LWT - Food Science and Technology 60 (2015) 773e780

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Microencapsulation of Lactobacillus plantarum (MTCC 5422) with

fructooligosaccharide as wall material by spray drying
R. Rajam, C. Anandharamakrishnan*
Department of Food Engineering, CSIR-Central Food Technological Research Institute, Mysore, 570 020, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 14 May 2014
Received in revised form
24 September 2014
Accepted 28 September 2014
Available online 7 October 2014

Microencapsulation is a promising technique for delivery of live microbial supplements through foods.
Prebiotics like fructooligosaccharide (FOS) can be used as a wall material to produce synbiotics. However, the low glass transition temperature (Tg) of FOS causes stickiness when used as wall material during
spray drying. This problem was alleviated by FOS in combination with whey protein isolate (WPI) or
denatured whey protein isolate (DWPI) for encapsulating the probiotic bacteria Lactobacillus plantarum
(MTCC 5422). Microencapsulation was performed with wall materials FOS, FOS WPI, FOS DWPI at
two different core-to-wall ratios of 1:1 and 1:1.5. FOS WPI and FOS DWPI microcapsules of 1:1 coreto-wall ratio exhibited higher encapsulation efciency, lower residual moisture content and narrow
range of particle size distribution than 1:1.5. However, microcapsules of 1:1.5 core-to-wall ratios
enhanced the storage stability and tolerance of probiotic cells in the simulated gastric and intestinal
conditions. FTIR analysis conrmed the presence of whey proteins and FOS in the microcapsules after
spray drying. On overall comparison, FOS DWPI microcapsules of 1:1 core-to-wall ratio had higher
encapsulation efciency (98.63%) but 1:1.5 ratio exhibited better storage stability and protection in
simulated gastric, as well as intestinal condition than the other encapsulates.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Microencapsulation
Spray drying
Probiotics
Prebiotics
Core-to-wall ratio

1. Introduction
Probiotics are live microorganisms which when administered in
adequate quantity confer health benet on the host. In recent decades, the strains of Lactobacillus plantarum have been identied in
many fermented foods and reported for their probiotic potential
and application into various types of functional food products (Lee,
Kim, Han, Eom, & Paik, 2014). The viability and dose levels are
important parameters for probiotic efcacy. Additionally, the
presence of prebiotics, i.e., various oligosaccharides has the potential to improve the probiotics viability and its survival in the
gastrointestinal tract (Burgain, Gaiani, Linder, & Scher, 2011). Prebiotics are resistant to gastric acidity and digestion by small intestinal enzymes and also supply a fermentable carbohydrate for
probiotic bacteria in the colon (Gibson, 1999). Hence both probiotics and prebiotics have the intestine as functional target, and
their combination may reinforce each other's effect (Ouwehand,
Tiihonen, M
akivuokko, & Rautonen, 2007). Thus, the synergistic
combination of probiotics and prebiotics, termed as synbiotics,

* Corresponding author. Tel.: 91 821 2513910; fax: 91 821 2517233.

E-mail addresses: anandhram@cftri.res.in, c.anandharamakrishnan@gmail.com
(C. Anandharamakrishnan).
http://dx.doi.org/10.1016/j.lwt.2014.09.062
0023-6438/ 2014 Elsevier Ltd. All rights reserved.

