LWT - Food Science and Technology 144 (2021) 111174

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LWT
journal homepage: www.elsevier.com/locate/lwt

Development of gastro-resistant coated probiotic granulates and evaluation

of viability and release during simulated upper gastrointestinal transit
Natashia Mai Yde Jacobsen a, Hanne Bjørn Nedergaard b, Anette Kock c, Ibrahim Caglayan c,
Marie Munch Laursen b, Eva-Marie Lange b, Martín Sebastián Marcial-Coba d,
Daniel Bar-Shalom a, e, Dennis Sandris Nielsen d, Anette Müllertz a, e, *
a
Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark
b
Danish Technological Institute, Gregersensvej 1, DK-2630, Taastrup, Denmark
c
Deerland Probiotics & Enzymes, Bogbinderivej 6, DK-3390, Hundested, Denmark
d
Department of Food Science, University of Copenhagen, Rolighedsvej 26, DK-1958, Frederiksberg, Denmark
e
Bioneer:FARMA, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark

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

Keywords: Probiotics have become one of the most consumed food supplements worldwide. Many probiotic strains are
Probiotics sensitive to low pH and bile concentrations encountered in the gastrointestinal tract upon oral ingestion. This
Hot-melt coating study aimed at developing gastro-resistant probiotic granulates releasing viable cells in the small intestine. Fatty
Fluid bed
alcohols were investigated as potential coating materials. Cetostearyl alcohol was selected and combined with
In vitro simulation
Probiotic delivery systems
different plasticizers to further optimize the coating properties. Combining cetostearyl alcohol with olive oil and
beeswax in selected concentrations was found promising, and the coatings were applied to L. acidophilus LA3 and
B. longum BB536, using hot-melt fluid bed coating. Viability in and release from the coated probiotic granulate
was investigated, using a physiological relevant in vitro model simulating conditions in the human stomach,
duodenum, jejunum and ileum. L. acidophilus LA3 coated with cetostearyl alcohol and olive oil in the ratio 95:5
(w/w) resulted in significantly higher viability after simulated gastrointestinal transit, compared to the uncoated
probiotic powder. Furthermore, the coating showed no release of viable cells after simulated gastric transit.
Released viable cells were detected after the remaining steps in the in vitro model, indicating that the coating
system provides gastric protection and release during intestinal transit.

1. Introduction
The human gut microbiota has in recent years received much
attention for its potential influence on the health and disease state of the
human host. Along with this, probiotic products have become one of the
most commonly consumed food supplements worldwide (Clarke, Black,
Stussman, Barnes, & Nahin, 2015). Probiotics are defined as “live mi­
croorganisms that, when administered in adequate amounts, confer a health
benefit on the host” (Hill et al., 2014). For probiotic products, lactobacilli
(previously known as Lactobacillus (Zheng et al., 2020)) and
Bifidobacterium strains are commonly used, and their potential beneficial
effects are very dependent on the specific strain applied (Vijaya Kumar,
Vijayendra, & Reddy, 2015). Many strains are sensitive to the environ­
ment found in the gastrointestinal tract (GIT), including low pH, high
bile levels and enzyme activities. Furthermore, viability can be influ­
enced by processing factors including moisture, heat and mechanical
stress, and hence, it can be a challenge to formulate oral delivery systems
where the probiotics reach the GIT alive (Ding & Shah, 2007).
Probiotics are often administered in capsules or tablets which can be
coated to achieve gastro-resistance and/or targeted delivery to the GIT

Abbreviations: aw, Water activity; B. longum, Bifidobacterium longum; BSM, Bifidus Selective Medium; C:O(95:5), Cetostearyl alcohol and olive oil in the ratio 95:5
(w/w); CFU, Colony forming unit; C:B(70:30), Cetostearyl alcohol and beeswax in the ratio 70:30 (w/w); C:B:O(65:30:5), Cetostearyl alcohol, beeswax and olive oil
in the ratio 65:30:5 (w/w); DSC, Differential scanning calorimetry; EC, Ethyl cellulose; GIT, Gastrointestinal tract; HPMC, Hydroxypropyl methylcellulose;
L. acidophilus, Lactobacillus acidophilus; RCA5.9, Reinforced clostridial agar with pH 5.9; SEM, Scanning electron microscopy; SiO2, Silicon dioxide; Tris, Tris
(hydroxymethyl)aminomethane; TSI, The Smallest Intestine; WG, Weight gain.
* Corresponding author. Department of Pharmacy, University of Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark.
E-mail address: anette.mullertz@sund.ku.dk (A. Müllertz).

