Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol.
52 (2012) 6-10
Microencapsulation of fish liver oil by
complex coacervation method with gelatin-arabic gum
Hiep X. Nguyen1, Chien N. Nguyen1,*
1Department of Pharmaceutial Industry, Hanoi University of Pharmacy, Hanoi, 10000, Vietnam
*Chiennnguyen@hup.edu.vn
Abstract
Fish liver oil was microencapsulated by complex
coacervation with a gelatin – arabic gum polymeric
wall system. The effect of core/wall ratio,
emulsification stirring speed, coacervation pH
value, concentration of formaldehyde solution, ratio
of gelatin/ arabic gum, weight of PVA, and
microencapsules stabilizing time on the
microcapsulation yield, encapsulation efficiency,
particle size and morphology of microcapsules were
investigated. The suitable conditions were
determined and rational formulation were
established.
Keywords: Fish liver oil, complex coacervation,
HPLC, extraction
Introduction
Microcapsule and microsphere have numerous
advantages over tablet namely more stable
bioavailabity amongst individuals since retention
time of microcapsule is less fickle in intestinal tract.
Microencapsulation especially takes active
ingredients to undergo the metamorphosis from
liquid state to solid form to be ultimately capsulated
or tabletted [3], and also conceals both ingredients’
stinky odour and foul taste. Be based on that and
the pharmaceutical project entitled
“Microencapsulation of fish liver oil by complex
coacervation method with gelatin-arabic gum” was
excecuted with fish liver oil as drug model in order
to investigate the effects of condition and
formulation parameters on microencapsulation of
fish liver oil.
Materials, equipments and methods
Materials
Vitamin A palmitate, gelatin, gum arabic, fish liver
oil, polyvinyl alcol (PVA): reach pharmaceutical
standards. Other chemicals: reach analytical
standards. Standard Vitamin A palmitate (500
mg/vial, 996,100 IU/g) is kindly provided by
National Institute of Drug Quality Control.
Equipments
IKA RW 20 overhead digital stirrer, CHRIST Freeze
Dryer, Refrigerated Centrifuge sigma 3 – 18K
sartorius AG, Hettich Zentrifugen Centrifuge Rotina
46, IKA vortex Genius 3, Optical microscope Carl
Zeiss, axiostar plus, High Performance Liquid
Chromatography system (HPLC) Shimadzu,
Scanning Electron Microscope (SEM) Hitachi S4800-
NIHE 10000kV.
Methods
Preparation of microcapsules
Microcapsules were prepared by complex
coacervation method with a gelatin – arabic gum
polymeric
wall system, analogous to the technique of Alavi
Talab H. [1]. The incipient formula came as follows:
30g 12.5% gelatin solution was mixed with 30g
12.5% gum arabic solution at room temperature,
stirring speed of 500rpm in 5 minutes (the first
stirring stage). Added 4.2g fish liver oil, 0.5g PVA,
0.04g BHT (Butylated hydroxytoluene), and 10 ml
distilled water to the mixture of gelatin solution and
gum arabic solution, then continued agitating in 15
minutes, at room temperature and the same stirring
speed as at the first stirring stage (the second
stirring stage). Increased the mixture’s temperature
to 500C and added 190ml distilled water, kept the
stirring speed on par with that at the first stirring
stage in 30 minutes (the third stirring stage).
Adjusted the mixture’s pH value to 4.5 by dropwise
addition of 10% acetic acid solution at 400C. Added
200ml distilled water and emulsified the mixture at
stirring speed of 250rpm, temperature below 100C
in 60 minutes (the fourth stirring stage). Altered pH
value of the mixture to 9.7 by 10% sodium
hydroxide solution addition, then added 20ml 20%
formaldehyde solution. Kept stirring at the speed of
250rpm, in 30 minutes, below 100C to stablize the
microcapsules. 22 hours later, centrifuged the
mixture at the centrifugation speed of 4000rpm, at
room temperature, in 15 minutes to collect
microcapsules. After that the microcapsules were
washed twice by 100ml distilled water below 100C
(centrifuged after each wash), vacuum filtered, and
then washed once by solution of 25ml isopropanol
and 25ml distilled water below 100C [4]. The
 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol. 52 (2012) 6-10

obtained microcapsules were desiccated by freeze Encapsulation Efficiency (EE)

