Journal of Food Engineering 116 (2013) 369–381

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Journal of Food Engineering

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Review

Microencapsulation of microbial cells

Sweta Rathore, Parind Mahendrakumar Desai, Celine Valeria Liew, Lai Wah Chan, Paul Wan Sia Heng ⇑
GEA-NUS Pharmaceutical Processing Research Laboratory, Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore

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

Article history: Microencapsulation involves coating or entrapping of a core material with a polymeric material to gen-
Received 11 October 2012 erate microspheres in the size range of 1–1000 lm. This versatile technology has been used to encapsu-
Received in revised form 6 December 2012 late a wide array of products such as pharmaceuticals, flavors, volatile oils, plant extracts, enzymes and
Accepted 7 December 2012
others. In the recent decades, this technology has also been applied to the area of microbial cell immo-
Available online 20 December 2012
bilization owing to its numerous advantages over other cell immobilization techniques such as higher cell
loading capacity, enhanced cell survival and increased production rate of the desired microbial products.
Keywords:
The confinement of microbial cells within a semipermeable polymeric matrix enables the physical isola-
Immobilization
Microencapsulation
tion of cells from the external environment while maintaining a hospitable internal micro-environment.
Microbial cells It has found application in various biotechnological processes such as probiotic encapsulation in food
Stability industries, in biotransformation and fermentation processes producing antibiotics, organic acids,
enzymes, and alcohols as well as environmental decontamination such as waste water treatment. The
judicious selection of materials and methods for the production of microspheres is critical for ensuring
minimum damage to the viability of the encapsulated microbial cells. The conventional methods used
for microencapsulation of microbial cells are reviewed along with the recent advances in the respective
methods. The effect of microencapsulation on the microbial cells, the stability of the microspheres as well
as the techniques for enumeration of the encapsulated cells are also discussed, followed by a summary of
recent applications of microencapsulation in different biotechnological processes.
Ó 2012 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
2. Microencapsulation as a cell immobilization method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
3. Materials used for microencapsulation of microbial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
4. Techniques used for microencapsulation of microbial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
4.1. Extrusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
4.2. Spray drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
4.3. Emulsification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
4.4. Coacervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
5. Drying of the microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
6. Stability of microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
7. Effect of microencapsulation on cell physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
8. Measurement of viability of encapsulated cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
9. Effects of microencapsulation techniques on cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376
10. Recent applications of microencapsulation of microbial cells in various biotechnological processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
11. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 378

⇑ Corresponding author. Tel.: +65 65162930; fax: +65 67752265.

E-mail address: phapaulh@nus.edu.sg (P.W.S. Heng).