https://doi.org/10.1016/j.lwt.2021.111174
Received 4 December 2020; Received in revised form 22 February 2021; Accepted 23 February 2021
Available online 26 February 2021
0023-6438/© 2021 Published by Elsevier Ltd.
N.M.Y. Jacobsen et al.
(Guarner, Khan, & Garisch, 2012). However, these dosage forms may
not be suitable for people having difficulties swallowing including
children and the elderly (Schiele, Quinzler, Klimm, Pruszydlo, & Haefeli,
2013). Alternative to this, probiotics may be administered in more
“easy-to-swallow” forms including powders or granulates, were the
product is taken directly from a stick pack or sprinkled onto food
(Saxelin, 2008). In these dosage forms, the probiotics are often found as
a freeze-dried powder, which ensures long-term stability during storage,
but does not entail any further protection upon oral intake (Broeckx,
Vandenheuvel, Claes, Lebeer, & Kiekens, 2016; Morgan, Herman, White,
& Vesey, 2006). This leads to a demand for easy-to-swallow dosage
forms ensuring the same protection and release as capsules or tablets.
To improve viability of the freeze-dried probiotic powder, one
strategy is to apply a functional coating layer. Hot-melt coating is a
technique were the coating material is supplied as a melt instead of a
solution or dispersion using a fluid bed coater (Lopes, Salar-Behzadi, &
Zimmer, 2017). This is an advantage for processing of freeze-dried
probiotic powder, sensitive to moisture, which furthermore has a large
surface area due to its small particle size. As solvent evaporation is not
necessary in hot-melt coating, a faster coating process can be achieved
(Jannin & Cuppok, 2013), and hereby the probiotics exposure time to e.
g. heat and moisture can be minimized.
To evaluate oral delivery systems, physiologically relevant in vitro
models, mimicking the conditions found in vivo are needed. For pro­
biotics, maintaining viability during GIT transit is important. During GIT
transit, factors influencing viability may include low pH levels, bile acid
concentrations and interactions with the present gut microbiota
(Govender et al., 2014). Hence, incorporation of these parameters in an
in vitro model will give a better indication of the delivery systems actual
performance in vivo.
The aim of this study was to develop probiotic containing easy-to-
swallow delivery systems, ensuring release of viable probiotic cells in
the GIT. Four fatty alcohols; stearyl alcohol, cetyl alcohol, myristyl
alcohol and cetostearyl alcohol were investigated as potential coating
materials, alone and in combination with selected plasticizing materials.
The cetostearyl coating with different combinations of plasticizers were
tested on two different strains; L. acidophilus LA3 and B. longum BB536,
used as model probiotics. The molten coating material was applied to
the freeze-dried powder by hot-melt fluid bed coating, using the same
material for both granulation and coating. Viability in and release from
the probiotic granulates was evaluated using an in vitro model simu­
lating transit through the stomach, duodenum, jejunum and ileum under
anaerobic conditions.
2. Materials and methods
2.1. Materials
Freeze-dried L. acidophilus LA3 was purchased from Sacco Srl.
(Cadorago, Italy) and B. longum BB536 was purchased from Morinaga
Inc. (Tokyo, Japan). KH2PO4, L-cysteine hydrochloride (cysteine chlo­
ride), bovine bile extract, gastric lipase (Rhizopus oryzae), pepsin,
pancreatin (from porcine pancreas), maleic acid, peptone, NaCl, CaCl2,
tris(hydroxymethyl)aminomethane (Tris), cetyl alcohol, myristyl
alcohol, stearyl alcohol, ethyl cellulose (EC), polysorbate 60 (Tween 60)
and Oil Red O were purchased from Merck (Darmstadt, Germany). Soy
phospatidylcholine (S-PC) was purchased from Lipoid (Ludwigshafen,
Germany). Suglets® sucrose spheres, mesh 25/30, was purchased from
Colocon (Dartford, UK). Rogosa agar, reinforced clostridial agar (RCA),
anaerogen compact sachets (Oxoid) and dialysis cassettes (Slide-A-
Lyzer, 10 kDa cut-off membrane) were purchased from Thermo Fisher
