Food Bioscience 21 (2018) 34–44

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Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Recent developments on encapsulation of lactic acid bacteria as potential T

starter culture in fermented foods – A review
Digambar Kavitakea,1, Sujatha Kandasamya,1, Palanisamy Bruntha Devib,
⁎
Prathapkumar Halady Shettya,
a
Department of Food Science and Technology, Pondicherry University, Pondicherry 605 014, India
b
Department of Microbial Biotechnology, Bharathiar University, Coimbatore 641 046, India

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

Keywords: Fermented foods are the first processed staple human diet that have been produced and consumed since de-
Encapsulation velopment of human civilizations. Majority of the fermented foods are made through controlled microbial
Lactic acid bacteria growth and enzymatic conversions of major and minor food components that gain high values because of its
Starter culture enhanced organoleptic properties. Ease of fermentation, risk in fermentation failure and several functional
Fermented foods
properties of lactic acid bacteria makes them as suitable starter culture in production of fermented foods. The
viability and stability of starter cultures in the fermented foods and gastro intestinal environment are key
challenges at industrial scale. Use of encapsulated starter cultures has been considered more in the recent years,
due to its improvement in survival and viability under adverse environmental conditions. This paper mainly
focuses on reviewing lactic acid bacteria as functional starter cultures in fermented foods including different
techniques and coating materials used for microencapsulation, factors affecting the microencapsulation,
methods for evaluating the efficiency of starter cultures and future perspectives to be overcome in this area.

1. Introduction relevant to health promotion and disease preclusion (Borresen,

Traditional fermented foods form an integral part of cultural heri-
tage and diet widely consumed since ancient civilization. Since ages,
this innovative practice of fermentation has extended and enriched to
preserve and increase the safety of existing food resources, mainly to
overcome the hidden hunger (Ray, Ghosh, Singh, & Mondal, 2016).
Traditionally, many of the fermented foods are produced under spon-
taneous fermentation using backslopping method which involves direct
addition of most dominant native microflora as selected starter culture.
This technique leads to reduction in fermentation time, prevention of
fermentation failure and standardization of final product. The fer-
mented food quality depends upon the population and microbial di-
versity in the raw material (Tamang, Watanabe, & Holzapfel, 2016).
Over the past two decades, the scientific interventions on fermented
foods revealed the health-beneficial concept by the native microflora or
starter culture roles in stimulating the probiotic functions, bio-avail-
ability of macro and micronutrients, production of antioxidant, anti-
nutritive and anti-microbial compounds and other functional compo-
nents during fermentation. This health promoting factors produce client
awareness for ingesting such ancient foods on functional basis in
Henderson, Kumar, Weir, & Ryan, 2012; Marco et al., 2017).
Lactic acid bacteria (LAB) are predominant microflora present in
most of the traditional fermented foods and their role in fermentation is
known since ages (Anandharaj & Sivasankari, 2013). Currently, for-
mulation of new functional starter culture of LAB that is industrially
important in food safety that deal with one or more organoleptic,
technological, nutritional or health benefits are being developed (Sathe
& Mandal, 2016). Maintenance and viability of starter culture in fer-
mented foods is still an immense challenge in the industrial process. At
the dawn of this situation, encapsulation of starter culture provides
protection to the cells and thus increases the viability of the delivered
amount. For successful encapsulation of viable cells, it is important to
preserve the bacterial viability under different handling processes along
with the type of encapsulation material compatible with food material
(Haffner, Diab, & Pasc, 2016). Fermentation using encapsulated starter
culture, offers numerous advantages compared to traditional cultiva-
tions, e.g., rapid fermentation, higher cell density, enhanced tolerance
of the cells towards high temperatures and toxic media, and selective
removal of toxic hydrophobic substances. However, production of ro-
bust capsules capable of withstanding several months or years, of

⁎
Corresponding author.
E-mail address: pkshalady@yahoo.co.uk (P.H. Shetty).
1
Two authors contributed equally.

https://doi.org/10.1016/j.fbio.2017.11.003
Received 27 June 2017; Received in revised form 7 November 2017; Accepted 7 November 2017
Available online 24 November 2017
2212-4292/ © 2017 Elsevier Ltd. All rights reserved.
D. Kavitake et al.
continuous use without deterioration of cell activity or capsule char-
acteristics, capsule size, large-scale production of capsules and eco-
nomic feasibility still remain challenges for industrial application of
encapsulated cells (de Prisco & Mauriello, 2016).
Although in recent years valuable and promising research on en-
capsulation of LAB as starter culture has been published and commer-
cialization of products are being made, the information exchange be-
tween industry and academia should noticeably intensify. This paper
aims to give an overview of the vast encapsulation of LAB as starter
culture in fermented foods on what has been done in the academia and
industry scenarios in the past few years, in terms of technologies em-
ployed and research insights. Here, we discuss the materials and tech-
niques used for encapsulation of LAB and its applications in terms of
biological production processes, as well as the effect of encapsulation
on cells and their viability.
2. Fermented foods
Fermented foods enrich the human diet as they offer and reserve
huge amounts of nutrition in a comprehensive mixture of flavor, aroma
and texture. Traditional fermentation improves the overall content or
availability of amino acid, vitamins, mineral profiles and therapeutic
potentialities that have profound effects directly on the consumer's
health (Steinkraus, 2002). Most of the global fermented foods are
known to be fermented by both functional and non-functional micro-
organisms that exist as native microflora in raw plant materials, con-
tainers, utensils and environment (Franz et al., 2014). These microbes
alter the biochemical constituents of raw materials, thereby improving
the flavor, digestibility and aroma while imparting nutritional and
pharmacological values in some fermented foods that are traditionally
and socially acceptable by the consumers. Most of these traditional
process of preparation remains majorly secretive being passed on from
generation to generation and tend to be specific for tribes and castes in
different provinces while many of them are made under home scale by
using back slopping (Tamang, Thapa, Tamang, Rai, & Chettri, 2015).
Each ethnic group region has its own unique food diversity and
beliefs including fermented foods that symbolize their heritage, tradi-
tion, agroeconomic and socio-cultural traits of the society. Although,
some fermented foods are popular as delicious daily dish and promoted
globally for their functional, nutraceutical and therapeutic properties.
Numerous reviews were published on the biological, chemical and
nutritional components of fermented foods from countries such as Asia
(Rhee, Lee, & Lee, 2011; Steinkraus, 2002; Tamang et al., 2015, 2016),
Africa (Benkerroum, 2013; Chelule, Mokoena, & Gqaleni, 2010) and
America (Nout, 2003; Oguntoyinbo, Tourlomousis, Gasson, & Narbad,
2011).
3. LAB as functional starter culture
LAB are a broad group of bacteria belonging to the genera (Table 1)
Bifidobacterium, Enterococcus, Lactobacillus, Leuconostoc, Lactococcus,
Propionibacterium, Pediococcus, Streptococcus, Tetragenococcus, Vago-
coccus and Weissella (Holzapfel & Wood, 2014; Tamang et al., 2015).
In traditional fermented foods, fermentation is mainly accomplished
by the native LAB from the available resources that proceed the fer-
mentation process in absence of an added industrial starter culture.
Selective addition of starter culture to raw materials controls the overall
fermentation process that mainly reduces the risk of fermentation
failure and fermentation period while improving the end product value.
Several functional properties of LAB have been reported widely in-
cluding antioxidant activity by Lactobacillus, Bifidobacteria (Mishra
et al., 2015), antimicrobial compounds from Aerococcus, Carno-
bacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pedio-
coccus, Streptococcus, Tetragenococcus, Vagococcus and Weissella
(Rattanachaikunsopon & Phumkhachorn, 2010), degradation of anti-
nutritive compounds by Lactobacillus plantarum, L. brevis, L. curvatus
35
Food Bioscience 21 (2018) 34–44
(Reale et al., 2004), fibrinolytic activity by Vagococcus carniphilus, V.
lutrae, Enterococcus faecalis, E. faecium, E. gallinarum and Pediococcus
acidilactici (Singh, Devi, Ahmed, & Jeyaram, 2014), peptide production
by Lactococcus sp. and Lactobacillus sp. (Brown et al., 2016), poly-lactic
acid by Lactobacillus delbrueckii and Lactobacillus bulgaricus (Ghaffar
et al., 2014) and probiotic effects of Lactobacillus acidophilus, L. casei, L.
johnsonii, L. fermentum, L. rhamnosus, L. plantarum, L. reuteri, L. sali-
varius, L. paracasei, L. delbrueckii subsp. bulgaricus, Streptococcus ther-
mophilus, Bifidobacterium lactis, B. longum and B. breve etc. (Quinto et al.,
2014). These properties play a major role in the selection of starter
culture for the production of functional food.
4. Encapsulation of LAB
Encapsulation process involves the entrapment of active substance
into another substance wall material that produces particles in various
scale. The encapsulated material is usually called as core, fill, active,
internal or pay load phase, whereas the material used for encapsulation
is called as coating membrane, shell, capsule, carrier material, external
phase, or matrix (Burgain, Gaiani, Linder, & Scher, 2011; Fang &
Bhandari, 2010). Encapsulation is intensively used in food sectors
(Fig. 1) as an effective barrier for liquid and solid ingredients against
several environmental (oxygen, light, free radicals etc.) parameters
(Desai & Park, 2005). Encapsulation involves wide area with en-
capsulating material, wall material, process, functionality and proper-
ties of encapsulated systems (John, Tyagi, Brar, Surampalli, & Prévost,
2011). Based on size of the beads produced, encapsulation can be
broadly classified into two types, i.e. macroencapsulation (millimeters
to centimeters) and microencapsulation (1–1000 µm) (Heidebach,
Först, & Kulozik, 2012). In case of macroencapsulation, bacterial cells
will normally grow on the beads surface due to depletion in nutrient
diffusion efficiency in depth of more than 300–500 µm as well as toxic
metabolites accumulation in center of the beads. Moreover, micro-
capsules are reported for higher mechanically robust than macro-
spheres (Park & Chang, 2000). There are several factors (Fig. 2) that
affect the microencapsulation effectiveness which can be further over-
come by selecting proper encapsulation method, coating material and
process conditions.
Different LAB were encapsulated in different polymers for different
purposes such as encapsulated Lactobacillus rhamnosus VTT E-97800
successfully increased the shelf life to at least 18 months during deep
freezing using liquid nitrogen (Mortazavian, Razavi, Ehsani, &
Sohrabvandi, 2007). Yogurt produced using encapsulated starter cul-
ture (S. thermophilus and Lb. delbrueckii) influenced its sensory and
flavor qualities as well as higher bacterial viability from initial to final
stages of the fermentation process (Krasaekoopt, Bhandari, & Deeth,
2004). Several studies on encapsulation of LAB such as Lactobacillus
acidophilus and Bifidobacterium lactis (Darukaradhya, Phillips, &
Kailasapathy, 2013), Lactococcus lactis and Lactobacillus paracasei
(Léonard et al., 2015), Lactobacillus curvatus MBSa2 (Barbosa, Todorov,
Jurkiewicz, & Franco, 2015) and Lactobacillus plantarum (Corbo et al.,
2016) were described using different coating materials with respect to
their functionality, viability and application.
5. Methods of encapsulation
Encapsulation of active substances into carrier materials can be
attained by several methods such as spray drying, spray chilling or
spray cooling, extrusion coating, fluidized-bed coating, liposomal en-
trapment, lyophilization, coacervation, centrifugal suspension separa-
tion, cocrystallization, inclusion complexation and thermal gelation
(Table 2) (Gibbs, Kermasha, Ali, & Mulligan, 1999; Poshadri & Kuna,
2010; Raymond, Neufeld, & Poncelet, 2004). Several factors such as
physical and chemical characteristics of core and carrier/coating ma-
terials and their proposed application in food matrix influence the
choice of suitable encapsulation process. Among all the techniques,
D. Kavitake et al. Food Bioscience 21 (2018) 34–44

