Trends in Food Science & Technology 109 (2021) 340–351

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Trends in Food Science & Technology

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

Novel approaches for co-encapsulation of probiotic bacteria with bioactive

compounds, their health benefits and functional food product development:
A review
Sourav Misra *, Pooja Pandey, Hari Niwas Mishra
Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal, 721 302, India

A R T I C L E I N F O A B S T R A C T

Keywords: Background: The growing awareness of functional food products for imparting potential health benefits has
Co-encapsulation focused on the co-encapsulation of probiotic bacteria with bioactive compounds in a single matrix. The co-
Probiotic bacteria encapsulation process with low cost and improved bioactivity is a novel approach for the development of
Bioactive compounds
therapeutic and nutraceutical food products.
Functional food products
Scope and approach: This review focuses primarily on the advantages of co-encapsulation of probiotic bacteria
with bioactive compounds such as omega-3 fatty acids, green tea extracts, curcuminoids, antioxidants from fruit
juice extracts, agroindustrial co-products, prebiotics in a single delivery format by employing different tech­
nologies. The health potentials of probiotics and bioactives have been emphasized as well as the desired char­
acteristics of the produced co-microcapsules for incorporation in the food products has been briefly discussed.
Key findings and conclusions: This review emphasizes that co-encapsulation of bioactive compounds and probiotic
bacteria in a single product provides synergistic health benefits and enhances the bioactivity of individual
components thus improve the adherence of probiotic bacteria to the intestinal wall during digestion. This process
has convenience and cost advantages over microencapsulation of individual ingredients. The co-encapsulation
process has also improved the storability of food products with a long term stabilization of probiotic bacteria
and bioactive compounds. A future scope is to be needed to explore the release mechanism of these ingredients in
a simulated animal model system along with the incorporation of co-microcapsules in the formulation of cost-
effective functional food products.

1. Introduction an alternative way to tackle these problems instead of taking antibiotic

Probiotics are defined as living organisms when administrated in an
adequate amount confer a health benefit on the host (FAO/WHO, 2006).
They reach the consumer either as food products (fermentable or
non-fermentable) or as dietary supplements (in powder, tablet, or
capsule forms). Consumption of probiotics is gaining popularity due to
increased awareness of consumers. The application of probiotics in
improving the health of human beings is an innovative and cheapest
invention in society. The human body comprises of thousand groups of
microorganisms, but the presence of bad microbes causes many diseases
and illnesses due to the accumulation of toxic substances during the
digestion process. This is the point where the immune system starts
depleting. In such cases, the consumption of probiotics in higher doses is
treatment (Reid et al., 2003). Lactobacillus and Bifidobacteria strains are
the most used probiotic microorganisms; to provide health benefits, the
population of viable bacteria should be more than 107 CFU/mL or g end
products at the time of consumption (Adhikari et al., 2000). The pro­
biotic food products are coming under the functional food category and
its global market has expanded from $33 billion in 2000 to $176.7
billion in 2013 which accounts for 60–70% of the total functional food
market (Hennessy, 2014). The probiotics market was valued at USD
46.17 billion in 2017 and is projected to reach USD 69.29 billion by
2023, at a compound annual growth rate (CAGR) of 7.0% from 2018
(Liu et al., 2020).
The growing awareness of functional food products for imparting
potential health benefits has focused on the incorporation of bioactive

* Corresponding author. Food Chemistry and Technology Laboratory Agricultural and Food Engineering Department Indian Institute of Technology Kharagpur,
Kharagpur, West Bengal, 721 302, India.
E-mail addresses: sourav.misra@iitkgp.ac.in, sourav.omprakash@gmail.com (S. Misra).

https://doi.org/10.1016/j.tifs.2021.01.039
Received 8 October 2020; Received in revised form 9 January 2021; Accepted 15 January 2021
Available online 21 January 2021
0924-2244/© 2021 Elsevier Ltd. All rights reserved.
S. Misra et al. Trends in Food Science & Technology 109 (2021) 340–351

compounds such as essential oil, flavors, antioxidants, vitamins, natural Table 1

extract, and prebiotics (Chawda et al., 2017). The supplementation of Potential benefits of different probiotic strains.
these ingredients and probiotic cells may affect their preserving poten­ Probiotic Strain Potential benefits References
tial and organoleptic properties of food products, as well as the com­
Lactobacillus • Greater acid tolerance Muhammad et al.
pounds and bacteria should remain in a metabolically active state during plantarum • Highly resistive to dehydration (2017)
processing and storage. In this context, encapsulation is an effective processes
approach to overcome the problems but till date entrapment of single • Greater potentiality to ruin the
species (Probiotic bacteria or bioactive compounds) is a successful stomach related ailment causing
microbes
preservation method by avoiding their deterioration and achieving a • Exhibits antifungal and antioxidant
targeted release in the gut in an adequate amount (Sultana et al., 2000). activities
Before the selection of probiotic strains, their desirable properties • Diminishes the hypolipidemic
(Table 1) such as exhibiting tolerance to acidic and bile salt condition, activity
Lactobacillus sp., • Removal of cholesterol Dimitrellou et al.
possessing antimicrobial activity against pathogenic bacteria, etc.
Lactobacillus casei • Activity against cancer cell (2016)
should be taken into consideration. Probiotics that are ingested orally proliferation
have to survive in unfavorable conditions during their transit through • Effective to prevent the risk
the gastrointestinal (GI) tract with the ability to affect the gut micro­ associated with osteoporosis
flora. The gut microbiota plays an important role in modulating physi­ • Used for the development of
functional dairy products like
ological functions in the human body. The consumption of probiotics yogurt, cheese, and fermented milk
with natural bioactive compounds is a “green” alternative to drugs for Lactobacillus reuteri • Tolerant to acidic conditions, and Liu et al. (2016)
maintaining blood pressure, good mental health, boosting immunity, can adhere to the GI tract
eliminating sleep disorders, etc. especially in the situation of the • Produces many of the essential
vitamin B complex, such as vitamin
COVID-19 outbreak.
B1, vitamin B2, vitamin B9, and
However, some researchers have reported that co-encapsulation of vitamin B12
more than one bioactive compounds provides the synergistic effect with • Producer of antimicrobial
improved bioactivity and functionality than that of a single component. compounds, such as reuterin, and
This concept has been widely popularized in the pharmaceutical in­ organic acids, which act as
inhibitors for Rotavirus
dustry and has possibilities to emerge as a potential single micro de­
(Helicobacter pylori) and other
livery vehicle for probiotic strains and bioactives in the targeted colon pathogens
through food products. Co-encapsulation of probiotics with prebiotics Lactobacillus • Beneficial changes during cheese Peralta et al.
such as inulin, polydextrose has been effective to preserve their viability paracasei ripening (2017)
• Increase of peptidolysis
during the GI transit and the addition of antioxidants such as tocopherol,
• Used as a flavor enhancer
ascorbic acid in microcapsules has prevented the bacteria from the • Capacity to Control adventitious
detrimental conditions such as low pH and oxidative stress during pro­ microflora
cessing and storage (Atia et al., 2017; Chen et al., 2017). This review Bifidobacteria • Improves human health by Fritzen-Freire
focuses on the co-encapsulation of probiotic bacteria and bioactive stabilizing the intestinal microflora et al. (2012)
• Reinforces mucosal defense system
compounds such as omega-3 fatty acids, green tea extracts, curcumi­
in opposition to pathogens
noids, antioxidants from fruit juice extracts, agroindustrial co-products, Lactococcus lactis • Wide application in industrial Dijkstra et al.
prebiotics in a single matrix by employing different technologies along fermented food product (2014)
with the impact on their stability and bioactivity in simulated GI con­ development, including
manufacturing of butter (milk) and
dition and storage. The health potentials of probiotics and bioactives
cheese
have been briefly emphasized as well as the desired characteristics of the • Shows proteolytic activity and other
produced co-microcapsules for incorporation in the formulation of functional properties such as the
functional food products have been briefly discussed. production of bacteriocin
Lactobacillus sakei • Provides resistant activity against Ferreira et al.
L. monocytogenes, a foodborne (2005)
2. Co-encapsulation technologies pathogen
• Decreases accumulation of biogenic
2.1. Emulsification amine during sausage fermentation
Lactobacillus • Effective to prevent diarrhea, and Li et al. (2016)
rhamnosus GG infectious diseases in the urogenital,
This technique is one of the common methods for the co-
respiratory, or GI tracts
encapsulation of probiotics. This method consists of two phases i.e. • Used as a vaccine adjuvant
dispersed phase which contains cell polymer suspension and oil (vege­ Enterococcus faecium • Shows potentiality in food Kanmani et al.
table oil/mineral oil) or organic solution is considered as a continuous preservation and exhibits immune (2011)
phase. The emulsion is prepared by homogenizing the mixture with the stimulant properties
Lactobacillus • Possess antagonistic properties Otero et al. (2007)
aid of surfactants. The particles are formed within the oil phase by the delbrueckii subsp against E. coli and A. pyogenes and
application of a cross-linking agent or cooling process to insolubilize the delbrueckii exhibits a good adherence capacity
water-soluble polymer. The produced microbeads are subsequently Lactobacillus gasseri • Exhibits inhibitory effect on Otero et al. (2007)
centrifuged or filtrated. The size of beads is controlled by the agitation Staphylococcus aureus, which causes
bovine mastitis
speed, rate of addition of cross-linking agents, surfactant concentration,
Lactobacillus • Improves the shelf-life of bread by Stefanello et al.
and water to oil ratio (Kailasapathy, 2009; Shah & Ravula, 2000) which fermentum reducing fungal spoilage (2019)
may vary between 25 μm and 2 mm. Emulsifiers improve the stability of Lactobacillus • Produces antimicrobial compounds Kailasapathy and
emulsions by lowering surface tension and result in a smaller size of acidophilus such as bacteriocin, lactacin B and Chin (2000)
microspheres (Krasaekoopt et al., 2003). serves as a curing agent against
intestinal and urogenital infections
Different types of emulsification processes have been demonstrated as well as upper GI tract diseases
by different researchers for the microencapsulation of probiotics. • Possesses lactase activity, which
Emulsification with ionic gelation involves hydrocolloids such as algi­ helps in the digestion of lactose in
nate, pectin, carrageenan, sodium carboxymethyl cellulose, cellulose lactase-deficient persons.