Scientific (Roskilde, Denmark). Acetylated monoglycerides were pur­
chased from CSM Deutschland GmbH (Bremen, Germany). Hydrox­
ypropyl methylcellulose (HPMC) was purchased from JRS Pharma
GmbH (Rosenberg, Germany). Beeswax was purchased from The British
Wax Refinery Company Ltd (Surrey, UK). Olive oil was purchased from
2
LWT 144 (2021) 111174
J. H. Müller (Rosengarten, Germany). Silica dioxide (SiO2) was pur­
chased from Grace GmbH & Co. KG (Worms, Germany). Bifidus Selec­
tive Medium (BSM) agar and BSM supplement was kindly donated from
the Gut, Microbes and Health group, Technical University of Denmark
(Lyngby, Denmark).
All other chemicals used were of analytical grade and all water used
was purified by a SG Ultra Clear UV apparatus (Evoqua Water Tech­
nologies, Pittsburgh, PA, USA) or a Milli-Q Integral water purification
system (Merck, Darmstadt, Germany).
2.2. Initial screening of fatty alcohols
To avoid the use of solvents for coating the freeze-dried probiotic
powder, fatty alcohols were chosen for further investigation. Four
different fatty alcohols; stearyl alcohol, cetyl alcohol, myristyl alcohol
and cetostearyl alcohol were evaluated.
2.2.1. Coating of model particles
To initially investigate the coating properties of the fatty alcohols,
sucrose containing spheres (in the following referred to as sugar spheres)
were used as model particles for coating. The fatty alcohols were melted
in a jacketed liquid container (Procept, Zelzate, Belgium), and the
molten mass was dispersed into a fluid bed coater (4M8-TriX Formatrix
fluid bed module, Procept, Zelzate, Belgium) by a jacketed two-fluid
nozzle. Prior to coating, the sugar spheres were fluidized and heated
and then coated to a weight gain of 50%. Process parameters used for
coating can be found in Appendix I.
2.2.2. In vitro release of sucrose from coated sugar spheres in simulated
gastric conditions
Release of sucrose from the coated sugar spheres gives an indication
of how well the coating layer functions. To investigate this, a simple in
vitro simulation of the stomach was performed. Gastric fluid was simu­
lated using 0.2 mol/L HCL and 0.2 mol/L KCL at pH 1.5. To 10 mL of
simulated gastric fluid, 1 g of particles were added and incubated at
37 ◦ C with shaking at 140 rpm for 1 h. After incubation, the samples
were immediately filtered through 0.22 μm nylon filters. The sucrose
concentrations in the collected samples were quantified using refractive
index detection (RID) (RefractoMax521, Thermo Fischer Scientific,
Waltham, MA, USA) on HPLC (Dionex UltiMate 3000, Thermo Fisher
Scientific, Waltham, MA, USA) with an Aminex HPX-87P (7.5 × 100
mm, 9 μm) column from Bio-Rad (Hercules, CA, USA). Purified water
was used as the mobile phase. Sucrose was quantified using a standard
curve ranging from 5 μg/mL–1 mg/mL.
2.2.3. Analysis of coated surfaces
To evaluate the coating layer on the sugar spheres, scanning electron
microscopy (SEM) was used. The coated sugar spheres where cut in half
and mounted on a carbon stub, sputter-coated (Polaron Emitech Sputter
Coater, Quorum Technologies, Lewes, UK) with a layer of approximately
5 nm gold under argon atmosphere and examined using SEM (Ultra55,
Carl Zeiss AG, Oberkochen, Germany) with 3 kV accelerating voltage.
2.2.4. Thermal analysis of coating materials
Differential scanning calorimetry (DSC) was used to analyze the
phase transition temperatures for melting and crystallization of the fatty
alcohols. Samples of 1–10 mg were placed in an aluminium pan, inserted
into the DSC apparatus (DSC 4000, Perkin Elmer, Waltham, USA) and an
empty pan was used as reference. Samples were heated and cooled in the
interval 0–70 ◦ C at a rate of 5 ◦ C/min to obtain DSC profiles.
2.3. Screening of plasticizers for further optimization of coating
To study if the properties of the fatty alcohol coating could be opti­
mized the effects of adding selected plasticizers were investigated.
Initially, acetylated monoglycerides, EC, HPMC, polysorbate 60, olive
N.M.Y. Jacobsen et al. LWT 144 (2021) 111174

