applied and
Environmental Microbiology 66, 869–873.
Lee, J.S., Cha, D.S., Park, H.J., 2004. Survival of freeze-dried Lactobacillus bulgaricus KFRI
673 in chitosan-coated calcium alginate microparticles. Journal of Agricultural and
Food Chemistry 52, 7300–7305.
Liong, M.T., Shah, N.P., 2005. Acid and bile tolerance and cholesterol removal ability of
lactobacilli strains. Journal of Dairy Science 88, 55–66.
Mandal, S., Puniya, A.K., Singh, K., 2006. Effect of alginate concentrations on survival of
microencapsulated Lactobacillus casei NCDC-298. International Dairy Journal 16,
1190–1195.
Manojlovic, V., Nedovic, V.A., Kailasapathy, K., Zuidam, N.J., 2010. Encapsulation of
probiotics for use in food products. In: Nedovic, N.J.Z.a.V. (Ed.), Encapsulation
Technologies for Active Food Ingredients and Food Processing. Springer, New York,
pp. 269–302.
Martinsen, A., Smidsrød, G.S.-B.O., 1989. Alginate as immobilization material: I.
Correlation between chemical and physical properties of alginate gel beads.
Biotechnology and Bioengineering 33, 79–89.
Mohammed, A.R., Weston, N., Coombes, A.G.A., Fitzgerald, M., Perrie, Y., 2004. Liposome
formulation of poorly water soluble drugs: optimisation of drug loading and ESEM
analysis of stability. International Journal of Pharmaceutics 285, 23–34.
Mokarram, R.R., Mortazavi, S.A., Najafi, M.B.H., Shahidi, F., 2009. The influence of multi
stage alginate coating on survivability of potential probiotic bacteria in simulated
gastric and intestinal juice. Food Research International 42, 1040–1045.
Mortazavian, A.M., Ehsani, M.R., Azizi, A., Razavi, S.H., Mousavi, S.M., Sohrabvandi, S.,
Reinheimer, J.A., 2008. Viability of calcium-alginate-microencapsulated probiotic
bacteria in Iranian yogurt drink (Doogh) during refrigerated storage and under
simulated gastrointestinal conditions. Australian Journal of Dairy Technology 63,
25–30.
Mumper, R.J., Huffman, A.S., Puolakkainen, P.A., Bouchard, L.S., Gombotz, W.R., 1994.
Calcium-alginate beads for the oral delivery of transforming growth factor-[beta]1
(TGF-[beta]1): stabilization of TGF-[beta]1 by the addition of polyacrylic acid
within acid-treated beads. Journal of Controlled Release 30, 241–251.
Peniche, C., Howland, I., Carrillo, O., Zaldivar, C., Arguelles-Monal, W., 2004. Formation
and stability of shark liver oil loaded chitosan/calcium alginate capsules. Food
Hydrocolloids 18, 865–871.
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.
Pimentel-González, D.J., Campos-Montiel, R.G., Lobato-Calleros, C., Pedroza-Islas, R.,
Vernon-Carter, E.J., 2009. Encapsulation of Lactobacillus rhamnosus in double
emulsions formulated with sweet whey as emulsifier and survival in simulated
gastrointestinal conditions. Food Research International 42, 292–297.
Shah, N.P., Lankaputhra, W.E.V., Britz, M.L., Kyle, W.S.A., 1995. Survival of Lactobacillus
acidophilus and Bifidobacterium bifidium in commercial yogurt during refrigerated
storage. International Dairy Journal 5, 515–521.
Shilpa, A., Agrawal, S.S., Ray, A.R., 2003. Controlled delivery of drugs from alginate
matrix. Polymer Reviews 43, 187–221.
Sugiura, S., Oda, T., Izumida, Y., Aoyagi, Y., Satake, M., Ochiai, A., Ohkohchi, N., Nakajima,
M., 2005. Size control of calcium alginate beads containing living cells using micro-
nozzle array. Biomaterials 26, 3327–3331.
Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., Kailasapathy, K.,
2000. Encapsulation of probiotic bacteria with alginate-starch and evaluation of
survival in simulated gastrointestinal conditions and in yoghurt. International
Journal of Food Microbiology 62, 47–55.
Truelstrup, H.L., Allan-Wojtas, P.M., Jin, Y.L., Paulson, A.T., 2002. Survival of Ca-alginate
microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal
conditions. Food Microbiology 19, 35–45.
Zuidam, N.J., Shimoni, E., 2010. Overview of microencapsulates for use in food products
or processes and methods to make them. In: Zuidam, N.J., Nedovic, V.A. (Eds.),
Encapsulation Technologies for Active Food Ingredients and Food Processing.
Springer, New York, pp. 3–29.