that enhances the viable counts of lactobacilli and bidobacteria

compared to either probiotic or prebiotic alone (Gibson, 1999;
Gonzalez, Adhikari, & Sancho-Madriz, 2011; Rodrigues et al., 2011).
Inulin type fructons such as inulin, fructooligosaccharides and
galactooligosaccharides exhibit prebiotic effect (Gibson, 1999).
Prebiotics are often incorporated to maintain the viability of probiotic cultures in foods. However, probiotic bacteria need to be
encapsulated to protect the cells from adverse environment and
also to avoid negative sensory impact on incorporation into foods
(Burgain et al., 2011). Spray drying is the most commonly used
encapsulation method in the food industry. It involves atomization
of feed solution into the hot air drying chamber, wherein evaporation of water takes place from the atomized droplets to form dry
powder (Anandharamakrishnan, Rielly, & Stapley, 2007). The advantages of spray drying are the production of owable powders,
ability to control particle size, rapid drying, easy scale up and
continuous production. Moreover, the dried encapsulated probiotic
bacteria
reduce
the
storage
and transportation
cost
(Santivarangkna, Kulozik, & Foerst, 2008). Carbohydrates such as
starch and maltodextrin are used for microencapsulation by spray
drying, however, only few studies have been reported the use of
fructooligosaccharide (FOS) as wall material. Stickiness behaviour
limits the application of FOS, which is mainly due to its low glass

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R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

transition temperature. Stickiness during spray drying can be

minimised by altering the glass transition temperature with the
introduction of high molecular weight agents (Adhikari, Howes,
Wood, & Bhandari, 2009). Studies have shown that the addition
of whey proteins overcome the stickiness of sugar rich solutions
due to their surface-active and lm forming properties (Adhikari,
Howes, Bhandari, & Langrish, 2009). Whey protein is an excellent
encapsulating material because of their emulsication, gelation and
lm forming properties (Perez-gago & Krochta, 2001). Denaturing
the whey protein ensures higher tensile property and lower oxygen
permeability which protects the probiotic cell from adverse
gastrointestinal conditions (Perez-gago & Krochta, 2001; Rajam,
Karthik, Parthasarathi, Joseph, & Anandharamakrishnan, 2012).
Besides the stability and viability of probiotics during spray drying
and subsequent storage, the core release characteristics are inuenced to a great extent by encapsulating wall matrix and core-towall ratio. Therefore to attain desired physical and functional
properties of microcapsules, core-to-wall ratio needs to be
considered (Lee & Rosenberg, 2000).
Hence, this study aimed to use fructooligosaccharide (FOS), FOS
in combination with whey protein isolate (WPI) or denatured whey
protein isolate (DWPI) as wall materials for microencapsulation of
L. plantarum at 1:1 and 1:1.5 core-to-wall ratios. The effect of wall
material combination and core-to-wall ratio on particle size and
morphology of microcapsules, encapsulation efciency, stability
and viability of cells during storage and in simulated gastric and
intestinal conditions were evaluated.

harvested L. plantarum cell concentrate (25 g) was mixed with each

wall material solutions to obtain a desired core-to-wall ratio of 1:1
and 1:1.5 (Table 1). All the feed solutions were homogenised using
high speed homogenizer (IKA-Ultra-Turrax T18basic, Germany) for
60 s at 7000 rpm before spray drying operation. All the glass wares
used in this experiment were sterilized at 121  C for 15 min.
2.2. Spray drying
Microencapsulation of probiotics was performed in a
laboratory-scale spray dryer (Jay Instruments & Systems Pvt Ltd,
Mumbai, India) equipped with twin uid nozzle atomization system (0.5 mm diameter nozzle), at constant air inlet temperature of
110 2  C and outlet temperature of 55 3  C. The feed solution
was kept under magnetic agitation at room temperature and was
fed into the spray chamber through a peristaltic pump, with feed
ow rate of 4 mL min1. The drying air ow rate and air pressure
were maintained as 25 m3 h1 and 0.196 MPa respectively. After
spray drying, dried microcapsules were collected from the bottom
of the cyclone, packed in polyethylene bags, sealed in aluminium
foil and stored at 4  C till the analysis.
2.3. Moisture content
The moisture content (% wet basis) of the spray dried microcapsules were determined by oven drying at 102 2  C until
reaching constant weight, according to the International Dairy
Federation Bulletin (IDF, 1993).