0260-8774/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.12.022
370 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
1. Introduction
Beneficial microbial cells have been used in wide range of fields
such as pharmaceutical, food processing, environmental protec-
tion, biofuel production, agricultural biotechnology and waste
water treatment for many years. However, for optimum activity
of these microbial cells, it is required that they are provided with
suitable conditions for growth and metabolism and at the same
time protected from harsh environmental conditions that they
are exposed to. In addition, in case of microbial cells that produce
substances of commercial importance, achievement of high cell
densities for higher product yields, retention of activity for longer
period of time and easy recovery of the cells from the products are
often desired. In order to attain these goals, immobilization of
microbial cells has been proposed (Zhu, 2007). Cell immobilization
can be defined as the physical confinement of whole cells to a cer-
tain defined space while preserving their viability (Karel et al.,
1985). The pioneering work in this field was carried out by Chibata
(1979) using immobilized microbial cells for the continuous pro-
duction of L-aspartic acid. Since then, cell immobilization has
gained significant importance in a wide range of ever expanding
applications in different industries such as food production, agri-
culture, waste water treatment, pest control, environmental
decontamination, production of therapeutic agents, enzymes and
biofuels (Amiet-Charpentier et al., 1999; Badr et al., 2001; Bashan,
1986; Byrd et al., 2005; De Castro-Cislaghi et al., 2012; Elibol and
Moreira, 2003; Häggström and Molin, 1980; Kim et al., 2012; Liu
et al., 2005; McLoughlin, 1994; Mensour et al., 1996; Ogbonna
et al., 2001; Olguín, 2003; Park et al., 1998; Quek et al., 2006; Qure-
shi and Maddox, 1987; Sipsas et al., 2009; Tripathi et al., 2010).
The utilization of immobilized microbial cells in various bio-
technological processes was found to be advantageous over the
use of free cells (Cassidy et al., 1996; Qureshi et al., 2004). Some
of these advantages include easy separation of the cells from the
products, greater productivity due to high cell concentrations
achieved, protection of cells against harsh environmental condi-
tions, possibility of the use of packed columns, reusability of the
immobilized cells and prevention of cell washout (Federici, 1993;
Park and Chang, 2000; Qureshi et al., 2004; Tripathi et al., 2010;
Wang et al., 1997).
Various techniques have been investigated for the purpose of
microbial cell immobilization such as adsorption or attachment
of cells to an inert substrate, self-aggregation by flocculation or
use of cross-linking agents and entrapment or encapsulation using
polymers (Jen et al., 1996; Kourkoutas et al., 2004). Adsorption of
microbial cells utilizes the natural ability of cells to adhere onto so-
lid supports to form biofilms which can exist as a single layer or
can be several millimeters thick (Rezaee et al., 2008; Vučurović
et al., 2008). For microbial cells that do not adhere naturally, strat-
egies like chemical cross-linking by glutaraldehyde, silanization
onto a silica support and metal oxide chelation can be used (Karel
et al., 1985). The advantages of this method include the simplicity
and low cost of cell immobilization process. However, it faces seri-
ous limitation of extensive cell leakage from the support, making it
difficult to obtain cell free effluent for downstream processing
(Kosseva, 2011; Zhu, 2007). The ability of some cells such as yeast
and fungal mycelium to flocculate, resulting in cell aggregates, has
been utilized as a technique of cell immobilization by aggregation
(Olguín, 2012; Yin et al., 2011). Under some conditions, non-floccu-
lating microbes can also be encouraged to flocculate (Deverell and
Clark, 1985; Vallejo et al., 2012). Cell immobilization by encapsula-
tion or entrapment involves coating or entrapping microbial cells
within a polymeric material to produce beads which are permeable
to nutrients, gases and metabolites for maintaining cell viability
within the beads (Ding and Shah, 2009b; John et al., 2011).
The encapsulation techniques used for immobilization of cells
can be broadly classified into two types based on the size of the
polymeric bead produced, i.e. macroencapsulation and microen-
capsulation (John et al., 2011). Macroencapsulation is the entrap-
ment of cells in polymeric beads of size ranging from few
millimeters to centimeters (Gentile et al., 1995; John et al.,
2011). On the other hand, microencapsulation produces beads in
the size range of 1–1000 lm (Byrd et al., 2005; Heidebach et al.,
2012). In the case of macroencapsulation, lower cell viability was
reported at the center of the larger beads after a relatively short
operation time due to depletion in the efficiency of nutrient diffu-
sion at a depth of more than 300–500 lm as well as accumulation
of toxic metabolites in the center of the bead (McLoughlin, 1994).
As a result, microbial cell growth is usually observed at the periph-
ery of larger beads (Park and Chang, 2000). Moreover, micro-
spheres are found to be more mechanically robust than
macrocapsules (Uludag et al., 2000). Many studies have reported
that smaller size beads permit high cell concentrations within
the beads by allowing efficient diffusion of nutrients, oxygen as
well as metabolites (Christenson et al., 1993; Dalili and Chau,
1987; Guiseley, 1989; Ogbonna et al., 1991). For example, cell
death due to hypoxic conditions in the center of larger beads was
reported (Christenson et al., 1993). Similarly, Ogbonna et al.
(1991) found that the optimum bead size for oxygen penetration
in different polymeric beads was 100–300 lm. Another report sug-
gested that the best size for beads used for immobilization of
microbial cells is 500–1000 lm (Guiseley, 1989). Thus, it can be
concluded that smaller size beads, less than 1000 lm, are usually
preferred over larger beads. These beads can be produced by the
process of microencapsulation.
This review focuses on the use of microencapsulation as a cell
immobilization strategy for various microbial cells used in differ-
ent fields. The salient features of the commonly used polymers to
encapsulate microbial cells are highlighted. Special emphasis is gi-
ven to the different microencapsulation techniques to form micro-
spheres along with some of the recent advances in the respective
techniques. In the later part of the review, the aspect of physical
stability of microspheres is reviewed, followed by an assessment
of the effect of microencapsulation process on microbial cells.
The significance of the encapsulated cell viability measurement
and the methods used for measuring cell viability are also de-
scribed. The effect of microencapsulation techniques on the cell
viability and storage stability is also described. Finally, the recent
applications of microencapsulation in various biotechnological
fields are summarized.
2. Microencapsulation as a cell immobilization method
Microencapsulation has found applications in many fields
including pharmaceutical, agricultural, nutritional and therapeu-
tics (Aghbashlo et al., 2012; Bringas-Lantigua et al., 2012; Byrd
et al., 2005; De Castro-Cislaghi et al., 2012; Frascareli et al.,
2012; Read et al., 2001; Rubilar et al., 2012; Shin et al., 2012). This
versatile technology has more recently been applied to microbial
cell immobilization in order to overcome the drawbacks encoun-
tered with other cell immobilization techniques such as limited
cell loading, cell leakage, low mechanical stability, contamination
and mass transfer limitations (Park and Chang, 2000). Depending
on the type of application, microbial cells can be encapsulated
for the purpose of isolation, protection and/or controlled release.
The confinement of microbial cells in microspheres offers protec-
tion against both mechanical as well as environmental stresses
while maintaining some growth and metabolic activities for ex-
tended periods of time (Anal and Singh, 2007; Uludag et al.,
 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
Fig. 1. Different novel techniques for droplet and subsequent microspheres formation by extrusion: (a) droplet formation by electrostatic force, (b) droplet formation by jet
cutter, (c) droplet formation by vibration frequency, (d) coaxial/co-extrusion droplet formation.
Fig. 2. Schematic diagram of a pneumatically driven micro-vibrator device
(adapted from Huang et al., 2010).
2000). Owing to their relatively small size, the microspheres have a
larger specific surface area for diffusion of nutrients into the micro-
spheres and diffusion of metabolites out of the microspheres. In
the fermentation industry, besides the above advantages, microen-
capsulation allows easy separation of cells and minimizes cell
wash out (Liu et al., 2010; Tan et al., 2011; Ylitervo et al., 2011).
It is possible to reuse the encapsulated microbial cells for continu-
ous operation for prolonged period of time due to constant cell
regeneration within the microspheres (Tan et al., 2011). Improved
tolerance or protection of cells from substrate and end-product
inhibition of microencapsulated cells has been reported in numer-
ous studies apart from the decrease in undesirable process effects
and contamination risks (Park and Chang, 2000). The fabrication of
microspheres can also be manipulated such that it allows for con-
trolled release of the microbial cells in a specific environment
along the gastrointestinal tract or in soil environment (Albertini
et al., 2010; Guérin et al., 2003; Sultana et al., 2000).
3. Materials used for microencapsulation of microbial cells
Producing stable microspheres for microbial cell immobiliza-
tion starts with the selection of an appropriate encapsulation
material. Studies have shown that polymer types play a dominant
role in determining the properties of the microspheres (Jen et al.,
1996). Microspheres are almost exclusively produced using
water-soluble polymers which provide a high degree of permeabil-
ity for low molecular weight nutrients and metabolites, thus pro-
viding optimal conditions for the functioning of immobilized
microbial cells. Both synthetic and natural water-soluble polymers
have been used for microencapsulation of microbial cells
(Groboillot et al., 1994; John et al., 2011). Though synthetic
polymers offer higher mechanical strength and better chemical
stability, natural polymers are preferred over their synthetic coun-
terparts as the formulation of microspheres using natural polymers
usually requires milder process and is less harmful to cell integrity
and viability (Rosevear, 1984). Gelation can occur by one or more
of the following mechanisms: ionotropic gelation, thermal
371
gelation, cross-linking and polymerization (Martins dos Santos
et al., 1998). The intramolecular and intermolecular interactions
brought about by gelation or cross-linking may occur through
hydrogen bonding, electrostatic interactions and/or hydrophobic
interactions which in turn are dependent on the chemical compo-
sition of the polymers (de Vos et al., 2009). Tikekar et al. (2011)
developed a real time in situ method for the measurement of
oxygen transport across oil/water interface in an emulsion. This
method was based on reversible fluorescence quenching of tris
ruthenium (II) bis (hexafluorophosphate) complex dye encapsu-
lated in the oil phase of an emulsion upon interaction with oxygen.
It could be used for the screening and evaluation of the barrier
properties of different polymeric membranes used for microencap-
sulation. Thus, based on the required permeability, a given
polymer could be selected using this method. Commonly used
polymers for microencapsulation of microbial cells are listed in
Table 1.
4. Techniques used for microencapsulation of microbial cells
For microencapsulation of microbial cells, it is essential that the
encapsulation process is performed under relatively mild condi-
tions to ensure high viability of the encapsulated cells. It is also
necessary for the microspheres to possess good mechanical stabil-
ity so as to support the growth and long-term culture of the micro-
bial cells. Various techniques for microencapsulation of microbial
cells have been investigated over the past few years for the protec-
tion and viability enhancement of microorganisms with varying
degrees of success. Some of the common techniques used include
extrusion, coacervation, spray drying and emulsification (de Vos
et al., 2010). Each methodology has its own characteristic features
and the selection of any particular method is based on the applica-
tion of the microspheres. For example, microspheres containing
probiotic cells are required to be smaller than 100 lm to avoid
gritty sensation when consumed (Hansen et al., 2002; Heidebach
et al., 2012). Similarly, microspheres containing fermenting micro-
organisms for use in bioreactors need to be mechanically strong to
withstand harsh mechanical and physical conditions like shear
forces, acidic environments, exposure to fermentation gases and
solvents. Thus, the selected method should be able to produce
microspheres with the necessary physical/chemical attributes
while causing minimal damage to cell integrity and viability and
be easy to scale up with acceptable processing costs. The major
techniques used for microencapsulation of microbial cells are dis-
cussed below along with their recent advances.
4.1. Extrusion
Extrusion is commonly employed for the microencapsulation of
microbial cells (Green et al., 1996; Koyama and Seki, 2004; ÖZer
372 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381

