LWT - Food Science and Technology 146 (2021) 111423

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LWT
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Encapsulation of Lactobacillus reuteri in W1/O/W2 double emulsions:

Formulation, storage and in vitro gastro-intestinal digestion stability
Ali Marefati a, *, Anastasios Pitsiladis a, Elin Oscarsson b, Niclas Ilestam c, Björn Bergenståhl a
a
Department of Food Technology, Engineering and Nutrition, Lund University, Box 124, 221 00, Lund, Sweden
b
The Diabetes and Celiac Disease Unit, Department of Clinical Sciences, Lund University, 205 02, Malmö, Sweden
c
BioGaia AB, Mobilvägen 10, 223 62, Lund, Sweden

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

Keywords: Encapsulation of Lactobacillus reuteri in the internal aqueous phase of W1/O/W2 emulsions was investigated.
Encapsulation of probiotics Microstructure of the emulsions was evaluated using a particle size analyzer and light microscopy. The encap­
Polyglycerol polyricinoleate sulation properties were evaluated in terms of encapsulation efficiency (EE) for freshly prepared samples and
Poloxamer 407
encapsulation stability (ES) during storage and in vitro digestion and the results were compared to control.
Emulsion microstructure
Viability of bacteria
Particle size analysis showed that encapsulation of the bacteria increased the droplet sizes of W1/O emulsions
significantly (P < 0.05) from 3.5 ± 0.2 to 4.8 ± 0.4 μm which was verified by microscopy. Neither encapsulation
nor storage changed (P > 0.05) the droplet sizes for double emulsions (14.9 ± 0.3 μm) compared to control (15.5
± 0.4 μm) which remained stable through storage period. The EE was 7.23 ± 0.07 Log CFU/mL. During cold
storage, the ES decreased with a higher pace for control (from 6.18 ± 0.03 to <1 Log CFU/mL) compared to
formulations with encapsulated bacteria (from 7.23 ± 0.07 to 2.82 ± 0.10 Log CFU/mL). Finally, during in vitro
digestion, the ES decreased with a higher rate for control (from 6.25 ± 0.36 to 2.69 ± 0.06 Log CFU/mL)
compared to encapsulated samples (from 6.69 ± 0.07 to 4.64 ± 0.06 Log CFU/mL). The results showed that
encapsulation of Lactobacillus reuteri using double emulsions can protect the probiotics during storage and in vitro
gastro-intestinal digestion.

1. Introduction
Recently the focus on food has changed from its primary role which
is a source of energy and nutrients to providing a complex system that
can promote human health and well-being. These complex systems are
referred to as “functional foods” or “nutraceuticals”. One way of
generating functional foods is by the inclusion of viable microorganisms
known as probiotics in the food systems which are claimed to have
beneficial effects on human health. In addition, there is a growing in­
terest in probiotics as remedies for gastro-intestinal diseases (Calinescu
& Mateescu, 2008). The most popular strains of probiotics are Lacto­
bacillus, Streptococcus, and Bifidobacterium, however, other organisms
such as yeasts have also been used as probiotics (Chow, 2002). It has
been reported that presence of probiotic bacteria is beneficial for pa­
tients with inflammatory bowel disease (Favier et al., 1997; Hartley
et al., 1992). In addition, probiotics have other nutritional and thera­
peutic benefits including improved digestion of lactose, controlling
some types of cancer and controlling cholesterol levels (Calinescu &
Mateescu, 2008). Therefore, colon delivery is the main goal in the
development of formulations for the prevention of inflammatory bowel
disease as well as other diseases associated with the colon. While the
dead bacteria and their components or fermentation byproducts can also
have probiotic properties, the delivery of live bacteria is considered to
be more effective (Shima, Morita, Yamashita, & Adachi, 2006). In order
to impose the beneficial effect, a sufficient number of >106–107 colony
forming units/mL (CFU/mL) or CFU/g of the viable microorganisms
should be present at the products, however, this recommended level is
not always delivered in commercial products (Anal & Singh, 2007; Ross,
Desmond, Fitzgerald, & Stanton, 2005; Zhao et al., 2020). Since some
strains of probiotics are sensitive towards different environmental fac­
tors, an efficient formulation for colon delivery should be in a way that
the microorganisms can survive the manufacturing, storage, and even
consumption conditions since they are sensitive to many environmental
stresses such as stomach acidity, oxygen, bile salts, and temperature
(Anal & Singh, 2007; Lucas, Sodini, Monnet, Jolivet, & Corrieu, 2004).
Lactobacillus reuteri is a Gram-positive bacterium which is commonly

* Corresponding author.
E-mail address: Ali.Marefati@food.lth.se (A. Marefati).