2. Materials and methods

2.4. Morphology
2.1. Preparation of wall materials and cell mixture
The probiotic strain, L. plantarum (MTCC 5422), isolated from
fermented cereals/legumes in our research institute (Roopashri &
Varadaraj, 2009), and kept as a certied stock at Microbial Type
Culture Collection and Gene Bank (MTCC) (Chandigarh, India) was
used as core material. The species L. plantarum was grown in sterile
MRS broth (HiMedia Laboratories Pvt Ltd, Mumbai, India) and the
cells were harvested in the early stationary phase as described in
our previous study (Rajam et al., 2012). The cell concentrate was
used on the same day for microencapsulation. High-purity fructooligosaccharide (FOS), (96.2% FOS and 3.8% Glucose Fructose
Sucrose) was purchased from Meiji Food Materia Co., Ltd, (Japan).
Whey protein isolate (WPI) powder (BN X-TRA Whey, containing
83.33% of pure WPI, 13.33% carbohydrates, 1.66% fat and 1.66% other
micronutrients) was obtained from British Nutritions, (Bangalore,
India). Denatured whey protein isolate (DWPI) solution was prepared by heating the WPI solution (10% w/v) at 90  C for 30 min in a
water bath. All the concentrations and compositions of FOS and
WPI mentioned were obtained from respective manufacturers.
Sterile Milli-Q water was used to prepare wall material solutions as
described in our previous study (Rajam et al., 2012). Freshly

The morphological examination of microcapsules was performed by Scanning Electron Microscope (Leo 435 VP, Leo Electronic
Systems, Cambridge, UK), where its images were systematically
observed at 15 kV under a vacuum of 9.75  105 Torr.
2.5. Particle size distribution and bulk density
The average particle size and size distribution of the microcapsules were determined by laser diffraction particle analyser
(S3500, Microtrac Inc., USA) using methanol as dispersing medium.
The volume distributions of samples were calculated, and the results were expressed as volume weighted mean particle size (D[4,3]).
The particle size distribution of the sample was represented by
span factor, and it is dened as (Tonon, Grosso, & Hubinger, 2011):

Span

dv;90  dv;10
dv;50

(1)

where, d[v,10], d[v,50], d[v,90] correspond to the diameters at which the

cumulative sample volumes were under 10%, 50% and 90%
respectively.

Table 1
Composition of feed solutions.
Wall material
formulation

Wall materials
ratio (w/w)

Core-to-wall
ratio

FOS (g)

WPI (g)

DWPI (g)

L. plantarum cell concentrate,

(wet weight, g)

Feed solution
concentration (% w/w)

FOS

e
e
1:1
1:1
1:1
1:1

1:1
1:1.5
1:1
1:1.5
1:1
1:1.5

25.00
37.50
12.50
18.75
12.50
18.75

e
e
12.50
18.75
e
e

e
e
e
e
12.50
18.75

25
25
25
25
25
25

20
20
20
20
20
20

FOS WPI
FOS DWPI

FOS e Fructooligosaccharide; WPI e Whey protein isolate; DWPI e Denatured whey protein isolate.

R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

The bulk density was determined by adding 2 g of sample into

an empty 10 mL measuring cylinder. The cylinder was tapped by
holding it on a vortex vibrator for 2 min. The bulk density was
calculated using the equation as follows (Goula & Adamopoulos,
2012):

Bulk density; rb

m
v

(2)

where rb is the bulk density (kg/m3), m is the mass of the sample

(kg), and v is the volume occupied in the cylinder (m3).
2.6. Fourier transform infrared (FTIR) measurement
The FTIR spectra of microcapsules with/without probiotic cells
were obtained with a Fourier transform infrared spectrometer (IFS
25, Bruker, Germany) using the KBr (potassium bromide) disk
method. The sample was mixed with KBr and then pressed into a
pellet. The spectra of the samples were obtained in transmission
mode from 400 to 4000 cm1 wave number range.