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S. Misra et al.
acetate phthalate, chitosan, and gelatin and their solution as the
continuous phase and solidifying agent (calcium chloride). This method
results in a smaller diameter of microcapsule along with higher survival
of probiotics and is easier to scale up (Chen & Chen, 2007). The major
disadvantages include the produced microcapsules of different shapes
and sizes; it also requires a second polymer solution for the additional
coating to the cell (Kailasapathy, 2009). Emulsification with gelification
involves the application of milk protein for the encapsulation of pro­
biotics as it exhibits gelation properties and acts as a natural vehicle for
probiotics (Livney, 2010). Application of rennet, a proteolytic enzyme
complex for gelation has been carried out due to the production of casein
micelles by splitting the k-casein molecules (Heidebach et al., 2009). At
a temperature above 18 ◦ C, the flocculating micelles form gels in the
presence of non-covalent cross-linkages and can protect the cells at low
pH conditions (Bansal et al., 2007). This method has proved to be a
suitable method for food application as the size of the microcapsule can
be controlled, which affects the organoleptic acceptability of the final
food product. Emulsification with interfacial polymerization process is
used to encapsulate microbes to increase their productivity during the
fermentation process and an organic solvent is used as the continuous
phase in which aqueous solution with the probiotic cell is dispersed
(Yáñez-Fernández et al., 2008). A biocompatible agent, which is soluble
in the continuous phase, is added to initiate polymerization and produce
membrane coated droplets (Kailasapathy, 2002).
This process has been effectively applied for the co-encapsulation of
probiotics and bioactive compounds. Gaudreau et al. (2016)
co-encapsulated probiotic strain Lactobacillus helveticus and green tea
extracts (GTE) by emulsification/internal gelation process with pectin
and whey protein isolate as coating materials. The encapsulation yields
of bacteria and polyphenol were observed as 95.5 and 79%, respectively
at an initial GTE concentration of 500 μg/mL. This technique causes a
slight loss of bacterial viability that can be prevented by the addition of
GTE. The electrostatic interaction between the amino group of whey
protein isolate and the carboxyl group of pectin protected core in­
gredients (only 0.5–1.1 log CFU/g loss of viability) in simulated gastric
conditions. Similarly, chitosan-coated alginate co-microcapsule beads
containing B. infantis and B. breve with GTE were developed by Vodnar
and Socaciu (2012) and resulted in higher bacterial viability of above
9.0 log CFU/mL with the incorporation of 10% (w/v) GTE. The
co-encapsulation process outcomes 5.05 and 7.38% increase in viability
of B. infantis and B. breve in simulated intestinal juice condition as
compared to encapsulating them individually. Potential by-products
from agroindustry like apple marc and cactus pear peel flour as pre­
biotics have been successfully co-encapsulated with four probiotic
strains i.e. Lactobacillus plantarum, Enterococcus faecium, Aerococcus vir­
idans, and Pediococcus pentosaceus in alginate ionotropic gel matrix
(Serrano-Casas et al., 2017). The symbiotic co-gelification with pre­
biotics improved the viability of probiotics with the highest and lowest
value were observed for Aerococcus viridans (>77%) and Lactobacillus
plantarum (56.61%), respectively after 120 min exposure in low pH
condition. The co-encapsulation of probiotics Staphylococcus succinus
and Enterococcus fecium by the emulsion process with sugar beet and
chicory resulted in higher survivability of 88.75–98.75% in the simu­
lated gastric environment.
2.2. Spray drying
This is an old established method of microencapsulation, which has
been applied in the food industry since the late 1950 to prevent oxida­
tive degradation of flavor oils by converting the liquid form to powder.
The selection of shell materials with solubility properties at an accept­
able level for the spray drying process is limited as aqueous material is
required for this drying process. Typically, maltodextrin, hydrophobi­
cally modified starch, and their mixtures, gum acacia, alginate, car­
boxymethyl cellulose, guar gum, soy protein, whey protein, and sodium
caseinate are employed as coating materials to encapsulate different
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Trends in Food Science & Technology 109 (2021) 340–351
probiotics. Spray drying is an economical as well as the commonly used
process which provides flexibility and produces good quality products.
In this process, the emulsion is formed by dispersing the core material
into a polymeric solution followed by homogenization, and finally, the
mixture is atomized in the drying chamber to produce matrix type mi­
crocapsules (Jackson & Lee, 1991).
This process can be operated continuously which requires proper
control and adjustment of the processing parameters such as inlet and
outlet temperature that can achieve the desired viability of the encap­
sulated culture. Spray drying involves atomization of the liquid feed into
fine droplets of size 10–150 μm followed by spraying into the hot and
dry air of temperature 150–250 ◦ C. Spray drying consumes less specific
energy as compared to freeze drying. The probiotic strain for spray
drying should be selected based on their resistance to osmotic, oxidative,
and thermal stress. Different species such as Lactobacillus, Lactococcus,
and Bifidobacterium are more sensitive to spray drying conditions due to
the application of high temperatures. Due to the reduction of water
activity, bacteria become resistant to osmotic stress and maintain their
viability by accumulating compatible solutes such as carbohydrates,
amino acids, or quaternary amines (Wood, 2011). Industrial-scale spray
dryers equipped with pneumatic devices have provision to maintain the
viability by cooling the powder immediately.
Different supporting materials have been used in various experi­
ments as protective agents to the probiotics. Among carbohydrates,
trehalose was found to be most effective by showing the stabilizing effect
on membranes and proteins (Morgan et al., 2006). Other carbohydrates
which include sorbitol, xylose, mannitol, sucrose, lactose, maltose,
glucose, dextrose, inulin, maltodextrin, galactooligosaccharide, potato
starch, and fructooligosaccharide have been used as drying matrices
(Perdana et al., 2014).
Spray drying has been proved as a low-cost technology for micro­
encapsulation of probiotics but limited research has been done on co-
encapsulation, which may be the negative impact of high temperature
on the stability of bioactives. The spray drying was carried out to co-
encapsulate resveratrol and probiotic Bacillus clausii at an inlet tem­
perature and outlet temperature of 210 and 70 ◦ C, respectively with a
pressure of 1.5 bar (Vázquez-Maldonado et al., 2020). The survivability
of bacteria in both lactose and inulin was found to be 8.62 and 8.52 log
CFU/g, respectively as well as both the carrying agents showed similar
antioxidant activity (23% for lactose and 21% for inulin). It may be a
hypothesis from the observation of this study that the culturability of
probiotic cells and their absence in scanning electron microscopic graph
indicates the presence of living cells inside the coating materials after
the spray drying process but further study is required to prove this ev­
idence. The probiotic strain Lactobacillus plantarum was co-encapsulated
by γ-aminobutyric acid (GABA), a major inhibiting neurotransmitter by
spray drying using exopolysaccharides such as inulin, dextran, and
maltodextrin at an air inlet temperature of 110 ± 2 ◦ C and outlet tem­
perature 50 ± 5 ◦ C with a feed rate of 2.5 mL min− 1. The drying pro­
duced thermostable powder with probiotic encapsulation and GABA
efficiency of 99.21 and 84.22%, respectively with a drying yield of more
than 50% as well as the co-microcapsule powder exhibited good flow­
ability and regular spherical shape. The thermal stability and desired
particle size (10–35 μm) of the co-microcapsule in the above study
shows its potent application in the formulation of thermally processed
foods.
2.3. Freeze drying
Freeze drying or lyophilization is also a widely used and convenient
method for enhancement of the storability of microencapsulated pro­
biotics. The process can be summarized in three major phenomena such
as freezing, primary and secondary drying. A high freezing rate avoids
extensive cellular damage than a slow freezing rate due to the formation
of small ice crystals (Fowler & Toner, 2006). The process conditions such
as freezing rate and temperature can be controlled that can affect the
S. Misra et al.
growth of ice crystal. During freezing, chemical and osmotic damage
happen due to the crystallization of water and concentration of solutes
in remaining unfrozen fraction. Removal of frozen water occurs due to
the sublimation process by applying vacuum conditions in the primary
drying and the remaining unfrozen water is desorbed in the secondary
drying process (Maltesen & van de Weert, 2008). Though it is a
well-known method of drying, this process carries several demerits such
as it can damage the cellular membrane, surface proteins, and cell wall,
thus declining the survivability after drying as water plays a crucial role
to maintain the cell stability and integrity. Freeze drying is an expensive
and time-consuming method. Production of powder particles also re­
quires an additional process (Maltesen & van de Weert, 2008).
