40
Research Article
Preparation of probiotic oil suspension containing
Lactobacillus acidophilus
Ngoc Khanh Lea, Khac Tiep Nguyena, Thi Thanh Duyen Nguyenb, Thi Phuong Linh Lea, Dieu Ly Nguyena,
Thi Hoa Nguyena, Thi Phuong Vua, Thanh Xuan Dama,*
aFaculty of Biotechnology, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam
bFaculty of Pharmaceutics and Pharmaceutical Technology, Hanoi University of Pharmacy, 13-15 Le Thanh Tong, Hanoi, Vietnam
Journal of Pharmaceutical Research and Drug Information, 2023, 14 (5): 40-46
A R T I C L E I N F O
Article history
Received 04 May 2023
Revised 17 Sept 2023
Accepted 25 Nov 2023
Keywords
Dispersion method
L. acidophilus
Oily suspension
Probiotic
Paediatric uses
A B S T R A C T
Probiotics are defined as microorganisms that, when administered in sufficient
quantities, confer a health benefit on the host. As they are very susceptible to
environmental conditions, solid dosage forms are used to increase their
stability. In pediatric use, liquid preparations of edible oily suspensions
containing probiotics are more favorable since they help overcome the
difficulty of oral administration in children. However, few research articles
related to the preparation process of these oily suspensions were reported in
Vietnam. Our study focuses on preparing an oily suspension containing L.
acidophilus at a laboratory scale, oriented for use in infants and children under
two years of age. The oily suspension was prepared using the dispersion
method. Different mixtures of oil and dispersing agents were studied for their
effect on the stability of the suspension in terms of sedimentation rate, drop
time, average particle size, and the amount of L. acidophilus. The results
showed that an oily suspension containing L. acidophilus could be made using
the dispersion method with a mixture of oils as the dispersion medium and
Aerosil and aluminum tristearate as dispersing agents. The prepared
suspension had an appropriate sedimentation rate of about 10–20%, an
average drop time of 1,0 drop/s, an average particle size of 3–3,5 µm, and an
amount of L. acidophilus above 3 x 108- 5 x 108CFU/ml. Our study
successfully prepared an oily suspension containing L. acidophilus using the
dispersion method. The prepared suspension had good physical stability, and
the number of L. acidophilus remained over 108CFU/ml during two months
of storage.
*Corresponding author: Thanh Xuan Dam; e-mail address: xuandt@hup.edu.vn
https://doi.org/10.59882/1859-364X/141
Journal homepage: jprdi.vn/JP
Journal of Pharmaceutical Research and Drug Information
An official journal of Hanoi University of Pharmacy
41
Introduction
The term "probiotics" refers to
microorganisms that, when administered in
sufficient amounts, provide a positive impact
on the health of the host organism [1]. By their
nature as microorganisms, probiotics exhibit a
high degree of vulnerability to environmental
factors. When administered orally and
subsequently passed through the
gastrointestinal tract, their viability is
significantly reduced. The use of solid dosage
forms incorporating protective layers is a
common approach to enhancing the durability
of probiotic products. Nevertheless, these solid
forms are difficult for small children to use,
initiating the study of liquid formulations such
as edible oily suspensions containing
probiotics as an approach to solving this issue.
The use of oily suspensions has also been
demonstrated to efficiently maintain the
stability of probiotics [2]. In Vietnam, it is
observed that the percentage of children aged
below 5 years constitutes around 10% of the
total population. Therefore, demand for a high-
quality probiotic product for this age group is
high. However, the current market consists of
a limited number of commercially available
products, with the majority of them being
imported. Moreover, there is a lack of research
articles related to the preparation procedure of
oily suspensions in Vietnam. Hence, to address
this research gap, our study aims to develop a
laboratory-scale oily suspension formulation
including L. acidophilus, with a focus on its
application for newborns and children aged
below two years.
Materials and methods
Materials
Lactobacillus acidophilus PROBIO-TEC
LA-5 BLEND-30 (L.a powder) was purchased
from Biogreen Pharmaceutical and
Biotechnology JSC. (3 x 1010 CFU/g). MRS
broth and MRS agar media were from Merck,
Germany. Edible oils for suspension include
sunflower oil (SF) from Ukraine, soybean oil
(SB), and medium-chain triglycerides (MCT)
from Vietnam. Other materials, such as Aerosil,
aluminum tristearate (AlS), and phosphate-
buffered saline (PBS), were from China.
Methods
Preparation
An oil suspension containing L.
acidophilus was prepared according to the
following procedure:
- Probiotic powder was sieved through a
125 µm sieve. Other excipients were sieved
through a 180 µm sieve.
- Preparation of dispersion medium: The
oil or oil mixtures were heated and
homogenized at 750 rpm at 121°C for 10
minutes.
- Disperse Aerosil and AlS evenly into the
dispersion medium by stirring at 750 rpm for
10 minutes and allow the suspension to cool
to room temperature.