oil and beeswax were tested together with cetostearyl alcohol in the
concentrations 3:100, 10:100 and 30:100 (w/w). The plasticizing com­
pounds were weighed in tin trays with a diameter of 9 cm. Melted
cetostearyl alcohol was then added, the mixtures were stirred and let
stand until solidification, resulting in molded plates. To evaluate the
combinations, contraction after solidification was measured, the surface
was visually inspected and the plates were broken into two, to inspect
the breakage surface. After this, selected combinations of plasticizers
were investigated (Table 1), using the same procedure.
2.4. Hot-melt coating of freeze-dried probiotic powder
Coated probiotic granulate was produced using hot-melt fluid bed
coating where freeze-dried probiotic powder was granulated and coated
using the same material in both process steps. A fluid bed (Ventilus® V
2.5, Romaco Innojet, Steinen, Germany) connected to a unit with an
isolated pump, tubes and a coating solution chamber with heat supply
(Hotmelt Device IHD 2.5, Romaco Innojet, Steinen, Germany) was used.
First, a mixture of freeze-dried probiotic powder and SiO2 in the ratio
99:1 (w/w) was blended for 5 min in a powder mixer (Turbula, Muttenz,
Switzerland). Three different coatings were prepared and for all coatings
cetostearyl alcohol was initially prepared by mixing cetyl alcohol and
stearyl alcohol in the ratio 30:70 (w/w). To the cetostearyl alcohol
mixture, plasticizers consisting of olive oil or beeswax were added in the
following concentrations; cetostearyl alcohol and olive oil in the ratio
95:5 (w/w) (C:O(95:5)), cetostearyl alcohol and beeswax in the ratio
70:30 (w/w) (C:B(70:30)) or cetostearyl alcohol, beeswax and olive oil
in the ratio 65:30:5 (w/w) (C:B:O(65:30:5)). The coating mixtures were
heated up to a molten mass at 100 ◦ C before use. The powder mixture
containing the freeze-dried probiotic powder was first granulated and
following coated in the fluid bed. In the granulation step the following
process parameters were used; spray air temperature: 100 ◦ C, pump-
head temperature: 100 ◦ C, airflow: 20–30 m3/h, nozzle air pressure:
0.6 bar, inlet air temperature: 20 ◦ C, product temperature: 25–30 ◦ C and
a pump speed set to 15 g/min. The granulation step was stopped, when a
weight gain of 30% was achieved. Hereafter a coating step was initiated
by changing the process parameters to the following; spray air temper­
ature: 100 ◦ C, pump-head temperature: 100 ◦ C, airflow: 30–45 m3/h,
nozzle air pressure: 1.0 bar, inlet air temperature: 20 ◦ C, product tem­
perature: 30–38 ◦ C and a pump speed set to 6.5 g/min. The coating was
stopped, when a weight gain of approximately 150% was achieved. All
coating combinations were applied to L. acidophilus LA3 and to test the
process on another strain B. longum BB536 was coated with the C:O
(95:5) combination.
2.4.1. Characterization of coated probiotic granulate
To evaluate the coating layer on the produced probiotic granulates,
SEM was used. The samples were mounted on an aluminium stub,
sputter-coated with a layer of approximately 5 nm gold (Cressington
sputter coater, 108 auto/SE, Watford, UK) under argon atmosphere and
Table 1
Compounds investigated for plasticizing properties together with cetostearyl
alcohol. Abb.: EC: Ethyl cellulose, HPMC: Hydroxypropyl methylcellulose.
Cetostearyl alcohol Beeswax Olive oil Polysorbate 60 EC HPMC
[% w/w]
85 10 5 ‒ ‒ ‒
65 30 5 ‒ ‒ ‒
45 50 5 ‒ ‒ ‒
‒ 95 5 ‒ ‒ ‒
‒ 100 ‒ ‒ ‒ ‒
87 10 ‒ ‒ 3 ‒
67 30 ‒ ‒ 3 ‒
96 ‒ ‒ 1 ‒ 3 a
94 3 3
‒ ‒ ‒
87 ‒ ‒ 10 ‒ 3
then examined using SEM (TM3030, Hitachi High Technologies Europe,
Krefeld, Germany) with 15 kV accelerating voltage.
2.5. In vitro evaluation of coated probiotic granulate
2.5.1. In vitro simulation of the upper gastrointestinal tract
To evaluate viability in and release from the coated probiotic gran­
ulates, a dynamic physiologically relevant in vitro model based on “The
Smallest Intestine” (TSI) model, previously setup and described by
Cieplak et al. (Cieplak, Wiese, Nielsen, Van de Wiele, van den Berg &
Nielsen., 2018), was used. The model simulates transit through the
stomach and small intestine including the duodenum, jejunum and
ileum segments, using physiologically relevant media, under anaerobic
conditions, at 37 ◦ C. Continuously through all steps, pH was adjusted,
being 2.5 during the gastric step, 6.5–6.8 during simulation of the du­
odenum, 6.8–7.5 during simulation of the jejunum and 7.5–8.0 during
simulation of the ileum. Absorption of bile acids was mimicked, by
continuously circulating the media through a dialysis cassette, during
simulation of the jejunum step. To simulate the ileum microbiota, a
small intestine microbiota inoculum consisting of Escherichia coli,
Streptococcus salivarius, Streptococcus luteinensis, Enterococcus faecalis,
Bacteroides fragilis, Veillonella parvula and Flavonifractor plautii, adjusted
to obtain a CFU ml− 1 of 107, was added at the ileum step (Cieplak,
Wiese, Nielsen, Van de Wiele, van den Berg & Nielsen., 2018). The
compositions of the used media are found in Table 2 and values are
based on previous work by Jacobsen et al. (Jacobsen, Caglayan,
Caglayan, Bar-Shalom, & Müllertz, 2020). Stock solutions of salts for the
gastric and concentrated intestinal media were prepared by mixing
NaCL, maleic acid and Tris (Table 2), with purified water. Furthermore,
stock solutions of CaCl2 and cysteine chloride were prepared. All solu­
tions were autoclaved before use.
The day before the in vitro experiment, bovine bile, phosphatidyl­
choline and CaCl2 were added to the gastric and intestinal solutions and
were left to dissolve overnight at 37 ◦ C under rotation at 225 rpm. The
following morning the gastric medium was adjusted to a pH of 2.5 and
the concentrated intestinal medium to a pH of 6.5, to reach the initial pH
levels used in the gastric and intestinal steps. The times and pH levels
used in each step in the simulated GIT transit is seen in Fig. 1. All steps
were simulated in one reaction vessel, by modifying the conditions in
this. The samples were run in duplicates.
2.5.2. Viability of probiotic cells in coated granulate before and after TSI in
vitro simulation
Maximum recovery diluent was prepared by mixing 8.5 g NaCl and 1
g peptone with 1 L of purified water and the solution was following
autoclaved.
To assess the initial viability in the coated probiotic granulates and
uncoated probiotic powders, samples of the granulates or powders were
Table 2
Content and pH in gastric and intestinal media.
Compounds Gastric medium Intestinal medium
a
Bile (mmol/L) 0.08 2.95
Phospholipidb (mmol/L) 0.02 0.26
NaCl (mmol/L) 34.2 50
Maleic acid (mmol/L) 2 2
Tris (mmol/L) - 2
CaCl2 (mmol/L) - 1.4
Enzymes
Gastric lipasec (U/mL) 50
Pepsin (U/mL) 450
Pancreatin (U/mL) - 600
pH 2.5 6.5
Bovine bile,
b
Phosphatidylcholine,
c
Rhizopus oryzae

