Food Structure 25 (2020) 100147

Contents lists available at ScienceDirect

Food Structure
journal homepage: www.elsevier.com/locate/foostr

Microencapsulation of probiotics in multi-polysaccharide microcapsules by T

electro-hydrodynamic atomization and incorporation into ice-cream
formulation
Davood Zaeima,c,*, Mahboobe Sarabi-Jamaba,*, Behrouz Ghoranib, Rassoul Kadkhodaeeb,
Weilin Liuc, R. Hans Trompd
a
Department of Food Biotechnology, Research Institute of Food Science & Technology, Mashhad, Iran
b
Department of Food Nanotechnology, Research Institute of Food Science & Technology, Mashhad, Iran
c
School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, 310018, China
d
NIZO Food Research, Flavor & Texture Department, Kernhemseweg 2, 6718 ZB, Ede, the Netherlands

A R T I C LE I N FO A B S T R A C T

Keywords: Ca-alginate/chitosan microcapsules were made by electro-hydrodynamic processing. Two different micro-
Alginate structures were formed through the application of single- or double-stage procedures. Inulin or resistant starch,
Chitosan as prebiotic components, and Lactobacillus plantarum, as a probiotic strain, were incorporated into the micro-
Inulin capsules and viability of bacteria was monitored during the storage for 90 days. Microcapsules containing
Resistant starch
probiotic-prebiotic blends were employed as an ingredient in the formulation of ice-cream. The bacterial survival
Electrospray
was also studied after the production and storage of the ice-cream. FTIR spectra confirmed the formation of
polyelectrolyte complexes between alginate and chitosan. SEM images revealed that a chitosan layer thoroughly
coated the Ca-alginate microcapsules formed through the double-stage procedure. After incorporation of either
inulin or resistant starch into the microcapsules, the signal of the inulin was detected at 935 cm−1, and signals of
the starch were traced at 1157 and 930 cm−1 in their FTIR spectra. Both polysaccharide matrices significantly
improved probiotic survival during the storage of microcapsules. However, inulin-containing microcapsules
showed a better performance than starch-containing ones as 7.23 ± 0.21 and 9.15 ± 0.33 log CFU g−1 of
probiotics remained viable in them after storage at 25 and 4 °C, respectively. The microcapsules also improved
the viability of probiotics after incorporating into the ice-cream as 7.37 ± 0.12 and 7.82 ± 0.39 log CFU g−1 of
bacteria were viable after 90 days in inulin- and starch-containing microcapsules, respectively. These findings
suggest that such microcapsules could be used as an ingredient for formulated functional foods.

1. Introduction capita consumption of these fermentable precursors, which ensure the

Nowadays, almost all researchers who work in the fields of medi-
cine, pharmacology, nutrition, and food are in a collective agreement
that gut microflora has undeniable effects on general health and pre-
vention of specific diseases in human (Ouwehand, Derrien, de Vos,
Tiihonen, & Rautonen, 2005; Rastall & Maitin, 2002; Wang, 2009).
Therefore, various strategies (e.g. specific diets, intermittent fasting,
application of antibiotics, bacteriocins, probiotics, and prebiotics) have
been proposed by researchers to modify the number, activity, and
variety of gut microflora into more health-beneficial species
(Ouwehand et al., 2005; Wang, 2009). Moreover, dietary fiber intake is
insufficient in the diet of most people around the world, and the per
health and balance of the gut microflora, is very low in developing
countries (Anderson et al., 2009). The application of probiotics and
prebiotics and their combination with dietary fibers have gained much
attention among food researchers to formulate and design food in-
gredients with health-promoting effects. Probiotics are natural gut mi-
croflora supplements that are administered in the form of living or-
ganisms, and prebiotics are a source of energy and growth stimulant for
such beneficial organisms (Crittenden & Playne, 2008). Maintaining the
viability and activity of probiotics is an integral part of their definition
and concept, so a variety of ideas have been put forward to keep them
alive until they reach the gut, of which microencapsulation has at-
tracted the most attention (Cook, Tzortzis, Charalampopoulos, &

⁎
Corresponding author at: School of Food Science and Biotechnology, Zhejiang Gongshang University, No. 18 Xuezheng Street, Xiasha Higher Education Park,
Hangzhou, 310018, Zhejiang, China.
E-mail address: d.zaeim@zjgsu.edu.cn (D. Zaeim).