https://doi.org/10.1016/j.lwt.2021.111423
Received 11 November 2020; Received in revised form 29 March 2021; Accepted 30 March 2021
Available online 2 April 2021
0023-6438/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
A. Marefati et al.
found in the gastro-intestinal tract as well as in different locations such
as the urinary tract, skin, and breast milk (Mu, Tavella, & Luo, 2018). It
is rod-shaped (0.7–1.0 in 2.0–5.0 μm) which grows between 15 ◦ C and
45 ◦ C and the pH range of 5.0–7.5 (Kandler, Stetter, & Köhl, 1980).
Several beneficial effects have been reported for Lactobacillus reuteri
including antibacterial properties due to producing anti-microbial sub­
stances, such as organic acids, ethanol, and reuterin which enables it to
inhibit the colonization of pathogenic microbes and optimize the
microbiota composition in the host (Axelsson, Chung, Dobrogosz, &
Lindgren, 1989; Mu et al., 2018). In addition, Lactobacillus reuteri can
improve the immune system of the host by reducing the production of
pro-inflammatory cytokines while promoting regulatory T cells (Mu
et al., 2018). Furthermore, colonization of Lactobacillus reuteri can
decrease the microbial translocation from the intestinal lumen to the
tissue and prevent inflammation by strengthening the intestinal barrier
(Mu et al., 2018). Therefore, supplementation of Lactobacillus reuteri is
an attractive way to prevent and/or treat inflammatory diseases.
Microencapsulation of probiotic bacteria can be used to protect their
viability during processing, storage, and gastro-intestinal conditions and
thereby, secure their delivery to the targeted sites in the gastro-intestinal
tract (Rodrigues, Cedran, Bicas, & Sato, 2020; Zam, 2020). Microen­
capsulation is a process where the cells are entrapped within a semi or
non-permeable, strong, thin matrix or membrane (Pimentel-González,
Campos-Montiel, Lobato-Calleros, Pedroza-Islas, & Vernon-Carter,
2009). Various formulation approaches have been successfully used
for the encapsulation of bacteria (Annan, Borza, & Hansen, 2008; Jan­
kowski, Zielinska, & Wysakowska, 1997; Khalil & Mansour, 1998; Lee &
Heo, 2000; Rodríguez-Huezo et al., 2007). In addition to those studies
mentioned above, emulsions have also been used as a formulation
approach for encapsulation of probiotic bacteria (Eslami, Davarpanah,
& Vahabzadeh, 2017; Pimentel-González et al., 2009; Satapathy et al.,
2019; Shima et al., 2006; Shima, Matsuo, & Adachi, 2007; Shima,
Matsuo, Yamashita, & Adachi, 2009). Due to the hydrophobic character
of the bacterial cells, dispersed aqueous phase (as in W/O or W1/O/W2
type emulsion) is preferred (Wang et al., 2020). Double emulsions have
shown to be suitable for encapsulation and delivery of bioactive com­
pounds (Dickinson, 2011; Marefati, Sjöö, Timgren, Dejmek, & Rayner,
2015). Previous studies have shown that the viability of probiotics
entrapped in the inner phase solution of W1/O/W2 double emulsions
was significantly higher than control samples at the initial state and also
during processing or simulated gastric and/or intestinal conditions
(Pimentel-González et al., 2009; Rodríguez-Huezo et al., 2014; Shima
et al., 2006).
Although the encapsulation properties of the W1/O/W2 emulsions in
the protection of probiotics has been evaluated using simulated gastric
juice (Eslami, Forootan, Davarpanah, & Vahabzadeh, 2020; Mardani
Ghahfarokhi, Pescarmona, Euverink, & Poortinga, 2020; Shima et al.,
2006; Shima et al., 2007) or intestinal juice (Shima et al., 2009) or both
(Pimentel-González et al., 2009) to see the stability against acid and bile
salts respectively, except for one case (van der Ark et al., 2017) a system
including gastro-intestinal conditions and lipase to promote the diges­
tion of lipids has not always been used. The objectives of this work were
to investigate the feasibility of encapsulation of Lactobacillus reuteri in
water-in-oil-in-water double emulsion type (W1/O/W2) and evaluation
of microstructural and encapsulation efficiency (EE) of the initial for­
mulations in addition to encapsulation stability (ES) during a 30-days
cold storage and simulated in vitro gastro-intestinal digestion. There
results were compared to control samples which were non-encapsulated
systems and had the bacteria dispersed in the external aqueous phase
(W2). In this study, Medium chain triglyceride (MCT) oil (C8–C12) was
used as the oil phase which has a low viscosity and liquid character with
a low melting point (− 12.5 ◦ C) and is fully saturated and does not
display oxidation. Polyglycerol polyricinoleate (PGPR) and Poloxamer
407 were used as hydrophobic and hydrophilic emulsifiers, respectively.
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LWT 146 (2021) 111423
2. Materials and methods
2.1. Material
Lactobacillus reuteri DSM 20016 (BioGaia, Sweden) was used for
encapsulation. De Man, Rogosa, and Sharpe (MRS) broth (Merk, USA)
was used for the cultivation of the bacteria. MRS agar (Merk, USA) was
used for plate counting. The freezing medium was consisted of 5 mmol/L
K2HPO4 (Merk, USA), 1.3 mmol/L KH2PO4 (Merk, USA), 2.3 mmol/L
Na-Citrate (Merk, USA), 1 mmol/L MgSO4 × 7 H2O (Sigma Aldrich,
USA) and 2 mol/L glycerol (Merk, USA). Peptone water was used for the
serial dilutions with 0.85% w/v NaCl (VWR, USA) and 0.1% w/v bac­