Table 1
LAB as starter culture in fermented foods.

Food products Country Raw material Starter culture Reference

Kivunde Tanzania Cassava Lb. plantarum Kimaryo, Massawe, Olasupo & Holzapfel, 2000
Sausages Italy Meat Pediococcus acidilactici, Leroy et al., 2006; Coppola, Mauriello, Aponte,
P.pentosaceus Moschetti & Villani, 2000
Lb. pentosus, Lb. plantarum
Thailand pork Pediococcus pentosaceus Kingcha et al., 2012
Kefir Iran Kefir grains Lb. kefir, Lb. casei, Assadi et al., 2000
Lb. brevis, Lb. plantarum,
S. lactis, Leu.mesenteroides
Spain Cow's milk Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris, Lc. lactis subsp. lactis Fontán, Martínez, Franco & Carballo, 2006
biovar diacetylactis, Leuconostoc mesenteroides subsp. cremoris, Lactobacillus
plantarum,Lb. casei
Kimchi Korea Cabbage Weissella cibaria, W. confusa, W. koreensis Lee et al., 2005
Sauerkraut China Cabbage Leu.mesenteroides, Lb. plantarum, Lb. casei, Xiong, Li, Guan, Peng & Xie, 2014
Lactococcus lactis
Fermented milk Europe Milk L. acidophilus, L.rhamnosus, Sodini, Lucas, Oliveira, Remeuf & Corrieu,
Streptococcus thermophilus, 2002
L.bulgarius, L. casei,
Lb. plantarum,
Switzerland Propionibacterium freudenreichii, Baer and Ryba, 1992
P. jensenii
Plaa-som Thailand Fish Lb. plantarum, Lb.reuteri, Saithong, Panthavee, Boonyaratanakornkit &
Sikkhamondhol, 2010
Sourdough Greece Wheat L. brevis, L. paralimentarius, P. pentosaceus, W. cibaria. Paramithiotis, Chouliaras, Tsakalidou &
Kalantzopoulos, 2005
South Korea Leu. citreum, Weissella koreensis Choi, Jung, Kim, Park, Kim & Han, 2003
Sauerkraut USA cabbage Leuc. mesenteroides Johanningsmeier, McFeeters, Fleming &
Thompson, 2007
Fermented olives Spain Green olives Enterococcus casseliflavus Lactobacillus pentosus De Castro, Montaño, Casado, Sánchez &
Rejano, 2002
Lactobacillus pentosus Blana, Grounta, Tassou, Nychas & Panagou,
Lb. plantarum 2014
Cheddar cheese Ireland Milk Streptococcus thermophilus Hou, Hannon, McSweeney, Beresford & Guinee,
2017
Australia Lactobacillus acidophilus, Lb. casei, Lb. paracasei Bifidobacterium spp. Ong et al., 2006
Yogurt Bulgaria Sheep Milk Streptococcus thermophilus, Michaylova, Minkova, Kimura, Sasaki & Isawa,
Lb. delbrueckii subsp. bulgaricus 2007
Sauce Thailand soybean Tetragenococcus Singracha, Niamsiri, Visessanguan, Lertsiri &
Assavanig, 2017
Fish Halophilus Udomsil, Rodtong, Choi, Hua &
Yongsawatdigul, 2011
Togwa Tanzania Maize & Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Mugula et al., 2003
sorghum Lactobacillus plantarum Pediococcus pentosaceus

Fig. 1. Application of microencapsulation of LAB.

Fig. 2. Factors affecting microencapsulation effectiveness.

36
D. Kavitake et al. Food Bioscience 21 (2018) 34–44

Table 2
Various techniques for microencapsulation.

Microencapsulation technique Phases in Encapsulation Technique

Extrusion Dispersion of active material in a molten carrier solution and forcing of the mixture into a dehydrating liquid
Centrifugal extrusions Co-extrusion of active and carrier solution through nozzles
Emulsion Polymers are used for coating oil-in-water emulsion droplets along with core material and homogenized well before chemically hardened to
form a shell
Spray-drying Atomize the infeed dispersion of the active and core mix and further dehydration of the atomized particles
Spray-chilling
Spray-cooling
Fluidized-bed coating Fluidization of active particles and covering them using coating material
Liposomal entrapment Micro fluidization, Ultra sonication & Reverse-phase evaporation of mixed active and carrier solution
Lyophilization Freeze-drying of a mixed active and carrier solution
Coacervation Polymers are used for coating oil-in-water emulsion droplets by coacervation that chemically hardened to form a shell
Centrifugal suspension separation Passing an active-coat mixture over a rotating disc to achieve tiny encapsulated particles
Cocrystallization Mixing of core in supersaturated sucrose solution
Inclusion complexation Grinding or spray-drying or mixing complexes
Thermal gelation Warm carrier solution (45 °C) is introduced into a temperature controlled dropping device, which drop the carrier solution in cold (10 °C) KCl
solution under gentle magnetic stirrer agitation.

extrusion, emulsion and spray drying are effectively used for en- Bifidobacterium BB-12 produced by emulsification/internal gelation
capsulation of LAB. using sodium alginate as coating material provided effective protection
under simulated GI condition and 120 days of frozen storage (Holkem
et al., 2017).
5.1. Extrusion method