Selection of suitable wall materials, optimization of the process pa­
rameters, pre-stressing of the bacterial sample before freeze drying
affect the survivability of probiotic strain during freeze drying. Appli­
cation of cryoprotectants such as trehalose and sucrose has been studied
to improve the survivability of probiotic strain during freeze-drying.
Probiotic cells present in the unfrozen fraction are subjected to
compression during the formation of ice crystals. The application of
cryoprotectants increases the unfrozen fraction that results in producing
more space to the probiotics thus preventing the cells from cellular
damage as well as osmotic or mechanical stress.
The co-encapsulation of probiotic strain Lactobacillus casei ssp. par­
acasei and black currant extract by freeze drying resulted in encapsu­
lation efficiency of 87.38 and 95.46%, respectively using inulin, whey
protein isolate, and chitosan as coating materials (Enache et al., 2020).
The produced dark purple powder showed good retention stability of
phytochemicals with antioxidant activity (63.64 ± 0.75 mg Trolox/g dry
weight) as well as storage stability with only 27% reduction in total
anthocyanin content after 3 months at 4 ◦ C. The chitosan-coated
co-microcapsules produced by freeze drying showed 85.72 and
82.74% survival rate of Lactobacillus acidophilus with prebiotics Jeru­
salem artichoke and inulin, respectively (Jantarathin et al., 2017). The
lower pressure and temperature during freeze drying results in higher
retention of bioactive compounds in the produced co-microcapsules but
the high operational cost limits its application in the co-encapsulation
context.
2.4. Extrusion
Extrusion is a simple, low-cost technology that is widely employed
for co-encapsulation of probiotic strains with higher cell viability
(Krasaekoopt et al., 2008). In this method, the polymeric solution
(generally hydrocolloid solution) is mixed with microbial cells, and
projecting the suspension through a syringe needle into a solution of a
cross-linking agent at high pressure that results in gelation. Several
factors affecting the size of produced microspheres are the viscosity and
flow rate of the polymeric solution, concentration, and temperature of
the polymer solution, the diameter of the orifice, and drop height or
distance from the orifice to the cross-linking solution (Brun-Graeppi
et al., 2011). The major drawbacks are involved in direct extrusion
processes such as this method is inefficient to produce microcapsules
lesser than 500 μm, the need for large diameter nozzle, and delays in the
production of microspheres for which it is difficult to scale up (Burgain
et al., 2011).
Different processes have been evolved to overcome these drawbacks.
Prilling is a technique when the droplets are formed at controlled
environment conditions (as opposed to spray drying) which is per­
formed by the vibration or pulsation of the jet nozzle. The process
involved in the formation of a charged stream of small droplets due to
disruption at the needle tip by the application of the electrostatic field is
known as coaxial flow or electrostatic field extrusion. The size of beads
can be governed by fluctuating the applied electric potential as well as
this method avoids the application of an organic solvent. Centrifugal
extrusion is another process, based on the co-extrusion principle that
involves a nozzle fitted with concentric orifices placed on the outer
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Trends in Food Science & Technology 109 (2021) 340–351
periphery of a rotating cylinder. Some advanced extrusion methods are
multi-nozzle systems, acoustic or vibration energy, rotating disc atom­
ization, and liquid jet cutting technique (Rathore et al., 2013).
Co-encapsulation of probiotic Lactobacillus plantarum with cis-4, 7,
10, 13, 16, 19-docosahexaenoic acid (DHA) rich oil was carried out by
extrusion technology and the highest survivability of probiotics
(88.66%) was found at the combination of 0.55% pectin, 0.39% gelatin,
and 1.06% alginate along with encapsulation efficiency of DHA-rich oil
in co-microcapsules was achieved as 69.37% (Vaziri et al., 2018). The
produced co-microcapsules were of high moisture content (13.67%) and
thermally stable. Kumherová et al. (2020) co-encapsulated Bifiobacte­
rium animalis subsp. lactis with inulin (1% w/w) and ascorbic acid (0.5%
w/w) with alginate by extrusion method. This process showed improved
viability of bacteria (10.3 log CFU/mL) than the emulsification process
with milk protein (8.2 log CFU/mL) as well as the alginate capsules are
bigger (1.7 mm) than the protein capsules (204 μm). The larger particles
produced by the extrusion method should be taken into consideration
for application in food product development.
2.5. Coacervation process
Co-encapsulation by the coacervation method has gained popularity
in the recent past owing to its application in many fields such as food,
agriculture, cosmetics, printing, pesticide, adhesive, and pharmaceu­
tical industries. This technique involves the formation of coacervate
resulted from phase separation of biopolymers, under specific compo­
sition, temperature, or pH of the solution and finally microsphere is
formed by the deposition of coacervate around the core ingredients
(Gouin, 2004). The microspheres can be strengthened by adding
different enzymatic or chemical cross-linking agents. The produced
liquid microspheres are an inconvenience to use as it is in the dispersed
phase, which alternatively provides lower stability of probiotics than
dry products as well as reduced shelf life and higher handling cost
minimizes the efficacy of the process. To overcome these problems,
liquid microspheres are subjected to a variety of dehydration methods to
only reduce the water content. The coacervation processes are classified
as two types viz: simple and complex. Simple coacervation involves
varying the condition of causing molecular dehydration of the macro­
molecules and different factors like change in temperature or pH, the
addition of non-solvent, or microions to the solution. Complex coacer­
vation is the ionic separation of macromolecules into two immiscible
liquid phases. The coacervation process results in good entrapment ef­
ficiency with controlled release of core active material when exposed to
changes in pH, and mechanical stress (Oliveira et al., 2007).
Eratte et al. (2015) co-encapsulated probiotic Lactobacillus casei and
omega-3 fatty acids rich tuna oil by complex coacervation-spray drying
and freeze drying in whey protein isolate-gum Arabic matrix. The spray
dried co-microcapsules exhibited lower residual moisture content
(3.19%), reduced water activity (0.26), higher oil encapsulation effi­
ciency (93.35%), and lower viability of probiotics (56.19%) as
compared to freeze dried coacervate co-microcapsules. The freeze dried
powder was characterized by high surface carbohydrate content while
high surface protein was found at spray dried co-microcapsules. Holkem
et al. (2019) performed co-encapsulation of probiotic Bifidobacterium
animalis subsp. lactis and proanthocyanidin-rich cinnamon extract by
coacervation followed by freeze drying with the aid of whey protein
concentrate and gum Arabic in the emulsion. This process produced a
reservoir-type co-microcapsule having a spherical shape, as well as the
homogenization process did not cause any rupture in cells. Later the
researchers incorporated the co-microcapsules in sugarcane juice which
has been discussed in the later part.
2.6. Electrospraying
There are different techniques such as coacervation, spray drying, or
emulsifying-cross linking technologies which involve the use of high
S. Misra et al.
temperature as well as organic agents which lead to the deterioration of
heat-sensitive encapsulated active substances. These above processes
may involve some problems due to toxicity associated with organic
chemicals (Birnbaum et al., 2000). Some alternative techniques should
be used which do not imply the application of high temperature and use
of organic agents. In this case, high voltage spinning, commonly known
as the electrospinning method is satisfying the above criteria. It is an
advantageous process due to its simplicity, high versatility, and pro­
duces capsules in the nano to the micro range. This method of producing
capsules is also called as electrospraying. This process involves an
application of the external electric field between two electrodes and
then imposed on a polymer solution without the requirement of tem­
perature. This method produces encapsulated particles of the large
surface to volume ratio. There is also a minimum requirement of organic
solvents for the dissolution of a polymeric substance followed by elec­
trospinning. Electrospinning of biopolymers from watery solutions can
be performed by controlling the process condition and varying the
properties of the solution by adding proper additives which shows the
potentiality in food applications (López-Rubio et al., 2012). Electro­
spinning or electrospraying is an electro-hydrodynamic atomization
process. This process involves high voltage either to produce particle
(electrospraying) or fibers (electrospinning) to avoid the thermal tech­
nologies which lead to damage to the bacterial cell.
Some natural biopolymers such as polysaccharides and proteins can
be used as encapsulation matrices as they are surface-active materials
and also categorized under amphiphilic macromolecules that have
greater importance in stabilizing food formulations. Some synthetic
encapsulating polymers such as Eudragit® or polyethylene oxide cannot