3
N.M.Y. Jacobsen et al. LWT 144 (2021) 111174

Fig. 1. Schematic overview of the TSI in vitro model.

The in vitro model simulates transit through the GIT in reactions vessels which are placed inside a PVC chamber under anaerobic conditions and a temperature of
37 ◦ C. All steps are performed in the same reaction vessel. In the first step, gastric medium, gastric lipase and pepsin is added to the reaction vessel containing the
probiotic sample. Automated addition of NaOH keeps pH at 2.5. After 30 min of incubation, concentrated intestinal medium and pancreatic solution is added to the
reaction vessel and pH is raised to 6.5. Following 30 min of incubation, a pump is started and the medium is continuously circulated through a dialysis cassette for 4 h
to reduce bile acid concentrations in the intestinal medium, while pH is slowly raised to 7.5. Finally, a bacteria inoculum simulating the ileum microbiota is added to
the reaction vessel, and over the following 2 h pH is raised to 8.0.

mixed with maximum recovery and placed in a stomacher (Stomacher
80, Seward, Worthing, UK) for 2 min, to release the probiotics from the
coating. The content was transferred to Falcon tubes and centrifuged for
5 min at 3500×g. Supernatant was discarded and the pellet was resus­
pended in maximum recovery diluent. From this, 10-fold dilution series
were prepared and spot plated onto agar plates in duplicates for eval­
uation of colony forming units (CFU). Rogosa agar plates were used for
samples containing L. acidophilus LA3 and BSM or RCA5.9 agar plates
were used for samples containing B. longum BB5369. After spot plating,
the agar plates were inverted and incubated anaerobically at 37 ◦ C in
anaerobic jars. Rogosa agar plates were incubated for 5 days, BSM agar
plate for 3 days and RCA5.9 agar plates for 2 days. To investigate if the
small intestine microbiota inoculum could grow on the used agar plates,
a 10-fold dilution series was plated on all three agar types and incubated
as described above.
To evaluate the remaining number of viable cells present in the
coated probiotic granulate, after in vitro simulated transit though the
upper GIT, the remaining content in the reaction vessels were placed in a
stomacher, as described above, and the following steps were also fol­
lowed to evaluate viability.
2.5.3. Release of viable cells during in vitro simulation in the TSI
To investigate the number of free viable cells released from the
coated probiotic granulate during an in vitro simulation, samples were
collected from the reaction vessels with a needle and syringe at selected
time points. As control, samples were also collected from the vessels
containing the uncoated probiotic powders, to investigate the remaining
number of viable cells present without a protective coating. The 1 mL
samples were centrifuged at 3500×g for 5 min, supernatant was
discarded and pellet was resuspended in 1 mL maximum recovery
diluent. From this, 10-fold dilution series were prepared and spot plated
onto agar plates in duplicates as described in section 2.5.2.
2.6. Statistical analysis
Statistical analysis was performed using Prism software version 8
(GraphPad Software, La Jolla, CA). The reductions in log CFU ml− 1 were
compared using t-test. Differences in number of released cells, at
different steps during the in vitro simulation, were compared using two-
way ANOVA with Bonferroni’s multiple comparisons post hoc test. For
both tests significance level was set at P < 0.05.
3. Results and discussion
3.1. Screening and selection of coating materials
3.1.1. Evaluation of fatty alcohols as coating material
In the initial screening, four fatty alcohols; stearyl alcohol, cetyl
alcohol, myristyl alcohol and cetostearyl alcohol, were tested as po­
tential coating materials, on sugar spheres.
After 1 h incubation in simulated gastric conditions, 65 ± 28% of the
sucrose was released from the stearyl alcohol and cetostearyl alcohol
coated sugar spheres (Fig. 2). For myristyl alcohol, a higher, but not
significantly different release of sucrose with a mean of 85 ± 6% was
detected, and for cetyl alcohol 100% release was detected.
The coating layer applied to the sugar spheres was furthermore
visually investigated using SEM (Fig. 3). Stearyl alcohol (Fig. 3 A)
showed crystalline properties, as the coating was found in layers around

4
N.M.Y. Jacobsen et al.
Fig. 2. Release of sucrose from coated sugar spheres in simulated gastric me­
dium. Data are represented as mean (±SD), n = 2.
the sugar spheres, and pores were found both between the sugar sphere
and the coating layer as within the coating layer. This result was ex­
pected, as long chained fatty alcohols display lamellar crystalline
structures when solidified (Valoppi, Calligaris, & Marangoni, 2016). The
cetyl alcohol coating layer (Fig. 3 B) and myristyl alcohol coating layer
(Fig. 3 C) both displayed less crystalline structures, with a more uniform
coating layer than the stearyl alcohol coating. Interaction between the
coating layer and the core material was seen for both cetyl alcohol and
myristyl alcohol, implying good contact between the molten droplets
during the coating process. For the cetostearyl alcohol coating layer, a
well-defined line between the coating layer and the sugar spheres was
detected (Fig. 3 D). In contrast to the stearyl coating layer, no pores were
observed, reveling good coating properties for the cetostearyl alcohol.
DSC was used to analyze the melting temperatures (Tm) and the so­
lidification temperatures (Ts) of the four fatty alcohols. Myristyl alcohol
had the lowest phase transition temperatures, with a Tm of 37.7 ◦ C and
5
LWT 144 (2021) 111174
Ts of 34.5 ◦ C. These temperatures are too low for ensuring optimal
protection after oral ingestion, as the coating is likely to melt when
encountering body temperature. Stearyl alcohol had the highest phase
transition temperatures with a Tm of 58.8 ◦ C and Ts of 55.5 ◦ C. As pro­
biotics are heat sensitive, these temperatures are too high for a coating
process. Cetyl alcohol and cetostearyl alcohol had similar phase transi­
tion temperatures with cetyl alcohol having a Tm of 48.8 ◦ C and Ts of
47.0 ◦ C and cetostearyl alcohol having two Tm of 43.0 ◦ C and 49.2 ◦ C
and a Ts of 46.8 ◦ C.
Combining sucrose release, SEM and DSC results, cetostearyl alcohol
was found to be the most promising coating material. However, as
crystallinity was seen in the cetostearyl alcohol coating layer, it had the
potential to be optimized. Hence, the use of plasticizing materials, to
increase the intermolecular separation of the fatty alcohols, resulting in
a more flexible coating layer (Zhu, Shah, Malick, Infeld, & McGinity,
2002) was investigated.
3.1.2. Evaluation of plasticizing materials
Selected plasticizers were evaluated by preparing molded plates of
cetostearyl alcohol with plasticizing materials in different concentra­
tions. The plasticizers were evaluated by measuring contraction of the
plates after solidification, and visually inspecting the outer surface area
and the breakage surface, after breaking the plates into two (Table 3).
For a successful coating, the criteria were to find as little contraction as
possible after solidification, as this was believed to result in a better
covering coating layer. Furthermore, smooth surfaces on the outer of the
molded plates and at the breakage surface was desired, as this indicates
less crystallinity in the material, which hereby also contributes to a
better covering coating layer.
In regards to contraction of the plates, the addition of HPMC, EC and
beeswax showed less contraction after solidification, compared to
addition of the other tested plasticizers. HPMC was not fully soluble in
cetostearyl alcohol, even at low concentrations. Acetylated mono­
glycerides showed increasing contractions with increasing amount of
plasticizer and was brittle in its structure. The addition of olive oil and
polysorbate 60, at all three concentrations, showed similar contraction
as the reference plate consisting of 100% cetostearyl alcohol.
The addition of beeswax resulted in flexible molded plates that had a
very smooth breakage surface. This result shows that beeswax contrib­
utes to a less crystalline structure in the material, indicating that this
combination would result in a more flexible coating layer. In contrast to
Fig. 3. SEM images of sugar spheres coated with
different fatty alcohols.
A) Stearyl alcohol coating layer, B) Cetyl alcohol
coating layer, C) Myristyl alcohol coating layer, D)
Cetostearyl alcohol coating layer. The coated sugar
spheres were cut in half prior to SEM investigation.
The coating layer on each particle is marked by an
orange double arrow. (For interpretation of the ref­
erences to colour in this figure legend, the reader is
referred to the web version of this article.)
N.M.Y. Jacobsen et al. LWT 144 (2021) 111174