terial peptone (Oxioid, UK). MilliQ water was used for all the
experiments.
Water-in-oil-in-water double emulsions (W1/O/W2) were produced
in ratios of 4:16:80 for W1:O:W2. This ratio was chosen based on pre­
vious studies (Kheynoor, Hosseini, Yousefi, Hashemi Gahruie, & Mes­
bahi, 2018; Matos, Gutiérrez, Coca, & Pazos, 2014; Mun, Choi, Shim,
Park, & Kim, 2011). Besides, this ratio provides a significant internal
phase but not too high to induce instability. In addition, this formulation
was chosen due to low viscosity that was essential for the digestion
experiments. The freezing medium was used as the internal water phase
(W1). 5% w/v polyglycerol polyricinoleate 90 (PGPR 90, Danisco,
Denmark), as the hydrophobic emulsifier, was dissolved in a MCT oil
(Miglyol® 812, Caesar & Loretz GmbH, Germany) using a magnetic
stirrer at 40 ◦ C and was used as the oil phase (O). 2.5% w/v Poloxamer
407 (Pluronic® F-127 produced by BASF Corp, Germany, supplied by
Sigma Aldrich, USA) as hydrophilic emulsifier was added to the freezing
medium using a magnetic stirrer at room temperature and was used as
the external water phase (W2).
The simulated in vitro digestion was performed using 1 mol/L HCl
(VWR, USA), 1 mol/L NaOH (VWR, USA), 0.3 mol/L CaCl2 (Sigma
Aldrich, USA), 10 mmol/L bile salts that were consisted of 50:50 com­
bination of sodium cholate (Applichem, Germany) and sodium deoxy­
cholate (Sigma Aldrich, USA) and lipase from porcine pancreas with the
activity of 500 U/mg (Sigma Aldrich, USA).
2.2. Methods
2.2.1. Cultivation and storage of bacteria
MRS broth was prepared in a volume of 150 mL, sterilized using an
autoclave (120 ◦ C for 20 min), cooled down, and inoculated with 1 mL of
Lactobacillus reuteri DSM 20016 culture before it was incubated in aer­
obic conditions at 37 ◦ C for 22 h. Subsequently, in order to harvest the
bacteria, the bacterial culture was centrifuged at 2800×g at 10 ◦ C using
a CL10 centrifuge (Thermo Scientific, USA) to pellet the bacteria, the
broth was discarded and substituted by an equal volume of freezing
medium. Thereafter, the pelleted bacteria were re-suspended in the
freezing medium using a vortex for approximately 10 min. The new
culture was transferred in sterile vials of 1.5 mL and the vials were
frozen at − 80 ◦ C. All steps of cultivating and freezing bacteria as well as
plate counting were performed under aseptic conditions.
2.2.2. Emulsion preparation and bacterial encapsulation
Initially, the oil phase was prepared by mixing 5% w/v PGPR as
hydrophobic surfactant into the MCT oil at 40 ◦ C using a magnetic bar
and magnetic plate. Thereafter, water-in-oil-in-water double emulsions
(W1/O/W2) were prepared using a two-step emulsification method. In
the first step, the primary water-in-oil emulsions (W1/O) consisted of
20% v/v freezing medium either including the bacteria for the encap­
sulated model or without bacteria for control model as the aqueous
dispersed phase (W1) and 80% v/v of MCT oil-PGPR mix (O) as the
continuous phase were prepared using IKA T50 rotor-stator high shear
homogenizer (IKA, Germany) with 6 mm dispersing tool at 13 000 rpm
for 5 min. Finally, 20% v/v of the primary water-in-oil emulsion (W1/O)
was dispersed into 80% v/v of the freezing media which contained 2.5%
A. Marefati et al.
w/v Poloxamer 407 as the external aqueous or continuous phase (W2) to
create double emulsions using IKA T50 rotor-stator high shear homog­
enizer (IKA, Germany) with 6 mm dispersing tool at 23 000 rpm for 30 s.
Control samples were prepared in the similar way without the bacteria
in W1, which was instead dispersed in the external aqueous phase (W2).
4 mL of both of the primary and double emulsions were transferred to a
test tube and pictures were taken after emulsification and after 30 days
of storage. Before any sampling, the emulsions were mixed using a
vortex and then by flipping the vials 3 times to make sure that the
content is completely mixed and the sampling was done from the middle
of the test tube using a pipette.
2.2.3. Evaluation of microstructure of the emulsions
2.2.3.1. Particle size distribution. The particle size distributions of the
primary and double emulsions were evaluated using a light scattering
particle size analyzer (Mastersizer 2000, Malvern Panalytical Ltd, UK).
The particle size measurements were performed by dispersing the pri­
mary emulsions (W1/O) in paraffin oil, while Milli-Q water was used as a
carrier phase for measurements of the double emulsions. The refractive
indices for the water and MCT oil were 1.33 and 1.46 respectively. All
samples were prepared in duplicates and the measurements were per­
formed three times for each sample (n = 6).
2.2.3.2. Microscopy. The microstructure of the emulsions was evalu­
ated using light microscopy (Olympus 50, Olympus, Japan) equipped
with a camera (DFK 41AF02, The Imaging Source, Germany). The
emulsions were diluted five times with Milli-Q water and then one drop
was placed on a glass microscopic slide. In order to prevent the defor­
mation of the droplets, no cover glass was used. The microscopic images
were taken using objective magnifications of 20× (LMPlanFI 20×/0.40,
Olympus, Japan) and 50× (LMPlanFI 50×/0.50, Olympus, Japan).
2.2.4. Evaluation of the viability of the bacteria