drying with the following parameters: temperature:
-500C, pressure: 0.007mbar, pre-freeze time: 4 Encapsulation efficiency is percentage of Vitamin A
hours and freeze drying time: 22-23 hours. in microcapsules in the total amount of Vitamin A in
fish liver oil material used.
Determination of microcapsules properties
Microcapsule Yield (MY)
Size and shape of microcapsules
Calculated microcapsule yield by dividing the
acquired microcapsules weight by the total weight
Used an optical microscope, each sample of 50
of materials.
microcapsules from 5% suspension of
microcapsules in cold water; and utilized Scanning
Results and discussion
Electron Microscope (SEM).
Chromatograms of Vitamin A in HPLC analysis
Vitamin A concentration
Ran HPLC with the Vitamin A treated samples to
Hydrolyzed fish liver oil samples (approximately obtain chromatograms in image 1. The
equivalent to 1740IU Vitamin A) in fish liver oil chromatograms gave a single, sharp and relatively
material, microcapsules or standard Vitamin A symmetrical Vitamin A peak on a flat, little-noisy
palmitate by 1ml saturated potassium hydroxide baseline with retention time of 2.6 min.
solution, 2ml ethanol and 0.02 g BHT in an
ultrasonic bath, in 30 minutes. Extracted Vitamin A
once by 6ml n-hexane, washed n–hexane extract by
distilled water, evaporated n-hexane entirely to
obtain solid deposit which was then dissloved in
isopropanol. Diluted the resulted solution to the
concentration range of 375IU/ml to 2350IU/ml. Ran
these samples on a High Performance Liquid
Chromatography system (HPLC) with the following
parameters [2]: Supelco Discovery C8
Chromatographic column, 15cm x 4.6mm (5μm);
Guard Column 2cm x 4.6mm (5μm); injected sample
volume: 20μl; column temperature: 300C; flow rate:
1.0ml/min; mobile phase: methanol:twice distilled
water=95:5; detection wavelength: 325nm.

400
750

400
500
200
m AU

m AU
m AU

200
250

0 0
0

0 2 4 6 0 2 4 6 0 1 2 3 4 5
Minutes Minutes
Minutes
A B C
Image 1: Chromatograms of the Vitamin A treated samples from fish liver oil microcapsules F–D (A, the final
formula, see below), standard Vitamin A palmitate (B) and fish liver oil material (C)

The effects of core/wall ratio C11) and 8.4g (F-C21)]. Properties of the resulted
microcapsules were demonstrated in table 1.
Prepared the formula F–C11, altered core/wall ratio
[the fish liver oil weight was 2.1g (F–C12), 4.2g (F-
 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol. 52 (2012) 6-10

Table 1: The effects of core/wall ratio on microcapsules properties

F Core/wall ratio C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

C12 1:2 326.33 51.27±11.4 82.09 65.32 Aggregation, hard to filter

C11 1:1 523.27 50.42±12.0 80.25 62.95 Scattering, easy to filter

C21 2:1 757.53 50.13±9.2 72.20 61.89 Aggregation, very hard to filter

Annotation: C was Vitamin A concentration in microcapsules samples, Φ= diameter, MC=microcapsules.

Table 1 indicated that when core/wall ratio varied
from 1:2, 1:1, to 2:1, the microcapsules’ average size
did not change (p>0.05), both encapsulation
efficiency and microcapsule yield reached their
maximums when the core/wall ratio was 1:2 (F-
C12), and arrived at their minimums when the
core/wall ratio was 2:1 (F-C21). These two fomulae
produced aggregated and hard-to-filter
microcapsules. On the other hand, microcapsules
Table 2: The effects of emulsification stirring speed on microcapsules properties
obtained by the formula core/wall ratio=1:1 (F-
C11) were spherical, scattering and easy-to-filter.
The effects of emulsification stirring speed
Prepared the formula F–C11, altered stirring speed
at the first, second and third stages from 300rpm to
1000rpm. Properties of the resulted microcapsules
were demonstrated in table 2.