https://doi.org/10.1016/j.foostr.2020.100147
Received 27 May 2020; Received in revised form 7 July 2020; Accepted 10 July 2020
Available online 17 July 2020
2213-3291/ © 2020 Elsevier Ltd. All rights reserved.
D. Zaeim, et al.
Khutoryanskiy, 2012).
Principally, microencapsulation is defined as a process in which a
highly sensitive component is coated by a semi-permeable membrane or
matrix (Paula et al., 2019). The process usually leads to the formation
of special particles/capsules which can release their content at con-
trolled rates under a particular condition (Nedovic, Kalusevic,
Manojlovic, Levic, & Bugarski, 2011). This is the core component that
determines the selection of a suitable carrier material and the method
being used for microencapsulation. Since probiotics are very sensitive
to organic solvents, Ca-alginate/chitosan hydrogel as a non-toxic, bio-
compatible biodegradable polymer network is prevalently used for
microencapsulation of these live cells (Zhao et al., 2015). Alginate is a
structural polysaccharide that is extracted from marine brown algae. It
consists of a homogeneous sequence of β-D-mannuronic acid (M-block),
α-L-guluronic acid (G-block), and heterogeneous sequence of both (MG-
block) (Izydorczyk, Cui, & Wang, 2005). Divalent cations (e.g. Ca2+)
could readily crosslink G-blocks in side chains through a gelation re-
action which forms a heat-stable network (Eslami, Shahedi, & Fathi,
2018). In contrast to negatively charged alginate, chitosan is a posi-
tively charged biopolymer that is obtained from alkaline de-acetylation
of chitin. Amine groups of chitosan spontaneously bind to the poly-
anionic chain of alginate through complex coacervation interaction
leading to the formation of strong polyelectrolyte complexes (PECs)
(Sankalia, Mashru, Sankalia, & Sutariya, 2007). These PECs improve
stability of Ca-alginate capsules at very low pH environments like sto-
mach, which may enhance the protective role of the microcapsules
(Bellich, Borgogna, Cok, & Cesàro, 2011; Cook, Tzortzis,
Charalampopoulos, & Khutoryanskiy, 2011). Alginate and chitosan are
not digested in the human upper GI tract and both are classified as
dietary fibers from the nutritional point of view (Draget, 2009;
Izydorczyk et al., 2005; Muzzarelli & Muzzarelli, 2009).
The method being used for microencapsulation of probiotics also
plays a significant role in their survival. Electro-hydrodynamic atomi-
zation or electrospray is a mild encapsulation process that can form
micron-sized capsules from highly-viscous solutions (Pérez-Masiá,
Lagaron, & López-Rubio, 2014; Zaeim, Sarabi-Jamab, Ghorani,
Kadkhodaee, & Tromp, 2017). In this process, a feed solution (e.g. Na-
alginate) is extruded through a capillary nozzle that is connected to an
electrode of a high voltage power supply. The solution is disintegrated
into small droplets due to high electrical repulsions. These droplets are
dispersed in the space and pulled instantly toward a collector connected
to the opposite electrode. The collector can be a solid surface like an
aluminum foil (dry-electrospray) or a gelling bath like CaCl2 solution
(wet-electrospray) (Nedovic et al., 2001; Zaeim, Sarabi-Jamab,
Ghorani, Kadkhodaee, & Tromp, 2018). High repulsive electrical forces
assist to make very fine droplets from the feed solution, which finally
gives rise to the formation of microparticles (Pérez-Masiá et al., 2014).
The experimental setup can also be horizontal or vertical, considering
the position of the capillary nozzle and the collector. Electro-hydro-
dynamic atomization has many advantages over common micro-
encapsulation methods. The size of droplets is relatively uniform and it
can be tuned simply by adjusting the feed flowrate and the voltage
between the nozzle and the collector. Since the droplets are electrically
charged, their motions such as deflecting or focusing on a particular
place can be controlled by imposing external electric fields. Charged
droplets were properly self-dispersed in space thanks to the electrical
repulsions, which prevents agglomeration and coagulation of the final
microparticles (Jaworek & Sobczyk, 2008). Moreover, no emulsion
preparation is required for microencapsulation by this method and in
contrast to spray- or freeze-drying methods, no extreme temperature is
applied during the process (Bock, Dargaville, & Woodruff, 2012). Since
the process is usually carried out at ambient conditions and no organic
solvent is used in the whole process, cell survival is higher than
common microencapsulation methods (Libran, Castro, & Lagaron,
2017).
To improve probiotics' survival under unfavorable gastric conditions
2
Food Structure 25 (2020) 100147
and support their proliferation at their target site, prebiotics are usually
incorporated into the microcapsules (Zaeim, Sarabi-Jamab, Ghorani, &
Kadkhodaee, 2019). It is worth mentioning that most colon cancers are
usually initiated in the distal colon and then develop into other parts.
Therefore, there is an urgent need to reduce the local pH by synthe-
sizing short-chain fatty acids like propionate or butyrate in this site.
High molecular weight prebiotics like long-chain inulin and resistant