Extrusion is one of the ancient and usual method to produce hy-

drocolloid capsules using a simple and low cost procedure that makes
minimal injuries to probiotic cells while maintaining comparatively
higher viability (Mortazavian et al., 2007). In this system (Fig. 3a), the
hydrocolloid solution is mixed with microorganisms and mixture is
delivered through an extruder (syringe) in a droplet form into hard-
ening solution (calcium chloride) that consists of multivalent cations.
After dripping, the cells are immediately entrapped by the polymers
leading to three-dimensional lattices that cross links with calcium ions
(Krasaekoopt et al., 2004). Mostly, alginate and calcium chloride con-
centration ranges from 0.5% to 4% and 0.05–1.5 M, respectively and
the beads size ranges from 2 to 3 mm in diameter which mostly depends
upon the distance between syringe and hardening solution, polymer
type, viscosity, concentration and mainly diameter of the extruder or-
ifice (Solanki et al., 2013). Probiotic Lactobacillus acidophilus en-
capsulated by ionic gelation with electrostatic extrusion showed im-
proved survival rate under refrigerated storage and simulated gastric
conditions (Kim et al., 2016).
5.2. Emulsion method
Emulsion is an extensively used chemical practice for encapsulating
live cells using hydrocolloids (alginate, carrageenan and pectin) as cell
wall materials, which are usually recovered by membrane filtration
technique (Fig. 3b). The diameter of microbeads is highly regulated by
the concentration and viscosity of the hydrocolloid solution and its
agitation rate (de Vos, Faas, Spasojevic, & Sikkema, 2010; Kailasapathy,
2006). In contrary to extrusion, easier scale-up, high bacterial survival
rate and capsules with smaller diameter (25 µm-2 mm) are added ad-
vantages, while the main disadvantage is large size range and shape and
higher cost for performance owed to usage of vegetable oil in emulsion
formation (Krasaekoopt et al., 2004; Kailasapathy, 2006).
Albertini et al. (2010) stated that encapsulation of Bifidobacterium
lactis BI07 and Lactobacillus acidophilus LA14 by extrusion using algi-
nates, cellulose acetate phthalate (CAP) and xanthan gum revealed
excellent stability at 5 °C up to 9 months of storage with improved re-
sistance to gastro acidic conditions. However, milk proteins and sugar
alcohols (sodium caseinate, skim milk, whey protein concentrate, soy
protein combined with glycerol, mannitol or maltodextrin) offer higher
protection for B. longum after freeze drying against acid and bile en-
vironment (Dianawati, Mishra, & Shah, 2013). Microparticles of
5.3. Spray drying
Spray drying is an ancient and widest encapsulation technique that
used for particle size less than 40 µm. The most used encapsulating
agents for spray drying are polysaccharides (maltodextrins, starches,
arabic gum and corn syrups), lipids (monoglycerides, diglycerides and
stearic acid) and proteins (casein, gelatin, soy, wheat and milk serum).
Spray drying process involves atomization of homogenized carrier
material (1:4) into a drying gas, resulting in capsules as dry powder,
that mainly are controlled by product feed, gas flow and temperature
(Fig. 3c). Spray drying is highly suitable for industrial applications due
to its rapidity, reasonable low cost and high reproducibility. Use of high
temperature is the main disadvantage in spray drying that is not com-
patible for bacterial viability (Ray et al., 2016).
For food applications, probiotic cultures are commonly use as spray
or freeze-dried powders (Holzapfel & Wood, 2014). Effective spray
drying has been described in L. paracasei (Desmond, Ross, O'callaghan,
Fitzgerald, & Stanton, 2002), Bifidobacterium ruminantium (O’Riordan,
Andrews, Buckle, & Conway, 2001) and L. rhamnosus (Corcoran, Ross,
Fitzgerald, & Stanton, 2004). However, extreme temperature and os-
motic conditions during the process damages cell membranes and as-
sociated proteins considerably reduce the probiotic activity within few
weeks of storage duration.
Behboudi-Jobbehdar, Soukoulis, Yonekura, and Fisk (2013) com-
pared the viability of spray dried L. acidophilus using sodium alginate,
chitosan and hydroxypropyl methylcellulose (HPMC) and found that
sodium alginate and HPMC did not affect cell viability. Chitosan en-
hanced the storage stability of L. acidophilus and higher viable counts
were recorded after 35-days of storage. Spray drying of Bifidobacterium
lactis Bb-12 in whey protein enhanced cell viability under bile condi-
tions and stability under storage for 12 weeks (de Castro-Cislaghi, Silva,
Fritzen-Freire, Lorenz, & Sant’Anna, 2012). Addition of thermo-
protectant such as trehalose prior to spray drying along with starch and
soluble fibre improves culture viability during storage (Vivek, 2013).
Spray chilling method was developed to overcome the difficulty that
occurs when microbial cells are subjected to high temperature in spray
drying. Similar equipment used for spray drying is used for this process,
where the hot chamber replaced with cold conveying air or cold
chamber (Champagne & Fustier, 2007). Spray chilling of B. lactis and L.
acidophilus was found to be appropriately mild without affecting the cell
viability (de Lara Pedroso, Thomazini, Heinemann, & Favaro-Trindade,

37
 D. Kavitake et al.

Table 3
Materials suitable for microencapsulation of the LAB.

Coating material Source Monomer linkage Method of Encapsulation Properties of polymer References

Starch Plant Glucose α (1→4) and α Emulsion Insolvable in cold water, ethanol and most of Sultana et al. (2000); Li, Turner, and
(1→6) the solvents, digestible, GRAS by FDA Dhital (2016)
Alginate Brown seaweeds, Microbial Mannuronic and guluronic β (1→4) Emulsion, extrusion Soluble in water (except ca+-alginate), GRAS Kailasapathy (2006); Zhang, Zhang,
(Pseudomonas aeruginosa) acids by FDA Zou, and Mc Clements (2016)
Xanthan Microbial (Xanthomonas campestris) Glucose, mannose, and β (1→4) Direct extrusion (complex Highly viscous, soluble in cold water, non- Argin, Kofinas, and Lo (2014)
glucuronic acid coacervation) gelling compound. Not GRAS but safe up to
15 g/day
Gellan Microbial (Sphingomonas elodea) Glucose -rhamnose and β (1→4) and α Emulsion Thickening, emulsifying and stabilizing Sun and Griffiths (2000);
glucuronic acid (1→3) property,
Not GRAS but agree to use in foods Rathore, Wan, Sia Heng & Chan
(2015)
Carrageenan K Marine algae, Galactose α (1→3) and β Emulsion, Thermal gelation Soluble in water, thickening, gelling agent, Oulad-Haddar Sifour, Idoui,