To determine the viability of the bacteria in the samples, the bacte­
rial population was evaluated using plate counting. MRS agar was pre­
pared according to the instructions given by the supplier and poured
into Petri dishes (18–20 mL/per dish). Serial dilutions of the samples
were made in peptone water in order to reach a suitable population that
can be counted on a plate. Thereafter, 100 μL of each of the relevant
dilutions was transferred into MRS agar plates and were spread using
sterile glass beads. The plates were incubated in anaerobic conditions
using Anaerocult A (Merk, USA) in an anaerobic jar at 37 ◦ C for 60 h and
the colonies were counted subsequently.
2.2.5. Evaluation of encapsulation properties of the emulsions
The encapsulation properties of the emulsions were evaluated in
terms of encapsulation efficiency (EE) for freshly prepared double
emulsions and encapsulation stability (ES) during storage and simulated
in vitro digestion of double emulsions. The resultes were presented as a
logarithm of colony forming units per mL of sample (Log CFU/mL).
2.2.5.1. Encapsulation efficiency. The encapsulation efficiency of emul­
sion samples was evaluated by transferring 1.5 mL of emulsions to a
sterile vial. Thereafter, the vials were centrifuged at 15 800×g for 10
min to disrupt the emulsion stability. Then, the mixture was mixed by
vortex and 1 mL of the aqueous phase was withdrawn and serially
diluted in peptone water to reach the desired concentration and 100 μL
of the relevant dilutions were applied on agar plates. Finally, the plate
counting process was performed as described in section 2.2.4. All sam­
ples were prepared in duplicates and cultivated in duplicates (n = 4).
2.2.5.2. Encapsulation stability during storage. The double emulsions
were prepared in 7 mL volume sterile test tubes of 12 mL volume with
screw caps and placed at a cold room at 6 ◦ C for evaluation of storage
3
LWT 146 (2021) 111423
stability at different time intervals (0, 3, 15, and 30 days). Thereafter,
1.5 mL of emulsions were transferred to a sterile vial and centrifuged at
15 800×g for 10 min to disrupt the emulsion stability. Then, the mixture
was mixed by vortex and 1 mL of the aqueous phase was withdrawn and
serially diluted in peptone water to reach the desired concentration and
100 μL of the relevant dilutions were applied on agar plates. Finally, the
plate counting process was performed as described in section 2.2.4.
Control samples were prepared with the bacteria dispersed in the
external aqueous phase. For each time interval samples were prepared in
duplicates and each of them was cultivated in duplicates (n = 4).
2.2.5.3. Encapsulation stability during in vitro digestion. The simulated in
vitro digestion was performed in a vessel with a thermostat jacket con­
nected to a water bath with the temperature set to 37 ◦ C according to a
previous method with some modifications (Minekus et al., 2014). Since
the formulation was liquid and did not contain starch and the food did
not need chewing nor it has a retention time in the oral phase of the
digestion, the oral phase of digestion was eliminated (Minekus et al.,
2014). In the gastric phase, 6 mL of the emulsions were transferred into
the digestion vessel along with 5.8 mL of sterile freezing medium and
since there was no protein in the system and addition of pepsin could
complicate further cultivation of the bacteria in MRS agar, no pepsin
was added. Then, the pH was adjusted to 3 using 1 mol/L HCl. The
amount of HCl was recorded and based on that additional amount of
freezing medium was added, to set the total volume in the pH-stat vessel
to 12 mL. Samples of 1 mL were taken at the beginning of gastric
digestion (time 0 min) and after 5, 30 and 60 min and transferred to
sterile centrifuge tubes.
After 60 min of in vitro gastric digestion, the intestinal digestion
initiated by the addition of 5.8 mL of freezing medium together with
0.018 mL of 0.3 mol/L CaCl2, 1.8 mL of 100 mmol/L bile salts was added
to the reaction vessel to set the final concentration of CaCl2, and bile
salts to 3 mmol/L and 10 mmol/L respectively. Then, the pH was set to
6.5 by the addition of 1 mol/L NaOH. Based on the amount of NaOH
added, an additional amount of freezing medium was added, to set the
total volume in the pH-stat vessel to reach 17.1 mL. Lastly, 0.9 mL of
lipase enzyme solution (100 mg/mL) was added in the vessel so that the
final volume reached 18 mL. Since there was no protein or starch in the
formulation and as we wanted to simplify the system instead of
pancreatin, only lipase was used for the intestinal digestion. Samples of
1.2 mL were taken at 5, 30, 60, 120, and 180 min and transferred to
sterile centrifuge tubes. Thereafter, 1.5 mL of emulsions were trans­
ferred to a sterile vial and centrifuged at 15 800×g for 10 min to disrupt
the emulsion stability. Then, the mixture was mixed by vortex and 1 mL
of the aqueous phase was withdrawn and serially diluted in peptone
water to reach the desired concentration and 100 μL of the relevant
dilutions were applied on agar plates. Lastly, the plate counting process
was performed as described in section 2.2.4. All samples were prepared
in duplicates and cultivated in duplicates (n = 4).
2.2.6. Statistics