- Disperse L.a powder into the above
suspension by stirring at 750 rpm for another
10 minutes to obtain an oil suspension
containing L. acidophilus.
Evaluation method
Sedimentation rate (S %): After preparation,
the product was left for 60 days until the solid
particles settled, leaving a transparent layer
above. The sedimentation rate was the ratio
between the height of the transparent layer and
the original suspension height [3].
Drop time (T drops/s): After preparation,
the suspension was packed into a 5 ml brown
glass bottle with a 2 mm diameter dropper
hole. Shake well for 10s and tilt the bottle at
a 45-degree angle to dispense the drop. The
drop time (T) was determined with a
stopwatch.
Ngoc Khanh Le et al, J.Pharm.Res-DI. 2023, 14(5): 40-46
42
Average particle size (D µm) and particle
distribution index (PDI): Spread a sufficient
amount of suspension on a glass slide and
take pictures at 40x magnification on an
upright microscope Eclipse Ci-L (Nikon,
Japan). The images were then processed and
analyzed using ImageJ 1.53 and Origin 8.0
software to determine the average particle
size (D) and particle distribution index (PDI)
in the suspension.
Method to determine the number of
microorganisms
Add exactly one dose-equivalent amount
(0.25mL) of the suspension and
approximately 0.8mL of PBS medium
containing 4% Tween 80 to an Eppendorf
tube, and then vortex for 1 min. Centrifuge
the mixture at 15,000 rpm for 2 min. Discard
the supernatant, add about 0.8 mL of PBS
medium containing 4% Tween 80 to the
pellet, vortex for 30s, and then continue to
centrifuge for a second time at 15,000 rpm for
2 min. Discard the supernatant and transfer
all the pellets from the Eppendorf tube to a
tube containing 5 mL of PBS medium with
4% Tween 80 and vortex for 30s. Transfer
0.5mL of the suspension from the first tube
to the second tube containing 4.5 ml of PBS
Ngoc Khanh Le et al, J.Pharm.Res-DI. 2023, 14(5): 40-46
Table 1. Formulas used in the study
Ingredient CT1 CT2 CT3 CT4 CT5 CT6 CT7 CT8 CT9 CT10
L.a powder
(g) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
AlS (g) 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75 1.75
Aerosil (g) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
SF (ml) 100 97.5 95 92.5 90 0 0 0 0 0
SB (ml) 0 0 0 0 0 100 97.5 95 92.5 90
MCT (ml) 0 2.5 5 7.5 10 0 2.5 5 7.5 10
Ingredient CT11 CT12 CT13 CT14 CT15 CT16 CT17 CT18 CT19 CT20
L.a powder
(g) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
AlS (g) 1.58 1.40 1.23 0.88 1.75 1.58 1.40 1.23 0.88 1.75
Aerosil (g) 0.50 0.50 0.50 0.50 0.45 0.45 0.45 0.45 0.45 0.40
SF (ml) 95 95 95 95 95 95 95 95 95 95
SB (ml) 0 0 0 0 0 0 0 0 0 0
MCT (ml) 5 5 5 5 5 5 5 5 5 5
Ingredient CT21 CT22 CT23 CT24 CT25 CT26 CT27 CT28 CT29 CT30
L.a powder
(g) 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
AlS (g) 1.58 1.40 1.23 0.88 1.75 1.58 1.40 1.23 0.88 1.58
Aerosil (g) 0.40 0.40 0.40 0.40 0.35 0.35 0.35 0.35 0.35 0.40
SF (ml) 95 95 95 95 95 95 95 95 95 95
SB (ml) 0 0 0 0 0 0 0 0 0 0
MCT (ml)
5
5
5
5
5
5
5
5
5
5
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medium with 4% Tween 80 and vortex for
30s. Repeat this dilution procedure until a
suitable concentration is obtained. Transfer
accurately 10 µL of the solution at the last
four concentrations to an MRS agar Petri
dish. Incubate in the 37°C incubator with 5%
CO2 and count the number of colony-
forming units (CFU) growing on the plate
after 48–72h.
Results and discussions
Through reference to several documents
[4-5], a number of factors in the formula were
investigated, including the type of oil used as
a dispersing medium and dispersing
excipients. The formulas used are shown in
Table 1. All formulas contain 2.0 grams of
L.a powder in a total volume of 100 mL.
Using a mixture of oils as a dispersion
medium helps increase the physical stability
of the oily suspension.
A suspension is considered physically
stable if the particle sedimentation rate is
minimized and the particles can be easily
redistributed before use [6]. The higher the
viscosity of the suspension, the lower the
sedimentation level, and the more physically
stable the suspension. However, it is difficult
to dispense the suspension if the viscosity is
too high. Also, if it is too low, the drop time
is too fast, and it is hard to obtain the exact
number of drops needed. Therefore, the
dispersion medium needs to have a suitable
viscosity, which reduces the level of
sedimentation and ensures a favorable drop
time during use.