starch are mostly fermented to short-chain fatty acids and reduce the
pH locally in the distal colon (Crittenden & Playne, 2008). Inulin is a
storage polysaccharide that consists of 2–60 fructose units terminated
by a glucose molecule (Kim, Faqih, & Wang, 2001). Resistant starch has
a physiological definition, as the starch and its degradation products
(i.e. dextrins and oligosaccharides) that are not absorbed in the small
intestine and arrive at the large colon, where they are mostly fermented
by beneficial microflora (Laurentin & Edwards, 2013).
The main purpose of this study is the application of a novel tech-
nique for co-spraying high-molecular-weight prebiotic polysaccharides
and live probiotic cells under ambient conditions. The potential ability
of the electrospray technique to encapsulate a high concentration of
probiotic-prebiotic blends directly into Ca-alginate/chitosan micro-
capsules, as a protective layer, was investigated. Probable interactions
occurring between wall and core materials in the complex poly-
saccharide structures were studied by FTIR. Besides, the surface mor-
phology of the microcapsules was investigated by SEM. Two different
molecular arrangements were proposed for the resultant micro-
structures, which is in agreement with our previous findings on the
effect of microcapsules` matrices on the viability of probiotics (Zaeim
et al., 2017). The viability of probiotics was monitored during the
storage at different environmental conditions. The survival of en-
capsulated strains was also monitored during the production and sto-
rage of ice-cream, as a food model.
2. Materials and methods
2.1. Materials
Na-alginate with a molecular weight of 80−120 kDa and man-
nuronic to guluronic acid ratio (M:G) of 3:2 was obtained from Sigma-
Aldrich (St. Louis, Missouri, USA). A highly pure ChitoClear® chitosan
with a molecular weight of 70−300 kDa and degree of deacetylation of
75–95 % was purchased from Primex (Siglufjordu, Iceland). Prebiotic
ingredients including Hi-maze® 260 resistant starch and Orafti® HP
inulin with a degree of polymerization ≥ 23 were provided by
Ingredion (Westchester, Illinois, USA) and Beneo (Mannheim,
Germany), respectively. Calcium chloride, tri-sodium citrate, and gla-
cial acetic acid were purchased from Merck (Darmstadt, Germany) and
used without further purification. Lactobacillus plantarum PTCC 1896™,
known as Lb. plantarum A7 isolated from the stool of breastfed infants
(Mirlohi, Soleimanian-Zad, & Sheikh-Zeinodin, 2008), was purchased
from the microbial culture collection of IROST (Iranian Research Or-
ganization for Science and Technology, Tehran, Iran) and used as a
probiotic model. MRS agar and broth were supplied by Merck (Darm-
stadt, Germany).
2.2. Fabrication of microcapsules
Two different procedures, previously developed in our lab, were
used for the fabrication of Ca-alginate/chitosan microcapsules as de-
scribed by Zaeim et al. (2017). Briefly, in the single-stage procedure,
the feed solution (50 mg g−1 Na-alginate) was electrosprayed into a
mixture of CaCl2 (0.3 mol L−1) and chitosan (2 g L−1) as the collector
solution. In the double-stage procedure, the feed solution was first
electrosprayed into a CaCl2 (0.3 mol L−1) solution; then the micro-
capsules were filtered out, washed, and immersed into a chitosan (2 g
L−1) solution to deposit the second layer on the Ca-alginate micro-
capsules. Electrospray was carried out vertically in a compartment
D. Zaeim, et al.
under the ambient conditions. The positive electrode was connected to
the feeding nozzle and the negative electrode was immersed into the
collector solution. The tip-to-collector distance was adjusted to 100
mm. Operating voltage (9.5 kV) was established by ANSTCO ES-Lab
RN/X high-voltage power supply (Tehran, Iran). All solutions were
extruded through a stainless-steel needle (Ø: 0.4 mm) supplied by B.
Braun (Melsungen, Germany) at a constant feed flow rate of 5 mL h−1
provided by New Era infusion pump (NY, USA).
2.3. Incorporation of probiotic and prebiotic
The active probiotic culture was inoculated into MRS broth and
incubated at 37 °C for 18 h. Cells were harvested by centrifugation at
6000 ×g and 4 °C for 10 min and the pellet was washed thoroughly
with sterilized distilled water (Shi et al., 2013). Prebiotic solutions were
simultaneously prepared by dispersing 5 g of either long-chain inulin or
resistant starch granules in 15 g sterilized water and vigorously agitated
for 10 min. Subsequently, probiotic cells were suspended in prebiotic
solutions and the probiotic-prebiotic blends were mixed gently with 80
g Na-alginate solution for 1 h to make homogeneous feed solutions
composed of 50 mg g-1 Na-alginate, 50 mg g−1 inulin or starch and 108
– 109 CFU g−1 live probiotic cells.
2.4. FTIR spectroscopy
Air-dried samples were ground together with KBr at a ratio of 1:10
and pressed into discs. IR transmission of each specimen was recorded
on an Infralum FT-08 spectrometer provided by Lumex Instruments®
(Mission, Canada) at 25 °C within wavenumber range of 4000 – 500
cm−1 at a resolution of 2 cm−1 (Sankalia et al., 2007). All the spectra
were normalized and examined by Essential FTIR® v3.50 software ob-
tained from Operant LLC (Burke, Virginia, USA).