Table 1
Commonly used polymers for microencapsulation of microbial cells.

Polymer Source Chemical structure Gelation mechanism Remark References

Agar Red algae Agarbiose backbone with Thermal gelation  Resistance to degradation by Olguín (2012) and Rosevear (1984)
alternating 1, 3-linked 3- most microorganisms
D-galactopyranose and 1,  Low mechanical strength
4-linked 3, 6-anhydro-a-  High cost
L-galactopyranose

j-Carrageenan Red algae Alternating 1–3 linked b- Thermal/ionotropic  Toxicity to cells due to excess of Adhikari et al. (2000), Olguín
D-galactopyranose and 1– gelation in the presence potassium used for cross-linking (2012), Rokka and Rantamäki
4 linked a-D- of potassium or calcium (2010) and Yu et al. (2002)
galactopyranose units ions
Starch Maize, potato, Amylose and Thermal gelation  Usually blended with alginate Mortazavian et al. (2007), Anal and
barley, oat, etc. amylopectin units joint microspheres so as to offer good Singh (2007), Jankowski et al.
together by glycosidic protection to the bacterial cells (1997), O’Riordan et al. (2001),
bonds and allow optimal diffusion of Sajilata et al. (2006) and Sultana
micronutrients and metabolites et al. (2000)
 Decomposes under acidic condi-
tions and presence of pancreatic
enzymes in the GIT
 Resistant starch, used as an
encapsulating polymer for pro-
biotics, is not digested by pan-
creatic enzymes (amylases) in
the small intestine and can be
fermented in the colon
Chitosan Crustacean Linear polysaccharide of Ionotropic gelation in  Found to reduce cell viability of Chávarri et al. (2010), Krasaekoopt
shell glucosamine units the presence of anions encapsulated cells et al. (2003) and Olguín (2003)
 Mostly used as a coating mate-
rial for microspheres at low
concentration
Alginate Brown algae Linear polysaccharide of Ionotropic gelation in  Mild and simple gelling condi- Chan et al. (2011), Chandramouli
D-mannuronic and L- the presence of divalent tions, non-toxicity, and excel- et al. (2004), Ding and Shah (2007),
guluronic acids cations, commonly lent biocompatibility properties Gouin (2004), Mandal et al. (2006),
calcium ions  Low cost Olguín (2012), Prevost and Divies
 Susceptible to cation chelating (1992), Qi et al. (2006), Rowley
agents and to antigelling cations et al. (1999), Shah and Ravula
as well as acidic environments, (2000) and Smidsrød and Skjåk-
which can cause capsule disrup- Bræk (1990)
tion or dissolution
Gellan gum Sphingomonas Linear polysaccharide Thermal/ionotropic  Two main types of gellan gum, Moslemy et al. (2002) and
elodea consists of repeating unit gelation in the presence acetylated (forms weak, less Muthukumarasamy et al. (2006)
of glucose, glucuronic of cations rigid, soft and elastic gels) and
acid, glucose and deacetylated (forms hard and
rhamnose brittle gels)
 Able to withstand the high tem-
perature of the autoclaving pro-
cess without significant loss of
gel strength
 Acid-resistant
Gelatin Collagen Consists of glycine Thermal/cross-linking  Useful as a thermal-reversible Annan et al. (2008), da Silva and
proline and 4- using formaldehyde or gelling agent for encapsulation Pinto (2012) and Hyndman et al.
hydroxyproline residues glutaraldehyde/physical either alone or in combination (1993)
cross-linking using high with other polymers
pressure, irradiation  Its amphoteric nature can be
useful to form strong interaction
with anionic polymer such as
gellan gum when the pH is
adjusted below its isoelectric
point causing the net charge of
gelatin to become positive
Xanthan gum Xanthomonas Composed of glucose, Ionotropic gelation in  Highly resistant to enzymatic Muthukumarasamy et al. (2006)
campestris mannose, and glucuronic the presence of divalent degradation, very resistant to and Sun and Griffiths (2000)
acid cations, commonly pH variations and stable at low
calcium ions pHs
 Used in combination with gellan
gum to form acid stable
microspheres
Milk proteins Milk Composed of lactose and Acid-induced gelation  Excellent gelation properties De Castro-Cislaghi et al. (2012),
(such as soluble proteins for caseins and heat-  Useful for encapsulation of pro- Doherty et al. (2011), Hébrard et al.
casein, induced gelation for biotic cells (2006), Livney (2010) and
whey whey proteins Millqvist-Fureby et al. (2001)
protein)
Polyacrylamide Chemical Composed of acrylamide Cross-linking using  Synthetic polymer Calvet et al. (2004) and Wyss et al.
synthesis subunits ammonium persulfate, (2004)
N,N-methylenebis
(acrylamide), N,N,N,N-
 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
Table 1 (continued)
Polymer Source Chemical structure Gelation mechanism
tetramethylenediamine
Polyvinyl Composed of vinyl Cross-linking using boric
alcohol alcohol subunits acid
(PVA)
et al., 2008). In employing extrusion, a polymeric solution is first
mixed with the microbial cells and then extruded through an ori-
fice as droplets into the solution of a cross-linking agent. Gelation
occurs by contact of the polymer solution with the cross-linking
agent, cooling or a combination of both. The factors affecting the
size of the microspheres produced include the diameter of the ori-
fice, the viscosity and flow rate of the polymeric solution, the drop
height or distance from the orifice to the cross-linking solution and
concentration and temperature of the polymer solution (Brun-Gra-
eppi et al., 2011).
The major advantages of the extrusion method are simplicity of
its operation, lower cost, and mild operational conditions ensuring
high cell viability (de Vos et al., 2010). However, there are also a
few drawbacks such as its inefficiency in producing microspheres
smaller than 500 lm, requirement of low to moderate viscosity
polymer solutions and relatively large diameter nozzles (Reis
et al., 2006). In addition, rapid cross-linking and hardening at the
surfaces of the microspheres delay the movement of cross-linking
ions into the inner core, resulting in less stable microspheres (Liu
et al., 2002). Although microspheres are conveniently produced
at laboratory-scale, the scaling up of the process is generally diffi-
cult due to the slow production of microspheres (Burgain et al.,
2011).
Numerous methods have been developed to overcome the lim-
itations of the simple direct extrusion technique. Some of these
techniques include the precision particle fabrication (PPF) method,
co-extrusion and coaxial flow method, multi-nozzle systems, rotat-
ing disc atomisation, droplet generation using electrostatic, vibra-
tion or acoustic energy, and liquid jet cutting technique
(Brandenberger and Widmer, 1998; Brun-Graeppi et al., 2011;
Cheng et al., 2010; Herrero et al., 2006; Kim et al., 2012; Ogbonna
et al., 1989; Piazza and Roversi, 2011; Prüße et al., 1998; Whelehan
and Marison, 2011). If the formation of droplets is carried out in a
controlled manner using pulse, jet or vibration of the nozzle, the
technique is referred as laminar jet break-up or prilling , as shown
in Fig. 1 (Brandenberger and Widmer, 1998; Del Gaudio et al.,
2005). In this technique, a vibration frequency is used to break
the liquid jet into droplets which are then gelled in a cross-linking
solution. The flow rate and viscosity of the polymer solution were
found to significantly affect the size distribution of the micro-
spheres (Del Gaudio et al., 2005). Graff et al. (2008) encapsulated
373
Remark References
 The content of the acrylamide
and the ratio of cells to acrylam-
ide affect the hardness of the gel
formed
 The acrylamide to cross-linker
ratio determines the pore size
of the gel, yields a very stable
gel
 Gel is very porous and has high
water content. However, due to
the toxicity of the reagents used
and the radical-dependent poly-
merization, serious damage to
the cell walls and loss of cell via-
bility are often observed
 Microorganisms enclosed in the Gao et al. (2004)
PVA matrix can be drastically
damaged by boric acid during
the bead preparation process
 Toxicity can be reduced by
substituting boric acid with
sodium sulfate for the cross-
linking reaction
the probiotic yeast, Saccharomyces boulardii using laminar jet break
up technique. The prepared microspheres were further coated with
chitosan solution. In vivo studies in rats showed that encapsulation
significantly reduced the degradation of yeast cells in the gastroin-
testinal tract after a single oral dose. However, chitosan coating did
not provide any additional benefit. In fact, uncoated microspheres
were found to be more effective in protecting the viability of S. bou-
lardii than coated microspheres. Modified precision particle fabri-
cation was used to encapsulate the bacterial strain Pantoea
agglomerans E325 using a coaxial nozzle (Kim et al., 2012). Alginate
