Vol.4, No.5B, 84-89 (2013) Agricultural Sciences
doi:10.4236/as.2013.45B016
Omega-3 emulsion of Rubber (Hevea brasiliensis)
seed oil
Siti Hamidah Mohd-Setapar*, Lee Nian-Yian, Wan Nuraisha Wan Kamarudin, Zuhaili Idham,
Abd-Talib Norfahana
Centre of Lipid Engineering and Applied Research (CLEAR), Department of Chemical Engineering, Faculty of Chemical Engineer-
ing, Universiti Teknologi Malaysia 81310 Skudai, Johor, Malaysia; *Corresponding Author:
Received 2013
ABSTRACT
The formulation of omega-3 emulsion using rub-
ber (Hevea Brasiliensis) seed oil based on the best
performance of the emulsion in terms of higher
viscosity, smaller droplet size, lower moisture
content and slightly acidic pH value supported
by degree of creaming in varying the type and
composition of emulsifier used. Rubber seed oil
contains significant value of alpha-linolenic acid
which plays an important role in maintaining
human health. Therefore, formulation of rubber
seed oil emulsion is important to become a new
source of omega-3 emulsion instead of fish oil.
Rubber seed oil was mixed with distilled water
and nonionic emulsifier which were lecithin and
span 80 by homogenizer. From the analysis
conducted, the best formulation was the emul-
sion with 50% of distilled water, 6% of lecithin
and 47% of rubber seed oil.
Keywords: Emulsion; Emulsifier; Lecithin; Span 80
1. INTRODUCTION
Rubber tree or its scientific name Hevea Brasiliensis is
a perennial plant from South America cultivated as an
industrial crop after being introduced to Southeast Asia
on 1876. This tree produced rubber seeds that compose
43% of oil [1]. Rubber seed oil (RSO) is a semi-dried
substance [2] that is rich in polyunsaturated fatty acids of
C18: 2 and C18:3, as it contributes 52% of its total fatty
acid composition [3]. RSO has already been shown to
have many industrial applications, including possible
uses for manufacturing fatty acids [4].
Malaysia has been a major rubber growing country as
estimated by Malaysian Rubber Board (2011) where
1,022,780 hectares are used for rubber plantation in 2011.
The production of rubber seeds by average in Malaysia is
about 1 million metric ton per year. Rubber kernel con-
tains 29.6% of fat and 11.4% of proteins per seed [5].
Thus, about 296,000 tones fats and 114,000 tones pro-
teins are wasted each year.
Aigbodion and Bakare [6] reported that rubber seed oil
contains oleic acid (22.95%), linoleic acid (37.28%) and
linolenic acid; also known as omega-3 (19.22%). Rubber
seed oil contains alpha-linoleic acid which is one of the
omega-3 fatty acids which is very important in our daily
diet. However, it cannot be produced by our body and
need to be taken through supplements or food. Normally,
omega-3 can be consumed from fish oil and vegetable oil.
Still, many people do not realize that rubber seed oil is
actually edible; instead they think rubber seed oil has
high toxicity. Nonetheless, Babatunde and Pond [7] have
once stated if the oil is rich in linolenic acid and linoleic
acids, it can be used for food and industrial market as it
contains high essential nutrients that is comparable to
buah perah and soy beans.
As in Jerantut, Pahang, the villagers consume rubber
seed as their daily dishes which is known as ‘asam rong’.
It is a traditional dish and normally the seeds are added
into sambal and curries. Therefore, this can be a proof
that rubber seed has become people’s choices and safe to
be eaten.
In this study, the rubber seed oil obtained was used to
formulate omega-3 emulsion. Emulsion is a heterogene-
ous system composed of at least two immiscible liquids,
water and oil and considered as liquid-liquid colloid type
[8]. Emulsion is commonly classified into oil in water
(O/W) or water in oil (W/O) depending on the continu-
ous phase.