2.5. Scanning electron microscopy (SEM)
Fresh hydrogel microcapsules were quickly frozen by liquid ni-
trogen and freeze-dried at -56 °C under 100 mTorr for 48 h by Operon
freeze-dryer (Gimpo, South Korea). Specimens were coated by a thin
layer of gold using Polaron E5200 sputter coater (Watford, UK) and
images were taken on Cambridge S360 scanning electron microscope
(Cambridge, UK) at an acceleration voltage of 9.5 kV and a working
distance of 38 mm (Wittaya-areekul, Kruenate, & Prahsarn, 2006).
2.6. Probiotic survival during storage
Freeze-dried microcapsules containing live probiotics and prebiotics
were maintained at 25, 4, and -18 °C for 3 months. The number of
viable cells was monthly determined by the plate count method. First,
microcapsules were fully disintegrated by vigorous agitation in 50 mM
tri-sodium citrate solution for 5 min. Then, a 1-mL sample was taken,
serially diluted, and spread on MRS agar plates. Colonies appeared after
24 h incubation at 37 °C (Chavarri et al., 2010). The feed solutions
containing all ingredients were freeze-dried and stored under the same
condition to be used as control samples.
2.7. Probiotic survival during ice-cream production and storage
The amount of each ingredient was weighed for preparing 500 g ice-
cream according to Table 1. Milk and cream were mixed and warmed to
45 ℃. Sugar, salep (flour obtained from milling tubers of some orchid
species using traditionally as a stabilizer or thickener in eastern Medi-
terranean foods), and skim milk powder were blended and gradually
mixed with the pre-warmed liquids. The mixture was stirred for 5 min
and the temperature was gently risen to 50 ℃ to obtain a homogeneous
mixture. Then it was pasteurized by heating at 80 ℃ for 20−30 s and
instantly cooling to 30 ℃ by an ice water bath. Subsequently, either free
3
Food Structure 25 (2020) 100147
Table 1
Ingredients of ice-cream formulation.
Ingredients wt.%
Sterilized milk (5% fat) 67.35
Sterilized cream (30 % fat) 9.60
Sugar 16.90
Skim milk powder 3.00
Salep 0.60
Vanilla 0.05
Rosewater 2.50
or encapsulated probiotics were dispersed in rosewater and vanilla
solution and quickly added to the rest of the ice-cream mixture. The
mixture was stirred for 20 min to ensure homogeneous distribution of
bacteria and then converted into a soft ice-cream by freezing and gently
scrubbing using a home ice-cream maker (Del Monte, Italy). The soft
ice-cream samples were dispensed into 50-ml plastic capped cups and
hardened at -18 ℃ for 24 h. The number of viable cells was counted
immediately after the ice-cream production and during the storage for 3
months as described in 2.6.
2.8. Statistical analysis
All experiments were carried out in triplicate and the data were
analyzed by a completely randomized design using SPSS v. 22 (IBM
corporation®, NY, USA) at a significance level of P ≤ 0.05. Significant
differences between the means were determined by Duncan’s multiple
range test at α = 0.05.
3. Results and discussion
3.1. Effect of encapsulation procedure on the structure of microcapsules
3.1.1. FTIR analysis
FTIR spectrum of Na-alginate and associated vibrations are shown
in Fig. 1-a. Characteristic peaks of Na-alginate at 1612, 1418, 1088, and
1032 cm−1 are respectively attributed to asymmetric and symmetric
stretching vibrations of carboxylic salt groups (COO) and stretching
vibrations of CeC and COe saccharide skeleton (Coates, 2006; Li, Dai,
Zhang, Wang, & Wei, 2008). Upon submerging Na-alginate droplets
into CaCl2 bath, Ca2+ ions readily substitute Na+ in G-blocks of algi-
nate and charge density, atomic weight and radius of cation were
modified. These changes lead to the formation of a new environment
around carbonyl groups. The band attributed to asymmetric stretching
(1612 cm-1) became broader but the band attributed to symmetric
stretching was shifted from 1418 to 1425 cm-1 and its intensity reduced
(Fig. 1-b). Sartori, Finch, Ralph, and Gilding (1997) observed the same
results and attributed them to the formation of crosslinks between al-
ginate molecules. According to Lawrie et al. (2007), a decrease in se-
paration between asymmetric and symmetric stretching vibrations (Δ)
indicates that carboxylate ligands act as a bridging or chelating agent.
Similar results were obtained in this study as Δ value of Na-alginate
biopolymer decreased from 197 to 188 cm-1 for Ca-alginate gel. In the
region of 1150 – 1000 cm-1 three sharp peaks can be observed that are
associated with CeC and COe stretching vibrations of saccharide ske-
leton. By replacing Na+ with Ca2+, these peaks were shifted to lower
wavenumbers (from 1088 and 1032–1084 and 1030 cm-1, respectively)
(Fig. 1-b). Such slight shifts indicate the weakening of CeC and COe
bonds, probably due to sharing the bonds with calcium ions (Sartori,
Finch, Ralph, & Gilding, 1997).
FTIR spectrum of chitosan and associated vibrations are shown in
Fig. 1-c. Chitosan characteristic peaks at 1600, 1424, and 1383 cm−1
are respectively attributed to NeH bending vibrations of amide I, NeH
stretching vibrations of amide or ether bonds and NeH stretching vi-
brations of amide II (Chandrasekar, Coupland, & Anantheswaran, 2017;
D. Zaeim, et al. Food Structure 25 (2020) 100147