All data were expressed as mean values ± standard deviation of the
mean. Student t-test (unpaired comparison) was used for the statistical
analysis of the results and differences were considered significant when
P < 0.05 was reached.
3. Results and discussions
3.1. Evaluation of macro and microstructure of the emulsions
Fig. 1 represents the primary and double emulsions after emulsifi­
cation and after 30 days of storage. The primary emulsions were stable
with no apparent creaming or sedimentation even upon prolonged
storage. Unlike primary emulsions, the double emulsions creamed after
emulsification which led to the formation of two distinct layers. The
A. Marefati et al.
Fig. 1. Representation of primary (W1/O) and double (W1/O/W2) emulsions after emulsification (day 0, a and b) and after 30 days (day 30, c and d).
upper layer included oil droplets containing the internal aqueous phase
(W1/O) and the lower layer that was consisted of the external contin­
uous aqueous phase (W2). Despite creaming, these emulsions did not
show any sign of instability (i.e. separated oil on the top) over the
storage stability test period (30 days) with constant opacity at the
continuous phase.
The droplet size distribution of primary emulsions (W1/O) with and
without encapsulated Lactobacillus reuteri are presented in Fig. 2a and
Table 1. The particle size distribution of the primary emulsions showed a
major peak between 1 and 10 μm representing water droplets in oil and a
minor peak at the range between 0.01 and 1 μm which is possibly due to
the presence of other particles (PGPR micelles) or the fitting error of the
scattering function (Matos, Timgren, Sjöö, Dejmek, & Rayner, 2013). In
addition, the particle size distribution of encapsulated W1/O emulsions
showed a second minor peak that could be due to the flocculation of
water droplets in oil. Moreover, the results showed that the inclusion of
bacteria increased the mode of volume-weighted diameter (D4,3) size
from 3.5 ± 0.2 μm to 4.8 ± 0.4 μm (P < 0.05). Considering the size of
Lactobacillus reuteri with a diameter of 0.7–1 μm and length of 2.0–5.0
μm, this increase in the size of primary emulsions seems reasonable
given the conditions of emulsification i.e. shear rate and emulsifier’s
concentration were the same. The increase in the size of the primary
emulsion can also be seen in the micrographs of double emulsions in
Fig. 3 where the bigger internal droplets are pointed out by red arrows.
These bigger internal droplets with sizes around 5 μm only can be seen in
encapsulated double emulsions and not in the control samples. PGPR
was used as the hydrophobic emulsifier in this study which is attractive
as a food and cosmetics ingredient and pharmaceutical excipient
approved by the EU and US Food and Drug Administration due to its
biocompatibility, biodegradability, and low toxicity (Behera et al.,
2015; Varshney et al., 2004). PGPR is a common hydrophobic emulsifier
which is described as one of the most potent oil-soluble emulsifiers
(Wilson, van Schie, & Howes, 1998). PGPR has shown to be highly
effective for water-in-oil emulsions made with triglyceride oils
4
LWT 146 (2021) 111423
(Ushikubo & Cunha, 2014). The required concentration of PGPR to
ensure the long-term stability of emulsions is 4–6 wt% but also the
concentrations as low as 0.5 wt% and as high as 10% have been reported
(Dickinson, 2011; Muschiolik & Dickinson, 2017).
The droplet size distributions of W1/O/W2 double emulsions with
and without encapsulated Lactobacillus reuteri are presented in Fig. 2b
and Table 1 and micrographs are presented in Fig. 3. The particle size
distribution of double emulsions showed a major peak representing
droplets and a minor peak that could be attributed to the micelles
formed by Poloxamer 407. The initial modes of D4,3 size of double
emulsions with and without encapsulated bacteria were 14.9 ± 0.3 μm
and 15.5 ± 0.4 μm where the difference was not statistically significant
(P > 0.05) showing that the inclusion of bacteria in the primary emul­
sions did not affect the droplet size distributions of double emulsions. In
addition, neither the droplet sizes for encapsulated double emulsions
nor the control samples changed over the cold storage period (P > 0.05),
and also there was no significant difference (P > 0.05) between the
droplet sizes of encapsulated and control double emulsions at any of
these time intervals (0, 3, 15, 30 days) as shown by superimposed par­
ticle size distributions. Poloxamer 407 is a highly efficient hydrophilic
emulsifier which is a US Food and Drug Administration-approved
polymer, and is used as a pharmaceutical excipient and is attractive
due to its biocompatibility, biodegradability, and low toxicity (Saxena &
Hussain, 2012). Formulation of double emulsions using Poloxamer 407
yields in the formation of droplets in the micron-sized range that can be
beneficial for this study due to the size of encapsulated Lactobacillus
reuteri (Varshney et al., 2004).
The micrographs in Fig. 3 confirmed that the internal droplets of
double emulsions with encapsulated bacteria (bottom row) seemed to be
larger than double emulsions without bacteria (top row) due to the size
of the bacteria and the volume they occupy in the internal aqueous
phase (W1).
A. Marefati et al. LWT 146 (2021) 111423

