J. Env. Bio-Sci., 2014: Vol.

28 (2):125-128
(125) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

ANALYSIS OF MICRO ENCAPSULATED PROBIOTIC BEADS

AND ITS PROPERTIES
N. Karthikeyan, G . Kumaresan and Elango. A
Department of Dairy Science
Veterinary College and Research Institute
Namakkal - 637 002, Tamil Nadu, India.
e-mail:karthitamil_2007@rediffmail.com

Received: 31-07-2014 Accepted: 25-08-2014

Micro encapsulation of probiotic culture Lactobacillus casei (NCDC-298) was done by extrusion and emulsion methods of
encapsulation using two different combination of wall materials viz., sodium alginate +starch and sodium alginate + whey protein
concentrate + starch with using different size of needles for introduction of coated microorganism mixture into hardening
solution. The developed microencapsulated beads were analysed for its size, shape, internal structure using Scanning electron
microscopy further the storage studies of prepared beads in two different solutions like water and calcium chloride were also
studied.

The addition of probiotic microorganisms to various foods in
order to enhance their nutritive value and potential health
benefits is currently of great interest. Modern consumers are
becoming health conscious and expect the food they eat not
only to be nutritive but also capable of preventing illness.
Considering the perceived health benefits, probiotics have been
incorporated into a range of dairy products including ice cream,
yoghurt, cheese, milk powder and frozen dairy desserts. An
effective probiotic must reside at the desired target sites
sufficiently long and at sufficient concentrations (106 - 107cfu/
ml) to elicit probiotic effects. However, due to processing
condition and also during the transit in the human digestive
system, there is a marked decline in the cells. Since the viability
and stability of probiotics pose marketing and technological
challenges, it was proposed to microencapsulate some proven
probiotics using extrusion and emulsion method to stabilize
the cells, enhance their viability and stability during production
and storage of dairy functional products.
The accurate size of microcapsules and their overall structure
was assessed by Electron microscopy however there are only
few publications regarding electron microscopy studies used
to know the micro structural description of bacteria loaded
microcapsules1-2.
The present study aimed to develop the microencapsulated
probiotic beads and analyse the size and shape of the beads
with - different wall materials and different size of needle. Further
concentration of emulsifiers used to prepare the optimum size
of the beads and stability of the beads in storage solutions will
be studied. The clear study of outer and inner structure of the
microencapsulated beads will be done by Scanning electron
microscopy (SEM).
MATERIALS AND METHODS
Propagation and maintenance of cultures: Pure freeze
dried probiotic culture were activated by inoculating into deMan
Rogosa Sharp (MRS) broth and incubated at 40oC for 24-48
hours for initial propagation. The probiotic biomass was
harvested by centrifugation at 3500rpm for 10 min at 4oC then
washed twice in sterile 0.9% saline under the same
centrifugation conditions, and used in the microencapsulation
process. Stock cultures of the probiotic bacteria were
maintained using MRS broth added with 15 per cent glycerol
and stored at -20oC.
Encapsulation methods of probiotics: The extrusion and
emulsion techniques described were followed in the present
study3.
Extrusion method: Probiotic cell suspension was prepared
by centrifuging 80 ml of 24 h old broth culture at 5000 X G for
15 minutes. The cells were washed twice with saline solution
(20ml). The wall materials used were sodium alginate (2.0%w/
 Analysis of Micro Encapsulated Probiotic Beads
v) + starch (0.5w/v) and sodium alginate (2.0%w/v) + whey
protein concentrate (1.0%w/v) + starch (0.5w/v). To form
capsules, a cell suspension was mixed with a 60ml of wall
material solution and the mixture was dripped into a solution
containing CaCl2 as the divalent cation. The droplets formed
gel spheres instantaneously, entrapping the cells in a three
dimensional lattice of ionically cross linked alginate. The
CaCl2 concentration was at 0.1M and dripping was achieved
with a sterile syringe with different size of needles (21G,
26G and insulin syringe). The distance between syringe and
CaCl2 solution was 30 cm.
Emulsion method: The probiotic bacterial cells were
harvested from 80 ml of a 24 h culture (late log phase) by
centrifugation at 5000X G for 15 minutes. The cells were
then washed twice under the same centrifugation conditions,
with 20 ml sterile saline and resuspended in 10 ml sterile
saline. The cell suspension (20ml) was then added to 60 ml
of wall material suspension, containing 0.9% NaCl to improve
the dispersability of alginate, and tempered in a water bath
at 470C. Sunflower oil 100ml was used as continuous phase
and different concentration of tween 80 (0, 0.1 and 0.5% w/
v) and sunflower oil was stirred at 400C for 2-3 minutes. The
capsules were formed by adding cell suspension/alginate
mixture to the sunflower oil/ tween 80 mixture quickly. The
emulsion was disturbed by adding 0.1M of CaCl2 solution
(150ml). The oil phase was removed, and the capsules
containing bacterial cells were separated from CaCl2 and
stored at 40C until use.
Analysis of encapsulated beads: Extrusion and emulsion
beads were observed under light microscope with oil
immersion (100X) for their size and shape. The size was
measured by using stage micrometer, for each sample 100
beads were measured the average bead size was recorded.
0.1M CaCl2 solution and water used for storage of beads
and were stored at 370C for one day. The size of the beads
were measured by fresh as well as after one day storage in
CaCl2 and recorded.
Scanning electron microscopy (SEM) was done in
Department of Geology, Alagappa College of Technology,
Chennai to examine the structure of the beads. The beads
samples were dried and coated with platinum to make it
conductive and then mounted on an aluminum stub.
(126)
Microscopy was performed under scanning electron microscope
at an accelerated voltage of 15 Kv and 20 Kv. For examining the
inner surface of the beads, after drying and fixing of the bead, it
was fractured with razor blade on a metal stage. The sliced
pieces were collected and dried. The dried specimens were
prepared and examined by SEM as for the beads.
RESULTS AND DISCUSSION
Size and shape of the beads in extrusion method of
encapsulation by using different wall materials and different size
of the needles used are shown in table-1. The beads obtained
by the extrusion method with different wall materials were regular
and spherical in shape. The respective mean ± SE values of
bead size (mm) by using sodium alginate + starch and sodium
alginate + whey protein concentrate + starch with different needle
size were 21G: 3.31 ± 0.03, 3.27 ± 0.04; 26G: 2.13 ± 0.03, 2.09
± 0.08 and Insulin syringe: 0.79 ± 0.08, 1.19 ± 0.01.
The range of extrusion bead size varied from 0.79 mm to
3.31mm. There was no significant difference observed among
the wall materials utilized. These results were in accordance
with the findings who observed a mean value of the bead size of
4.0±0.3mm, when alginate and gelatin were used as wall
materials and other workers opined that the bead size ranged
from 0.5-1.0 mm diameter when 0.6 mm syringe was used for
dripping in extrusion method4-5.
The effect of wall materials and percentage of emulsifier (Tween
80) used in emulsion method of encapsulation on the size of the
beads are shown in Table-2. The respective mean ± SE values
of bead size (µm) by using sodium alginate + starch and sodium
alginate + starch + whey protein concentrate of different
percentage of emulsifier are as follows; 0% : 205 ± 1.2, 247 ±
1.8; 0.1% : 81 ± 1.2, 102 ± 2.2; 0.5%: 53 ± 1.1, 57 ± 1.3. There
was no significant difference observed among different wall
materials.
The size of emulsion beads obtained in this study ranged from
53 µm to 247 µm with 0, 0.1 and 0.5 per cent of emulsifier used.
When the emulsifier percentage was maximum, the size of the
bead was minimum. Tween 80 was selected as an emulsifier.
Tween 80 was essential to prevent spheres from coalescing
before the breaking of the emulsion. The structure of the beads
was irregular, spherical, rough outer surface under electron
microscope.
These findings concurred with the findings of researchers who
(127) Karthikeyan, Kumaresan and A