Formulations of emulsion require which can influence
the properties of emulsion in several ways for example
the stability of dairy emulsion [9]. In foods sold in the
Europe, emulsifiers are given E numbers beginning with
3 or 4 [10]. Emulsifiers will reduce the dynamic surface
tension, thus leading to the formation of small fat drop-
lets during homogenization; displace protein that may
otherwise be available for bridging flocculation, from the
fat globule surface; interact with interfacial protein,
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S. H. Mohd-Setapar et al. / Agricultural Sciences 4 (2013) 84-89 85
leading to a thicker and stronger adsorbed layer and in-
crease the viscosity of the aqueous phase through the
formation of self bodying mesophase structures.
In this experiment, two types of emulsifiers were used,
which were lecithin and span 80. Common food applica-
tions of lecithin include use as an emulsifier, a stabilizer,
a dispersing aid, and an incidental additive, such as a
release agent for baked goods. Regardless of its food
application, lecithin is generally used in small amounts,
with the result that it is, according to one lecithin manu-
facturer, present in finished foods at levels rarely ex-
ceeding 1% by weight of the final food product. On the
other hand, span 80 also known as sorbitan monooleate,
a commercial Span 80 does contain monoesters, diesters,
as well as triesters and tetraesters. Furthermore, the polar
head group of all esters present in Span 80 is not sorbitol,
but more likely one of the different forms of anhydrized
sorbitol.
Therefore, this study aims to determine the best for-
mulation of omega-3 emulsion using rubber seed oil to
obtain the most stable emulsion.
2. MATERIALS AND METHODS
2.1. Preparation of Rubber Seed Oil
Rubber seeds were prepared by removed the outer skin.
The seeds were dried in oven and ground into fine pow-
der. The rubber seed oil was produced using soxhlet ex-
traction method.
2.2. Formulation of Emulsion
The rubber seed oil was mixed with distilled water and
emulsifier at the temperature of 50℃ to 60℃. The mix-
ture was homogenized at 10,000 rpm. The lecithin and
span 80 were used as emulsifier in this experiment.
Lecithin is a food ingredient that is derived from plant
sources, including soy. Meanwhile, span 80 is a molecu-
larly heterogonous non-ionic emulsifier which is cheap
and normally is used as food emulsifier and in oral
pharmaceuticals. The concentrations of emulsifiers were
varied according to Table 1.
Table 1. Emulsion composition and codes.
Code O:W
(v/v) Type of emulsifier Emulsifier (v/v)% / (w/w)%
E1 50:50 lecithin 3.0
E2 50:50 lecithin 6.0
E3 50:50 lecithin 9.0
E4 50:50 span 80 10.0
E5 50:50 span 80 8.5
E6 50:50 span 80 7.0
E7 50:50 span 80 6.0
v/v. volume/volume; w/w. weight/weight
The further analyses done on the emulsion were vis-
cosity, pH value, droplet size, moisture content and de-
gree of creaming.
2.3. Stability Test
The fresh prepared emulsion was transferred to a
graduated cylinder. The amount of free water or oil was
separated from the emulsion and observed everyday for a
period of 10 days. The volume of water inside the emul-
sion was calculated using eq.1 to determine the stability
of the emulsion with the respect to days.
Degree of creaming = v1 – v2 (1)
In Eq.1, v1 represents the total volume of the fresh
emulsion while v2 is the volume of the crude emulsion
after a few days.
2.4. Physical Characteristics
The prepared emulsions were observed at room tem-
perature for 24 hours. Observations were done based on
the phase separation of the emulsion, and then the emul-
sion was used for further analysis. The emulsion which
stable for 24 hours is usually stable for several days [11].
The viscosity is a measure of the ratio of shearing
stress to rate of shear as shown in the Eq.2. Viscosity
was performed at 25℃ using viscosmeter LVDV-II +P
CP Brookfield with the use of Rheocalculator V3.3 Build
49-0 software.
Viscosity (cP) = 100/rpm × TK × SMC × Torque (2)
where RPM is current viscometer spindle speed, TK is
viscometer torque constant, SMC is current viscometer
torque % expressed as a number between 0 and 100.