To determine the cell viability, the cells were completely

released from microcapsules as described in our previous study
(Rajam et al., 2012). After appropriate serial dilutions in 0.85% saline (extra pure sodium chloride, HiMedia Laboratories Pvt Ltd,
Mumbai, India) solution, the samples (1 mL) were pour plated on
MRS agar (HiMedia Laboratories Pvt Ltd, Mumbai, India) plates and
incubated at 37  C for 24 h. All enumerations were performed in
duplicate and the plates containing 20e350 colonies were counted
and expressed as CFU per g of dried microcapsules or per mL of
solution. The log cell number of viable cells released from the microcapsules before (N0) and after (N) drying was evaluated and
expressed as Encapsulation Efciency (EE)

N
 100
N0

(3)

2.8. Survival of microencapsulated L. plantarum during storage

The viability of microencapsulated L. plantarum MTCC 5422
during storage for 60 days at 4  C was determined by enumeration
on MRS agar, as described in Section 2.7. To evaluate the encapsulated cell viability during storage, the plot of the logarithmic value
of the relative cell viability (log Nt/Ni) versus storage time (t, day)
was tted to a rst order reaction kinetics model as described by
the Eq. (4).

log

Nt
kT t
Ni

SGF, MRS broth solution pH was adjusted to 2.0 using 1 M HCl and
sterilized by autoclaving at 121  C for 15 min. Pepsin (1:3000 m/g,
extra pure; SD ne chemicals, Boisar, India) was ltered through
0.22 mm sterile membrane lter, then suspended in sterile MRS
broth to a nal concentration of 0.3% (v/v). To prepare SIF, bile salt
(HiMedia Laboratories Pvt Ltd, Mumbai, India) was dissolved in
MRS broth solution to a concentration of 2% (v/v) and pH was
adjusted to 7.5 using sodium bicarbonate (1 N), then sterilized by
autoclaving. Spray dried microcapsules (0.5 g) were suspended in
9.5 mL of sterile SGF/SIF and incubated at 37  C with constant
agitation at 100 rpm. Aliquots of 1000 mL were taken at 0, 1, 2, 3 and
4 h and evaluated for the cell viability. The survival of encapsulated
L. plantarum released in SGF/SIF was expressed in terms of relative
viability (a ratio of viable cells at time t to that at time zero h).
2.10. Statistical analysis
Student's t-test was used to compare groups, and the level of
statistical signicance was set at p < 0.05.
3. Results and discussion

2.7. Enumeration of bacteria and encapsulation efciency

Encapsulation Efficiency

775

(4)

where Nt is the number of viable bacteria at a particular storage

period (in CFU/g), Ni represent the number of viable cells at the
beginning of storage (in CFU/g) and t is the storage time (in days). kT
is the specic rate of viability loss at 4  C per day.
2.9. Survival of encapsulated bacteria in simulated gastric and
intestinal conditions
Simulated gastric uid (SGF) and simulated intestinal uid (SIF)
were prepared and survival of encapsulated cells in SGF/SIF was
determined by the procedure described in the previous studies
with slight modications (Dolly, Anishaparvin, Joseph, &
Anandharamakrishnan, 2011; Kim et al., 2008; Rajam et al., 2012;
Sohail, Turner, Coombes, Bostrom, & Bhandari, 2011). To prepare