be used for food formulations but have a wide scope in the pharma­
ceutical application (Sobel et al., 2014). The selected biopolymers not
only have an impact on the vitality but also decide the solubility as well
as the thermal and mechanical stability of the probiotic cells.
Limited findings on the applicability of this technology for co-
encapsulation of probiotic and bioactive compounds have been
explored. Zaeim et al. (2019) showed 98, 79, and 99% of encapsulation
yield for probiotic bacteria (Lactobacillus plantarum and Bifiobacterium
lactis), inulin, and resistant starch, respectively in
Ca-alginate/chitosan-coated electrosprayed co-microcapsules. The pro­
longed storability at room temperature (6.33 log CFU/g of viability after
90 days), smaller particle in micron size as well as protected capability
of carrier materials in simulated GI condition (>6.0 log CFU/g for both
probiotics) showed the potentiality of this technique in food
applications.
3. Co-encapsulation of probiotic bacteria and bioactive
compounds
3.1. Co-encapsulation of probiotic bacteria and omega-3 oil
Omega-3 fatty acids have been proved as physiologically essential
fatty acids from the point of human health benefits. The main bioactive
compounds of the polyunsaturated fatty acids (PUFAs) are cis-5, 8, 11,
15, 17- eicosapentaenoic acid (EPA), and cis-4, 7, 10, 13, 16, 19-docosa­
hexaenoic acid (DHA). These bioactive compounds are crucial for
numerous physiological functions such as improving memory processing
and immune power, preventing cardiovascular diseases, combating
neural disorders, diabetes, and some forms of cancer, helping in brain
and vision development in infants, and preventing Alzheimer’s disease
(Eratte et al., 2017). The global demand for omega-3 fatty acids has been
raised to 4.0 billion USD in 2018 from 1.6 billion USD in 2010 (Trans­
parency Market Research, 2013, p. 2018). An adequate amount of
omega-3 content should be consumed through foods (Kris-etherton
et al., 2002). The problem associated with omega-3 rich foods is lipid
oxidation which impairs nutritional value and flavor as well as shortens
the shelf life of the product and ultimately causes loss in bioactivity
(Barrow & Shahidi, 2007).
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A higher amount of omega-3 fatty acids are present in fish oil as
compared to microalgae and seed oils (Rubio-rodríguez et al., 2010).
Therefore, stabilization of omega-3 fatty acids by microencapsulation
technology has been employed for successful fortification in a range of
food products such as bread, fruit juices, chocolate, milk, yogurt drinks,
and tortillas (Eratte et al., 2017) and the inherent fishy odor of fish oil
can also be prevented (Nickerson et al., 2013).
Microencapsulation of DHA fatty acids and probiotic bacteria have
been studied broadly in separate matrices, but co-encapsulation of these
ingredients as a single entity provides synergistic effects and enhances
the bioactivity of individual components (Halwani et al., 2008). The
synergistic effect also helps probiotic strains attach to the intestinal
linings during digestion (Das, 2002). This process has convenience and
cost advantages over the microencapsulation of individual ingredients.
This cost-effective approach has been widely employed in the pharma­
ceutical industries.
Co-encapsulation of Lactobacillus plantarum with DHA rich oil
resulted in higher thermal stability and entrapment efficiency of pro­
biotics but the retention of DHA fatty acids was not significantly affected
by the bacterial activity (Vaziri et al., 2018). This study revealed that the
presence of DHA rich oil caused a lower swelling rate in gastric condi­
tions due to the presence of oil in or on the surface of capsules as well as
the addition of gelatin and pectin in the alginate matrix significantly
enhanced the resistance of encapsulated materials in GI conditions. The
co-encapsulation process of L. casei with coconut oil and omega-3 rich
tuna oil resulted in higher viability due to the less prone to oxidative
reaction (Eratte et al., 2015). The produced co-microcapsules also pro­
vided greater oxidative stability and thus high retention of omega-3 fatty
acids than that of microcapsules (Eratte et al., 2016). The retention of
omega-3 fatty acids can be improved by the combination of phytosterol
esters and fish oil as core ingredients in co-microcapsules due to the
antioxidant property. The application of other antioxidants such as
limonene and eugenol can also be employed for the protection of
omega-3 fatty acids and probiotics in the microcapsules. Eratte et al.
(2017) observed that microencapsulation resulted in higher viability
loss in the simulated gastric fluid as compared to the
co-microencapsulation process which might be due to the shielding ef­
fect of omega-3 oil and whey protein isolate during the gastric simula­
tion. The dietary PUFA, particularly EPA improves the viability of
probiotic bacteria by promoting the secretion of immune signaling
protein TGF-β1 (Transforming Growth Factor β1) by gut microbiota
(Bentley-hewitt et al., 2015). The co-encapsulation process also
increased the adherence ability of bacterial cells to intestinal linings in
terms of surface hydrophobicity than that of microencapsulated cells but
the degree of cholesterol assimilation was not affected by these pro­
cesses significantly (Eratte et al., 2017). The stability of bioactive in­
gredients and biocompatibility of co-microcapsules during storage
encourages the scaling up the food products of high nutritional status
with lower processing cost (Fig. 1).
3.2. Co-encapsulation of probiotic bacteria and green tea extract
Green tea (Camelia sinensis) is a popular beverage in the world and a
rich source of polyphenols. It possesses numerous health-promoting
properties. The primary polyphenolic compounds in green tea or
green tea extracts (GTE) are epicatechin-3-gallate (ECG),
epigallocatechin-3-gallate (EGCG), epicatechin, and epigallocatechin
(EGC) (Iyer & Kailasapathy, 2005). Green tea catechins exhibit antiox­
idant, cancer chemopreventive, anti-inflammatory, and anti-aging
properties (Gaudreau et al., 2016). EGCG is the most active and abun­
dant component of green tea leaves with various biological functional­
ities such as antitumorigenesis, antimutagenicity, antimicrobial activity
against gut pathogens, and free radical scavenging activity (Du et al.,
2012, pp. 1679–1691; Si et al., 2006). It was reported that catechins also
increased the survival of probiotic bacteria in simulated GI conditions
(Vodnar & Socaciu, 2012) as well as food products during the storage
S. Misra et al.
Fig. 1. Schematic diagram of different co-encapsulation technologies for producing microcapsules containing probiotic bacteria and bioactives in a single matrix and
their incorporation to formulate functional food products with the potential health benefits in the human body.
period (Shah et al., 2010).
Co-encapsulation of GTE with probiotic bacteria could provide a
synergistic effect. GTE being an antioxidant could be used to protect the
oxygen-sensitive probiotic bacteria from the combined stresses of oxy­
gen and acid during the transit through the upper part of the GI tract and
potential health benefits to the host. It could be effectively supple­
mented as food ingredients to deliver those compounds to the targeted
colon.
Gaudreau et al. (2016) co-encapsulated probiotic bacteria Lactoba­
cillus helveticus and GTE by the emulsification/internal gelation tech­
nique. It was found that the addition of 500 μm/mL GTE prevented
bacterial loss and resulted in the highest bacterial and polyphenol
encapsulation yield as well as increased radical scavenging activity of
microparticles. In this study, the pectin-whey protein complex enhanced
the viability of entrapped bioactive compounds in simulated GI condi­
tions. The produced co-microparticles can be directly ingested or sup­
plemented in food products as these contain higher polyphenol
concentration than the level of green tea catechins (10–100 μM) to
provide in vitro biological effects (Liao et al., 2001). The hydrophilic
nature of catechins may result in better stability and bioavailability of
probiotic strains when co-encapsulated with a hydrophobic compound
such as curcumin in optimized processing and storage conditions.
Co-encapsulation of B. breve and B. infantis with GTE in chitosan-coated
alginate beads was studied by Vodnar and Socaciu (2012). The survival
rate of bacteria increased in 10% GTE samples than the microcapsules
without GTE in simulated GI conditions. The ability of green tea to
stimulate the growth and multiplication of probiotic bacteria can be
justified by the mechanism which involves antioxidant and antiradical
properties of polyphenols that modulate the oxidative stress in the me­
dium thus preventing the degradation of cells in the co-encapsulation
process (Molan et al., 2009). Some studies also revealed that quercetin
resulted in lower survivability of probiotic bacteria in microcapsules due
to the interaction with flavonoid (Iyer & Kailasapathy, 2005).
3.3. Co-encapsulation of probiotic bacteria and curcuminoids
Curcuminoids are the collective compounds comprising of three
molecules namely, curcumin (76–80% w/w), demothoxycurcumin
(12–15% w/w), and bisdemethoxycurcumin (2–5% w/w), generally
obtained from turmeric rhizomes by the solvent extraction process.
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Curcumin possesses antidiabetic, antimicrobial, anticancer, anti-
inflammatory, antioxidant, anti-arthritic, antidepressant, anti-
atherosclerotic, anti-aging, memory enhancing, and wound healing