First of all the viscometer was auto zero with no spindle
attached for calibration. Then after 10 seconds, the main
display flashed at 00.0. The spindle was attached to the
viscometer and spindle must not to touch the bottom or
sides and should be centered. After that, the spindle was
immersed in the sample cup and switched on the button
for agitation process. The speed was adjusted from 0 to
10 rpm until the stable reading appeared. Lastly, the dial
reading and rpm were recorded after the reading was
stabilized.
The moisture content was determined using drying
method. Firstly, the sample was weighed and recorded
before dried in the oven at 105℃ for 6 hours. After dry-
ing process, the weight of sample was measured. The
eq.3 showed the calculation of moisture content.
% Moisture = [(Mwet - Mdry) / Mwet ] × 100% (3)
where Mwet is the weight of wet sample while Mdry is the
weight of dry sample.
The pH value was determined by homogenized the
emulsion using vortex to ensure the sample was mixed
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S. H. Mohd-Setapar et al. / Agricultural Sciences 4 (2013) 84-89
86
well and measured by a digital 3505 pH meter (model
Jenway) for 3 times and average pH value was calcu-
lated.
The diameter of the droplets was measured by using
Olympus BX 5 optical microscope (x40) equipped with a
calibrated eyepiece micrometer. The emulsion was put
into a glass plate and measurements were taken after 3 to
4 days. The images of size and distribution of particles
were taken using digital camera and analyzed by CC12
soft imaging system software.
2.5. Analysis of Variance (ANOVA)
In this study, the parameters were analyzed using
Analysis of Variance (ANOVA). The results of ANOVA
and degree of creaming were used to determine the best
formulation.
3. RESULTS
3.1. Stability of Emulsion
The stability of emulsion was studied through the de-
gree of creaming for a period of 10 days by observing the
volume of the oil as a dispersed phase. The results were
shown in the Tabl e 2. The value 0.0 indicates the emul-
sion maintain its stability.
Based on the results shown in Table 2, the E4 and E5
of span 80 did not form creaming at all in 10 days. They
were very stable compared to the others. Meanwhile, E6
and E7 started to form creaming at day 2. The E6
emulsion showed the higher rate of creaming formation
initially but it had been slowed down and the degree of
creaming was the same as E7 in the end
3.2. Physical Characteristics
3.2.1. Effect of Concentration and Type of
Emulsifier on Viscosity
The formulation of emulsion with lecithin as emulsifier
had the higher viscosity compared to formulation of
emulsion with span 80 as shown in Figure 1. The maxi-
mum value for lecithin was 30 cP while for span 80 was
8 cP.
Table 2. Effect of lecithin and span 80 percentages on degree of
creaming.
Day
Code 1 2 3 4 5 6 7 8 910
E1 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.2 0.4
E2 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.00.0
E3 0.0 0.0 0.0 0.0 0.00.5 1.4 1.8 2.0 2.4
E4 0.0 0.6 0.9 1.1 1.21.4 1.5 1.6 1.6 1.7
E5 0.0 1.0 1.1 1.1 1.31.3 1.4 1.5 1.6 1.7
E6 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.00.0
E7 0.0 0.0 0.0 0.0 0.00.0 0.0 0.0 0.00.0
Figure 1. Viscosity of the formulation of emulsion with the
increasing percentage of emulsifiers.
Figure 2. Interaction of pH value with increasing percentage
and type of emulsifier.
3.2.2. Effect of Concentration and Type of
Emulsifier on pH value
The pH value showed in the Figure 2 increased as the
amount of span 80 increased while the pH values were
dereased when the amount of lecithin was increased. The
formulation of span 80 mostly approximately neutral
while lecthin produced slightly acidic emulsion.
3.2.3. Effect of Concentration and Type of
Emulsifier on Moisture Content
Figure 3 showed the effect of the percentage and type
of emulsifier on moisture content. The use of span 80,
the moisture content increased from 6% to 8.5% and then
decreased when the span 80 amounts was further increased.