3.1. Moisture content

Moisture content of the spray dried microcapsules varied from
5.52 to 9.47% (Table 2) and it was signicantly inuenced by inlet
and outlet drying air temperature, droplet formation mechanism,
composition and concentration of feed solution (Anandharama
krishnan et al., 2007). As the outlet air temperature in the present study was 55  C, microcapsules retained slightly higher
moisture content. Moreover, the result shows that the partial
replacement of FOS with WPI or DWPI exhibited signicant
(p < 0.05) reduction in the moisture content than the cells encapsulated with FOS alone. This may be due to the migration of proteins to water/air interface, and rapid formation of lm around the
particle surface that favours faster rate of moisture removal
(Adhikari, Howes, Bhandari, et al., 2009). All the microcapsules of
1:1.5 core-to-wall ratio exhibited higher moisture content than 1:1
ratio. This is due to the increased wall material concentration that
facilitates strong crust formation which in turn restricts the diffusion of water vapour from the interior of droplet to the surface
during spray drying (Anandharamakrishnan et al., 2007). The results obtained are in agreement with our previous study on WPI
encapsulated L. plantarum (Dolly et al., 2011).
3.2. Morphology of spray dried microcapsules
Morphology directly inuences the bulk density, owability and
rehydration characteristics of encapsulated powders (Walton,
2000). Fig. 1 shows the SEM micrographs of the microcapsules
produced with three different combinations of wall materials in 1:1
and 1:1.5 core-to-wall ratios. Micrographs revealed that there were
no visible fractures or cracks on the surface of the microcapsule
indicating the less fragility of wall system. This helps to with stand
the mechanical forces associated with expansion and ballooning
during spray drying (Jafari, Assadpoor, Bhandari, & He, 2008).
Moreover it can reduce the air permeability that provides better
protection of probiotic microorganisms. FOS encapsulated microcapsules were spherical in shape with more aggregation than
FOS WPI and FOS DWPI encapsulates. This aggregation of
particles is due to stickiness caused by low glass transition temperature of FOS (Adhikari, Howes, Wood, et al., 2009). Thus, the
addition of high molecular weight whey proteins caused a signicant increase in the Tg that resulted in the absence of aggregation in
FOS WPI and FOS DWPI encapsulates. Moreover, migration of

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R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

Table 2
Effect of wall material and core-to-wall ratio on nal moisture content, particle size, bulk density of spray dried microcapsules and viability of L. plantarum cell before and after
drying.
Wall material

FOS
FOS WPI
FOS DWPI

Core-to-wall ratio

1:1
1:1.5
1:1
1:1.5
1:1
1:1.5

Final moisture content, % (w.b.)

7.62
9.47
5.52
6.83
6.21
7.43

0.85
0.57
0.34
0.26
0.27
0.29

Particle size analysis

Span

D[4,3] (mm)

1.63
2.11
1.89
2.65
1.76
2.15

15.44
23.89
7.34
8.97
6.68
13.62

Bulk density kg/m3

Cell viability (Log10 CFU/mL)

Before drying

564.72
512.87
434.29
405.11
473.56
429.21

3.64
3.18
3.83
2.75
4.23
3.69

10.4
10.3
10.0
9.9
10.0
9.9

0.1
0.1
0.1
0.1
0.1
0.1

After drying
7.3
7.6
9.6
8.7
9.8
9.1

0.3
0.4
0.1
0.2
0.0
0.0

FOS e Fructooligosaccharide; WPI e Whey protein isolate; DWPI e Denatured whey protein isolate.

whey proteins to the droplet surface and their rapid skin forming
properties overcome the particle-to-particle stickiness during
spray drying (Adhikari, Howes, Bhandari, et al., 2009). The surface
irregularities such as concavities/dents on the surface of FOS WPI,
and wrinkles on FOS DWPI encapsulates may be associated with
the low drying temperature as well as combined effect of atomization mechanism and different lm forming properties of wall
materials used for encapsulation (Rajam et al., 2012). However,
increase in the core-to-wall ratio did not inuence the morphology
of microcapsules.
3.3. Particle size characterization and bulk density
Particle size and its distribution are important physical properties that directly affect the use of microcapsules into food formulations. Microcapsules exhibited a wide range of sizes varying
from 6.68 mm to 23.89 mm diameter (Table 2). The microcapsules of
1:1.5 core-to-wall ratio had larger particle size than 1:1, due to an
increase in wall material concentration in the feed (Kurozawa, Park,
& Hubinger, 2009). Moreover, there was a signicant variation in
mean diameter D[4,3] of the particles between the samples with
different wall materials for the same core-to-wall ratio. All the feed
solutions were spray dried under same conditions, therefore the
variations in the particle size distribution between the samples of
same core-to-wall ratio might be due to the different lm forming
and gelling properties of the wall materials used for encapsulation.
The microcapsules obtained with 1:1.5 core-to-wall ratio exhibited
higher span value indicating wider range of particle size distribution than 1:1 ratio (Table 2.).
Knowledge of the bulk density is important during processing,
storage, and packaging of encapsulated microcapsules. According
to Walton (2000) the parameters such as moisture content, particle
size distribution and morphology can affect the bulk density of
spray dried powders. Results showed signicant variation in the
bulk density, according to the composition of wall materials and
core-to-wall ratio used for encapsulation (Table 2). Bulk density of
spray dried microcapsules ranged from 405.11 to 564.72 kg/m3
(Table 2). FOS encapsulates exhibited higher bulk density due to
particle aggregation and less interspace between particles as seen
in Fig. 1 a & b (Goula & Adamopoulos, 2012). Moreover, FOS WPI
microcapsules had lower bulk density than FOS DWPI due to skin
forming nature of whey protein isolate that caused voids inside
particles (Walton, 2000). In addition, microcapsules of 1:1.5 coreto-wall ratio had lower bulk density than 1:1 for all samples
(p < 0.05) due to larger particle sizes of samples (Kurozawa et al.,
2009).
3.4. FTIR analysis
The FTIR spectra of microcapsules with/without probiotics are
shown in Fig. 2. The FTIR spectrum of FOS exhibited distinct bands