Fig. 1. FTIR spectra of Na-alginate (a), Ca-alginate (b), chitosan (c), Ca-algi-
nate/chitosan microcapsules obtained from single- (d) and double-stage pro-
cedures (e). Characteristic peaks of alginate and chitosan are shown on re-
spective spectra.

Li et al., 2008). As shown in Fig. 1-d and -e, the chemical structure of
Ca-alginate/chitosan microcapsules principally consists of Ca-alginate.
Steric hindrance of alginate macromolecules and the formation of al-
ginate-chitosan PECs on the surface of droplets inhibited penetration of
chitosan molecules into the core of alginate droplets (Gaserod,
Smidsrod, & Skjak-Brak, 1998; Zaeim et al., 2017). The addition of
chitosan led to the formation of new hydrogen bondings between −OH
and NeH2 groups of chitosan and eCO] and −OH groups of alginate
molecules (Kulig, Zimoch-Korzycka, Jarmoluk, & Marycz, 2016;
Sankalia et al., 2007). After adding chitosan, the characteristic peaks of Fig. 2. Surface morphology of uncoated Ca-alginate microcapsules (a) and Ca-
Ca-alginate were shifted from 1612 and 1425 cm−1 to 1610 and 1422 alginate/chitosan microcapsules obtained from single- (b) and double-stage (c)
cm−1, respectively (Fig. 1-d and -e). Slight shifting of carboxylate bands procedures. Scale bars represent 50.0 μm. Inset figures on top right show optical
to lower frequencies occurred possibly due to sharing the bonds with images of single hydrogel microcapsules fabricated by electrospray technique.
amine groups of chitosan on the surface of microcapsules as previously
reported by Sarmento, Ferreira, Veiga, and Ribeiro (2006). Increasing through the single-stage procedure increased surface roughness of the
the intensity of the peak at 1610 cm−1 confirms the formation of PECs Ca-alginate microcapsules (Fig. 2-b). In contrast, the double-stage
between carbonyl groups of alginate and amine groups of chitosan procedure resulted in the microcapsules with even and smooth surfaces.
(Ribeiro, Silva, Ferreira, & Veiga, 2005; Sarmento, Ferreira, Veiga, & In other words, a slight roughness of the Ca-alginate network was
Ribeiro, 2006). thoroughly covered by chitosan through the double-stage procedure
and a smoother surface is obtained (Fig. 2-c). FTIR analysis confirmed
the presence of chitosan in the structure of microcapsules regardless of
3.1.2. Surface morphology the fabrication procedure. However, SEM images revealed that the
The surface morphology of the microcapsules was considered as a chitosan molecules were not adequately deposited on the surface of
sign of chitosan deposition on the surface of Ca-alginate microcapsules. microcapsules fabricated through the single-stage procedure, as the
SEM images revealed chitosan coating on Ca-alginate microcapsules