properties.
Co-encapsulation of both curcuminoids and probiotics offers a syn­
ergistic effect and acts as a beneficial health supplement that will pro­
vide targeted delivery of both the compounds efficiently.
Madhavamenon and Maliakel (2019) observed that bioavailability, ab­
sorption, and half-life of the encapsulated curcuminoids increased after
oral administration, compared to that of non-encapsulated ones and
claimed that a dose of 250–1000 mg of encapsulated powder would
provide 106–1012 CFU/g of probiotic bacteria as well as more than 20%
concentration of curcuminoids. In this context, the gel formed by the
galactomannan-rich fenugreek fiber strongly bound the curcuminoids in
the hydrophobic pockets whereas, ragi matrix enhanced the viability of
probiotic organisms and acted as a growth medium. The targeted de­
livery capacity of the formulated powder in the gut indicates an
emerging process to incorporate curcuminoids along with the probiotic
strains in food products.
3.4. Co-encapsulation of probiotic bacteria and bioactive compounds
from fruit juice
Food products enriched with functional properties such as antioxi­
dants (anthocyanins and polyphenols) and essential nutrients have high
consumer demand in this recent decade to prevent many diseases, such
as cancer, inflammation of internal organs, and congestive heart failures
(Ivanovic et al., 2014). Fruits, tubers, and legumes are extensively
applied to fulfill the above requirements as natural sources of bioactive
compounds as well as to reduce the risk and secondary effects (Dimi­
trios, 2006). Black currant (Ribes nigrum) is an abundant source of ca­
rotenoids, anthocyanins, phenolic acids, flavonoids, quercetin,
myricetin, vitamin C, and proanthocyanidins (Enache et al., 2020). Due
to the presence of these bioactive compounds, these berry fruits are
prescribed for consumption to prevent cancer (Bishayee et al., 2011).
Among these phenolic compounds, anthocyanins are the prominent
group of Black currants, which include cyanidin-3-O-glucoside, delphi­
nidin-3-O-glucoside, cyanidin-3-O-rutinoside, and delphinidin-3-O-r­
utinoside (Enache et al., 2020).
Co-encapsulation of blackberry juice and Lactobacillus acidophilus has
S. Misra et al.
been carried out by spray drying (at 130 ◦ C air inlet and 60 ◦ C outlet
temperature with 40–50 mBar atomizing pressure) in the blend of bio­
polymeric materials (whey protein concentrate (WPC), maltodextrin
(MD), and gum Arabic (GA) (Colín-Cruz et al., 2019). In this study,
GA-MD microcapsules resulted in higher retention of phenolic com­
pounds (98.4%) and anthocyanins (99%) than other combinations. The
hydroxyl group (OH-) of GA forms a hydrogen bond with water from
blackberry juice thus protecting the encapsulating materials in the
coating matrix. The electrostatic interaction between
anthocyanin-GA-MD and hydrophilic nature of GA and MD protects
anthocyanins from degradation. WPC exhibited the highest encapsula­
tion efficiency for probiotic bacteria (81.2%) due to the interaction of
phospholipid of polar groups of the probiotic membrane with WPC thus
causing limiting the excessive loss of water, whereas the lower encap­
sulation efficiency was achieved in MD-WPC microcapsules. Enache
et al. (2020) co-encapsulated black currant extract and Lactobacillus
casei ssp. paracasei using inulin, whey protein isolate, and chitosan by
freeze drying and satisfactory stability of bioactive compounds and
bacteria was achieved up to 3 months of storage at 4 ◦ C. The carrier
materials also showed a protective effect on anthocyanins even after
90–120 min of exposure to gastric condition but the total liberation of
bioactive compounds from microcapsules was observed during intesti­
nal digestion. These results also demonstrated an inhibitory effect of
co-microencapsulated powder for α-amylase (87.10%) and α-glucosi­
dase (36.96%), the major enzymes for carbohydrate metabolism. Hence,
it is proved that the co-microencapsulation process is a potent way to
deliver these ingredients with greater stability and exhibits
cholesterol-lowering and anti-diabetic potential.
3.5. Co-encapsulation of probiotic bacteria and agroindustrial co-
products
In the food processing chain, the transformation step produces agro
waste products that cover nearly 39% of the loss in developed countries
(Serrano-Casas et al., 2017). The non-consumed parts of plants are
generally discarded due to low consumer acceptance, like peels and
seeds. Apple skin is also discarded as waste products, which is a rich
source of bioactive compounds such as pectin, polyphenols, and vitamin
C. The utility of these by-products is now getting a concern as these are
an abundant source of carbohydrates, proteins, dietary fiber, phenolic
compounds, and carotenoids as well as the recovery of these compounds
from by-products will have great economic potential (van der Goot
et al., 2016). Fruit polyphenols also possess preventing capacity against
chronic diseases with potential anti-inflammatory, antioxidant, and
anticarcinogenic effects (Shinde et al., 2013).
Serrano-Casas et al. (2017) studied on co-encapsulation of four
probiotic strains with agricultural co-products such as apple marc or
cactus pear peel flour as alternate prebiotic ingredients. Cactus pear peel
is generally discarded as waste but cactus pear is a cheap source of di­
etary fiber and exhibits a hypoglycemic effect (SAGARPA, 2016). Inulin
and apple marc presented higher viability of probiotics due to the
presence of insoluble carbohydrates as well as co-encapsulation pro­
vided better structural integrity with prebiotics to protect cells by
increasing their tolerance to acidic conditions (Serrano-Casas et al.,
2017). Symbiotic co-gelification of alginate with prebiotics can be
employed for food product development with therapeutic effects by
achieving targeted delivery of probiotic bacteria in the colon through
the GI tract. There was only 0.13 log reduction of probiotic count in milk
after 50 days when co-encapsulating L. acidophilus with apple skin
polyphenol extracts by co-extrusion technology and the higher viability
(>106 CFU/mL) was achieved due to the antioxidant property of poly­
phenols but a higher loss of viability was found (1.1 log CFU/mL
reduction in cell count) for unencapsulated probiotic bacteria and these
microcapsules can be supplemented in acidic beverages like fruit juices
and yogurt (Shinde et al., 2013). In this study, the method of polyphenol
extraction was one of the major influential factors for retaining the
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viability of bacteria such as ethanolic apple skin extracts showed a
higher reduction of cell count (0.16 log CFU/mL) as compared to
aqueous apple skin extracts (0.13 log CFU/mL).
3.6. Co-encapsulation of probiotic bacteria and resveratrol (RSV)
Resveratrol (3, 4’, 5-trans-trihydroxy-stilbene) is a polyphenolic
compound generally found in berries, peanuts, grapes, and medicinal
plants. Among these, berries juices, and red wine are the most abundant
dietary source of resveratrol (Banez et al., 2020). The regular intake of
RSV containing food products may prevent many diseases such as
obesity, cancer, cardiovascular, aging, diabetic, inflammatory, and
neuropathies effects (Huang et al., 2019). It also possesses antioxidant,
antibacterial, antiparasitic, antifungal, and antiviral activity (Bostan­
ghadiri et al., 2017). The RSV rich food supplements have gained lots of
attention these days due to its enormous health benefits.
Co-encapsulation of probiotic bacteria and resveratrol may preserve
the bioactivity in the harsh condition of the intestine and low pH as well
as it may result in a higher stabilization with controlled-release during
the GI transit. Vázquez-Maldonado et al. (2020) emphasized on the
synergistic effect of co-encapsulated probiotics on the conservation of
antioxidants by employing inulin and lactose as protective agents. It was
observed that both the carrier materials provided a similar
thermo-protective effect on probiotics as protein denaturation was
prevented due to the hydrogen bonding interaction. In this study,
co-encapsulation resulted in lower survivability of bacteria as compared
to the encapsulation process but the concentration of bacterial cells was
above the recommended value to exhibit probiotic effect while incor­
porating in food products. This result can be correlated with the inhib­
itory ability of RSV to transport electron, which ultimately degrades cell
proliferation and finally inhibition of cell division (Vázquez-Maldonado
et al., 2020). Inulin as wall material protected much better to encap­
sulated RSV than lactose and proved as a prominent encapsulating
material for bioactive compounds due to the higher antioxidant activity
(Silva et al., 2016). The release profile of RSV and other bioactives such
as curcumin, gallic acid may also depend on the presence of functional
groups and their interaction with the wall materials. For example, RSV
has one more hydroxyl group than curcumin that results in strong
interaction with the coating materials. So, suitable encapsulating poly­
mers play a major role to assure the functionality of bioactives.
3.7. Co-encapsulation of multiple probiotics
Han et al. (2020) co-encapsulated L. bulgaricus and L. paracasei using
whey protein isolate and sodium alginate by internal/external gelation
method and higher encapsulation efficiency was achieved in whey
protein isolate (90.54%) as compared to calcium-alginate (84.46%)
coated microcapsules. The addition of trehalose also improved the
viability from 3 to 41.26% of encapsulated probiotics during freeze