in three spectral regions such as 1200e900 cm1, 3000e2700 cm1

and 900e600 cm1. The bands observed in the spectral region
between 1200 and 900 cm1 corresponds to CeC, CeO stretching
and CeOeH, CeOeC deformation modes of oligosaccharides
(Grube, Bekers, Upite, & Kaminska, 2002). The spectrum of WPI
shows two major peaks attributed to the amide I and amide II of
protein structure. From a molecular point of view, the peaks seen at
1700e1600 cm1 attributed to the C]O stretching vibrations of
amide I band and the peaks at 1550e1480 cm1 due to CeN
stretching and NeH bending vibrations of amide II band (Bagheri,
Madadlou, Yarmand, & Mousavi, 2013). Denaturation of whey
protein showed no marked difference for the band in the region
between 1600 cm1 and 1700 cm1. In all the samples, the
appearance of the band at 2800e3000 cm1 is due to CeH
stretching and a broad band at ~3300 cm1 is due to OeH
stretching (Bagheri et al., 2013). FTIR analyses showed that the
whey proteins and FOS were retained as such in the microcapsules
after spray drying.
3.5. Encapsulation efciency
The viability of L. plantarum encapsulated with three different
wall materials for core-to-wall ratio of 1:1 and 1:1.5 before and after
drying given in Table 2. During spray drying, the outlet temperature
has more dominant effect on the particle drying than inlet air
temperature (Anandharamakrishnan, Rielly, & Stapley, 2008;
Oldeld, Taylor, & Singh, 2005). Upon atomisation, the droplets
contact hot inlet air and results in water evaporation that quickly
cools droplets to wet bulb temperature of around 40e50  C (Oldeld
et al., 2005). However, as the particle moves near the bottom of the
drying chamber, the particle temperature increases away from their
wet bulb temperature and approaches the outlet air temperature
(Anandharamakrishnan et al., 2008). The higher outlet temperature
and rapid drying inuences the cell survival after drying as well as
during storage (Dolly et al., 2011; Rajam et al. 2012). Hence in order
to retain the maximum cell viability, lower outlet temperature
(55  C) has been maintained in this study. Cell viability after the
drying process is expressed in terms of encapsulation efciency
(Fig. 3). Results showed that FOS microcapsules of both core-to-wall
ratios had almost similar encapsulation efciency (70.77e72.82%).
However, the combination of FOS with WPI or DWPI shows significant increase (p < 0.05) in the encapsulation efciency. This is due
to the presence of whey proteins and FOS in the wall matrix which
provides dual advantages such as formation of protective coating on
bacterial cell wall and partial replacement of sites of water molecules in cells during drying respectively. This helps in prevention of
cell membrane disruption during spray drying process which in turn
increases the encapsulation efciency (Adhikari, Howes, Bhandari,
et al., 2009; Schwab, Vogel, & Ganzle, 2007). On comparison,
FOS DWPI encapsulates exhibited signicantly (p < 0.05) higher
encapsulation efciency than FOS WPI for both core-to-wall