38
(1→4) texture enhancer stabilizing property, GRAS Bouridane, and Arid (2016);
by FDA
Chitosan Crustacean shells, insects and fungi Galactose β (1→4) Coaxial technique Insoluble, biodegradable biocompatible, Klaypradit and Huang (2008);
GRAS by FDA Tylingo, Mania, and Szwacki
(2016);
Gelatin Animal (cattle, chicken, pigs and Complex mixture of single – Emulsion, extrusion insoluble in cold water but readily hydrates in Annan, Borza, and Hansen (2008);
fish) /multi-stranded polypeptides hot water
Cellulose Plants, Microbial, Glucose β (1→4) Freezing-thawing Insoluble in water, non-digestible in intestine Fijałkowski, Peitler, Rakoczy, and
Żywicka (2016); Li et al. (2016).
Guar gum Plants Galactose and mannose β (1→4), α Spray drying and freeze Soluble, indigestible, GRAS by FDA Kuck and Noreña (2016)
(1→3) drying
Cellulose Acetate Phthalate Partially substituted cellulose acetate (CA) with phthalic anhydride in – Spray drying, freeze drying Soluble in mildly acidic to slightly alkaline Mortazavian et al. (2007); Antunes
(CAP) presence of acetic acid, acetone, or pyridine condition et al. (2013);
Milk Proteins Caseins, whey proteins, milk fat globule membrane (MFGM) proteins – Spray drying widely available, inexpensive, natural and Livney (2010); Ying et al. (2013)
and total milk protein GRAS
Other possible polymers, prebiotics and microbial Resistant starch, levan, galactan, dextran,
exopolysaccharides (EPS) which can be used for encapsulation
Food Bioscience 21 (2018) 34–44
D. Kavitake et al.
2012).
6. Coating material for LAB encapsulation (Table 3)
6.1. Starch and starch derivatives
Starch is a storage polysaccharide made of α-D-glucose units linked
by α−1→4 and α−1→6 glycosidic bonds. It consists of two different
polysaccharide molecules, one is the linear amylose (20–30%) and
other is high branched amylopectin (70–80%) (Li et al., 2016). Alginate
starch gel beads are stated to prevent oxygen diffusion over the gel by
forming anoxic conditions in the center of the beads thereby preventing
cell death of L. acidophilus and B. lactis (Talwalkar & Kailasapathy,
2004). Addition of Hi-Maize starch enhanced the survival of bacteria
compared to encapsulation without starch (Iyer & Kailaspathy, 2005).
Symbiotic Lactobacilli (Lactobacillus reuteri, L. acidophilus and L. plan-
tarum) with pure native starches (corn, potato and tapioca) and pure
pullulan systems reported 70–80% viability with pullulan and pullulan/
potato starch films even after storage for 2 months at 4 °C (Kanmani &
Lim, 2013). Addition of Hi–maize (1%) and chitosan (0.4%) in alginate
beads of Lactobacillus acidophilus provided better protection during both
GI resistance tests and under storage of moist and freeze-dried micro-
particles (de-Araujo et al., 2016). Microencapsulation of Lactobacillus
plantarum 299 v in native or partially hydrolyzed maize starches after
enzymatic modification results in significantly higher total probiotic
numbers after exposure to acid (pH = 2.0, 1 h), bile salt (3% w/v, 4 h)
and heat treatment (60 °C, 15 min). This seems as an effective method
39
Food Bioscience 21 (2018) 34–44
Fig. 3. Schematic representation of encapsulation
methods: (a) Extrusion (b) Emulsion (c) Spray
drying.
to increase encapsulation yields when starch-based material is used for
encapsulation and further ensures that the number of viable bacteria
remain above the minimum dosage as requirement during food pro-
cessing and digestion in the system (Li et al., 2016).
6.2. Alginate
Alginate is an extensively used biopolymer for encapsulation, which
is derived from brown algae (Kelp), mainly composing β-D-mannuronic
and α-L-guluronic acids. Alginate bead gelation occurs by cross-linking
of Ca2+ gelling ions with the alginate and calcium alginate is ideal at
concentrations of 0.5–4% for encapsulating probiotics. Alginate is
widely used for its simplicity, biocompatibility, low cost, non-toxicity,
easier gel matrice formation around bacterial cells and mild processing
condition (Krasaekoopt et al., 2004). Difficulty in scaling-up of the
process and porous microparticles are major drawbacks in protecting
the cells from its environment which might be overwhelmed by com-
bining alginate with other polymers or by coating capsules with other
compounds or using different additives for structural modification of
the alginate (Gouin, 2004). Alginate gel matrices enclose the bacterial
cells to produce beads in the range of 1–3 µm diameter and pore sizes of
the surface up to 7 nm. Bacteria encapsulated using alginate improve
their survival rates in skim milk by one log compared to free cells until
storage of 24 h (Talwalkar & Kailasapathy, 2004). Encapsulation using
blend of alginate-glycerol significantly increases the viability of LAB
even after deep freezing process (Sultana et al., 2000).
Cui, Goh, Kim, Choi, and Lee (2000) prepared poly-L-lysine cross
D. Kavitake et al.
linked alginate microparticles for encapsulating bifidobacteria to pro-
tect the cells without losing their survivability in gastric fluid, due to
stability of cross linked microparticles. Probiotic cultures such as L.
casei, L. acidophilus, B. bifidum and B. longum encapsulated using a blend
of prebiotics (fructooligosaccharides or isomaltooligosaccharides),
growth promoter (peptide) and sodium alginate in the ratio of 3:1:1
reported the highest counts (Chen, Chen, Liu, Lin, & Chiu, 2005). Edible
coating using sodium alginate or sodium alginate/whey protein con-
centrate combined with Lactobacillus rhamnosus in probiotic pan bread
significantly improved its viability until 7 days storage (Soukoulis,
Singh, Macnaughtan, Parmenter, & Fisk, 2016). Microencapsulation of
many LAB was studied using alginate in combination with other coating
materials such as L. lactis using composite alginate/pectin (Bekhit,
Sánchez-González, Messaoud, & Desobry, 2016), L. plantarum and Sta-
phylococcus xylosus using alginate-starch mixture (Bilenler, Karabulut, &
Candogan, 2017) and Lactobacillus rhamnosus GG in chitosan-coated
alginate bead (Gandomi, Abbaszadeh, Misaghi, Bokaie, & Noori, 2016).
L. plantarum encapsulated using inulin-sodium alginate-skim milk (ISA)
by freeze-drying method enhanced the viability as well as probiotic
functionality compared to free cells under both gastric and bile condi-
tions (Wang, Yu, Xu, Aguilar, & Wei, 2016).
6.3. Gellan and Xanthan Gum
Gellan is a bacterial polysaccharide from Pseudomonas elodea,
composed of repetitive units of glucose, glucuronic acid, glucose and
rhamnose. Upon cooling, gellan gum produces thermo-reversible gel
and gelation temperature depends upon polymer concentration, ionic
strength and cation type in the solution (Vivek, 2013).
Xanthan is another bacterial polysaccharide produced by
Xanthomonas campestris, that constitutes repeating units of penta-
saccharide (glucose, mannose and glucuronic acid in the molar ratio of
2:2:1) (Garcia et al., 2000). This polymer is hydrated quickly in cold
water without lump formation by proper dispersion into the solvent.
The solution of xanthan gum shows a very pronounced pseudoplastic
behavior due to its semi-rigid conformation and higher viscosity that
lead to form a network by hydrogen bonding and polymer chain en-
tanglement (Kang & Petitt, 1993). A major disadvantage is high gel
setting temperature (80–90 °C for 60 min) that damages the probiotic
cells due to heat (Sun & Griffiths, 2000). In contrast to alginate, en-
capsulation of probiotic cells using mixture of xanthan-gellan gum of-
fers high resistance towards acid conditions (Sultana et al., 2000). B.
lactis encapsulated using gellan/xanthan improved their storage survi-
vability for 21 days at 4 or 22 °C in Mahewu (fermented, non-alcoholic
maize beverage) (McMaster, Kokott, Reid, & Abratt, 2005).
6.4. Carrageenan K
Carrageenan are natural biopolymer with sulphated galactans,
commonly used as a food additive and mainly extracted from marine
macroalgae. Microbial cells are mixed with the polymer when tem-
perature ranges between 40 and 50 °C followed by cooling to room
temperature for gelation and finally potassium ions are added for sta-
bilization of microparticles (Krasaekoopt et al., 2004). Probiotic en-
capsulation using carrageenan can be done by both extrusion and
emulsion methods. Doleyres, Fliss, and Lacroix (2004) demonstrated
ionotropic gelation technique for immobilizing bifidobacteria cells
using carrageenan and locust bean gum for better tolerance under si-
mulated GI conditions, freeze drying and hydrogen peroxide.
6.5. Chitosan
Chitosan is a linear polysaccharide consisting of glucosamine units
that polymerise by cross-linking in presence of anions and polyanions
(polyphosphates and sodium alginate). This polymer is mostly utilized
as a coating material but cannot be used as a capsule in encapsulation,
40
Food Bioscience 21 (2018) 34–44
since it is less efficient in increasing cell viability (Mortazavian et al.,
2007). Krasaekoopt et al. (2004) revealed higher probiotics survival (L.
acidophilus, L. casei and B. bifidum) using chitosan-coated alginate beads
in yogurt and milk during storage conditions. Encapsulation of L. bul-
garicus by alginate and chitosan coating effectively delivers viable cells
to the colon as well as upholding their survival under refrigerated
conditions (Lee, Cha, & Park, 2004). Encapsulated Lactobacillus plan-
tarum and Bifidobacterium longum using alginate and pectin coated with
chitosan and glucomannan showed increased survival in pomegranate
and cranberry juices during storage (Nualkaekul, Cook, Khutoryanskiy,
& Charalampopoulos, 2013).
6.6. Gelatin
Gelatin is a complex blend of single or multi-stranded polypeptides