3.2. Evaluation of the viability of the bacteria after cultivation and

freezing

The viability of Lactobacillus reuteri after cultivation, before and after

freezing were analyze and presented as a logarithm of colony forming
units present per mL of sample (Log CFU/mL). The initial population
was 10.69 ± 0.19 Log CFU/mL and it was decreased after frozen storage
at − 80 ◦ C to 8.98 ± 0.04 Log CFU/mL which corresponds to a viable
bacterial count decline of 1.7 Log CFU/mL or 98% of the initial bacterial
population. This reduction is obviously due to the conditions imposed by
freezing and that the freezing solution did not provide the protection
necessary for the preservation of the bacteria. Nevertheless, the popu­
lation after freezing was still high and this population was used for the
continuation of the study.

3.3. Evaluation of encapsulation properties of the emulsions

3.3.1. Initial encapsulation efficiency

The viable number of the bacteria calculated theoretically (based on
the number of viable bacteria after frozen storage and the dilution fac­
tor) and the actual survival rate after encapsulation in W1/O/W2 double
emulsions were 7.59 ± 0.04 and 7.23 ± 0.07 Log CFU/mL, respectively.
This reduction in population is likely due to the sensitivity of Lactoba­
cillus reuteri to oxygen that is incorporated into the system while mixing
during the preparation of double emulsions and not due to the me­
chanical stress during the homogenization since the elasticity of the cell
membrane helps the bacteria to resist mechanical stress (Schär-Zam­
maretti & Ubbink, 2003). A previous study showed that homogenization
(in the range of 10 000–24 000 rpm for 2 min) used in the preparation of
double emulsions did not affect the viability of Lactobacillus acidophilus
(Shima et al., 2006). This study also showed that the viability of the
bacteria was correlated to the droplet size of the double emulsions, and
larger droplets showed a higher rate of viability perhaps due to a thicker
Fig. 2. The particle size distribution of primary W1/O emulsions with and oil barrier around them and better exclusion from the external envi­
without bacteria in W1 (a), The particle size distribution of encapsulated W1/O/ ronment or smaller total surface area, however, they did not explain
W2 double emulsions with bacteria in W1 and control W1/O/W2 double emul­
why. In addition, since cytotoxicity of PGPR is not reported, the
sions with the bacteria dispersed in W2 after emulsification (day 0) and over the
reduction of the viability of the bacteria compared to the bacterial cul­
storage stability test period (day 3, 15, and 30) (b).
ture could not be due to the anti-bacterial activity of the hydrophobic
surfactant. On the other hand, the difference in the results could be due
Table 1 to the fact that the viability may be different between different vials or
Particle size values of W1/O and W1/O/W2 emulsions with encapsulated bac­ the viable population may have decayed during the additional days of
teria and control immediately after emulsification (day 0) and over the storage frozen storage at − 80 ◦ C. Nevertheless, the viability was in the range
stability test period (day 3, 15, and 30), the values are presented as mean ± proposed for probiotic products (i.e. >7 Log CFU/mL).
standard deviation (n = 6) where different letters represent significant differ­
ence between the samples (P < 0.05) while same letter means there were no 3.3.2. Encapsulation stability during cold storage
statistically significant difference present between the samples (P > 0.05). The encapsulation stability was evaluated during 30-days storage in
W1/O emulsions the cold room at 6 ◦ C and compared with the control samples where the
Sample D4,3 [μm] Span of Mode of D4,3 bacteria was inserted in the outer aqueous phase (W2) and shown in
D4,3 [μm] Fig. 4. There was a slight decay in the encapsulation during the first 3
Encapsulated W1/O 8.1a±0.5 2.4a±0.9 4.8a±0.4 days from 7.23 ± 0.07 Log CFU/mL to 7.04 ± 0.10 which was not sta­
Control W1/O 2.3b ± 0.2 2.7a±0.4 3.5b ± 0.2 tistically significant. In control samples, the initial number of viable
W1/O/W2 double emulsions
bacteria was lower at the beginning of the storage (day 0) with 6.18 ±
Sample D4,3 [μm] Span of Mode of D4,3 0.03 Log CFU/mL which decreased to 5.67 ± 0.29 Log CFU/mL, how­
D4,3 [μm] ever, this change was not statistically significant either (P > 0.05). The
Encapsulated W1/O/W2 day 13.4a±0.3 1.7a±0.0 14.9a±0.3 viability of the bacteria, however, decayed drastically (P < 0.05) after
0 15 and 30 days. The number of colony-forming units decreased to 5.20
Control W1/O/W2 day 0 13.7a±0.4 1.7a±0.0 15.5a±0.4 ± 0.04 and 3.84 ± 0.10 Log CFU/mL for encapsulated and control
Encapsulated W1/O/W2 day 13.5a±0.0 1.7a±0.0 15.0a±0.0 samples after 15 days, respectively. Finally, after 30 days of storage, the
3
Control W1/O/W2 day 3 13.6a±0.4 1.7a±0.0 15.4a±0.3
number of bacteria decreased to 2.82 ± 0.10 Log CFU/mL for encap­
Encapsulated W1/O/W2 day 13.0a±0.3 1.7a±0.0 14.6a±0.3 sulated samples, whereas, no colonies of Lactobacillus reuteri formed for
15 control samples (i.e. <1 Log CFU/mL). The results showed that the
Control W1/O/W2 day 15 13.5a±0.4 1.7a±0.0 15.2a±0.3 encapsulation of Lactobacillus reuteri in double emulsions improved the
Encapsulated W1/O/W2 day 13.3a±0.1 1.7a±0.0 15.0a±0.1
viability of the bacteria in all cases and this improvement seemed to be
30
Control W1/O/W2 day 30 13.4a±0.2 1.7a±0.0 15.1a±0.2 more obvious in longer terms (i.e. 15 and 30 days). The low storage
stability of bacteria could be due to their poor storage stability, pre­
sumably due to low oxygen tolerance in the non-encapsulated samples