R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

777

Fig. 1. SEM micrographs of FOS (a & b) encapsulates, showing particle aggregation; FOS WPI (c & d) encapsultes showing spherical particles with concavities/surface dents;
FOS DWPI (e & f) encapsulates showing spherical particles with wrinkled surface; unencapsulated cells (g). A-particle aggregation, D-dents, W- wrinkles.

ratios, which indicates the effective protection of DWPI due to the

formation of stronger gel network as reported by Parthasarathi,
Ezhilarasi, Jena, and Anandharamakrishnan (2013). In particular,
FOS DWPI encapsulates of 1:1 exhibited higher encapsulation
efciency than 1:1.5 core-to-wall ratio.

3.6. Survival of microencapsulated L. plantarum during storage

The viability loss during storage is mainly due to membrane
lipid oxidation; thus storage temperature and moisture content are
key factors that affect the cell viability (Santivarangkna et al., 2008).

778

R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

Fig. 2. FTIR spectra of cells, wall materials and probiotic microcapsules.

The effect of wall materials on storage stability of encapsulated

L. plantarum stored at 4  C is shown in Fig. 4. The rate of viability
loss of encapsulated cells followed rst-order kinetics (R2
0.962e0.991) during storage. Cells encapsulated with prebiotic
(FOS) alone showed a higher rate of viability loss than combination
of FOS with WPI or DWPI over 60 days of storage. Higher moisture
retaining capacity of FOS prevents excessive drying, that deteriorate the cell survival of FOS encapsulates due to higher residual
moisture content (Table 2), (Crittenden & Playne, 1996). Hoobin
et al. (2013) suggests that moisture content and molecular
mobility of the matrix composition have a considerable impact on
survival of probiotics during storage. FOS encapsulates exhibited no
signicant difference in the viability loss for both core-to-wall ratios during storage (Fig. 4A). However, increase in the core-to-wall
ratio (1:1.5) enhanced the storage stability of FOS WPI, and
FOS DWPI microcapsules as shown by a lower rate of viability
loss. Comparatively, FOS DWPI favours better stability of encapsulated probiotic cells during storage than FOS WPI in both the

core-to-wall ratios (1:1 and 1:1.5) due to low water vapour and
oxygen permeability of DWPI (Perez-gago & Krochta, 2001).
3.7. Survival of encapsulated probiotic cells in simulated gastric
condition
The effect of gastric conditions on the relative viability of
microencapsulated L. plantarum is shown in Fig. 5A. The results
revealed that different wall matrix composition provided signicantly (p < 0.05) different degree of protection to the encapsulated
L. plantarum cells. High loss in relative viability was observed for
FOS encapsulated cells, indicating the poor protection of FOS to the
cells on exposure to gastric medium. This may be due to water
soluble nature of FOS (Crittenden & Playne, 1996) encapsulates that
completely released the cells when suspended in gastric medium.
Contradictorily, combination of FOS WPI and FOS DWPI
exhibited slight reduction in the relative cell viability after 1 h incubation in gastric medium, after which a stable viability was
observed. This may be due to formation of a protective coating on
the surface of the particles by whey proteins (Adhikari, Howes,
Bhandari, et al., 2009). Moreover, FOS DWPI microcapsules
exhibited less reduction in relative viability (Crittenden & Playne,
1996) than FOS WPI. Exposure of hydrophobic and sulfhydryl
groups due to denaturation may decrease the rate of swelling
which in turn reduced the diffusion of cells from FOS DWPI microcapsules than FOS WPI (Perez-gago & Krochta, 2001). Moreover, FOS WPI or FOS DWPI encapsulates of 1:1.5 core-to-wall
ratio had improved relative cell viability than 1:1 due to increased
wall material concentration and larger particle size (Lee &
Rosenberg, 2000). These results suggest that FOS DWPI provided better protection to L. plantarum in the simulated gastric
conditions.
3.8. Survival of encapsulated probiotic cells in simulated intestinal
condition

Fig. 3. Encapsulation efciency of microencapsulated L. plantarum with different wall

material and core-to-wall ratio of 1:1 and 1:1.5. Error bars represent standard deviation of means (n 3).