solutions with two different concentrations were introduced sepa-
rately into the inner and outer chambers of the coaxial nozzle. The
polymer droplets, produced using acoustic excitation, were al-
lowed to cross-link in calcium chloride solution. This method pro-
duced size-controlled alginate microspheres. Release of the
microbial cells from the microspheres could be controlled by
adjusting the concentrations of the shell and core materials. An-
other innovative device fabricated by Huang et al. (2010) consisted
of a pneumatically driven micro-vibrator which could continu-
ously generate alginate microspheres in the size range of 30–
70 lm (Fig. 2). This device offered a number of advantages such
as compactness, flexibility and use of a mild process to minimize
damage to the encapsulated cells. Co-extrusion technique uses
two coaxial nozzles, with the core material extruding from the in-
ner nozzle and the polymer solution from the outer nozzle. A rotat-
ing fluid jet cutter breaks the liquid stream into droplets which fall
into the cross-linking solution to form microspheres (Piazza and
Roversi, 2011). Brandenberger and Widmer (1998) used a multi-
nozzle system (13 nozzles) to produce microspheres in the size
range of 200–1000 lm. A sterilisable encapsulation device employ-
ing the laminar jet break-up technique was developed for commer-
cial application to continuously produce beads of size range from
250 to 1000 lm (Serp et al., 2000).
Hirsutella rhossiliensis, an endoparasitic fungus used against
many commercially important plant-parasitic nematodes was
encapsulated in hollow microspheres containing corn gluten and
yeast extract as nutrients for fungal growth (Patel et al., 2011).
The microencapsulation process was carried out by layer by layer
approach. The nutrients and the fungal biomass were dispersed
in sulfoethyl cellulose (SEC). The resultant suspension was then
dripped into polydiallyldimethylammoniumchloride (PDADMAC)
374 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
to form the microspheres. The fungal cells were also encapsulated
in calcium alginate beads (for comparison purpose) prepared by
extrusion method. The water retention property of the SEC-PDAD-
MAC membrane allowed better mycelial growth of the fungus
compared to alginate beads. Encapsulation enabled protection of
the fungus from adverse environmental conditions, allowed con-
trolled release into the environment and extended shelf life of
the fungus.
Although the techniques mentioned above are advantageous
over the simple, conventional extrusion technique, many have
not been proven to be feasible for large scale production due to
technical difficulties related to the requirement for a large number
of nozzles and their associated operational problems such as nee-
dle blockage, need for regular cleaning and the maintenance of ste-
rility. In addition, none of the techniques have been shown to
possess satisfactory processing throughput rates, low processing
cost and reproducibility for industrial application.
4.2. Spray drying
Spray drying is commonly used for the microencapsulation of
probiotics and it involves the atomization of a suspension of micro-
bial cells in a polymeric solution into hot drying air, followed by ra-
pid evaporation of water (Corcoran et al., 2004; Zhao et al., 2008).
The microencapsulated product is then separated as a dry powder
from the conveying air in a cyclone. Various spray drying condi-
tions such as product feed rate, air flow, feed temperature, inlet
air temperature, and outlet air temperature need to be optimized
in order to produce discrete well-formed microspheres (O’Riordan
et al., 2001; Vega and Roos, 2006). The appropriate adjustment of
the inlet air temperature is important as a low air temperature re-
duces the rate of water evaporation, leading to the formation of
aggregated microspheres with high density membranes and poor
flow properties while excessively high air temperature can ad-
versely affect cell viability. In addition, feed temperature adjust-
ment is crucial to modify the viscosity of the polymer solution
and in turn, its capacity to be sprayed homogeneously (Brun-Gra-
eppi et al., 2011). Microencapsulation of the probiotic bacteria Bifi-
dobacterium lactis Bb-12 in whey protein using spray drying
improved the viability of the cells when exposed to bile. There
was also an improvement in the stability of the probiotic cells on
storage for 12 weeks (De Castro-Cislaghi et al., 2012)
Compared to other conventional techniques, spray drying offers
the attractive advantage of producing microspheres in a relatively
simple continuous processing operation (Brun-Graeppi et al., 2011;
Gouin, 2004). However, when applied on a large scale, the high
installation and operational costs as well as considerable floor area
required by the equipment present as major challenges of the pro-
cess (John et al., 2011). The range of polymers that can be used for
encapsulation is also rather limited (Brun-Graeppi et al., 2011;
Gouin, 2004). Moreover, the use of a high inlet air temperature
can lead to excessive rapid moisture evaporation, resulting in
cracks in the polymeric membrane (Brun-Graeppi et al., 2011).
The use of high temperatures has also been reported to severely
impact the viability of encapsulated cells due to dehydration of
the cells as well as the inactivation of essential enzymes that main-
tain cellular balance (Ananta et al., 2005; Doherty et al., 2010; John
et al., 2011). According to some studies, the survival of probiotic
cultures subjected to the spray drying process was inversely pro-
portional to the outlet air temperature used in the spray dryer
(Ananta et al., 2005; Fávaro-Trindade and Grosso, 2002; Oliveira
et al., 2007b). Apart from the above factors, the microorganism
strain and the type of encapsulating polymer used have been
known to affect the survival rate of the encapsulated cells during
the spray drying process as well as during storage (Desmond
et al., 2002). Certain materials may be incorporated into the
polymer matrix to improve the viability of the microbial cells dur-
ing spray drying. For example, improved survival of Lactobacillus
paracasei NFBC 338 in spray dried powders containing gum acacia
during drying and storage has been reported by Desmond et al.
(2002).
The spray chilling method was devised to overcome the prob-
lem of cell damage due to the exposure of microbial cells to high
temperature during spray drying. The process is performed using
equipment similar to that used for spray drying except that a cold
conveying air or cold chamber is used instead of hot air (Cham-
pagne and Fustier, 2007; Pedroso et al., 2012). Pedroso et al.
(2012) encapsulated B. lactis and Lactobacillus acidophilus in solid
lipid microcapsules using spray chilling technique. An emulsion
containing probiotic cells, molten fat and lecithin was prepared
by homogenisation. This emulsion was then atomized into a cold
chamber where the solid lipid microspheres were formed. The con-
ditions used in the spray chilling method were sufficiently mild
and did not affect the viability of the encapsulated cells.
4.3. Emulsification
The emulsification technique is widely used for encapsulation
of various microbial cells (Adhikari et al., 2000; Ding and Shah,
2009b; Özer et al., 2008). It involves dispersion of the cell/polymer
suspension (dispersed phase) in an oil/organic phase (continuous
phase). The mixture is homogenized to form a water-in-oil emul-
sion with the aid of surfactant and stirring. Congealation of the dis-
persed phase is initiated by cooling or addition of a cross-linking
agent to the emulsion. The microspheres produced are subse-
quently harvested by filtration or centrifugation. Thus, it is essen-
tially a chemical technique involving microencapsulation by
interfacial polymerization. The size of the microspheres is affected
by the stirring speed and the rate of addition of the cross-linking
solution. The concentration of the surfactant used also has an effect
on the droplet size of the dispersed phase which eventually affects
size of the microspheres (Shah and Ravula, 2000).
Emulsification usually results in microspheres with wide size
distribution. The shear forces used in emulsification disperse the
cells heterogeneously within the microspheres (Rabanel et al.,
2009). Emulsification can be utilized to produce microspheres of
size below 300 lm which is difficult to achieve with extrusion
method due to clogging of the orifice (Burgain et al., 2011). The
main drawback of the emulsification technique is the potential
toxicity of organic solvents to the encapsulated cells. Although oils