drying due to the high glass transition temperature. Whey protein isolate
can improve the buffering capacity of the medium and protect the
probiotics from the adverse impact of the acidic environment but
calcium-alginate is more effective in bile salt condition though both the
wall materials are reducing the diffusivity of bile salts into microcap­
sules (Vargas et al., 2015). Alginate forms a continuous,
thermo-irreversible, and stable three-dimensional network by interact­
ing with calcium thus avoiding heat shock to probiotics (Rather et al.,
2017).
3.8. Co-encapsulation of probiotics and prebiotics
Kumherová et al. (2020) prepared capsules containing probiotic
strain Bifiobacterium animalis subsp. lactis Bb12 co-encapsulated with
ascorbic acid and inulin by emulsion and extrusion method. The
co-encapsulation process resulted in a lower reduction in the surviv­
ability of probiotic bacteria in both protein and alginate capsules and
S. Misra et al.
showed greater resistance to the conditions of the simulated digestive
tract. The combination of prebiotic inulin with an antioxidant ascorbic
acid may be applicable for the formulation of bioactive compounds
enriched food products with higher preservation of sensitive bacteria
during processing and storage. A lower amount of inulin (5%) in
co-encapsulated alginate beads not only provided the mucoadhesive
properties of probiotic strains Lactobacillus salivarius, Lactobacillus reu­
teri, and Pediococcus acidilactici Ul5 but also improved the stability of
core ingredients in simulated intestinal condition but the destabilization
of the polymeric network was observed at 20% inulin content (Atia
et al., 2017). Another research revealed that the viability of
chitosan-coated probiotic bacteria Lactobacillus acidophilus TISTR 1338
with Jerusalem artichoke was significantly higher than the
co-encapsulated cells with inulin and showed improved stability in
thermal processing as well as freeze drying. Jerusalem artichoke is a rich
source of fructooligosaccharides and different factors such as the degree
of polymerization, degree of branching and glycosidic linkage affect the
encapsulated bacterial viability (Jantarathin et al., 2017). Sathyabama
et al. (2014) explored renewable carbohydrate sources such as sugar
beet and chicory, which are rich in potential oligosaccharides for
co-encapsulation of probiotic strains Enterococcus fecium and Staphylo­
coccus succinus. This study reported that sugar beet beads were more
stable in bile condition but chicory beads resulted in higher viability of
probiotic strains in intestinal conditions. The co-encapsulation of strains
with these cheap sources of prebiotics can be incorporated into food
products which will be economically feasible. Zaeim et al. (2019)
applied the electro-hydrodynamic atomization technique for
co-encapsulation of Lactobacillus plantarum and Bifiobacterium lactis with
high molecular weight prebiotics such as resistant starch and inulin into
a single alginate/chitosan matrix. Microcapsules containing inulin
showed improved viability during storage but resistant starch performed
higher preservation of probiotics in GI conditions. Among different ol­
igosaccharides such as isomalto-oligosaccharides, xylo-oligo­
saccharides, fructo-oligosaccharides, and galactooligosaccharides,
fructo-oligosaccharides was the best co-encapsulating agent for the
protection of Lactobacillus fermentum L7 when exposed to the simulated
GI conditions and storage condition at 4 ◦ C (Liao et al., 2019). The
increasing in the concentration of inulin from 0 to 2.63% increased the
encapsulation efficiency of γ-aminobutyric acid (GABA) as well as the
dextran and inulin as prebiotics had a positive effect on the survival of
Lactobacillus plantarum but a negative result was found at higher con­
centrations due to their stickiness properties (Pandey & Mishra, 2020, p.
110293). The co-encapsulation of GABA with probiotics has the poten­
tiality to prevent anxiety, stress, diabetes, cancer as well as improve the
gut health of human kinds but the stability of GABA in acidic conditions
of the GI tract is to be verified yet.
4. Desirable characteristics of co-microcapsules for
development of functional food products
The residual moisture content of microencapsulated powder is a
crucial indicator for participating to affect the survivability of pro­
biotics. It has been reported that residual water content within the range
of 2.8–5.6% is preferable for long term preservation of probiotics in
powder particles (Khem et al., 2016) as many deteriorative biochemical
reactions are limited in this range (Dianawati et al., 2013). Water ac­
tivity is an important parameter for the assessment of the shelf life of the
microencapsulated probiotics. Water activity lower than 0.3 restricts the
biochemical reactions and extends the shelf life of products (Tonon
et al., 2009).
Glass transition temperature (Tg) is defined as the temperature at
which a state of polymeric material transforms from a glassy amorphous
state to a rubbery state. This temperature is affected by moisture con­
tent, molecular weight, and chemical structure of the material. Low
molecular weight disaccharides such as trehalose maintain the integrity
of cellular membrane lipids and reduce the mechanical stresses by
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Trends in Food Science & Technology 109 (2021) 340–351
minimizing the size of crystal in the inter-membrane space (Koster et al.,
2000). The presence of probiotics in the glassy state restricts molecular
mobility and thus slows down various detrimental processes such as the
formation of large crystals that cause membrane damage and unfolding
of protein during freezing. This provides prolonged protection to the
immobilized probiotics (Broeckx et al., 2016). Maltodextrin has a higher
molecular weight and thus higher Tg than trehalose, so less efficient for
protecting bacterial strains during both freezing and lyophilization
stages (Semyonov et al., 2010).
Morphological behavior impacts the density, flowability, and bulk
rehydration characteristics of the encapsulated powder. The spherical
form of particles facilitates good flowability and processability (Kabra
et al., 2013). The glassy state maintains the storability of powder so a
thumb rule has been proposed that Tg should be 10–20 ◦ C higher than
the storage temperature (Grasmeijer et al., 2013). The smaller particle
size of microcapsules is desirable for application in the food industry
from the point of improving the food texture and optical characteristics.
The textural and organoleptic properties of foods are influenced by the
large size of the particles (exceeding 100 μm), but higher viability of
probiotics was observed in larger microcapsules (Muthukumarasamy
et al., 2006). The characterization of co-microcapsules produced by
different technologies with the encapsulation yield of core ingredients
has been summarized in Table 2.
5. Research and development of functional food products by the
application of the co-encapsulation concept
There is very limited research that has been done on the formulation
of functional food products based on the co-encapsulation of probiotic
bacteria with different bioactive compounds. Sahitya et al. (2013)
developed ice cream by supplementing co-encapsulated probiotic strains
Lactobacillus helveticus 194 and Bifidobacterium bifidum 231 with prebi­
otic fructooligosaccharides and reported a higher probiotic counts (>7.9
log CFU/g) at the end of the storage study (90 days at − 20 ◦ C) without
affecting the color, texture, appearance and overall acceptability.
Enache et al. (2020) incorporated co-microencapsulated powder con­
taining Lactobacillus casei and black currant extract at a concentration of
5% in 50 g of yogurt and found a 12 and 26% increase in anthocyanins
and polyphenol content, respectively but flavonoids content decreased
with 12% with higher viability of probiotic bacteria up to 21 days of
storage period at 4 ◦ C. The co-encapsulation of L. acidophilus with the
prebiotic Hi-maize coated with chitosan resulted in only a 1.0-log cycle
decrease in probiotic counts in synbiotic yogurt and co-microcapsules
showed a higher efficacy to preserve bacterial cells than micro­
encapsulated and free cells (Iyer & Kailasapathy, 2005). Incorporation
of co-encapsulated proanthocyanidin-rich cinnamon extract and pro­
biotic bacteria Bifidobacterium animalis subsp. lactis into sugarcane juice
was successfully performed by Holkem et al. (2019). This study revealed
that the addition of co-microcapsules resulted in a decrease in pH due to
the presence of bacteria on the surface of microcapsules, increase in
density and soluble solids, increase in phenolic content (1.73–2.22 mg
GAE/mL), decrease in proanthocyanidins content (0.19–0.12 mg
B2/mL) due to the action of probiotics with polyphenols within the
particle, increase in viscosity and higher viability of bacteria (>7 log
CFU/mL) without changing the organoleptic properties of juice during
the storage period of 28 days at 4 ◦ C.
6. Negative aspects of these recent approaches
The encapsulation efficiency of probiotics and bioactive substances
depends on morphological properties, swelling behavior, crystallinity
nature, and presence of functional groups in the co-microcapsules. The
co-encapsulation of two nutrients as a single entity has been shown to be
more hygroscopic that resulted in the readily release of core ingredients
due to their amorphous nature (Vaziri et al., 2018). More extensive
research is to be needed for scientific evidence of the detrimental effect
S. Misra et al. Trends in Food Science & Technology 109 (2021) 340–351