5
A. Marefati et al.
Fig. 3. Light microscopy of control W1/O/W2 double emulsion with bacteria dispersed in W2 (a) and W1/O/W2 double emulsions with encapsulated bacteria in
W1 (b).
Fig. 4. Viability of bacteria encapsulated in the internal phase (W1) of W1/O/
W2 double emulsions or the external water phase (W2) in control samples
during a 30-days storage stability test, the values are presented as mean ±
standard deviation (n = 4) where different letters represent significant differ­
ence between the samples (P < 0.05) while same letter means there were no
statistically significant difference present between the samples (P > 0.05).
or absence of essential nutrients such as amino acids and vitamins that
the microorganisms need to survive and grow during storage in the inner
aqueous phase of the double emulsions.
3.3.3. Encapsulation stability during in vitro digestion
Viability of Lactobacillus reuteri during simulated in vitro digestion for
the bacteria encapsulated in the double emulsions and control samples,
where the bacteria were dispersed in the external aqueous phase, (W2) is
shown in Fig. 5. Compared to the steady decline in the number of viable
6
LWT 146 (2021) 111423
bacteria throughout the gastric digestion when the bacteria were
encapsulated in double emulsions, the population in the control samples
with non-encapsulated bacteria tends to decrease significantly at the
very beginning of gastric digestion (first 5 min) and remained relatively
stable for the remainder of exposure to gastric digestion. After initiation
of the gastric digestion (during the first 5 min), the number of viable
bacteria only decreased from 6.69 ± 0.07 Log CFU/mL to 6.59 ± 0.04
Log CFU/mL (0.10 Log CFU/mL decrease) for encapsulated bacteria and
remained stable until the end of the gastric phase (6.53 ± 0.07 Log CFU/
mL). Whereas, in control samples during the first 5 min of the gastric
phase, the number of viable bacteria dropped from 6.25 ± 0.36 Log
CFU/mL to 4.76 ± 0.08 Log CFU/mL (1.49 Log CFU/mL decrease) and
then remained relatively stable until the end of the 1 h gastric phase
(4.70 ± 0.08 CFU/mL). Some digestion models consider the gastric
residence time to be 2 h (Shima et al., 2006), although others consider it
to be shorter, like 30 min (Jacobsen et al., 2021). However, this may
depend on the type and the physical state of the formulation, and the
presence or absence of proteins and other components sensitive to
gastric digestion in the evaluated systems. In addition, in the present
study, the initial reduction of the viability of the bacteria that took place
during the first 5 min of the gastric digestion in the non-encapsulated
control samples, further gastric exposure did not result in a statisti­
cally significant change in the viability of the bacteria (p > 0.05), and
therefore, it does not seem likely that extending the gastric digestion
from 1 h to 2 h could have an important impact on the results of the
present study. These results showed that the current formulation can
provide the desired gastric protection for Lactobacillus reuteri. The results
of the present study showed that around 70% of the bacteria survived
the gastric condition in encapsulated formulation compared to 2.8%
survival rate in the control samples. The results also showed that the
survival rate in the present study was more than what was observed in a
previous studies by Shima et al. (2006) or Rodríguez-Huezo et al. (2014)
with 49% survival rate for encapsulation of L. acidophilus and up to 2.2%
A. Marefati et al.
Fig. 5. Viability of the bacteria during simulated in vitro gastrointestinal digestion for the encapsulated bacteria in the W1/O/W2 double emulsions and control
samples where the bacteria were dispersed in the W2 of the emulsions, The values are presented as mean ± standard deviation (n = 4).
survival rate for encapsulation of L. plantarum for similar formulations.
However, the former study showed that lower internal phase fractions
(0.03 and 0.15) had a lower survival rate compared to when higher
internal phase fractions (0.3 and 0.45) was used (Shima et al., 2006).
A similar trend was observed when the intestinal digestion started.
Compared to the relatively gradual drop in the viability of the bacteria
for encapsulated formulation in the beginning of the intestinal digestion
(from 6.53 ± 0.07 to 6.28 ± 0.07 Log CFU/mL during the first 5 min)
and throughout the intestinal digestion (4.64 ± 0.06 Log CFU/mL after
180 min), a steep drop occurred at the beginning of the intestinal
digestion for control samples (4.70 ± 0.08 to 3.05 ± 0.07 Log CFU/mL
during the first 5 min) and continued with a slower pace until the end of
the intestinal digestion (ending at 2.69 ± 0.06 Log CFU/mL after 180
min). The results showed that these encapsulated formulations can
provide protection in the intestine and delay the release throughout
small intestinal digestion and result in complete release in the colon.
Although Lactobacilli are generally considered rather tolerant in low
pH (Jiménez-Pranteda et al., 2012), the reduction of non-encapsulated
bacteria in control samples during gastric digestion can be attributed
to the direct exposure to low pH and higher exposure to oxygen during
the gastric digestion. Therefore, the inclusion of the Lactobacillus in the
internal aqueous phase of double emulsions provided a protective bar­
rier against low pH and oxygen, and thereby, resulted in higher survival
rates which have been noted in previous studies (Lian, Hsiao, & Chou,
2003; Pimentel-González et al., 2009). On the other hand, the sharp
reduction in the population of the bacteria at the threshold of the in­
testinal digestion is attributed to the effect of lipolysis in digestion and
disruption of the formulation which, in turn, will expose the bacteria to
the bile salts. Bile salts, in addition to their well-known functionality in
the solubilization and absorption of the lipids, are known as highly
effective physiological antimicrobials particularly on Gram-positive
bacteria (Begley, Gahan, & Hill, 2005). The anti-microbial effect of