Table-1. Size of encapsulated beads (mm) with different wall materials (Extrusion method)

[Different superscripts in same row differ significantly at P>0.05]

Table-2. Size of encapsulated beads (µm) with different wall materials using Tween 80 (Emulsion method)

[Different superscripts in same row differ significantly at P>0.05]

Table-3. Effect of rate of addition of CaCl2 on the formation of emulsion beads

Table-4. Effect of storage of encapsulated beads in CaCl2 and water (on size of beads)
 Analysis of Micro Encapsulated Probiotic Beads (128)

reported that the size of emulsion beads were 20-100µm and beads could be stored in water up to 24 hrs without any
100µm - 1mm diameter, when alginate + starch and K- appreciable reduction in size7-8.
carrageenan + gellan were used as wall materials In this study, the size of beads were increased with increase
respectively6.Further the findings of this study were also in the size of needle used and there was no significant difference
supported by other researchers who found that incorporation in the bead size of extrusion method with different wall materials
of emulsifier at the rate of 0.5% yielded better bead size in used. But, there was a highly significant difference observed
emulsion method, which is the preferred bead size for addition between different size of needle used with regard to the size of
in dairy products7. beads. Beads prepared by using insulin syringe was
The effect of speed of addition of hardening solution (CaCl2) comparatively smaller. It is revealed from the present study
on the shape of emulsion beads is presented in Table-3.The that the shape of beads formed was influenced by the
speed of addition of hardening solution at the level of more percentage of emulsifier added and speed of addition of
than 20 ml/sec produced uniform drop shaped beads. If the hardening solution. The present study showed that there was
speed of addition of hardening solution is less than 10ml/sec, n o c h a n g e i n sand water when they were
i z e o f b e a d s i n C a C l

2
the beads were of string type. If the hardening solution is added fresh. But after 24 hrs, the bead size got reduced in CaCl2,
at a speed of more than 100 ml / sec, the emulsion got clumped when compared with the size of beads stored in water. Hence,
instead of giving beads. the emulsion beads could be stored in water up to 24 hrs
Storage studies of encapsulated beads in CaCl2 and water on without any appreciable reduction in size.
the size during 24 h is shown in Table-4. The respective mean
± SE value of fresh beads in CaCl2 and water were, extrusion REFERENCES
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