are less toxic than organic solvents, removal of oil from the micro-
spheres may be more difficult compared to organic solvents. More-
over, its use in emulsification also increases the overall cost of
process (Krasaekoopt et al., 2003). The probiotic yeast, S. boulardii
was encapsulated in calcium alginate microspheres prepared by
emulsification method with inulin and mucilage as coating materi-
als. The encapsulated cells were found to have longer shelf life
(6.17-fold higher) compared to free cells (Zamora-Vega et al.,
2012).
A two-step emulsification technique for preparing water-in-oil-
in-water (w/o/w) emulsion, known as microchannel emulsifica-
tion, was studied by Sugiura et al. (2004). In the first step, water-
in-oil emulsion (w/o) was prepared using a homogenizer. This
emulsion was subsequently forced through a silicon plate, consist-
ing of uniform microsized channels, along with an external water
phase. This led to the formation of w/o/w droplets which could
be cross-linked to form the microspheres. The mild emulsification
technique used in this method could be employed to encapsulate
microbial cells. Moreover, no leakage of the internal water phase
was observed with this technique, indicating the applicability of
this technique to encapsulate microbial cells within this phase.
However, limitations such as low fluxes and volume fraction of
 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
the dispersed phase resulted in low production rate. These issues
need to be addressed so as to enable large scale application of this
method (Maan et al., 2011; van der Zwan et al., 2006).
4.4. Coacervation
Microencapsulation using the coacervation technique has been
attempted to encapsulate flavor oils, preservatives, enzymes as
well as microbial cells (John et al., 2011; Oliveira et al., 2007a,b;
Park and Chang, 2000). This technique utilizes phase separation
of one or more incompatible polymers from the initial coating
polymer solution under specific pH, temperature or composition
of the solution. The incompatible polymer(s) is added to the coat-
ing polymer solution and the dispersion is stirred. Changes in the
physical parameters, as described earlier, lead to the separation
of incompatible polymer and deposition of dense coacervate phase
surrounding the core material resulting in formation of micro-
spheres (Gouin, 2004; John et al., 2011; Nihant et al., 1995; Oliveira
et al., 2007a,b). If required, chemical or enzymatic cross-linking
agents can be used for strengthening the microspheres. The more
important processing factors to be considered for the coacervation
technique are the volume of the dispersed phase, addition rate of
the incompatible polymer to the coating polymer solution, stirring
rate of the dispersion and core material to be encapsulated (Nihant
et al., 1995). Apart from these factors, the composition and viscos-
ity of the coacervate and supernatant phases are known to affect
the size distribution, surface morphology and internal porosity of
the final microspheres (Nihant et al., 1994, 1995). Oliveira et al.
(2007b) utilized the coacervation technique to encapsulate B. lactis
(BI 01) and L. acidophilus (LAC 4) using a casein/pectin complex.
Coacervation is a highly promising encapsulation technology in
view of its good encapsulation capacity and controlled liberation of
core material from the microspheres by mechanical stress, temper-
ature and pH changes (Oliveira et al., 2007a). The latter is espe-
cially useful for encapsulation of probiotics which are required to
be released when exposed to higher pH in the large intestine. How-
ever, higher costs and control of different critical conditions asso-
ciated with composition and kinetics of reaction limit its
usefulness (Freitas et al., 2005; Nihant et al., 1995; Park and Chang,
2000). Moreover, the coacervation method may not be useful for
producing microspheres that are very small (John et al., 2011; Fre-
itas et al., 2005).
5. Drying of the microspheres
Microspheres can be easily handled and stored for a long time if
they are isolated as dry powder. Spray drying can be used to pro-
duce and dry microspheres in a single step or can be used sepa-
rately as a drying method for microspheres prepared using other
techniques. Apart from spray drying, other drying methods include
spouted-bed drying and freeze-drying (Cook et al., 2012). Although
the spray drying method is relatively cheap and capable of higher
productivities, high temperature used in the process has been re-
ported to be decrease cell viability due to the rapid dehydration
of the cells as well as thermal and oxygen stresses to the cells (Anal
and Singh, 2007; Knorr, 1998; Ross et al., 2005). Oliveira et al.
(2007a,b) encapsulated probiotic bacteria using a complex coacer-
vation method, which was accompanied either by spray drying or
spouted bed drying. In spouted-bed drying, the microspheres are
fed onto a bed of glass beads in the drying chamber by dripping
or atomization. An air jet with controlled temperature and pres-
sure is used to dry the microspheres contained in the chamber.
In case of freeze-drying, the microspheres are subjected to very
low temperature, resulting in the formation of ice crystals from
the residual water present. Sublimation of the ice crystals under
375
reduced pressure results in the formation of porous dried product.
Freeze-drying is relatively more effective than spray drying at
overcoming the limitations associated with the high temperature
in spray drying (Dolly et al., 2011). The viability of freeze-dried Lac-
tobacillus helveticus had been found to be significantly higher than
of the spray dried form (Johnson and Etzel, 1995). However, even
low temperatures have been known to cause cold injuries to the
microbial cells and hence the use of cryoprotectants is necessary
for freeze-drying and this will escalate cost for the already energy
expensive process. In a study by Albertini et al. (2010), micro-
spheres containing L. acidophilus and B. lactis for intestinal delivery
prepared by the extrusion method were freeze-dried using 10% (w/
v) of glycerol as cryoprotective additive. Other limitations of
freeze-drying include high capital and operating costs due to low
temperatures, high vacuum and long residence times required
(Chen and Wang, 2007; Oliveira et al., 2007a). Dolly et al. (2011)
investigated a spray-freeze-drying (SFD) technique by spraying
the polymeric solution containing the microbial cells into a cold
medium (spray-freezing) and subsequent dehydration by freeze-
drying. It was reported that the viability of the probiotic cells
encapsulated by the SFD technique was higher than those prepared
by spray drying alone.
6. Stability of microspheres
The attribute of mechanical stability of the microspheres is very
important for its successful application in various processes.
Microspheres should possess sufficient mechanical resistance to
withstand the various external forces during the whole duration
of application. For example, despite the suitability of sodium algi-
nate as an encapsulating material, low mechanical stability of algi-
nate in the presence of chelating agents, such as phosphate, lactate
or citrate, is its major limitation (Smidsrød and Skjåk-Bræk, 1990).
Mechanical disruption of microspheres due to high density growth
of encapsulated cells as well as production of fermentation gases
has also been observed. Therefore, special treatments, such as coat-
ing/cross-linking with other polymers, mixing with starch and
other additives and incorporating surfactant, have been investi-
gated for improving the stability of the beads produced (John
et al., 2011; Krasaekoopt et al., 2004).
Coating with polymers is one of the more common approaches
for improving both mechanical and chemical stability of the micro-
spheres, resulting in better retention and higher viability of encap-
sulated microbial cells. Chitosan is a commonly used polymer and
effective coating material for microspheres. The mechanical resis-
tance of alginate beads was shown to be enhanced by treatment
with 5–10 kDa chitosan, thereby resulting in decreased cell leakage
(Serp et al., 2000). Chávarri et al. (2010) used chitosan as the coat-
ing material for alginate microspheres containing probiotics to re-
duce porosity of the alginate beads and to subsequently decrease
the cell leakage from the microspheres. In another study, chitosan
coat improved the stability of the alginate microspheres and a sub-
sequent improvement in the viability of the encapsulated cells
(Krasaekoopt et al., 2004). Chitosan coating was also effective at
protecting alginate microspheres from degradation in the presence