utilized in probiotic encapsulation. In pure form, the polymer is
translucent, brittle, colorless or slightly yellow, tasteless and odorless.
This thermo reversible gel can be used as alone or in cooperation with
gellan gum like polysaccharides due to amphoteric nature (Krasaekoopt
et al., 2004). Both gellan and gelatin carries net negatives charges that
repels each other and form homogenous mixture at a pH higher than 6.
However, at lower pH the net charge of gelatin becomes positive and
bind strongly gellan with a negative (Anal & Singh, 2007).
Encapsulation of Bifidobacterium adolescentis 15703T by alginate-
coated gelatin microspheres enhanced their survival in gastro-intestinal
tract during exposure to adverse conditions (Annan et al., 2008).
Nawong, Oonsivilai, Boonkerd, and Hansen (2016) developed and
characterized novel food-grade gelatin–maltodextrin microspheres
cross-linked with transglutaminase (TGase) that protect the en-
capsulated LAB under simulated GI conditions.
6.7. Cellulose Acetate Phthalate (CAP)
CAP is utilized for encapsulation of probiotic bacteria due to its
good protection, safety nature and being physically inert characteristics
under simulated GI conditions. This polymer is insoluble at acidic pH
(< 5) but soluble at pH > 6 because of its ionizable phthalate groups
(Mortazavian et al., 2007). Addition of spray dried Bifidobacterium an-
imalis encapsulated in CAP along with inulin significantly enhanced
probiotic viability during storage at 5 °C for 30 days (Antunes et al.,
2013).
6.8. Milk proteins
Milk proteins are remarkable encapsulation materials because of its
biocompatibility and their gel forming ability similar to gelatin under
suitable conditions. Due to its structural and physico-chemical char-
acteristics, they are suitable as natural vehicles in probiotics cell de-
livery system (Livney, 2010). Whey proteins has been revealed to
provide better protection for L. rhamnosus GG in apple juice compared
to resistant starch alone during storage at 4 °C (Ying et al., 2013). En-
capsulation of Bifidobacterium lactis and Lactobacillus paracasei, in a
casein gel obtained by action of transglutaminase or rennet increased
the encapsulation efficiency as well as viability of probiotics in simu-
lated gastric condition as well as during freeze drying and storage
(Heidebach et al., 2012). Ranadheera, Evans, Adams, and Baines (2015)
described that encapsulation of mixed cultures of L. acidophilus LA5,
Bifidobacterium animalis subsp. lactis BB12, and Propionibacterium jen-
senii 702 using goats’ milk did not affect the viability during storage at
4 °C, while a significantly reduction occurred at 30 °C.
6.9. Prebiotics and microbial exopolysaccharides (EPS)
The viability of encapsulated probiotic cells (LAB) have been re-
ported to be enhanced using prebiotic components (resistant starch) or
cryoprotectants (glycerol) in food products. Cryoprotectants protects a
D. Kavitake et al.
microbial cell from dehydration, osmotic stress and membrane damage
that leads to loss in cell viability and acidification activity. There are
several cryoprotectants such as glucose, sucrose, trehalose, lactose,
sorbitol, proteins and skim milk which has been used in order to im-
prove the cell viability and survival during freeze drying and sub-
sequent storage (Hubalek, 2003). Addition of prebiotics along with
calcium alginate provides better protection to LAB in food systems as
well as GI tract (Sultana et al., 2000). Co-encapsulation using a pre-
biotic Hi-maize starch along with chitosan or alginate coating sig-
nificantly improved the probiotic survival rate compared to free cells
(Iyer & Kailaspathy, 2005). Encapsulated B. bifidum 235 with prebiotics
gives improvement in sensory attributes in fermented food like dahi
compare to free cells (Vivek, 2013). Addition of non-resistant waxy
starch (0.6% w/v) to alginate-chitosan matrix of L. rhamnosus GG im-
proved stress tolerance upon freeze drying, two-week storage at 4 °C
and heat treatment at 100 °C for 30 min compared to resistant starch
(prebiotic Hi-Maize) (Cheow, Kiew, & Hadinoto, 2016). Peredo,
Beristain, Pascual, Azuara, and Jimenez (2016) evaluated the influence
of three natural prebiotics potato starch (PS), Plantago psyllium (PSY)
and Inulin (INU) along with alginate on the viability of L. casei and L.
plantarum. Higher encapsulation yield was obtained when PSY and INU
were used in co-encapsulation with alginate, which offers more viabi-
lity during storage and better protection in GI conditions. Shamekhi,
Shuhaimi, Ariff, and Manap (2013) demonstrated that B. lactis en-
capsulated with prebiotics mixed to calcium alginate matrix was ef-
fective for preparation of symbiotic powder nutraceutical for paediatric
use.
LAB are known to produce different types of EPS that differ in
chemical, structural and functional properties dependent upon strain
type, culture conditions and media composition. Microbial EPS plays a
vital role in protection of microbial cells in their natural environment
against severe conditions like desiccation, osmotic stress, antibiotics or
toxic compounds (Patel & Prajapat, 2013). Incorporation of β-glucan
could act as both prebiotic and encapsulating material which will be
favorable in stimulating the growth protecting beneficial probiotic
microflora (lactobacilli and bifidobacteria) in GI tract (Mitsou,
Panopoulou, Turunen, Spiliotis, & Kyriacou, 2010). Co-encapsulation of
probiotic cultures (Lactobacillus casei, L. brevis and L. plantarum) along
with β-glucan as matrix displayed improved tolerance towards acidic
pH, high temperature, simulated GI conditions and under storage
(Shah, Gani, Ahmad, Ashwar, & Masoodi, 2016).
7. Methods for evaluating the microencapsulation efficiency of
starter culture
The efficiency of the encapsulated starter culture is evaluated by
various methods such as viability and stability of LAB, capsule density
and micro geometrical properties of the beads, cell concentration, dif-
fusion property, encapsulation / entrapment yield (EY), moisture con-
tent and texture analyzer. The viability of encapsulated starter cultures
can be measured by inoculating the encapsulated beads in suitable
media and microbial load is determined by spread plate method (Annan
et al., 2008). While surface density (g/cm2) of the capsules is calculated
by breaking/dissolving the capsules, drying and weighing them and
dividing the result by total surface of the beads (Picot & Lacroix, 2004).
Total surface of the beads can be assessed with respect to the average
bead diameter and its proportion to the surface. Capsule density can be
done by compressing the beads and then measured using scanning
electron microscopy (SEM) (Sultana et al., 2000). Stability of en-
capsulated LAB can be determined by storing the beads at different
temperatures for different time duration and then the number of sur-
vived/ live cells calculated by performing spread plate technique.
Usually, encapsulated LAB shows more stability than free cells during
fermentation (Ding & Shah, 2009).
Micro geometrical properties comprise average bead shape, size/
diameter, integrity and uniformity in shape and size. Generally, beads
41
Food Bioscience 21 (2018) 34–44
size are measured using light scattering technique or laser dif-
fractometer (Picot & Lacroix, 2004). Spherical or elliptical shape of the
beads are observed directly by microscopic techniques. Efficiency of
beads depends upon integrity and uniformity of the beads which are
determined using SEM and light scattering technique. Uniformity of
beads size are done by sieving or membrane filtration method (Sultana
et al., 2000). Cell concentration in encapsulated beads is determined by
plate counting method using MRS agar after suspending the beads in
phosphate buffer (0.1 M, pH 7.0) and homogenization at 300 rpm on
orbital shaker (Martin, Lara-Villoslada, Ruiz, & Morales, 2013).
Entrapment yield gives an idea about the % cells that is successfully
entrapped into beads. It is calculated as follows: EY = (Log 10 N) / (Log
10 N0) × 100, where N – number of viable entrapped cells released
from the beads, N0 - number of free cells added to the polymer mixture
immediately before entrapment (Reid et al., 2005). Moisture content is
determined by subjecting the microcapsules to oven-drying at 100 °C
for 24 h and measured gravimetrically. Texture analysis show clear
differences in structure of the beads. Gel strength of the coating ma-
terial can be measured using a texture analyzer that will measure the
hardness, cohesiveness, springiness and resilience of the encapsulated
beads (Sandoval-Castilla, Lobato-Calleros, García-Galindo, Alvarez-
Ramírez, & Vernon-Carter, 2010).
8. Conclusion and future perspectives
Encapsulation is one of the most practical application that have
great possibility in prolonging the shelf life of food thereby availing
consumers with benign and healthier foods. Although the existing
technologies for encapsulation on a laboratory scale are effective, still
there are several challenges in producing food grade microencapsulated
microorganisms at a large scale large-scale. One significant challenge in
cell encapsulation is the size of encapsulated microbial cells (1–4 mm)
or freeze-dried culture particles (> 100 mm). Capsules with larger size
lean towards negative effect on textural and sensory qualities of food
products. Selection of appropriate technique and encapsulating mate-
rials are other main challenges. The foremost approach of micro-
encapsulation is to tackle the problem in survivability of starter culture
under storage environment as well as in food matrix. Use of electrical