Several formulas were monitored for
sedimentation rate, drop time (results are in
Table 2), and average particle size to select
the suitable dispersion medium. After 60
days, the sedimentation rate of those using
sunflower oil in combination with less than
7.5% MCT was about 8–15% (CT1 – CT4),
significantly lower than formulas using a
higher rate of MCT (CT5 with over 50%).
The trend was similar to the formulas using
soybean oil in combination with MCT. The
sedimentation level of those with less than
7.5% MCT was about 20–30% (CT6–CT9),
while the sedimentation rate of those with a
higher MCT ratio was about 41% (CT10).
With the same MCT ratio, sunflower oil-
containing formulas had a lower
sedimentation level. The sedimentation result
was because sunflower oil has the highest
viscosity, followed by soybean oil, and MCT
viscosity is significantly lower than the other
oils [7,8]. Therefore, when the proportion of
MCT in the mixture increased, the viscosity
of the dispersion medium decreased, leading
to an increase in the settling level of the
suspension. Our study proposed that the
appropriate level of sedimentation was below
35% after 60 days to ensure that the
microorganisms could still be evenly
redispersed when shaking the suspension. It
also minimized the phenomenon of particle
sedimentation and agglomeration. Thus,
sunflower (or soybean) oil in a mixture with
MCT (the MCT rate was below 7.5%) was
chosen as a dispersion medium for further
study.
To ensure accurate dosing and flowability
of the product, we also surveyed the drop
times of the above formulas and compared
them with the BioGaia Protectis baby drops
(BioGaia, Sweden). The results showed that
as the MCT ratio increased, drop time de-
creased. When using the product, the drop
time can affect dosing accuracy and
convenience for the user. A long drop time
will cause inconvenience, while a short drop
time will make it harder to get the exact
number of drops, affecting the dosing.
Ngoc Khanh Le et al, J.Pharm.Res-DI. 2023, 14(5): 40-46
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Therefore, the drop time of the product must
be optimal to meet both requirements. The
drip process of the reference product
(BioGaia Protectis baby drops) occurred
smoothly with a drop time of 1.5 drops/s,
though the drip speed was too fast.
Therefore, the proposed suitable drop time
for the suspension in the study was
approximately 1.0 drop/second. Among all
formulas, CT3 (the sunflower oil: MCT ratio
was 95:5) and CT7 (the soybean oil: MCT
ratio was 97.5:2.5) had a drop time of 1.2
drops/s and 1.0 drops/s, respectively. These
formulas were subsequently evaluated for
the average particle size and particle
distribution index.
The results showed that CT3 and CT7
had an average particle size of 3.61 ± 0.34
µm and 3.51 ± 0.14 µm, in comparison to
the BioGaia product (4.02 ± 0.11 µm)
(Table 3 and Figure 1). All these formulas
had good particle size distribution index of
below 0.5.
Immediately after preparation, the
number of L. acidophilus in both CT3 and
CT7 was about 5 x 108CFU/ml (log CFU/mL
reached 8.7), meeting the required standards
of a probiotic product. After two months at
room temperature (30 oC), the number of
microorganisms in both formulas decreased
sharply to 1.36 x 107CFU/mL and 6.95 x 105
CFU/mL, respectively (log CFU/mL reached
7.13 and 5.84, respectively). For storage
conditions of 0-5 oC, the number of L.
acidophilus in the two samples also decreased
but remained at approximately 108 CFU/ml
(log CFU/mL about 8.3 - 8.6).
Table 3. Average particle size and particle distribution
index of Biogaia, CT3, and CT7 suspensions
The above results showed that a dispersion
medium containing sunflower oil and MCT
with a ratio of 95:5 (CT3) was optimal to
ensure the stability of the suspension.
Ngoc Khanh Le et al, J.Pharm.Res-DI. 2023, 14(5): 40-46
Table 2. Sedimentation rate and drop time of CT1 to CT10 formulas
Ingredient CT1 CT2 CT3 CT4 CT5 CT6 CT7 CT8 CT9 CT10
S (%)
8.3 ±
0.12
11.1 ±
0.06
10.1 ±
0.20
15.5 ±
0.06
53.0 ±
0.12
21.2 ±
0.06
25.3 ±
0.06
23.9 ±
0.10
31.3 ±
0.15
41.0 ±
0.20
T (drops/s)
0.5 ±
0.01
0.7 ±
0.02
1.2 ±
0.09
1.4 ±
0.05
1.8 ±
0.12
0.7 ±
0.05
1.0 ±
0.04
1.3 ±
0.07
1.5 ±
0.15
2.2 ±
0.17
Figure 1. Picture of solid particle dispersed in an oily suspension of A. BioGaia, B. CT3 and C. CT7.
Taken at 40x magnification on an upright microscope Eclipse Ci-L (Nikon, Japan)
BioGaia CT3 CT7
D (μm)
4.02 ± 0.11 3.51 ± 0.14 3.61 ± 0.34
PDI 0.48 0.33 0.46