4
D. Zaeim, et al. Food Structure 25 (2020) 100147

Fig. 3. The process diagram of Ca-alginate/chitosan microcapsules formation by electrospray through single- or double-stage procedures (a). Immersion of a Na-
alginate micro-droplet into a mixture of CaCl2-chitosan solution and the resultant microstructure – single-stage procedure (b). Immersion of a Na-alginate micro-
droplet into a CaCl2 solution and formation of a Ca-alginate microcapsule followed by its immersion into a chitosan solution and the resultant microstructure –
double-stage procedure (c).

rough surface of the Ca-alginate network was still visible. Hari, Chandy,
and Sharma (1996)) showed Ca-alginate capsules have a rough surface
with some wrinkles. Coating the Ca-alginate network by chitosan pro-
vides a relatively smooth surface on the capsules. Similar findings were
also reported by Gaserod et al. (1998). In a study carried out by
Wittaya-areekul et al. (2006) on the content of chitosan in micro-
capsules formed by single- or double-stage procedures, it was reported
that chitosan content of Ca-alginate/chitosan microcapsules obtained
from the single-stage procedure was almost half of those fabricated
through the double-stage procedure. They attributed it to a competition
between chitosan molecules and Ca2+ ions to link to alginate molecules
in the collector solution of the single-stage procedure.
In the single-stage procedure, once a Na-alginate droplet is im-
mersed into the collector solution, a porous gel membrane is instantly
formed around it, through which both Ca2+ and chitosan, mainly short-
chain molecules, are diffused gradually into the droplet. However, in
the double-stage procedure, a dense Ca-alginate network is formed
during the first stage and then a layer of chitosan is deposited on the
surface by linking to the remaining negatively charged carboxylate
groups (Gaserod et al., 1998; Wittaya-areekul et al., 2006; Zaeim et al.,
2017). Our hypothesis on the formation of microcapsules through
single- and double-stage procedures are represented in Fig. 3. An
overall scheme of the single- and double-stage procedures is illustrated
in Fig. 3-a. Chitosan molecules and Ca2+ are competing to interact with