Table 2
Characterization of co-microcapsules with the encapsulation yield of different technologies.
Co-encapsulation Core material Wall material Characteristics of microcapsules Encapsulation yield References
technology (Probiotics-bioactives)
Particle size Morphological
characteristics

Electro- Lactobacillus plantarum
hydrodynamic and Bifiobacterium lactis;
atomization- inulin or resistant
Freeze drying starch
Spray drying Lactobacillus plantarum
and γ-aminobutyric acid and maltodextrin
(GABA)
Emulsification Staphylococcus succinus
and Enterococcus fecium;
Sugarbeet and chicory
Spray drying Lactobacillus acidophilus Gum Arabic (GA),
and blackberry juice
External gelation Lactobacillus delbrueckii
method-Freeze subsp. bulgaricus and
drying Lactobacillus paracasei
subsp. Paracasei (SA) and trehalose
Extrusion-Freeze Lactobacillus plantarum
drying and DHA-rich oil
Complex Lactobacillus casei and
coacervation- tuna oil
spray drying/
Freeze drying
Emulsification/ Lactobacillus helveticus
internal and green tea extracts
gelation (GTE)
Ionotropic Lactobacillus plantarum,
gelation Enterococcus faecium,
Aerococcus viridans and
Pediococcus pentosaceus;
Inulin, cactus pear peel
flour or apple marc
flour
Extrusion Bifiobacterium animalis
subsp. lactis, inulin,
ascorbic acid
Freeze drying Lactobacillus acidophilus, Alginate-chitosan
Jerusalem artichoke
and inulin
Spray drying Bacillus clausii and Inulin and lactose
resveratrol
Freeze drying Lactobacillus casei ssp.
paracasei and isolate, inulin, and
anthocyanins from
black currant extract
Ca-alginate/ Probiotics: 460–710 Less wrinkles on the Inulin: 78.9%; Zaeim et al. (2019)
chitosan μm; surface Resistant starch: 99.2%;
Probiotics-inulin:830 Lactobacillus plantarum
μm; -inulin: 6.33 log CFU/g at
Probiotics- resistant 25 ◦ C; Bifiobacterium lactis-
starch: 1 mm inulin: 7.01 log CFU/g at
− 18 ◦ C after 90 days
Inulin, dextran, 10–35 μm Spherical shape with Lactobacillus plantarum: Pandey and Mishra
some 99.21%; GABA: 84.22% (2020)
Concavities on the after immediate drying
surface
Alginate – Rough internal and 8.1–7.9 log CFU/mL after Sathyabama et al.
external surfaces 30 days at 4 ◦ C (2014)
GA: 11.15 μm; GA and MD: compact Total phenolic compounds: Colín-Cruz et al.
maltodextrin WPC: 5.05 μm; microstructure with a 98.4%; Total monomeric (2019)
(MD), and whey MD-WPC: 5.65 μm coarse porous network anthocyanin content:
protein and large cavities; WPC: 99.0%; Lactobacillus
concentrate granular particles with acidophilus: 81.2% after 10
(WPC) spherical shape weeks at 20 ◦ C
Whey protein – Regular morphology and Probiotics-WPI: 90.54%; Han et al. (2020)
isolate (WPI), intensified structure. Probiotics-SA: 84.46%
sodium alginate
Alginate, pectin, – Completely entrapment Lactobacillus plantarum: Vaziri et al. (2018)
and gelatin of cells in the matrix and 88.66%; DHA-rich oil:
particles with smooth 69.37%
and compact surface
Whey protein – Amorphous structure Lactobacillus casei: 84.95%; Eratte et al. (2015)
isolate-gum tuna oil: 93.35%
Arabic
Whey protein 81–130 μm – Lactobacillus helveticus: Gaudreau et al.
isolate-pectin 71–95%; Polyphenol: (2016)
36.1–79.0%
Sodium alginate Lactobacillus – Lactobacillus plantarum: Serrano-Casas et al.
plantarum: 83.78–88.94%; (2017)
69.23–92.45 μm; Enterococcus faecium:
Enterococcus faecium: 85.94–90.91%; Aerococcus
85.71–89.01 μm; viridans: 85.24–90.00%;
Aerococcus viridans: Pediococcus pentosaceus:
75.82–82.42 μm; 86.14–90.50%
Pediococcus
pentosaceus:
82.42–95.60 μm
Alginate 1.7 ± 0.1 mm – Bifiobacterium animalis Kumherová et al.
subsp. lactis-inulin- (2020)
ascorbic acid: 9.9 log CFU
mL− 1 after 6 week of
storage at 6 ◦ C.
– – Lactobacillus acidophilus- Jantarathin et al.
Jerusalem artichoke: (2017)
89.65%; Lactobacillus
acidophilus- inulin: 89.93%
2–25 μm Regular spherical Bacillus clausii Vázquez-Maldonado
morphology with –resveratrol-inulin: 8.52 et al. (2020)
smooth surfaces Log10 CFU/g; Bacillus
clausii –resveratrol-lactose:
8.62 ± 0.06 Log10 CFU/g
Whey protein 147.60 μm Irregular shape Lactobacillus casei: 87.38%; Enache et al. (2020)
anthocyanins: 95.46%
chitosan