The survival of microencapsulated L. plantarum in simulated

intestinal uid (SIF) containing 2% bile salt at 37  C over 4 h incubation period was studied and the results are shown in Fig. 5B. The

R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

779

Fig. 4. Viability loss of spray dried L. plantarum expressed as a function of storage time, (day), at a storage temperature of 4  C. Linear regression analysis was used to obtain the rate
constant, k.

results revealed a similar trend that was found in simulated gastric

conditions. FOS encapsulates exhibited stable reduction in the
relative viability irrespective of the core-to-wall ratio. The relative
viability of FOS DWPI microcapsules for both core-to-wall ratios
was signicantly higher during 4 h of incubation in SIF. Moreover,
microcapsules of 1:1.5 core-to-wall ratios were less susceptible to
viability loss in bile condition compared to 1:1. This may be due to
the increased wall material concentration which offered better
protection to cells.
This study indicates that FOS DWPI encapsulated microcapsules of both core-to-wall ratios can provide better tolerance to
probiotic cells in both gastric and intestinal conditions. Moreover,
variations in the viability of L. plantarum in simulated gastric

medium and simulated intestinal medium may be due to the direct

consequence of the methodology applied. Besides, it would be
interesting to investigate the survival of the encapsulated probiotic
using a dynamic model of the gastrointestinal tract and bacterial
ltration method for the evaluation of cell viability in simulated
gastrointestinal conditions would be worth to explore in our future
studies.
4. Conclusions
Low molecular weight and highly hygroscopic nature restrict
the use of fructooligosaccharide (FOS) as encapsulating matrix due
to difculty in converting into dry powder form through spray

Fig. 5. (A) Viability of L. plantarum after exposure to simulated gastric juice at pH 2.0 for 4 h at 37  C. (B) Viability of L. plantarum after exposure to simulated intestinal juice for 4 h
at 37  C. Error bars represent standard deviation of means (n 3).

780

R. Rajam, C. Anandharamakrishnan / LWT - Food Science and Technology 60 (2015) 773e780

drying. However, combination of FOS with WPI or DWPI reduced

the particle stickiness and aggregation. Increase in core-to-wall
ratio (1:1.5) did not inuence the morphology of microcapsules
but resulted in high residual moisture content, large particle size,
wide range of particle size distribution and low bulk density
regardless of wall material combinations. On overall comparison,
FOS DWPI microcapsules of 1:1 core-to-wall ratio had higher
encapsulation efciency and 1:1.5 ratio yielded better storage stability and protection in simulated gastric as well as intestinal
condition than the other encapsulates. Hence, microencapsulation
of L. plantarum with FOS and DWPI as wall material was found to be
most effective in retaining the viability of bacteria after drying,
during storage and in simulated gastric and intestinal conditions.
Therefore, production of synbiotic microcapsules with the combination of FOS and DWPI wall matrix by spray drying method has
potential applications in the functional food industry.
Acknowledgements
The authors wish to thank Prof. Ram Rajasekharan, Director,
CSIR-CFTRI for the support and encouragement. We gratefully
acknowledge the Department of Science and Technology (SR/WOSA/ET-50/2010), Government of India for the nancial support and
Women Scientist Fellowship (WOS-A) to Rajam, which enabled this
work to be carried out. Authors wish to thank Dr. M.C. Varadaraj,
Chief Scientist, CFTRI, and Mr. K. Anbalagan, CIFS, CFTRI for their
help.
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