Table 3
Properties for molded plates of cetostearyl alcohol with plasticizing materials at different concentrations.
Plasticizer Acetylated EC HPMC Polysorbate Olive oil Beeswax Reference (no
concentrations monoglycerides 60 plasticizers)

[% w/w]

3 Diameter [cm] 8.8 8.8 8.7 8.8 8.6 8.7 8.6

Surface of molded Irregular Smooth Smooth Irregular Irregular Irregular Irregular
plate
Breakage surface Irregular Irregular Very Irregular Irregular Smooth Irregular
irregular

10 Diameter [cm] 8.7 8.8 8.9 8.6 8.8 8.8 ‒

Surface of molded Irregular Smooth Irregular Irregular Irregular Irregular
plate
Breakage surface Irregular Irregular Very Irregular Irregular Smooth
irregular

30 Diameter [cm] 8.3 8.7 Not soluble 8.9 8.8 8.8 ‒

Surface of molded Irregular Irregular Irregular Irregular Irregular
plate
Breakage surface Irregular Irregular Irregular Irregular Very
smooth

Abb. EC: Ethyl cellulose, HPMC: Hydroxypropyl methylcellulose.

this, the addition of EC resulted in very strong molded plates that were
hard to break.
From this initial screening, all plasticizers, with exception of acety­
lated monoglycerides, had a positive influence on the properties of
cetostearyl alcohol and where therefore investigated in different com­
binations and concentrations, to further improve the cetostearyl alcohol
coating material (Table 4).
To evaluate the contrary effects of beeswax and EC, these two ma­
terials were combined with cetostearyl alcohol. However, the results
showed that the molded plates still were very hard to break, despite the
addition of beeswax, and this combination would therefore not function
as a flexible coating layer. Olive oil mainly consists of triacylglycerides
of oleic acid, and it has been found that oleic acid in combination with
beeswax has a positive effect on mechanical properties of a coating layer
(Thomas, Visakh, & Mathew, 2013). The combinations of beeswax (30
and 50% w/w) and olive oil resulted in flexible plates. This flexibility
was not seen for beeswax at 10% w/w. To aid the solubility of HPMC, it
was combined with polysorbate 60, however, HPMC did still not show
complete solubility.
Based on the screening of selected plasticizers effect on the proper­
ties of cetostearyl alcohol, beeswax alone and in combination with olive
oil was found promising. Furthermore, cetostearyl alcohol and olive oil
in the ratio 95:5 (w/w) had previously shown good viability in coated
strains, L. acidophilus LA3 and B. longum BB536, used as model
probiotics.
The coated probiotic granulates were visually investigated using
SEM (Fig. 4). The C:O(95:5) coating on B. longum BB536 (Fig. 4. A + B)
showed an even coating layer with small pores that seemed to only
perforate the top of the coating layer. An even coating layer was
furthermore seen for the L. acidophilus LA3 strain coated with C:O(95:5)
(Fig. 4. C + D). Coating of the B. longum BB536 strain resulted in smaller
particles compared to the L. acidophilus LA3 strain, which may be caused
by a difference in the structure of the freeze-dried powder between the
two. For L. acidophilus LA3 coated with C:B(70:30) and C:B:O(65:30:5),
small pores that seemed to perforate further into the coating layer than
the ones in the C:O(95:5) coating were found (Fig. 4. E-H), indicating
that the addition of beeswax has an impact on the functionality of the
coating layer.
Table 5
Coated probiotic granulates produced using hot-melt fluid bed coating.
Strain Cetostearyl alcohol Beeswax Olive oil Abbreviations
[% w/w]

granulate after the hot-melt coating process (data not shown). L. acidophilus LA3 95 ‒ 5 C:O(95:5)
70 30 ‒ C:B(70:30)
65 30 5 C:B:O
(65:30:5)
3.2. Surface properties of coated probiotic granulate
B. longum BB536 95 ‒ 5 C:O(95:5)
Based on the above, the combinations seen in Table 5 were used for
producing coated probiotic granulates, with two different freeze-dried

Table 4
Properties for molded plates of cetostearyl alcohol with different combinations of plasticizing materials.
Abb. EC: Ethyl cellulose, HPMC: Hydroxypropyl methylcellulose.
Cetostearylalcohol Beeswax Olive oil Polysorbate 60 EC HPMC Surface of molded plate Breakage surface Breakage Properties