of bile salts (Murata et al., 1999). The ion exchange reaction be-
tween the chitosan coating and bile salts took place on the surface
of the microspheres. This in turn could have limited the diffusion of
the bile salts into the microspheres. The use of palm oil and poly-
lysine coating to improve the stability of the alginate microspheres
containing probiotic cells was investigated by exposing the micro-
spheres to acidic conditions and bile salts. For the coated micro-
spheres, improvement in acid tolerance by about 1 log unit
higher than uncoated alginate microspheres was observed while
no significant improvement in stability was seen when the coated
376 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
microspheres were exposed to bile salts. The authors postulated
that bile salts may have emulsified palm oil, thereby releasing
the coating matrix from the microspheres under such conditions.
Coating also conferred microspheres with smoother and less por-
ous surface (Ding and Shah, 2009b).
7. Effect of microencapsulation on cell physiology
The microenvironment as well as the external environment of
the cells forms a complex system along with the encapsulated cells
(Sun et al., 2007). Immobilization may result in changes in the var-
ious physico-chemical properties of the microenvironment of the
microbial cells such as the presence of ionic charges, cellular met-
abolic products, reduced water activity, altered osmotic pressure,
high temperature and modified surface tension (Groboillot et al.,
1994; Sun et al., 2007). These changes can affect the physiology
and function of the immobilized cell system. Tolerance for hydro-
gen peroxide, simulated gastric and intestinal juices, antibiotics or
freeze-drying was improved due to the immobilization of lactic
acid bacteria (Doleyres et al., 2004). It is crucial to monitor the
changes in the microenvironment as well as the external environ-
ment, and investigate their effects on the cells and the biotechno-
logical process of interest. Methods to investigate the effect of the
microencapsulation process on the encapsulated cells need to be
developed. Heat output by microbial cells was found to change
when cells were encapsulated. Recently, Ma et al. (2007) utilized
microcalorimetry to measure heat output of both free and encap-
sulated cells. The study revealed that the encapsulated cells pro-
duced more heat than the free cells and this was attributed to
the rapid metabolization of the substrates.
8. Measurement of viability of encapsulated cells
For a given application, it is necessary that the microspheres
contain sufficient number of viable microbial cells in order to carry
out the desired function efficiently. It has been found that the via-
bility of the encapsulated cells is affected by the various physico-
chemical properties of the microspheres as well as the processing
parameters used for microencapsulation (Burgain et al., 2011; Gro-
boillot et al., 1993; O’Riordan et al., 2001; Weinbreck et al., 2010).
The severity of cell damage depends on the materials and the
method used as well as the strain of microbial cell to be encapsu-
lated. Measurement of cell viability within the microspheres is
thus crucial to assess the effect of the microencapsulation process
on the cell viability. Measurement of the viability of encapsulated
cells is usually conducted by first disrupting the microspheres
using physical or chemical methods, followed by culturing and
counting the released cells on agar surface (Desmond et al.,
2002; Doria-Serrano et al., 2001; Lian et al., 2003; ÖZer et al.,
2008; Young et al., 2006). However, apart from being repetitive
and time-consuming, this method can cause false negative results
for cells which are metabolically active but non-cultivable (Amor
et al., 2002). In addition, some microspheres are quite rigid and
may be difficult to break up and while breaking the microspheres,
the viability of the encapsulated cells may be affected. Develop-
ment and standardization of rapid and non-destructive methods
for the determination of viability of encapsulated microbial cells
is therefore necessary.
Viability of encapsulated cells has been assessed using different
fluorescent dyes such as SYTO-green (green fluorescent dye), ethi-
dium bromide (red fluorescent dye) and 6-carboxyfluorescein
(Ding and Shah, 2009b; Kim et al., 2012; Lahtinen et al., 2007; Yang
et al., 1998). The fluorescent dyes stain the nucleic acid of dead
bacteria with damaged cytoplasmic membrane so as to distinguish
from live bacteria with intact cytoplasmic membranes. Kim et al.
(2012) investigated the viability of P. agglomerans E325 using
SYTO-green and ethidium bromide under fluorescence microscope
(Kim et al., 2012). The viable cells were stained green while the
dead cells appeared red based on the different permeabilities of
the dyes into living and non-living cells. Ding and Shah (2009b)
used a slightly modified method wherein the water-soluble fluo-
rescent dye, 6-carboxyfluorescein, was encapsulated along with
the microbial cells. Microspheres with superior retention of the
dye indicated better retention of water-soluble nutrients which
translated to higher cell viability. However, the effect of the type
of polymer and its concentration on the penetration of fluorescent
dye into the microspheres was not investigated.
A luminometric method was proposed to estimate the encapsu-
lated viable cell biomass (Navrátil et al., 2000). Ethanol producing
yeast strains were immobilized in alginate, calcium pectate and j-
carrageenan. Based on the fact that dead cells possessed negligible
adenosine triphosphate (ATP), the amount of ATP determined was
attributed to the living cells. The extraction of ATP from the immo-
bilized cells was carried out using an ATP releasing buffer. The re-
leased ATP was subsequently allowed to react with luciferin which
led to emission of bioluminescent radiation. This radiation was
measured by a luminometer in relative light units. Comparison of
the results obtained by bioluminometry and the conventional
gravimetric method showed excellent correlation between the
two methods.
9. Effects of microencapsulation techniques on cell viability
The purpose of microencapsulation is to preserve the viability of
encapsulated microbial cells against detrimental environmental
features, such as changes in pH, damaging metabolic products, os-
motic stress and changes in temperature as well as improve stor-
age stability of the microencapsulated cells (Mortazavian et al.,
2007). However, there is loss of cell viability during the processing
steps of microencapsulation itself. For example, microencapsula-
tion by spray drying is known to adversely affect cells viability ow-
ing to the high temperatures during processing steps. Spray drying
encapsulation of L. acidophilus reduced the viability by about 100
times than before encapsulation owing to the high temperature
used in the process (Zhao et al., 2008). Nonetheless, the viability
of the encapsulated cells was much higher compared to free cells
after 8 weeks of storage at 4 °C.
Various factors such as the type and concentration of carriers,
temperature time combinations and the heat resistance of the
microbial cells are known to affect the viability of cells encapsu-
lated using spray drying (Gardiner et al., 2002; Lian et al., 2002;
Muthukumarasamy et al., 2006). Incorporation of certain compo-
nents such as sugars, gum acacia, maize starch have been shown
to improve the storage stability of encapsulated cells (Desmond
et al., 2002; Kailasapathy, 2002; Leslie et al., 1995; Reid et al.,
2007; Rokka and Rantamäki, 2010). Spray drying microencapsula-
tion of four strains of lactic acid bacteria viz. Lactobacillus
plantarum, Pediococcus pentosaceus, Aerococcus viridans and Enete-
rococcus faecium strains was carried out. It was found that spray
drying did not have any significant adverse effect on the viability
of the for strains of four lactic acid bacteria owing to their thermo-
tolerant nature (Pérez-Chabela et al., in press). The high inlet and
outlet air temperature also significantly affect the cell viability dur-
ing the spray drying process (Gardiner et al., 2000; Mauriello et al.,
1999). For example, an inlet air temperature >120 °C and subse-
quent high air outlet temperature (60 °C) during spray drying re-
sulted in significant loss of the cell viability of bifidobacteria
(O’Riordan et al., 2001). The inlet air temperature was then ad-
justed to 100 °C such that the outlet air temperature was 45 °C.
The selected temperature settings allowed good bacterial cell
 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381 377