bile salts as highly surface-active components on the Gram-positive
bacteria is due to the higher permeability of their cell walls to the bile
salts that will result in imposing extensive membrane disruption and
DNA damage in addition to protein unfolding and aggregation (Cremers,
Knoefler, Vitvitsky, Banerjee, & Jakob, 2014; Ruiz, Margolles, &
Sánchez, 2013). The membrane damage can also results in leakage of
intracellular material and cell shrinkage (Begley et al., 2005). In addi­
tion, since L. reuteri is sensitive to oxygen, the conditions of the in vitro
digestion model is deemed to be severe compared to the actual anaer­
obic in vivo intestinal conditions, due to the presence of oxygen. Shima
et al. (2009) showed that upon 2 h exposure to 6 mM/L sodium taur­
ocholate as a bile representative, the viability of encapsulated
7
LWT 146 (2021) 111423
L. acidophilus in W/O/W type emulsion declined around 10%, however,
in control samples, this reduction was more than 60%. Although the
present results of the survival rate of encapsulated bacteria in the double
emulsions during in vitro gastro-intestinal digestion are not extremely
high, it should be pointed out that these results include the effect of the
presence of intestinal lipase which results in gradual digestion of tri­
glycerides which also results in the solubilization of the oil phase and
disruption of the emulsions and causes higher exposure of the bacteria to
the bile as well as oxygen. In comparison, in the studies mentioned
above, addition of bile salts in the smilated gastric fluid which will
merged with the external water phase of the double emulsion (W2) could
hardly affect the encapsulated bacteria that is in the W1 phase. There­
fore, the present study provides a better representation of
gastro-intestinal conditions.
Coencapsulation of probiotics and prebiotics is suggested to preserve
the viability of prebiotics (Rashidinejad et al., 2020). The lower survival
rates bacteria in both storage stability test and in vitro digestion in this
study compared to the previous study by Pimentel-González et al.
(2009) could be due to the absence of nutrients in the system specifically
amino acids and vitamins as these nutrients are essential for the growth
of bacteria (Tseng & Montville, 1993). In the above-mentioned study
with Lactobacillus rhamnosus, the bacteria were supplied with sweet
whey that contained high amounts of protein and lactose and showed a
higher survival rate, something that could be investigated in the future.
In addition, Shima et al. (2007) also claimed that utilization of MRS
broth as the internal water phase could inhance the vibility of the bac­
teria since the broth include the nutrient needed for the growth of the
cells. Another option could be to freeze or freeze dry such a formulation
to inactivate the bacteria and eliminate the need for the nutrients;
however other strategies must be accounted for since emulsions are
sensitive to freezing and can face destabilization. In addition, the strain
used in this study seemed to be highly sensitive to the environmental
conditions such as presence of oxygen, low pH and bile salts. This higher
sensitivity could be optimized by the selection of acid and bile tolerant
strain of probiotic bacteria. The proposed formulation, can be used in
specific delivery products for probiotics for liquid, semi-liquid or dried
forms of products such as beverages, capsules or tablets.
4. Conclusions
Double emulsions of W1/O/W2 type were successfully formulated for
the encapsulation of probiotic bacteria of Lactobacillus reuteri. The initial
population of the cultivation was 10.69 Log CFU/mL which was reduced
to 8.98 Log CFU/mL as a result of freezing during storage. The
A. Marefati et al.
emulsification process appears to have a small impact on the population
of the bacteria resulting in a 7.23 Log CFU/mL survival rate in the
bacterial population. Encapsulation of the bacteria had a positive impact
on the survival rate during storage compared to control samples,
although it was lower than expected at the end of the storage period
given the lack of growth medium, resulting in 2.82 Log CFU/mL
compared to no colony forming units (<1 Log CFU/mL) in the control
samples. Furthermore, the simulated in vitro digestion showed that the
encapsulation of bacteria in W1/O/W2 double emulsion protects the
bacteria against the acidic condition in the stomach (6.53 Log CFU/mL
for encapsulated compared to 4.70 Log CFU/mL for control samples). In
addition, compared to control samples, encapsulation displayed a much
higher survival rate at the end of intestinal digestion (4.64 Log CFU/mL
for encapsulated samples compared to 2.69 Log CFU/mL for control
samples) resulting in a delayed release of the bacteria in the small in­
testine and allowing a complete release in the colon. Since the encap­
sulated bacteria needs nutrients in order to be able to survive, provision
of nutrient in the internal phase of the double emulsions could be a
subject for future studies. In addition, an idea for the future could be to
design pH-sensitive formulation in order to release/deliver the bacteria
in/to a specific location of the gastro-intestinal tract.
CRediT authorship contribution statement
Ali Marefati: Conceptualization, Methodology, Formal analysis,
Investigation, Data curation, Writing – original draft, Writing – review &
editing, Visualization. Anastasios Pitsiladis: Formal analysis, Investi­
gation, Data curation, Writing – original draft. Elin Oscarsson: Meth­
odology, Formal analysis, Investigation, Data curation, Review &
editing. Niclas Ilestam: Conceptualization, Methodology, Review &
editing. Björn Bergenståhl: Conceptualization, Methodology, Formal
analysis, Investigation, Data curation, Writing – review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This research was financed through NextBioForm Competence
Centre, funded by Vinnova Swedish Governmental Agency for Innova­
tion and The Swedish Research Council under grant number 2017-
02155.
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