devices and advances in nanotechnology also lead to more sophisticated
developments in encapsulation. Nanoencapsulation can take on im-
portance in near future for developing starter culture that could over-
come on bead size and stability.
Another key factor in future will involve the combined use of pre-
biotic and EPS in encapsulation, so that self-encapsulation can be
achieved using EPS producing microorganisms in which they will be
more compatible. However, microencapsulation and delivery of com-
bined/mixed starter culture in food system seems to underdeveloped.
Factors such as change in pH, mechanical stress, temperature, enzyme
activity, time and osmotic force can stimulate the rapid release of en-
capsulated cultures or ingredients. In such cases, release of en-
capsulated active agents inside a polymer matrix might be controlled
based on food constituents such as microbial load, pH and water ac-
tivity. Upcoming research must also govern the process cost, optimal
size of encapsulated cells that offers extreme defense to probiotics while
improving the sensory qualities of the product. As now, there are no
probiotic products stable at high temperatures, which are available in
markets. Since it is a great challenge to increase the survivability of
probiotics during the heat treatment process. Development of heat
stable starter cultures and an encapsulation system which effectually
can be an “insulation material” are needed in further research for
benefits in food industries.
Conflict of interest
The authors of this article declare that they have no conflicts of
interest of any kind.
D. Kavitake et al.
References
Albertini, B., Vitali, B., Passerini, N., Cruciani, F., Di Sabatino, M., Rodriguez, L., &
Brigidi, P. (2010). Development of microparticulate systems for intestinal delivery of
Lactobacillus acidophilus and Bifidobacterium lactis. European Journal of Pharmaceutical
Sciences, 40, 359–366.
Anal, A. K., & Singh, H. (2007). Recent advances in microencapsulation of probiotics for
industrial applications and targeted delivery. Trends in Food Science & Technology, 18,
240–251.
Anandharaj, M., & Sivasankari, B. (2013). Isolation of potential probiotic Lactobacillus
strains from human milk. International. Journal of Research and Development in
Pharmacy & Life Sciences, 1, 26–29.
Annan, N. T., Borza, A. D., & Hansen, L. T. (2008). Encapsulation in alginate-coated
gelatin microspheres improves survival of the probiotic Bifidobacterium adolescentis
15703T during exposure to simulated gastro-intestinal conditions. Food Research
International, 41, 184–193.
Antunes, A. E. C., Liserre, A. M., Coelho, A. L. A., Menezes, C. R., Moreno, I., Yotsuyanagi,
K., & Azambuja, N. C. (2013). Acerola nectar with added microencapsulated pro-
biotic. LWT – Food Science and Technology, 54, 125–131.
de Araújo Etchepare, M., Raddatz, G. C., de Moraes Flores, É. M., Zepka, L. Q., Jacob-
Lopes, E., Barin, J. S., & de Menezes, C. R. (2016). Effect of resistant starch and
chitosan on survival of Lactobacillus acidophilus microencapsulated with sodium
alginate. LWT-Food Science and Technology, 65, 511–517.
Argin, S., Kofinas, P., & Lo, Y. M. (2014). The cell release kinetics and the swelling be-
havior of physically crosslinked xanthan-chitosan hydrogels in simulated gastro-
intestinal conditions. Food Hydrocolloids, 40, 138–144.
Assadi, M. M., Pourahmad, R., & Moazami, N. (2000). Use of isolated kefir starter cultures
in kefir production. World Journal of Microbiology and Biotechnology, 16(6), 541–543.
Baer, A., & Ryba, I. (1992). Serological identification of propionibacteria in milk and
cheese samples. International Dairy Journal, 2(5), 299–310.
Barbosa, M. S., Todorov, S. D., Jurkiewicz, C. H., & Franco, B. D. (2015). Bacteriocin
production by Lactobacillus curvatus MBSa2 entrapped in calcium alginate during
ripening of salami for control of Listeria monocytogenes. Food Control, 47, 147–153.
Behboudi-Jobbehdar, S., Soukoulis, C., Yonekura, L., & Fisk, I. (2013). Optimization of
spray-drying process conditions for the production of maximally viable micro-
encapsulated L. acidophilus NCIMB 701748. Drying Technology, 31, 1274–1283.
Bekhit, M., Sánchez-González, L., Messaoud, G. B., & Desobry, S. (2016). Encapsulation of
Lactococcus lactis subsp. lactis on alginate/pectin composite microbeads: Effect of
matrix composition on bacterial survival and nisin release. Journal of Food
Engineering, 180, 1–9.
Benkerroum, N. (2013). Traditional fermented foods of North African countries:
Technology and food safety challenges with regard to microbiological risks.
Comprensive Reviews in Food Science and Food Safety, 12, 54–89.
Bilenler, T., Karabulut, I., & Candogan, K. (2017). Effects of encapsulated starter cultures
on microbial and physicochemical properties of traditionally produced and heat-
treated sausages (sucuks). LWT Food Science and Technology, 75, 425–433.
Blana, V. A., Grounta, A., Tassou, C. C., Nychas, G. J. E., & Panagou, E. Z. (2014).
Inoculated fermentation of green olives with potential probiotic Lactobacillus pentosus
and Lactobacillus plantarum starter cultures isolated from industrially fermented ol-
ives. Food Microbiology, 38, 208–218.
Borresen, C. E., Henderson, A. J., Kumar, A., Weir, T. L., & Ryan, E. P. (2012). Fermented
foods: Patented approaches and formulations for nutritional supplementation and
health promotion. Recent Patents on Food, Nutrition and Agriculture, 4, 134–140.
Brown, L., Pingitore, E. V., Mozzi, F., Saavedra, L., Villegas, J. M., & Hebert, E. M. (2016).
Lactic acid bacteria as cell factories for the generation of bioactive peptides. Protein
and Peptide Letters, 24, 146–155.
Burgain, J., Gaiani, C., Linder, M., & Scher, J. (2011). Encapsulation of probiotic living
cells: From laboratory scale to industrial applications. Journal of Food Engineering,
104, 467–483.
de Castro-Cislaghi, F. P., Silva, C. D. R. E., Fritzen-Freire, C. B., Lorenz, J. G., & Sant’Anna,
E. S. (2012). Bifidobacterium Bb-12 microencapsulated by spray drying with whey:
Survival under simulated gastrointestinal conditions, tolerance to NaCl, and viability
during storage. Journal of Food Engineering, 113, 186–193.
Champagne, C. P., & Fustier, P. (2007). Microencapsulation for the improved delivery of
bioactive compounds into foods. Current Opinion in Biotechnology, 18, 184–190.
Chelule, P. K., Mokoena, M. P., & Gqaleni, N. (2010). Advantages of traditional lactic acid
bacteria fermentation of food in Africa. Current Research, Technology and Education
Topics in Applied Microbiology and Microbial Biotechnology, 2, 1160–1167.
Chen, K. N., Chen, M. J., Liu, J. R., Lin, C. W., & Chiu, H. Y. (2005). Optimization of
incorporated prebiotics as coating materials for probiotic microencapsulation.
Journal of Food Science, 70, 260–266.
Cheow, W. S., Kiew, T. Y., & Hadinoto, K. (2016). Effects of adding resistant and waxy
starches on cell density and survival of encapsulated biofilm of Lactobacillus rham-
nosus GG probiotics. LWT-Food Science and Technology, 69, 497–505.
Choi, I. K., Jung, S. H., Kim, B. J., Park, S. Y., Kim, J., & Han, H. U. (2003). Novel
Leuconostoc citreum starter culture system for the fermentation of kimchi, a fermented
cabbage product. Antonie Vanăto- Leeuwenhoek, 84(4), 247–253.
Coppola, S., Mauriello, G., Aponte, M., Moschetti, G., & Villani, F. (2000). Microbial
succession during ripening of Naples-type salami, a southern Italian fermented sau-
sage. Meat Science, 56(4), 321–329.
Corbo, M. R., Bevilacqua, A., Speranza, B., Di Maggio, B., Gallo, M., & Sinigaglia, M.
(2016). Use of alginate beads as carriers for lactic acid bacteria in a structured system
and preliminary validation in a meat product. Meat Science, 111, 198–203.
Corcoran, B. M., Ross, R. P., Fitzgerald, G. F., & Stanton, C. (2004). Comparative survival
of probiotic lactobacilli spray-dried in the presence of prebiotic substances. Journal of
42
Food Bioscience 21 (2018) 34–44
Applied Microbiology, 96, 1024–1039.
Cui, J. H., Goh, J. S., Kim, P. H., Choi, S. H., & Lee, B. J. (2000). Survival and stability of
bifidobacteria loaded in alginate poly-L-lysine microparticles. International Journal of
Pharmaceutics, 210, 51–59.
Darukaradhya, J., Phillips, M., & Kailasapathy, K. (2013). Effect of encapsulation on the
survival of probiotic bacteria in the presence of starter and non-starter lactic acid
bacteria in Cheddar cheese over a 6-month ripening period. International Journal of
Fermented Foods, 2, 63–76.
De Castro, A., Montaño, A., Casado, F. J., Sánchez, A. H., & Rejano, L. (2002). Utilization
of Enterococcus casseliflavus and Lactobacillus pentosus as starter cultures for Spanish-
style green olive fermentation. Food Microbiology, 19(6), 637–644.
Desai, K. G. H., & Park, H. J. (2005). Encapsulation of vitamin C in tripolyphosphate
cross-linked chitosan microspheres by spray drying. Journal of Microencapsulation, 22,
179–192.
Desmond, C., Ross, R. P., O'callaghan, E., Fitzgerald, G., & Stanton, C. (2002). Improved
survival of Lactobacillus paracasei NFBC 338 in spray-dried powders containing gum
acacia. Journal of Applied Microbiology, 93, 1003–1011.
Dianawati, D., Mishra, V., & Shah, N. P. (2013). Survival of Bifidobacterium longum 1941
microencapsulated with proteins and sugars after freezing and freeze drying. Food
Research International, 51, 503–509.
Ding, W. K., & Shah, N. P. (2009). Effect of various encapsulating materials on the sta-
bility of probiotic bacteria. Journal of Food Science, 74(2).
Doleyres, Y., Fliss, I., & Lacroix, C. (2004). Continuous production of mixed lactic starters