[% w/w]

85 10 5 ‒ ‒ ‒ Smooth Irregular Hard to break

65 30 5 ‒ ‒ ‒ Smooth Smooth Flexible
45 50 5 ‒ ‒ ‒ Smooth Smooth Flexible and soft
‒ 95 5 ‒ ‒ ‒ Smooth Smooth Very flexible, hard to break
87 10 ‒ ‒ 3 ‒ Smooth Irregular Difficult to break
67 30 ‒ ‒ 3 ‒ Very smooth Smooth Very difficult to break
96 ‒ ‒ 1 ‒ 3 Smooth Irregular Hard to break
94 ‒ ‒ 3 ‒ 3 Smooth Irregular Hard to break
87 ‒ ‒ 10 ‒ 3 Smooth Irregular Very hard to break

6
N.M.Y. Jacobsen et al.
3.3. In vitro evaluation of coated probiotic granulates
The physiologically relevant TSI in vitro model, simulating transit
through the stomach, duodenum, jejunum and ileum, under anaerobic
conditions, was used to investigate viability in and release from the
coated probiotic granulates. It was found that the small intestinal
inoculum was not able to grow on rogosa or BSM agar and these agars
could therefore be used for enumeration of released viable cells after
simulation of the ileum.
3.3.1. Viability in coated probiotic granulates before and after TSI in vitro
simulation
The number of viable cells present in C:O(95:5) coated probiotic
granulates and uncoated probiotic powders of L. acidophilus LA3 and
7
LWT 144 (2021) 111174
Fig. 4. SEM images of coated probiotic granulate.
A þ B) B. longum BB5369 coated with C:O(95:5), C þ
D) L. acidophilus LA3 coated with C:O(95:5), E þ F)
L. acidophilus LA3 coated with C:B(70:30), G þ H)
L. acidophilus LA3 coated with C:B:O(65:30:5). The
orange arrows indicate pore formations. Abb.: C:
Cetostearyl alcohol, O: Olive oil, B: Beeswax. Ratios
are given in w/w. (For interpretation of the references
to colour in this figure legend, the reader is referred
to the web version of this article.)
B. longum BB536, was investigated prior to and following in vitro simu­
lation in the TSI, after the ileum step (Table 6).
Coated L. acidophilus LA3 showed a 2.1 log CFU ml− 1 reduction after
simulated GIT transit, which was significantly lower than the reduction
of 4.9 log CFU ml− 1 seen for the uncoated L. acidophilus LA3 powder.
Both the coated and uncoated B. longum BB536 showed high stability
during simulated stomach and small intestinal transit, with no signifi­
cant difference in viability before and after. The results show an
important impact of coating L. acidophilus LA3 with C:O(95:5), as a
significant higher number of viable cells were found in the coated
granulate after simulated GIT transit, compared to the uncoated powder.
This is in line with other studies that have investigated viability of
coated and uncoated L. acidophilus strains in simulated gastric and in­
testinal fluids (de Araújo Etchepare et al., 2020; Ding & Shah, 2007; Du
N.M.Y. Jacobsen et al. LWT 144 (2021) 111174

Table 6 Table 8
Viability (log CFU ml-1) of C:O(95:5) coated probiotic granulates and uncoated Number of free viable cells after the stomach, evaluated for
probiotic powders (uncoated powder) before and after in vitro simulated transit the B. longum BB536 strain on RCA5.9 agar. Data is repre­
through the GIT. Data is represented as mean (±SD), n=2. sented as mean (±SD), n=2.
[log CFU ml-1] [log CFU ml-1]