F v (v/ph) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

A 300 452.77 X X 59.42 Scattering, nonspherical, hard to filter

B 400 496.43 X X 60.73 Scattering, nonspherical, hard to filter

C 500 523.27 50.41±12.0 80.25 62.95 Scattering, spherical, easy to filter

D 750 519.53 40.13±10.2 81.94 64.11 Scattering, spherical, easy to filter

E 1000 556.79 26.46±9.1 79.33 63.27 Scattering, spherical, very easy to filter

Annotation: X = undetermined

As the emulsification stirring speed was 300rpm (F-
A) and 400rpm (F-B), microcapsules were
nonspherical, hard-to-filter. Be based on that and
only encapsulation efficiency was investigated for
these formulae. If the stirring speed increased from
500rpm to 1000rpm, then the microcapsules’
average size decreased correspondingly, EE and MY
did not change significantly. In addition, the
formulae with stirring speed of 500rpm (F-C) and
Table 3: The effects of the microencapsules stabilizing time on microcapsules properties
750rpm (F-D) created scattering microcapsules. EE
of F-D was maximum.
The effects of microencapsules stabilizing time
Prepared the formula F–D, altered the
microencapsules stabilizing time from 8h, 15h to
22h. Properties of the resulted microcapsules were
demonstrated in table 3.

F T (hours) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

T8 8 484.06 41.32±9.2 80.24 64.82 Aggregation, hard to filter

T15 15 484.46 41.10±10.1 80.92 65.34 Scaterring, hard to filter

D 22 476.15 40.82±9.7 81.94 64.11 Scaterring, easy to filter

 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol. 52 (2012) 6-10

When the microcapsules stabilizing time changed
from 8h to 15h and 22h, the microcapsules’ average
size, microcapsule yeild and encapsulation
efficiency did not change (p>0.05). As the stabilizing
time was more than 15h, microcapsules were
scattering and easy to filter. As a result, the
microcapsules stabilizing time 22h was selected to
serve the study.
The effects of coacervation pH value
Prepared the formula F–D (the microcapsules
stabilizing time was 22h), altered coacervation pH
value from 3.0 to 5.0. Properties of the resulted
microcapsules were demonstrated in table 4.

Table 4: The effects of coacervation pH value on microcapsules properties

F pH C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

H1 3.02 587.14 X X 53.91 Nonspherical, hard to filter

H2 4.11 548.24 38.54±6.8 73.76 60.67 Scattering, spherical, easy to filter

D 4.50 531.53 40,13±10.2 81.94 64.11 Scattering, spherical, easy to filter

H4 5.06 525.40 40.02±8.6 77.40 61.05 Scattering, spherical, easy to filter

When the coacervation pH value was 3.02, the
microcapsules clustered, nonspherical, hence only
encapsulation efficiency was studied. When the
coacervation pH value varied from 4.11; 4.50 to
5.06, the microcapsules’ average size changed
insignificantly. The pH value 4.50 brought about the
maximum microcapsule yield (81.94%), the
maximum encapsulation efficiency (64.11%), easy-
to-filter microcapsules. Be based on that and the
coacervation pH value 4.50 (F-D) was choosed.
The effects of concentration of formaldehyde
solution
Prepared the formula F–D (the coacervation pH
value was 4.50), altered concentration of
formaldehyde solution from 10%, 20% to 30%.
Properties of the resulted microcapsules were
demonstrated in table 5.

Table 5: The effects of concentration of formaldehyde solution on microcapsules properties

C formaldehyde
F C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties
(% w/v)

F1 10 535.76 41.27±7.3 75.42 60.98 Scattering, easy to filter

D 20 519.53 40.13±10.2 81.94 64.11 Scattering, easy to filter

F3 30 520.11 42.45±8.9 83.03 65.13 Aggregation, hard to filter

When the concentration of formaldehyde solution
altered from 10%, 20% to 30%, the microcapsules’
average size changed insignificantly (p>0.05). The
formula F-D (20% formadehyde) accounted for
microcapsule yield and encapsulation effiency
higher than F-F1 and lower than F-F3, but
scattering microcapsules.
The effects of ratio of gelatin/arabic gum
(gel:gum)
Prepared the formula F–D (the concentration of
formaldehyde solution was 20%), altered the gel:
gum ratio: 3.75g:7.50g (1:2); 3.75g:3.75g (1:1);
7.50g:3.75g (2:1). Properties of the resulted
microcapsules were demonstrated in table 6.
 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol. 52 (2012) 6-10