containing probiotics using immobilized cell technology. Biotechnology Progress, 20,
145–150.
Fang, Z., & Bhandari, B. (2010). Encapsulation of polyphenols–a review. Trends in Food
Science and Technology, 21, 510–523.
Fijałkowski, K., Peitler, D., Rakoczy, R., & Żywicka, A. (2016). Survival of probiotic lactic
acid bacteria immobilized in different forms of bacterial cellulose in simulated gastric
juices and bile salt solution. LWT-Food Science and Technology, 68, 322–328.
Fontán, M. C. G., Martínez, S., Franco, I., & Carballo, J. (2006). Microbiological and
chemical changes during the manufacture of Kefir made from cows' milk, using a
commercial starter culture. International Dairy Journal, 16(7), 762–767.
Franz, C. M., Huch, M., Mathara, J. M., Abriouel, H., Benomar, N., Reid, G., & Holzapfel,
W. H. (2014). African fermented foods and probiotics. International Journal of Food
Microbiology, 190, 84–96.
Gandomi, H., Abbaszadeh, S., Misaghi, A., Bokaie, S., & Noori, N. (2016). Effect of
chitosan-alginate encapsulation with inulin on survival of Lactobacillus rhamnosus GG
during apple juice storage and under simulated gastrointestinal conditions. LWT-Food
Science and Technology, 69, 365–371.
Garcıa-Ochoa, F., Santos, V. E., Casas, J. A., & Gomez, E. (2000). Xanthan gum:
Production, recovery, and properties. Biotechnology Advances, 18, 549–579.
Ghaffar, T., Irshad, M., Anwar, Z., Aqil, T., Zulifqar, Z., Tariq, A., & Mehmood, S. (2014).
Recent trends in lactic acid biotechnology: A brief review on production to pur-
ification. Journal of Radiation Research and Applied Sciences, 7(2), 222–229.
Gibbs, B. F., Kermasha, S., Ali, I., & Mulligan, C. N. (1999). Encapsulation in the food
industry: A review. International Journal of Food Science and Nutrition, 50, 213–224.
Gouin, S. (2004). Microencapsulation: Industrial appraisal of existing technologies and
trends. Trends Food Science and Technology, 15, 330–347.
Haffner, F. B., Diab, R., & Pasc, A. (2016). Encapsulation of probiotics: Insights into
academic and industrial approaches. AIMS Materials Science, 3, 114–136.
Heidebach, T., Först, P., & Kulozik, U. (2012). Microencapsulation of probiotic cells for
food applications. Critical Reviews in Food Science and Nutrition, 52, 291–311.
Holkem, A. T., Raddatz, G. C., Barin, J. S., Flores, É. M. M., Muller, E. I., Codevilla, C. F., &
de Menezes, C. R. (2017). Production of microcapsules containing Bifidobacterium BB-
12 by emulsification/internal gelation. LWT-Food Science and Technology, 76,
216–221.
Holzapfel, W. H., & Wood, B. J. (Eds.). (2014). Lactic acid bacteria: biodiversity and tax-
onomy (pp. 447–455). John Wiley Sons, Ltd.
Hou, J., Hannon, J. A., McSweeney, P. L., Beresford, T. P., & Guinee, T. P. (2017). Effect of
galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a
starter culture adjunct on the properties of Cheddar cheese made with low or high pH
at whey drainage. International Dairy Journal, 65, 44–55.
Hubalek, Z. (2003). Protectants used in the cryopreservation of microorganisms.
Cryobiology, 46(3), 205–229.
Iyer, C., & Kailaspathy, K. (2005). Effect of co-encapsulation of probiotics with prebiotics
on increasing the viability of encapsulated bacteria under in vitro acidic and bile salt
conditions and in yogurt. Food Microbiology and Safety, 70, 1–6.
Johanningsmeier, S., McFeeters, R. F., Fleming, H. P., & Thompson, R. L. (2007). Effects
of Leuconostoc mesenteroides starter culture on fermentation of cabbage with reduced
salt concentrations. Journal of Food Science, 72(5).
John, R. P., Tyagi, R. D., Brar, S. K., Surampalli, R. Y., & Prévost, D. (2011).
Bioencapsulation of microbial cells for targeted agricultural delivery. Critical Reviews
in Biotechnology, 31, 211–226.
Kailasapathy, K. (2006). Survival of free and encapsulated probiotic bacteria and their
effect on the sensory properties of yogurt. LWT Food Science and Technology, 39,
1221–1227.
Kang, K. S., & Petitt, D. J. (1993). Xanthan, gellan, welan and rhamsan. In R. L. Whistler,
& J. N. BeMiller (Eds.). Industrial gums. Polysaccharides and their derivatives (pp. 343–
397). (3rd ed.). New York and London: Academic Press Inc.
Kanmani, P., & Lim, S. T. (2013). Development and characterization of novel probiotic-
residing pullulan/starch edible films. Food Chemistry, 141, 1041–1049.
Kim, J. U., Kim, B., Shahbaz, H. M., Lee, S. H., Park, D., & Park, J. (2016). Encapsulation
of probiotic Lactobacillus acidophilus by ionic gelation with electrostatic extrusion for
enhancement of survival under simulated gastric conditions and during refrigerated
storage. International Journal of Food Science and Technology, 52, 519–530.
D. Kavitake et al.
Kimaryo, V. M., Massawe, G. A., Olasupo, N. A., & Holzapfel, W. H. (2000). The use of a
starter culture in the fermentation of cassava for the production of “kivunde”, a
traditional Tanzanian food product. International Journal of Food Microbiology, 56(2),
179–190.
Kingcha, Y., Tosukhowong, A., Zendo, T., Roytrakul, S., Luxananil, P., Chareonpornsook,
K., & Visessanguan, W. (2012). Anti-listeria activity of Pediococcus pentosaceus BCC
3772 and application as starter culture for Nham, a traditional fermented pork sau-
sage. Food Control, 25(1), 190–196.
Klaypradit, W., & Huang, Y. W. (2008). Fish oil encapsulation with chitosan using ul-
trasonic atomizer. LWT Food Science and Technology, 41, 1133–1139.
Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). Evaluation of encapsulation techni-
ques of probiotics for yogurt. International Dairy Journal, 13, 3–13.
Kuck, L. S., & Noreña, C. P. Z. (2016). Microencapsulation of grape (Vitis labrusca var.
Bordo) skin phenolic extract using gum Arabic, polydextrose, and partially hydro-
lyzed guar gum as encapsulating agents. Food Chemistry, 194, 569–576.
de Lara Pedroso, D., Thomazini, M., Heinemann, R. J. B., & Favaro-Trindade, C. S. (2012).
Protection of Bifidobacterium lactis and Lactobacillus acidophilus by microencapsula-
tion using spray-chilling. International Dairy Journal, 26, 127–132.
Lee, J. S., Cha, D. S., & Park, H. J. (2004). Survival of freeze-dried Lactobacillus bulgaricus
KFRI 673 in chitosan-coated calcium alginate microparticles. Journal of Agriculture
and Food Chemistry, 52, 7300–7305.
Lee, J. S., Heo, G. Y., Jun, W. L., Oh, Y. J., Park, J. A., Park, Y. H., ... Jong, S. A. (2005).
Analysis of kimchi microflora using denaturing gradient gel electrophoresis.
International Journal of Food Microbiology, 5(102), 143–150.
Léonard, L., Beji, O., Arnould, C., Noirot, E., Bonnotte, A., Gharsallaoui, A., & Oulahal, N.
(2015). Preservation of viability and anti-Listeria activity of lactic acid bacteria,
Lactococcus lactis and Lactobacillus paracasei, entrapped in gelling matrices of alginate
or alginate/caseinate. Food Control, 47, 7–19.
Leroy, F., Verluyten, J., & De Vuyst, L. (2006). Functional meat starter cultures for im-
proved sausage fermentation. International Journal of Food Microbiology, 106(3),
270–285.
Li, H., Turner, M. S., & Dhital, S. (2016). Encapsulation of Lactobacillus plantarum in
porous maize starch. LWT Food Science and Technology, 74, 542–549.
Livney, Y. D. (2010). Milk proteins as vehicles for bioactives. Current Opinion in Colloid
and Interface Science, 15, 73–83.
Marco, M. L., Heeney, D., Binda, S., Cifelli, C. J., Cotter, P. D., Foligné, B., & Smid, E. J.
(2017). Health benefits of fermented foods: Microbiota and beyond. Current Opinion
in Biotechnology, 44, 94–102.
Martin, M. J., Lara-Villoslada, F., Ruiz, M. A., & Morales, M. E. (2013). Effect of un-
modified starch on viability of alginate-encapsulated Lactobacillus fermentum
CECT5716. LWT Food Science and Technology, 53, 480–486.
McMaster, L. D., Kokott, S. A., Reid, S. J., & Abratt, V. R. (2005). Use of traditional African
fermented beverages as delivery vehicles for Bifidobacterium lactis DSM 10140.
International Journal of Food Microbiology, 102, 231–237.
Michaylova, M., Minkova, S., Kimura, K., Sasaki, T., & Isawa, K. (2007). Isolation and
characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermo-
philus from plants in Bulgaria. FEMS Microbiology Letters, 269(1), 160–169.
Mishra, V., Shah, C., Mokashe, N., Chavan, R., Yadav, H., & Prajapati, J. (2015).
Probiotics as potential antioxidants: A systematic review. Journal of Agriculture and
Food Chemistry, 63, 3615–3626.
Mitsou, E. K., Panopoulou, N., Turunen, K., Spiliotis, V., & Kyriacou, A. (2010). Prebiotic
potential of barley derived β-glucan at low intake levels: A randomised, double-
blinded, placebo-controlled clinical study. Food Research International, 43,
1086–1092.
Mortazavian, A., Razavi, S. H., Ehsani, M. R., & Sohrabvandi, S. (2007). Principles and
methods of microencapsulation of probiotic microorganisms. Iranian Journal of
Biotechnology, 5, 1–18.
Mugula, J. K., Narvhus, J. A., & Sørhaug, T. (2003). Use of starter cultures of lactic acid
bacteria and yeasts in the preparation of togwa, a Tanzanian fermented food.
International Journal of Food Microbiology, 83(3), 307–318.
Nawong, S., Oonsivilai, R., Boonkerd, N., & Hansen, L. T. (2016). Entrapment in food-
grade transglutaminase cross-linked gelatin–maltodextrin microspheres protects
Lactobacillus spp. during exposure to simulated gastro-intestinal juices. Food Research
International, 85, 191–199.
Nout, M. R. (2003). Traditional fermented products from Africa, Latin America and Asia.
In T. Boekhout, & V. Robert (Eds.). Yeasts in food - beneficial and detrimental aspects
(pp. 451–473). Behr's-Verlag.
Nualkaekul, S., Cook, M. T., Khutoryanskiy, V. V., & Charalampopoulos, D. (2013).
Influence of encapsulation and coating materials on the survival of Lactobacillus