On the other hand, moisture content for lecithin for-
mulation decreased slightly when lecithin increased. All
the formulations used lecithin had lower moisture content
compared to span 80. The 9% of lecithin emulsifier gave
the lowest moisture content which was 23% moisture
content.
3.2.4. Effect of Concentration and Type of
Emulsifier on Droplet Size
The analysis results of optical microscope were shown
in Figure 4 for span 80 and Figure 5 for lecithin. The
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S. H. Mohd-Setapar et al. / Agricultural Sciences 4 (2013) 84-89 87
microstructure of the emulsions indicated that most of
the dispersed phase were present as circular droplets and
had many range of sizes. This generally occurred for the
emulsions, which were prepared by physical methods
like vortex method and mechanical homogenizers [12].
Figures 4(a) and (b) show the microstructure of the short
term stable emulsions after 48 hours which span 80 as an
emulsifier. This condition also applied to Figure 5(c)
which lecithin as an emulsifier.
In addition, Figures 4(c) and (d) show the microstructure
for the most stable emulsion with the percentage of span
80 were 8.5% (E5) and 10%(E4) respectively. Figures
5(a) and (b) also show the microstructure of the more
stable emulsion.
The maximum droplet size in Figure 6 of lecithin
slightly increased as the percentage of emulsifier increased.
The span 80 with 8.5% shows the smallest value of
maximum droplet size between all the emulsifier con-
centrations followed by the 10% of span 80 and 3% of
lecithin.
Figure 3. The effect of percentage and type of emulsifier on
moisture content.
(a) (b)
(c) (d)
Figure 4. The microstructure of emulsion with different
concentration of span 80 (a) E7-6%; (b) E6-7%; (c) E5-8.5%;
(d) E4-10%.
3.2.5. Effect of Concentration and Type of
Emulsifier on Moisture Content
The one-way ANOVA test was carried out to deter-
mine the best formulation of omega-3 emulsion in terms
of moisture content, pH value, viscosity and droplet size
for various formulations and it was expressed as mean ±
SD. Abbreviation (a, b, c) were arranged according to
small value to the big value. The data tabulated in Table
3 and the results of stability analyses were used to de-
termine the best formulation of omega-3 emulsion.
(a) (b) (c)
Figure 5. The microstructure of emulsion with different
concentration of lecithin (a) E1-1%, (b) E2-6%, (c) E3-9%.
Figure 6. Maximum droplet size based on the concentration of
emulsifiers.
Table 3. The analysis of omega-3 emulsion samples for various
formulations.
Formula
Parameter
E7 E6 E5 E4 E1 E2 E3
%(w/w)
Moisture 16.748 a25.187bc34.800d 25.134bc 30.006cd 29.647cd 23.662b
pH 5.15 a6.07c6.25 d 6. 35d 6.01 c 5.67b 5.69b
Viscosity
(cP) 7.887 d4.2767 a6.0833c 5.4300 b 8.2167 d 30.580 f16.613e
Drop Size
(μm) 105.13 f106.42 g13.54 a 37.41 b 42.57 c 99.27 e
94.81 d
The average of all the analysis conducted were expressed as mean of three
replication ± standard deviation. Abbraviation (a, b, c) represent statistical
comparison made for each measurement for each formualtion. The mean
with different letters for each formulation and samples were significantly
different (p < 0.005).
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4. DISCUSSION
Based on the statistical analysis and the results of sta-
bility test, the best formulation was determined. The
formulation with lower moisture content was needed
because it could prevent microbiological present in the
sample. E4 showed a good stability among the emulsions
and E4 is the best formulation in term of moisture con-
tent while E1 showed the lowest moisture content but
this emulsion is not stable.
The emulsion must be slightly acidic to prevent the
growth of microbial which will cause the emulsion being
spoiled easily. This is because most bacteria grow at pH
range 6 to 8 and pathogens do not grow well below pH
4.5. The pH of the Scott Emulsion was taken to be
compared with the experimental rubber seed oil emulsion.