Table 6: The effects of gel:gum ratio on microcapsules properties

F gel:gum C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

Aggregation, nonspherical, very hard to

M 1:2 425.03 X X 58.89
filetr

D 1:1 519,53 40.13 ± 10.2 81.94 64.11 Scattering, spherical, easy to filter

P 2:1 472,13 41.91 ± 13.0 78.32 61.81 Aggregation, spherical, easy to filter

When the gel:gum ratio was 1:2 (F-M), the product The effects of weight of PVA
was nonspherical, cluster of polymers containing
many fish liver oil droplets, encapsulation efficiency Prepared the formula F–D (the gel:gum ratio was
was lowest (58.89%). As the gel:gum ration was 2:1 1:1), altered weight of PVA from 0.21g to 0.50g and
(F-P), microcapsules were spherical with relatively 0.80g. Properties of the resulted microcapsules
equal size, but clustered with low MY and EE. When were demonstrated in table 7.
the gel:gum ratio was 1:1 (F-D), microcapsules were
spherical, scattering, with relatively equal size and
maximum encapsulation efficiency.
Table 7: The effects of weight of PVA on microcapsules properties

F PVA (g) C (IU/g) Φ MC (μm) MY (%) EE (%) MC properties

P1 0,21 468.17 42.22±10.1 62.79 47.90 Scattering, easy to filter

D 0,50 519.53 40.13±10.2 81.94 64.11 Scattering, easy to filter

P3 0,80 450.12 40.89±9.4 83.30 64.05 Aggregation, easy to filter

When the weight of PVA varied, the microcapsules’
average size changed insignificantly (p>0.05). As
the weight of PVA increased from 0.21g (F-P1) to
0.80g (F-P3), microcapsule yield increased.
Encapsulation efficiency reached its maximum
(64.11%) when the weight of PVA was 0.50g (F-D)
and its minimum (47.90%) when the weight of PVA
was 0.21g (F-P1). Continuous augmentation of PVA
weight to 0.80g (F-P3) did not lift the encapsulation
efficiency, let alone made clustered, hard-to-filter
microcapsules.
F-D was selected as the final formula (F-D was
similar to F-C11, only changed the emulsification
stirring speed at the first, second and third stages to
750 rpm).
The fundamental structure and size of
microcapsules were displayed in image 2.

A (optical microscope, 100X) B (SEM, magnification of 3000X)

Image 2: Images of microcapsules (formula F-D)
 Hiep X. Nguyen et al. / Pharmaceutical Journal ISSN 0866-7225 No 437 Vol. 52 (2012) 6-10
Discussion
Microencapsulation by complex coacervation
method with gelatin-arabic gum is complicated,
multi-stages, each stage affects the quality of
microcapsules. In an alike study, Alavi et.al [1]
demonstrated that as stirring speed increased,
microcapsules’ average size decreased
correspondingly. Besides, using 25%
glutaraldehyde as the cross linking agent instead of
formaldehyde brought the microcapsules more
spherical shape, smooth surface with no obvious
dents and narrower particle size distribution [1]. The
project of Junyaprasert et. al [4] illustrated that the
freeze drying method and the gel:gum ration=1:1
were appropriate to prepare Vitamin A palmitate
microcapsules by complex coacervation method.
Conclusion
The study investigated the effects of some
condition, formulation parameters on
microencapsulation of fish liver oil by complex
coacervation method, determined the suitable
conditions and established rational formulation.
References
1. Alavi T. H. et al. (2010), "Optimization of
morphology and geometry of encapsulated
Hypophthalmichthys molitrix oil", Iranian Journal of
Fisheries Sciences, 9(2), p. 199-208.
2. Vietnam Ministry of Health. (2009), Appendix
10.10: "Vitamin A quatitative analysis, Method 5:
High Performance Liquid Chromatography (HPLC)
method after Vitamin A extraction", Viet Nam
pharmacopoeia IV, Hanoi, p. PL 190-PL193.
3. James S. et al. (2007), "Microencapsulation
Technology, Encyclopedia of Pharmaceutical
Technology", Informa Healthcare, New York, 3(4), p.
2315–2332.
4. Junyaprasert, V. B. et al. (2001), "Effect of Process
Variables on the Microencapsulation of Vitamin A
Palmitate by Gelatin-Acacia Coacervation", Drug
Development and Industrial Pharmacy, 27(6), p.
561–566.