of high concentrations of GTE on the viability of probiotics although
some studies have concluded that interaction of flavonoid with quer­
cetin causes the lowering the bacterial viability (Iyer & Kailasapathy,
2005) and reduction in polyphenol yield have been achieved in the
co-microcapsules containing high levels of GTE (Gaudreau et al., 2016).
The higher concentration of prebiotics such as inulin and dextran has
resulted in lower viability of probiotics and produced sticky powders
after spray drying. During the preparation of emulsions, the homoge­
nization process at a higher rpm may cause cell injury or complete
breakage of the matrix and ultimately result in lower entrapment effi­
ciency. The extrusion process is unsuitable for large-scale production of
co-microcapsules due to the lower payload whereas, freeze drying
method is so expensive that limits its application on an industrial scale.
The larger particles (containing probiotics with inulin and ascorbic acid)

348
S. Misra et al.
produced by the extrusion method restrict its application in liquid and
semi-solid foods due to the alteration in sensory properties (Chen et al.,
2017). The freeze dried co-microcapsules containing probiotics and
black currant extract has an irregular shape with a larger size (>100 μm)
indicates the inability of wall materials to retain the core substances
during processing and storage as well as a negative impact on organo­
leptic properties of food materials. The application of co-encapsulation
in food formulations is directly connected with consumer acceptance.
Starting from the selection of probiotics, encapsulating materials to the
development of products, each step requires great attention with clinical
evidence. The researchers should look upon the stability of core mate­
rials during thermal processing of food products as well as the interac­
tion of these active substances with the food components which may
alter the color, flavor, and other physicochemical properties during
storage as many studies has found that the stability of probiotics and
bioactive substances is maintained for a short period even if storing at a
refrigerated condition. This is a great hurdle for the marketability of the
food materials which may be sorted by selecting suitable packaging
materials as a barrier to oxygen, light, and other environmental pa­
rameters. This challenge has to be resolved in order to maintain the
functionality accompanied by the longer shelf life of food products.
7. Benefits with respect to less recent approaches and their
possible advances
The limited research on this field provides a wide scope for the re­
searchers to develop functional food products by incorporating the
bioactive and probiotics as co-encapsulation materials to provide multi
functionalities and better delivery of active ingredients in the human
gut. The advantages of the existed research on co-encapsulation con­
cepts are intending the application of essential oil, vitamins, antimi­
crobial compounds such as garlic extract and nisin, and flavors such as
menthol and xylitol along with the probiotics in food formulations at
low cost. In order to provide synergistic effects, suitable wall materials
should be selected for enhancing the functionality. This review directs
the employment of dietary fiber enriched agro-industrial waste products
as a potential vehicle for the targeted delivery of probiotic strains in a
metabolically active state. The mechanism of improved viability of
probiotics with the aid of bioactive compounds is still unclear. The
bioavailability of bioactive compounds with respect to the preservation
of beneficial probiotic properties during the passage of the GI tract can
be proved by an in-vivo study. This will help in the discovery of novel
encapsulating wall materials and technologies for improving the sta­
bility and bioactivity of active ingredients. The storage study of func­
tional foods and the release mechanism in simulated GI conditions will
further strengthen the existing findings.
8. Conclusions and future scopes
Generally, identification and selection of probiotics is a wise alter­
native to the ingestion of medicine and other treatments to control the
health imbalance in human beings as well as other animals. Microen­
capsulation is an effective technology to preserve the viability of mi­
croorganisms during storage, manufacturing, and gastrointestinal
digestion. Different technologies have been implemented for the
encapsulation of probiotics but the production of desired microcapsules
with less damage to probiotics is a still challenging factor for commer­
cialization purposes.
Encapsulation of bioactive compounds and probiotics as a single
entity has been extensively studied before but co-encapsulation of pro­
biotics with bioactives will be a still low cost and efficient methodology
to deliver these ingredients to the targeted colon with increased viability
and bioactivity to improve the health status in the host. Application of
prebiotics and fiber is gaining popularity which exploits co-
encapsulation concepts that can provide an enhanced growth and
multiplication of bacteria through a synbiotic effect when they are
349
Trends in Food Science & Technology 109 (2021) 340–351
released in the GI tract. Encapsulating materials that are not certified
with GRAS status or of animal origin are critical concerns for consumers
whereas, the properties of produced microcapsules should be thor­
oughly investigated to avoid alteration in the organoleptic properties of
developed products. Different technologies such as spray chilling, vac­
uum drying, impinging aerosol technology can be employed for co-
encapsulation technology. The mechanism of improving the viability
of probiotics by the bioactive compounds is still unclear so there is a
need for in-vivo stability evaluation of these bioactives and the simu­
lation in an animal model system.
The leading players in the global market eg. Danone, DuPont, Chr.
Hansen, Morinaga, Yakult Honsha, Probi, Nestlé, Lallemand, Bifodan,
Probiotics International, BioGaia, Nebraska Cultures are manufacturing
probiotic fortified dairy products, non-dairy beverages, ready to eat
cereal, and meat-based products. A persistent effort should be put in for
the expansion of co-encapsulated probiotic and bioactives enriched fruit
beverages, milk products, bakery, and extruded products for all age
groups. From the overview, it is concluded that co-encapsulation tech­
nology has a future profit-making potential despite the extra cost by
incorporating the co-encapsulated probiotic bacteria and bioactive
compounds for the development of a higher value product.
Declaration of competing interest
The authors have declared no conflict of interest.
Acknowledgment
The authors would like to acknowledge the financial support of the
National Agricultural Science Fund (NASF) (Grant No. F No. NASF/AE-
6017/2016-17/49), Indian Council of Agricultural Research (ICAR),
Ministry of Agriculture and Farmers Welfare, Govt. of India.
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