The pH value was 3.14 which were very acidic. E2 that
gave the best stability showed the pH ranges between 5.67
- 5.69 for rubber seed oil omega-3 emulsion. Flavoring
that is high acidic such as lemon oil can be included to
improve the pH of emulsion.
Viscosity can be defined as an important aspect of
product quality, the ability to detect differences in the
viscosity of beverages allows more satisfying and delicious
food product to be created [13]. For the purpose of
commercialization, the properties of high viscosity with
low oil content and fine droplet size are required. As E5
had the best viscosity value in this experiment when
added with span 80 as emulsifier while E2 showed the
best viscosity with added lecithin, so their viscosity value
were used to compare with Scott’s Emulsion. E5 and E2
had lower viscosity compared to the Scott’s Emulsion
which has 37 cP, but E2 showed the nearest viscosity
with the commercial product and it can be concluded that
lecithin is the best type of emulsifier for omega-3 emulsion
of rubber seed oil in term of viscosity. Therefore, in order
to improve the viscosity of emulsion, additives such as
thickeners, co-surfactants and so forth for specific appli-
cation can be added into the emulsion during formula-
tion.
Furthermore, E5 was the best formulation in term of
droplet size. In this case, E2 which had the moderate droplet
size but it gave the most stable emulsion. Theoretically,
when the droplet diameter is large, bacteria reproduce more
easily than smaller droplet diameter, as the bacterial growth
is reduced due to the lack of nutrients inside the droplets.
The droplet sizes are expected to not substantially exceed-
ing 1000 μm for microbiologically stable emulsion [14].
Hence, the less stable emulsion which were E7 and E6
added with the emulsifier of span 80 will not affected
with the growth of microorganism as the maximum
droplet size was only about 110 μm but the stability will
be affected due to the creaming, flocculation of small
droplet particles.
5. CONCLUSIONS
From the analysis conducted, there were two best
formulations which using 6% (v/v) lecithin and 8.5%
(v/v) span 80 as the emulsifier. For span 80, the best
formulation was E5 with 50% (v/v) distilled water, 8.5%
(v/v) span 80 of the total volume of emulsion and 41.5%
(v/v) rubber seed oil (RSO). The other formulation which
considered was 50% (w/w) distilled water, 6% (w/w)
lecithin of the total weight of oil only and 47% (w/) of
RSO. These formulations had nice texture and the
emulsion can be kept longer.
However, from this two formulation of emulsion,
formulation with lecithin as an emulsifier was the best as
the viscosity, stability, pH value and its moisture content
were achieved the requirement for the commercial
purposed. It can be used as a benchmark for formulation.
From this study, the concentration of span 80 in the
formulation will be affecting the omega-3 emulsion. The
range between 8.5% to 10% of the span 80 is suggested
to produce the most stable emulsion that can be main-
tained up to a week with a fine droplet size. So, further
study should be conducted to use combination of emulsi-
fiers according to hydrophilic-lipophilic balance (HLB)
value to improve the physical stability. Besides, the fur-
ther studies also may include what type and how much of
preservatives, antioxidants and other food additives are
necessary to increase the shell life of this product.
As a conclusion, the omega-3 emulsion of rubber (Hevea
Brasiliensis) seed oil has the potential to be commercial-
ized as omega-3, alpha-linolenic acid supplement which
is extracted from local source and sustainable. Therefore,
more research should be carried out to study the potential
of rubber seed oil so that can be applied into different
industries such as food, cosmetics and pharmaceutical.
6. ACKNOWLEDGEMENTS
The authors gratefully acknowledge the Centre of Lipid Engineering
and Applied Research (CLEAR) and also Universiti Teknologi Malay-
sia, Johor for the laboratory experiment and instruments provided.
Acknowledgement also extended to Malaysia Government (Escience
Fund) Vot 4S020 for the financial support.
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