plantarum and Bifidobacterium longum in fruit juices. Food Research International, 53,
304–311.
O’Riordan, K., Andrews, D., Buckle, K., & Conway, P. (2001). Evaluation of micro-
encapsulation of a Bifidobacterium strain with starch as an approach to prolonging
viability during storage. Journal of Applied Microbiology, 91, 1059–1066.
Oguntoyinbo, F. A., Tourlomousis, P., Gasson, M. J., & Narbad, A. (2011). Analysis of
bacterial communities of traditional fermented West African cereal foods using cul-
ture independent methods. International Journal of Food Microbiology, 145, 205–210.
Ong, L., Henriksson, A., & Shah, N. P. (2006). Development of probiotic Cheddar cheese
containing Lactobacillus acidophilus, Lb. casei, Lb. paracasei and Bifidobacterium spp.
and the influence of these bacteria on proteolytic patterns and production of organic
acid. International Dairy Journal, 16(5), 446–456.
Ouled-Haddar, H., Sifour, M., Idoui, T., Bouridane, H., & Arid, S. (2016). Lactobacillus
plantarum G1 Microencapsulation enhanced its viability during storage and gastro-
intestinal transit. Sains Malaysiana, 45, 1049–1055.
Paramithiotis, S., Chouliaras, Y., Tsakalidou, E., & Kalantzopoulos, G. (2005). Application
of selected starter cultures for the production of wheat sourdough bread using a
43
Food Bioscience 21 (2018) 34–44
traditional three-stage procedure. Process Biochemistry, 40(8), 2813–2819.
Park, J. K., & Chang, H. N. (2000). Microencapsulation of microbial cells. Biotechnology
Advances, 18, 303–319.
Patel, A., & Prajapat, J. B. (2013). Food and health applications of exopolysaccharides
produced by lactic acid bacteria. Advances in Dairy Research, 1–8.
Peredo, A. G., Beristain, C. I., Pascual, L. A., Azuara, E., & Jimenez, M. (2016). The effect
of prebiotics on the viability of encapsulated probiotic bacteria. LWT-Food Science and
Technology, 73, 191–196.
Picot, A., & Lacroix, C. (2004). Encapsulation of bifidobacteria in whey protein-based
microcapsules and survival in simulated gastrointestinal conditions and in yogurt.
International Dairy Journal, 14, 505–515.
Poshadri, A., & Kuna, A. (2010). Microencapsulation technology: A review. Journal
Resource of ANGRAU, 38, 86–102.
de Prisco, A., & Mauriello, G. (2016). Probiotication of foods: A focus on micro-
encapsulation tool. Trends in Food Science and Technology, 48, 27–39.
Quinto, E. J., Jiménez, P., Caro, I., Tejero, J., Mateo, J., & Girbés, T. (2014). Probiotic
lactic acid bacteria: A review. Food Nutrition and Science, 5, 1765–1775.
Ranadheera, C. S., Evans, C. A., Adams, M. C., & Baines, S. K. (2015). Microencapsulation
of Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12 and
Propionibacterium jensenii 702 by spray drying in goat's milk. Small Ruminant Research,
123, 155–159.
Rathore, S., Wan, Sia Heng, P., & Chan, L. W. (2015). Microencapsulation of Clostridium
acetobutylicum ATCC 824 spores in gellan gum microspheres for the production of
biobutanol. Journal of Microencapsulation, 32, 290–299.
Rattanachaikunsopon, P., & Phumkhachorn, P. (2010). Lactic acid bacteria: Their anti-
microbial compounds and their uses in food production. Annals of Biological Research,
1(4), 218–228.
Ray, M., Ghosh, K., Singh, S., & Mondal, K. C. (2016). Folk to functional: An explorative
overview of rice-based fermented foods and beverages in India. Journal of Ethnic
Foods, 3, 5–18.
Raymond, M. C., Neufeld, R. J., & Poncelet, D. (2004). Encapsulation of brewers yeast in
chitosan coated carrageenan microspheres by emulsification/thermal gelation.
Artificial Cells, Blood Substitutes and Biotechnology, 32(2), 275–291.
Reale, A., Mannina, L., Tremonte, P., Sobolev, A. P., Succi, M., Sorrentino, E., & Coppola,
R. (2004). Phytate degradation by lactic acid bacteria and yeasts during the whole-
meal dough fermentation: A 31P NMR study. Journal of Agricultural and Food
Chemistry, 52(20), 6300–6305.
Reid, A. A., Vuillemard, J. C., Britten, M., Arcand, Y., Farnworth, E., & Champagne, C. P.
(2005). Microentrapment of probiotic bacteria in a Ca2+-induced whey protein gel
and effects on their viability in a dynamic gastro-intestinal model. Journal of
Microencapsulation, 22(6), 603–619.
Rhee, S. J., Lee, J. E., & Lee, C. H. (2011). Importance of lactic acid bacteria in Asian
fermented foods. Microbial Cell Factories, 10, S5.
Saithong, P., Panthavee, W., Boonyaratanakornkit, M., & Sikkhamondhol, C. (2010). Use
of a starter culture of lactic acid bacteria in plaa-som, a Thai fermented fish. Journal
of Bioscience and Bioengineering, 110(5), 553–557.
Sandoval-Castilla, O., Lobato-Calleros, C., García-Galindo, H. S., Alvarez-Ramírez, J., &
Vernon-Carter, E. J. (2010). Textural properties of alginate–pectin beads and survi-
vability of entrapped Lb. casei in simulated gastrointestinal conditions and in yogurt.
Food Research International, 43(1), 111–117.
Sathe, G. B., & Mandal, S. (2016). Fermented products of India and its implication: A
review. Asian Journal of Dairy and Food Research, 35, 1–9.
Shah, A., Gani, A., Ahmad, M., Ashwar, B. A., & Masoodi, F. A. (2016). β-Glucan as an
encapsulating agent: Effect on probiotic survival in simulated gastrointestinal tract.
International Journal of Biological Macromolecules, 82, 217–222.
Shamekhi, F., Shuhaimi, M., Ariff, A., & Manap, Y. A. (2013). Cell viability of micro-
encapsulated Bifidobacterium animalis subsp. lactis under freeze-drying, storage and
gastrointestinal tract simulation conditions. Folia Microbiologica, 58, 91–101.
Singh, T. A., Devi, K. R., Ahmed, G., & Jeyaram, K. (2014). Microbial and endogenous
origin of fibrinolytic activity in traditional fermented foods of Northeast India. Food
Research International, 55, 356–362.
Singracha, P., Niamsiri, N., Visessanguan, W., Lertsiri, S., & Assavanig, A. (2017).
Application of lactic acid bacteria and yeasts as starter cultures for reduced-salt soy
sauce (moromi) fermentation. LWT – Food Science and Technology, 78, 181–188.
Sodini, I., Lucas, A., Oliveira, M. N., Remeuf, F., & Corrieu, G. (2002). Effect of milk base
and starter culture on acidification, texture, and probiotic cell counts in fermented
milk processing. Journal of Dairy Science, 85(10), 2479–2488.
Solanki, H. K., Pawar, D. D., Shah, D. A., Prajapati, V. D., Jani, G. K., Mulla, A. M., &
Thakar, P. M. (2013). Development of microencapsulation delivery system for long-
term preservation of probiotics as biotherapeutics agent. BioMed Research
International. http://dx.doi.org/10.1155/2013/620719.
Soukoulis, C., Singh, P., Macnaughtan, W., Parmenter, C., & Fisk, I. D. (2016).
Compositional and physicochemical factors governing the viability of Lactobacillus
rhamnosus GG embedded in starch-protein based edible films. Food Hydrocolloids, 52,
876–887.
Steinkraus, K. H. (2002). Fermentations in world food processing. Comprehensive Reviews
in Food Science and Food Safety, 1, 23–32.
Sultana, K., Godward, G., Reynolds, N., Arumugaswamy, R., Peiris, P., & Kailasapathy, K.
(2000). Encapsulation of probiotic bacteria with alginate - starch and evaluation of
survival in simulated gastrointestinal conditions and in yogurt. International Journal
of Microbiology, 62, 47–55.
Sun, W., & Griffiths, M. W. (2000). Survival of bifidobacteria in yogurt and simulated
gastric juice following immobilization in gellan-xanthan beads. International Journal
of Microbiology, 61, 17–25.
Talwalkar, A., & Kailasapathy, K. (2004). The role of oxygen in the viability of probiotic
bacteria with reference to L. acidophilus and Bifidobacterium spp. Current Issues on
D. Kavitake et al.
Intestinal Microbiology, 5, 1–8.
Tamang, J. P., Thapa, N., Tamang, B., Rai, A., & Chettri, R. (2015). Microorganisms in
fermented foods and beverages. In J. P. Tamang (Ed.). Health benefits of fermented
foods and beverages (pp. 1–110). CRC Press.
Tamang, J. P., Watanabe, K., & Holzapfel, W. H. (2016). Review: Diversity of micro-
organisms in global fermented foods and beverages. Frontiers in Microbiology, 7.
Tylingo, R., Mania, S., & Szwacki, J. (2016). A novel method for drop in drop edible oils
encapsulation with chitosan using a coaxial technique. Reactive and Functional
Polymers, 100, 64–72.
Udomsil, N., Rodtong, S., Choi, Y. J., Hua, Y., & Yongsawatdigul, J. (2011). Use of
Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce
fermentation. Journal of Agricultural and Food Chemistry, 59(15), 8401–8408.
Vivek, K. (2013). Use of encapsulated probiotics in dairy based foods. International Journal
of Food, Agriculture and Veterinary Sciences, 3, 188–199.
de Vos, P., Faas, M. M., Spasojevic, M., & Sikkema, J. (2010). Encapsulation for
44
Food Bioscience 21 (2018) 34–44
preservation of functionality and targeted delivery of bioactive food components.
International Dairy Journal, 20, 292–302.
Wang, L., Yu, X., Xu, H., Aguilar, Z. P., & Wei, H. (2016). Effect of skim milk coated
inulin-alginate encapsulation beads on viability and gene expression of Lactobacillus
plantarum during freeze-drying. LWT – Food Science and Technology, 68, 8–13.
Xiong, T., Li, X., Guan, Q., Peng, F., & Xie, M. (2014). Starter culture fermentation of
Chinese sauerkraut: Growth, acidification and metabolic analyses. Food Control, 41,
122–127.
Ying, D., Schwander, S., Weerakkody, R., Sanguansri, L., Gantenbein-Demarchi, C., &
Augustin, M. A. (2013). Microencapsulated Lactobacillus rhamnosus GG in whey
protein and resistant starch matrices: Probiotic survival in fruit juice. Journal of
Functional Foods, 5, 98–105.
Zhang, Z., Zhang, R., Zou, L., & Mc Clements, D. J. (2016). Protein encapsulation in
alginate hydrogel beads: Effect of pH on microgel stability, protein retention and
protein release. Food Hydrocolloids, 58, 308–315.