survival. The storage stability of free and starch encapsulated cells
in dry malted beverage powder and muesli was monitored over a
period of 20 and 14 days respectively. However, it was found that
both free and encapsulated cells could not survive during the stor-
age period. The authors speculated that hydrophilic starch might
have absorbed the intracellular fluid of the bacteria resulting in
loss of viability. In another study, L. acidophilus La-05 and B. lactis
Bb-12 were encapsulated using spray drying technique with cellu-
lose acetate phthalate as the wall material (Fávaro-Trindade and
Grosso, 2002). It was found that an inlet air temperature of
130 °C and outlet air temperature of 75 °C did not significantly af-
fect the viability of B. lactis cells. On the other hand, the cell count
of L. acidophilus reduced by two log cycles at the same temperature
settings. Similarly, there was a decrease in viability of conidia of
the biocontrol fungus, Beauveria brongniartii, when the outlet air
temperature was increased to approximately 53 °C ± 2 °C (Hor-
aczek and Viernstein, 2004). Thus, it is obvious that use of high
temperature in spray drying process is detrimental to cell viability
and optimisation studies should be undertaken so as to obtain
microspheres with desirable properties and yet retain maximal cell
viability.
Other microencapsulation techniques have been found to be
relatively less harsh to the microbial cells in comparison to spray
drying technique. Microencapsulation of B. lactis and L. acidophilus
by spray chilling method was found to be sufficiently mild and the
viability of the microbial cells was unaffected (Pedroso et al.,
2012). Encapsulation of cells in PEG microbeads using microfluidic
device was also found to be mild with no effect on cell viability
(Lee et al., 2010). Extrusion method used for encapsulation of L. aci-
dophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus
casei 01 had no adverse effect on the viability of the cells (Kra-
saekoopt et al., 2004). P. agglomerans E325 encapsulated by preci-
sion particle fabrication experienced minimal stress during the
encapsulation process (Kim et al., 2012). In another study by
ÖZer et al. (2008, encapsulation of probiotic culture B. bifidum
and L. acidophilus by extrusion and emulsification improved the
shelf life in comparison to free cells during 90 days of storage. Dry-
ing of the microspheres prepared by complex coacervation by