L. acidophilus LA3 Before After B. longum BB536 After stomach

C:O(95:5) 9.2±0.2 7.0a±0.1 C:O(95:5) 4.3±1.6

Uncoated powder 10.1±0.0 5.3±0.1 Uncoated powder 5.9±1.9

B. longum BB536 Before After

C:O(95:5) 8.4±0.0 8.0±0.1 indicating that this coat does not provide gastric protection to the same
Uncoated powder 8.8±0.1 9.1±0.0 degree as the C:O(95:5) coat. After simulation of the duodenum,
a
Log reduction significantly lower than for the uncoated powder at the 95% jejunum and ileum, 7.0, 8.4 and 7.6 log CFU ml− 1, respectively, was
confidence level. detected. These numbers were higher than for the uncoated powder,
showing 5.0, 4.7 and 4.5 log CFU ml− 1 after the same steps. This in­
Le & Trinh, 2018). For the B. longum BB536 strain, the results showed dicates that the coating does provide some protection and continuous
that the uncoated cells themselves were stable during simulated GIT release during simulated transit through the small intestine.
transit, and no significant effect of coating was found. B. longum strains In regards to the C:B:O(65:30:5) coated granulate, a low release of
have previously been shown to be relatively resistant to low pH; a study 2.0 log CFU ml− 1 after simulation of the gastric step was found, showing
by Ding & Shah showed a reduction of only ~2 log units after 30 min better gastric protection than the C:B(70:30) coating. That cetostearyl
incubation in a simulated gastric medium at pH 2.0 (Ding & Shah, alcohol in combination with beeswax is able to decrease release has
2007). previously been shown by Gowda & Shivakumar (Gowda & Shivakumar,
2007), were release of lithium carbonate from the core of the micro­
3.3.2. Release of viable cells from coated probiotic granulates during TSI in spheres was investigated in simulated gastric and intestinal conditions.
vitro simulation We, however, only found a decrease in release when the cetostearyl
The number of viable cells released during simulated transit through alcohol and beeswax was combined with olive oil. As for the C:B(70:30)
the stomach, duodenum, jejunum and ileum in the TSI in vitro model was coating, a higher number of free viable cells was seen with the C:B:O
evaluated for L. acidophilus LA3 coated with C:O(95:5), C:B(70:30) and (65:30:5) coating after simulation of the duodenum, jejunum and ileum,
C:B:O(65:30:5) and for B. longum BB536 coated with C:O(95:5) compared to the uncoated samples.
(Table 7). For both the coated and uncoated B. longum BB5369, no viable cells
From the C:O(95:5) coated L. acidophilus LA3 strain, no release of were detected after simulated gastric transit. As increasing numbers of
viable cells were detected after the gastric step. As 8.1 log CFU ml− 1 viable cells were detected for the uncoated strain after simulation of the
viable cells were detected for the uncoated sample, these results confirm remaining steps in the in vitro model, the results imply that the B. longum
that the C:O(95:5) coating provides gastric protection of the probiotics. BB5369 cells were stressed and not cultivable rather than dead, at the
After the duodenum step, a lower number of 2.8 log CFU ml− 1 viable given time point, on the BSM agar used for enumeration (Lahtinen,
cells were detected for the C:O(95:5) coated granulate, compared to 5.0 Gueimonde, Ouwehand, Reinikainen, & Salminen, 2006). To further
log CFU ml− 1 for the uncoated powder. After the jejunum and ileum investigate the lack of viable cells, the samples were evaluated on the
steps, a similar number in the range of 5 log CFU ml− 1 was found for non-selective RCA5.9 agar, which showed viable cells for both the
both the coated granulate and the uncoated powder. This indicates that coated and uncoated samples after the gastric step (Table 8). BSM is a
a slow release from the C:O(95:5) coated granulate occurs during bifidobacteria selective medium, which may result in lack of growth
simulated GIT transit, which also has been observed in a study by da with stressed cells and thus, other non-selective medium may give better
Silva et al. (da Silva et al., 2019) where release of L. acidophilus micro­ growth conditions for stressed cells (Ferraris, Aires, Waligora-Dupriet, &
encapsulated in gelatin and gum arabic was investigated in simulated Butel, 2010). The RCA5.9 agar could, however, not be used for
GIT conditions. enumeration after the ileum step, as some strains from the small intes­
For the C:B(70:30) coated granulate, a number of 6.8 log CFU ml− 1 tinal inoculum were able to grow on this agar. After simulation of the
released cells was found already after simulation of the gastric step, duodenum, jejunum and ileum, similar numbers of free viable cells with
the coated and uncoated samples were found, indicating no significant
effect of the C:O(95:5) coating on the B. longum BB536 strain.
Table 7 To conclude, L. acidophilus LA3 coated with cetostearyl alcohol and
Released number of viable cells after each step in the TSI in vitro simulation. Data olive oil in the ratio 95:5 (w/w), resulted in significantly higher viability
is represented as mean (±SD), n=2 in comparison to the uncoated probiotic powder, after simulated GIT
[log CFU ml-1] transit. Furthermore, this coating showed gastric protection as no
release of viable cells were detected after simulation of the gastric
L. acidophilus After After After After
LA3 stomach duodenum jejunum ileum
compartment. The same protection of this coat was not seen when
applied to the B. longum BB536 strain, and hence, the coating process
C:O(95:5) 0.0a±0.0 2.8±0.1 5.4±0.3 5.5±0.1
needs to be optimized when used on different strains. To further
C:B:O(65:30:5) 2.0±0.0 4.9±0.3 6.5±0.4 6.4±0.0
C:B(70:30) 6.8±0.4 7.0±0.3 8.4±0.4 7.6±0.1 investigate the potential of the coating on the L. acidophilus LA3 strain, in
Uncoated 8.1±0.3 5.0±0.1 4.7±0.3 4.5±0.1 vivo evaluations in humans would be interesting.
powder

B. longum BB536 After After After After Funding

stomach duodenum jejunum ileum

C:O(95:5) 0.0±0.0 2.8±0.6 6.3±0.3 8.1±0.2 This work was supported by the Innovation Fund Denmark, Grand
Uncoated 0.0±0.0 3.3±0.5 7.0±0.3 8.3±0.1 solutions project “Stable Probiotic Powder Targeted Children and the
powder Elderly” and the Department of Pharmacy, University of Copenhagen.
a
Log CFU ml-1 significantly lower than for the uncoated powder at the 95%
confidence level.

8
N.M.Y. Jacobsen et al.
CRediT authorship contribution statement
Natashia Mai Yde Jacobsen: Writing – original draft, Methodology,
Investigation, Validation. Hanne Bjørn Nedergaard: Methodology,
Investigation, Validation. Anette Kock: Methodology, Investigation,
Validation. Ibrahim Caglayan: Investigation. Marie Munch Laursen:
Investigation. Eva-Marie Lange: Conceptualization, Methodology,
Funding acquisition. Martín Sebastián Marcial-Coba: Investigation.
Daniel Bar-Shalom: Conceptualization, Supervision, Funding acquisi­
tion. Dennis Sandris Nielsen: Methodology, Writing – review & edit­
ing, Supervision, Funding acquisition. Anette Müllertz:
Conceptualization, Writing – review & editing, Supervision, Funding
acquisition.
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.
Appendix A. Supplementary data
Supplementary data related to this article can be found at htt
ps://doi.org/10.1016/j.lwt.2021.111174.
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