5
D. Zaeim, et al.
Fig. 4. FTIR spectra of pure inulin (a), Ca-alginate/chitosan microcapsules
containing inulin (b), unloaded Ca-alginate/chitosan microcapsules (c) Ca-al-
ginate/chitosan microcapsules containing resistant starch (d) and pure resistant
starch (e). Characteristic peaks of inulin and resistant starch are shown on re-
spective spectra.
the alginate molecules in the collector solution of the single-stage
procedure (Fig. 3-b). It led to the formation of Ca-alginate micro-
capsules, which are not appropriately coated by chitosan molecules.
Moreover, Ca2+-mediated crosslinks did not form the internal structure
of the Ca-alginate network orderly (Fig. 3-b). In contrast, in the double-
stage procedure, as illustrated in Fig. 3-c, an ordered structure of the
Ca-alginate network was formed properly during the first step. Subse-
quently, by submerging the Ca-alginate microparticles in the chitosan
solution, a layer of chitosan precipitated on the surface of the particles
during the second step (Fig. 3-c). The microcapsules formed by the
double-stage procedure were employed for co-encapsulation of pre-
biotics and probiotics in the subsequent experiments.
6
Food Structure 25 (2020) 100147
3.2. Incorporation of prebiotics into microcapsules
FTIR spectra of inulin and its characteristic bands are shown in
Fig. 4-a. The main signal of inulin appears at 935 cm−1 that is attrib-
uted to CH2 out-of-plane deformation (Araujo et al., 2013; Jain, Sood,
Bora, Vasita, & Katti, 2014). The peak at 1645 cm−1 is related to vi-
brations of H2O molecules indicating hygroscopicity of long-chain in-
ulin. Similar spectra were reported for inulin by López-Molina et al.
(2005), and Rahul, Jha, Sen, and Mishra (2014)). After incorporation of
inulin to Ca-alginate/chitosan microcapsules, no new peak appeared
(Fig. 4-b). However, the intensity of the band associated with OeH
stretching was increased and shifted from 3405 to 3418 cm−1 (Fig. 4-
b). Inulin contains many hydroxyl groups that may interfere and
overlap with alginate/chitosan bands. Besides, some new hydrogen
bonding may be formed between polysaccharides. Widening the band
at 1030 cm−1 was also brought about by the interference of the peaks
ascribed to the saccharide skeleton in the range of 1150 – 1000 cm−1.
Similar results were reported by Lim and Ahmad (2017). The presence
of inulin in the microcapsules is confirmed by the peak that was ap-
peared at 935 cm−1, as indicated by an arrow in Fig. 4-b.
The characteristic peaks of starch appear in the region 1300 – 800
cm−1, which is sensitive to its molecular conformation and crystalline
structure (Kizil, Irudayaraj, & Seetharaman, 2002; Zhang et al., 2013).
As shown in Fig. 4-e, the peaks at 1157 and 1081 cm−1 are attributed to
CC and CO stretching and COHeeee bending vibrations, respectively
(Kizil et al., 2002; López-Córdoba, Deladino, & Martino, 2014). The
peak at 1015 cm−1 is indicative of the amorphous region of starch
(Xiong, Li, Shi, & Ye, 2017), and the band at 930 cm−1 is associated
with α-1,4 glycosidic bonds (Kizil et al., 2002). The incorporation of
starch caused no new peak in the spectrum of Ca-alginate/chitosan
microcapsules (Fig. 4-d). However, hydrogen bondings between these
polysaccharides may intensify the band in the region of 3700 – 3100
cm−1 as previously reported by Wang, Hu, Du, and Kennedy (2010).
Loading resistant starch granules into Ca-alginate/chitosan micro-
capsules increased the separation (Δ) between asymmetric and sym-
metric stretching vibrations of carboxylic salts from 188 to 200 cm−1.
This means that COO– ligands of alginate molecules coordinated to the
metal center in a monodentate mode (Lawrie et al., 2007). In other
words, the distribution of starch granules among Na-alginate chains
prevented Ca2+-mediated bridging by steric hindrance and thus the
number of calcium bridges was reduced in the gel network. In agree-
ment with our findings, López-Córdoba et al. (2014) reported that the
stretching vibration of asymmetric carboxyl groups was shifted from
1610 to 1643 led to an increase in Δ value after incorporation of starch
granules into the Ca-alginate gel. As indicated in Fig. 4-d, a sharp peak
was observed at 1157 cm−1 in the spectrum of starch-containing mi-
crocapsules which is attributed to CeC and COe stretching vibrations
of starch. Moreover, another peak was observed at 930 cm−1, which is
exclusively attributed to α,1–4 glycosidic bonds of amylose. These
findings confirm physical entrapment of the resistant starch granules in
Fig. 5. Viability of Lb. plantarum PTCC 1896 during the sto-
rage at 25 ( ), 4 ( ) and -18 ( ) ˚C for 90 days. The number of
viable cells encapsulated in inulin-containing Ca-alginate/
chitosan microcapsules (a) and free cells in the feed solution
(control) (b). Each point is the mean of three separate ex-
periments.
D. Zaeim, et al.
Fig. 7. Viability of Lb. plantarum PTCC 1896 in ice-cream samples during the
storage at -18 °C for 90 days. Encapsulated cells in inulin-containing Ca-algi-
nate/chitosan microcapsules ( ), encapsulated cells in starch-containing Ca-
alginate/chitosan microcapsules ( ), free cells in the feed solution containing
inulin ( ) and free cells in the feed solution containing starch ( ). Each point is
the mean of three separate experiments.
Ca-alginate/chitosan microcapsules (Siddaramaiah, Swamy, Ramaraj,
& Lee, 2008; Wang, Hu, Du, & Kennedy, 2010). The encapsulation ef-
ficiency and release profile of inulin and resistant starch from the Ca-
alginate/chitosan microcapsules were previously reported by Zaeim
et al. (2019).
3.3. Determination of probiotics survival during storage
3.3.1. Inulin-containing microcapsules
Probiotic cultures often undergo a long storage time before being
applied to formulated functional foods. Therefore, successful micro-
encapsulation could be claimed when the number of viable cells re-
mains higher than an acceptable level of 106 – 107 CFU g−1 after the
storage (Peredo, Beristain, Pascual, Azuara, & Jimenez, 2016). As
shown in Fig. 5, the number of viable Lb. plantarum PTCC 1896 in in-
ulin-containing microcapsules did not change meaningfully (P ≤ 0.05)
during the storage at 4 and -18 °C for 90 days. However, it showed 2 log
cycles reduction at 25 °C over the same period. The number of viable
cells in the control samples reduced almost 1 log cycle during the sto-
rage at 4 and -18 °C; it underwent 4.5 log cycles reduction at 25 °C after
90 days. These results confirm the positive influence of inulin-con-