Table 2
Application of microencapsulation of microbial cells in various biotechnological processes.

Microorganism Materials used for encapsulation Microencapsulation Purpose of microencapsulation References

technique
Pantoea agglomerans Sodium alginate, calcium chloride Modified precision Protection against harsh environmental conditions Kim et al. (2012)
E325 particle fabrication and controlled release for use as bio control agent
Lactobacillus Sodium alginate, calcium chloride, Emulsification Protection against harsh environmental conditions Takei et al. (2008)
delbrueckii subsp. polyvinyl alcohol
bulgaricus NBRC
13953
Bifidobacterium BB- Reconstituted skim milk (RSM), inulin, Spray drying Protection against harsh environmental conditions Fritzen-Freire et al.
12 oligofructose, and oligofructose-enriched (2012)
inulin
Enterococcus faecium Sodium alginate, calcium chloride Extrusion Enhanced production of bacteriocin Ivanova et al.
A 2000 (2000–2002)
Bifidobacterium lactis Gellan/xanthan gum blend, calcium Extrusion Protection of the probiotic against harsh GIT McMaster et al.
chloride conditions (2005)
Pseudomonas species Polyvinyl alcohol Extrusion Enhanced biodegradation of phthalic acid esters Wang et al. (1997)
Saccharomyces Xanthan gum, calcium chloride Extrusion Improved invertase activity and stability Chang et al. (1996)
cerevisiae SEY
2102
Bifidobacterium lactis Interesterified fat with palm and palm Spray chilling Protection of the probiotic against harsh GIT Pedroso et al. (2012)
and Lactobacillus kernel conditions
acidophilus
Lactobacillus and Sodium alginate, palm oil, polylysine Emulsification Protection of the probiotic against harsh GIT Ding and Shah
Bifidobacterium conditions (2009b)
species
Lactic acid bacteria Acacia gum Spray drying Protection of lactic acid bacteria to improve Pérez-Chabela et al.
microbial safety in cooked meat food products (in press)
Lactobacillus species Xanthan gum, gellan gum, pullulan gum, Extrusion Protection of the probiotic from bile salts Jiménez-Pranteda
jamilan et al. (2012)
Lactobacillus and Alginate, guar gum, xanthan gum, locust Emulsification Protection of the probiotic against harsh GIT Ding and Shah
Bifidobacterium bean gum, and carrageenan gum conditions (2009a)
species
Genetically modified Sodium alginate, cellulose sulfate Extrusion Preservation of high cell viability for long term Hucík et al. (2010)
Escherichia coli storage of the cells and
Schenkmayerová
et al. (2012)
Shewanella Sodium alginate, calcium chloride Extrusion Prolongation of viability of cells when exposed to Rosas-Ledesma et al.
putrefaciens harsh conditions (2012)
Lactobacillus reuteri Sodium alginate, calcium chloride Extrusion Protection of the probiotic against harsh GIT Zhao et al. (2012)
DPC16 conditions
Escherichia coli Mixture of potassium persulfate and d Microfluidic Encapsulation of cells for various applications Lee et al. (2010)
(+) sorbitol,G-oil, Abil EM 90 emulsification including biotransformation, biosensing,
bioremediation, and engineering of artificial cells
Saccharomyces Trimethylolpropane trimethacrylate, Emulsification Achievement of high encapsulation efficiency Takei et al. (2010)
cerevisiae 2,20-azobis(4-methoxy-2,4-
dimethylvaleronitrile), poly(vinyl
alcohol)
Pseudomonas species Sodium alginate, calcium chloride, Extrusion Enhancement of phenol degradation ability of the Mollaei et al. (2010)
SA01 chitosan microbial cells
378 S. Rathore et al. / Journal of Food Engineering 116 (2013) 369–381
spouted bed drying was found to have insignificant effect on the
cell viability (Oliveira et al., 2007a)
In case of microbial cells encapsulation, retention of cell viabil-
ity during storage holds special importance especially in the food
industry. Microencapsulation aids in improving the stability as
well as viability of the probiotic cells during processing and storage
conditions (Fávaro-Trindade and Grosso, 2002; Oliveira et al.,
2007b). The log count of encapsulated of L. acidophilus MJLA1 in
fermented frozen desserts was higher than free cells after12 weeks
of storage (Shah and Ravula, 2000). Storage temperature has been
found to be indirectly proportional to viability of encapsulated
cells. Decrease in temperature leads to increase in storage stability
(Lievense et al., 1994; Pedroso et al., 2012). For example, encapsu-
lated B. lactis encapsulated by spray chilling method showed log
reduction in cell viability 4.83 (37 °C), 4.82 (7 °C) and 1.85 log c-
fu/g (18 °C) after 90 d of storage whereas the free cells had a log
reduction of 7.80 (37 °C), 7.47 (7 °C) and 4.53 log cfu/g (18 °C)
(Pedroso et al., 2012).
Thus, the selection and optimization of a suitable microencap-
sulation process should be carried out such that the viability of
encapsulated cells is maintained during both processing and
storage.
10. Recent applications of microencapsulation of microbial cells
in various biotechnological processes
Owing to its significant advantages and versatility with respect
to a wide range of materials and techniques available, microencap-
sulation has been used to immobilize cells for a variety of pur-
poses. However, commercial success has been obtained mostly
for microencapsulation of probiotics in the food industry. Apart
from probiotics, utilization of encapsulated cells has also been car-
ried out for fermenting microorganisms and for environmental
protection/decontamination. A list of some recent applications of
this versatile technology in various biotechnological applications
is shown in Table 2.
11. Conclusion
A wide range of materials has been used for the purpose of
microbial cell encapsulation. There are many innovative microen-
capsulation techniques reported for microbial cells. However, the
applicability of these methods for large scale production still needs
to be assessed. Stability of microspheres is also a very important
consideration for their utilization in various applications and this
should be carefully considered. Cell physiology was found to be
significantly altered after confinement within the microspheres.
Various methods for detection and measurement of the altered
physiology need to be developed and standardized. Another impor-
tant challenge in this field is to develop non-destructive, accurate
and rapid methods for the enumeration of viable encapsulated
cells.
Acknowledgment
The authors acknowledge support by the Singapore Ministry of
Health’s National Medical Research Council under IRG NMRC/
1187/2008 (R-148-000-114-213) and GEA-NUS PPRL fund
(N-148-000-008-001).
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