taining Ca-alginate/chitosan microcapsules on maintaining the viability
of Lb. plantarum PTCC 1896 during the storage. Such protective effects
of a polysaccharide matrix on probiotic bacteria were reported for the
co-encapsulation of inulin and Lb. casei (Krasaekoopt & Watcharapoka,
2014), inulin, and Lb. plantarum (Wang, Yu, Xu, Aguilar, & Wei, 2016)
and inulin and Lb. rhamnosus in Ca-alginate/chitosan capsules
7
Food Structure 25 (2020) 100147
Fig. 6. Viability of Lb. plantarum PTCC 1896 during the sto-
rage at 25 ( ), 4 ( ) and -18 ( ) ˚C for 90 days. The number of
viable cells encapsulated in starch-containing Ca-alginate/
chitosan microcapsules (a) and free cells in the feed solution
(control) (b). Each point is the mean of three separate ex-
periments.
(Gandomi, Abbaszadeh, Misaghi, Bokaie, & Noori, 2016). Okuro,
Thomazini, Balieiro, Liberal, and Fávaro-Trindade (2013) reported the
addition of inulin to probiotic-containing microcapsules could sig-
nificantly enhance the survival of Lb. acidophilus for 120 days. The re-
duction of probiotic viability during the storage at 25 °C is attributed to
the metabolic activity of the cells at this temperature and the produc-
tion of organic acids in the microcapsules (Holkem et al., 2017).
3.3.2. Starch-containing microcapsules
Viability of Lb. plantarum PTCC1896 in starch-containing micro-
capsules is demonstrated in Fig. 6. After 2 months of storage at 4 °C, the
number of viable cells in the microcapsules was reduced from
9.18 ± 0.30–8.21 ± 0.36 log CFU g−1, corresponding to almost 1 log
cycle loss, while no significant decrease was observed in the control
sample under the same condition. In the next month, the number of
viable cells was decreased more than 0.8 log cycles in the control
sample but it did not change significantly (P ≤ 0.05) in the micro-
capsules. Similar trends were also observed for the samples stored at
-18 °C. In agreement with our results, other researchers reported that
the main reduction of viable cells in the microcapsules occurred at the
very first months of storage and the number of viable cells remained
almost constant during the following months (Holkem et al., 2017;
Okuro, Thomazini, Balieiro, Liberal, & Fávaro-Trindade, 2013; Wang
et al., 2016). Similar results were reported on improving the viability of
probiotics by the addition of starch into Ca-alginate/chitosan micro-
capsules (Chan et al., 2011; Etchepare et al., 2016; Martin, Lara-
Villoslada, Ruiz, & Morales, 2013). After 90 days of storage at 25 °C,
5.33 ± 0.18, and 5.95 ± 0.27 log CFU g−1 of the cells remained viable
in the control sample and microcapsules, respectively. The adverse ef-
fect of room temperature on the viability of encapsulated Lb. plantarum
during the storage was recently reported by Paula et al. (2019).
3.4. Determination of probiotics survival during ice-cream production and
storage
As shown in Fig. 7, the microencapsulation of Lb. plantarum
PTCC1896 in Ca-alginate/chitosan microcapsules containing either in-
ulin or resistant starch significantly (P ≤ 0.05) improved the bacterial
survival in the ice-cream samples after 90 days of storage. By in-
corporating inulin-containing microcapsules into the ice-cream for-
mulation, the number of viable cells reduced from
8.1 ± 0.39–7.37 ± 0.12 log CFU g−1 after 90 days storage, corre-
sponding to 0.7 log cycle. However, the number of viable cells in the
control sample dropped to 6.63 ± 0.21 log CFU g-1 under the same
condition, corresponding to 1.5 log cycle. Starch-containing micro-
capsules even showed a better performance than inulin-containing ones
in maintaining probiotic viability during the storage of ice-cream. The
number of viable cells in the ice-cream samples after 90 days storage
reduced from 8.37 ± 0.09–7.82 ± 0.39 log CFU g−1, equal to 0.5 log
cycle. Nevertheless, the number of viable cells declined to 6.75 ± 0.31
D. Zaeim, et al.
log CFU g−1 in the control sample, corresponding to 1.6 log cycle. The
positive role of probiotic-prebiotic co-encapsulation on improving the
survival rate of probiotics in various food products was frequently re-
ported by other researchers (Homayouni, Azizi, Ehsani, Yarmand, &
Razavi, 2008; Iyer & Kailasapathy, 2005). Co-encapsulation provides a
prebiotic-rich microenvironment around the live probiotic cells. Such
microstructures make prebiotics available to probiotic cells, which may
improve their viability under unfavorable conditions as well as enhance
their competitiveness in super-complex gut microbiota by accelerating
their growth and proliferation (Okuro et al., 2013; Sathyabama,
Ranjith, Bruntha, Vijayabharathi, & Brindha, 2014).
4. Conclusions
Electrospray technology, as a novel technique for microencapsula-
tion of food components, was used for the incorporation of live pro-
biotics and high-molecular-weight prebiotics (i.e. long-chain inulin or
resistant starch) directly into double-layer Ca-alginate/chitosan mi-
crocapsules. This technique successfully made complex microstructures
from highly-viscous feed solutions. Two single- and double-stage pro-
cedures were examined for making those peculiar microstructures. SEM
images revealed that the microcapsules fabricated through the double-
stage procedure were thoroughly coated by a chitosan layer and FTIR
spectra confirmed the presence of inulin or resistant starch in the mi-
crocapsules. Polysaccharide matrices significantly enhanced the viabi-
lity of probiotics during the storage, especially at room temperature.
Inulin-containing microcapsules improved the survival of Lb. plantarum
PTCC1896 during the storage more efficiently than starch-containing
ones. However, by incorporating microcapsules into a real food system
(i.e. ice-cream), starch-containing microcapsules efficiently enhanced
cell survival. Such multi-component microcapsules can be used as a
synbiotic ingredient in the formulation of functional foods. They could
enhance the health-promoting effects of the foods without any impact
on their organoleptic properties. Moreover, electro-hydrodynamic
processing provided the possibility of forming micron-sized spherical
capsules loaded with a high concentration of probiotics and prebiotics.
Declaration of Competing Interest
There are no conflicts to declare.
Acknowledgments
The authors would like to acknowledge the Research Institute of
Food Science and Technology (RIFST) and NIZO Food Researchfor fi-
nancial support of this project.
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