DOI: 10.21776/ub.jpacr.Year.Vol.No.XXX J. Pure App. Chem. Res.

, Year, Volume (Number), page X

Day month Year

FORMULATION AND PHYSICOCHEMICAL PROPERTIES

OF KAFFIR LIME OIL-IN-WATER BEVERAGE EMULSIONS
CONTAINING VITAMIN E :
STABILITY AND PERCEPTION TEST
Vincentius Johar 1,2, Elvina Dhiaul Iftitah 1,2, Uswatun Hasanah 1

1
Chemistry Department, Faculty of Sciences, University of Brawijaya
2
Essential Oil’s Institute, University of Brawijaya
Email author 1: vincentiusjohar@gmail.com
Email author 2: vin_iftitah@yahoo.com
Email author 3: uswahas55@gmail.com

Received day month year; Accepted day month year (will be given)

ABSTRACT
Essential oil inside beverage emulsion became an interesting subject to get a new value for
the fast-moving customer goods (FMCG) market. In this study, kaffir lime oil (KFO)
antibacterial test on formulation and physicochemical properties of kaffir lime oil in water
beverage emulsions was investigated. The main ingredients of beverage emulsions are
water, high fructose corn syrup (HFCS), Arabic gum as an emulsifier, vitamin E, and kaffir
lime oil as flavoring and antibacterial. KFO composition used instrument with Stability of
oil-in-water emulsion was observed by measuring physicochemical properties: turbidity,
alkalinity, and conductivity. GC-MS characterization on kaffir lime essential oils revealed
the top three components, i.e., citronella (46,47%), citronellol (12,22%), and citronellyl
acetate (6,48%). FT-IR KFO spectrum had absorbance at 1726 (C=O stretching), 2922, and
2874 cm-1 (C-H stretching from aldehyde). Absorbance was also present on 1454 and
1379 cm-1 (C=C stretching) wavelengths, resembling citronella. Gum arabic with
R−S(=O)₂−OH group addition reduced pH value to 1.1 on each composition addition and
increased conductivity by 317.3 μS/cm. Fructose addition reduced the value by 153.4
μS/cm. Turbidity value increased averagely by 46,9 NTU, then reduced by 14,4 NTU.
Citronellal in KFO could hinder e-coli bacterial growth and had an alt value >72 CFU/mL.
This formulation study produces a beverage product with pH, conductivity, and turbidity
values following the standard and has criteria under the microbiological contamination
limit of BPOM and SNI standards.

Keywords: emulsified beverage, kaffir lime oil, physicochemical properties, stability,

perception

INTRODUCTION
Emulsified beverages are used to illustrate the emulsion system of oil in water (O/W)
consisting of flavors, coloring, sweeteners, and others. An emulsified beverage is an unstable
system of a liquid insoluble within other materials in a system. The colloidal/emulsion system
in the beverage industry should meet the requirements that all ingredients should be
environmentally friendly, affordable, and free of adverse effects to optical properties,
rheology, or sensory abilities of the final product.
If oil is the dispersed phase and water is the dispersing phase, this system is termed oil
emulsion in water. Conversely, if water is the dispersed phase and oil is the dispersing phase,
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 J. Pure App. Chem. Res., Year, Volume (Number), page X
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the system is termed water emulsion in oil. A suitable emulsifier or surfactant is often added
(Nasution et al. 2005). The stability character in an emulsion should present a constant value
in a time unit during emulsion solution storage.
One of the most exciting Indonesian essential oils applied to an emulsion system is
the kaffir lime (Citrus hytrix, DC). The volatile kaffir lime essential oil is utilized as flavor,
perfume ingredient, antioxidant, and good bacteria. Several precedent studies mentioned that
kaffir lime crude extract has an active component as cytotoxic (Chueahongthong, F. et al.
2011), anti-cancer ( Tunjung, W. A. S. et al. 2015), and antibacterial (Wannissorn, B. et al.
2005).
The current study developed a kaffir lime oil emulsion system in water. It also examined the
stability character and biological activities of kaffir lime essential oil components as
alternative preservative support, delivering fresh drink taste and aroma.

EXPERIMENT
Chemicals and instrumentation
We have created formulations with software to get variable composition of the
emulsion drink. We used design expert10 RO-Water, Gum arabic, kaffir lime oil (was
purchased from Institut Atsiri Brawijaya), HFCS. was installed from stat-ease.Inc to get
formulation of water: Fructose: Gum arabic: vitamin E : kaffir lime oil. Each formulation
mix on 1000 rpm with 60˚C mixing temperature until 3 minutes [8].

Organoleptic Preception Test

Thirty people use the panel to assay organoleptik. The panelist are trained and have the
ability well-adapted flavorings and tasting food and baverage. The panel is an employee of
PT CS2 Pola Sehat start from operational QC until managerial level. We use two stages test,
first scoring test and we used a hedonic interval test according SNI 2346:2006

Stable physicochemical analysis

All formulation were analyzed by Refractive index of sugar` dissolved with handled
refractometer and acidity with pHmeter seven Go Melter toledo , Turbidity with eutech
TB1000, TDS with TDSmeter KL731, conductivity with conductimeter PE02 and make sure
all formulation was measurement day by day until 5 days

RESULT AND DISCUSSION

Formulation calculate
Software design experts 10 chosen because having features to calculate the
determination of the formulation based on the upper limit composition and lower limit to use.
User asked admit value a lower limit and the upper limit with specific units will be used in
each material. Like for example water a key component , prosentase lower and upper limit of
used between 800 ml to 900 ml of the total composition while for kaffir lime use the upper
limit and lower more small because it requires a little bit smaller.

Table 1. Design Expert 10 formulation calculation.

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RO KFO HFCS GA Vit E

water

F1 800 0.125 ml 100 ml 15 gram 0,5 gram

ml

F2 900 0.2 ml 200 ml 10 gram 0,5 gram

ml

F3 900 0.125 ml 200 ml 10 gram 1 gram

ml

F4 800 0.125 ml 100 ml 5 gram 0,5 gram

ml

F5 800 0.5 ml 200 ml 10 gram 0,5 gram

ml

F6 800 0.125 ml 300 ml 10 gram 0,5 gram

ml

F7 800 0.125 ml 200 ml 10 gram 0,5 gram

ml

F8 800 0.2 ml 200 ml 10 gram 1 gram

ml

F9 800 0.125 ml 300 ml 10 gram 1 gram

ml

F1 800 0.125 ml 300 ml 15 gram 0,5 gram

0 ml

Water as the main components to making an emulsion drink. water composition

about 800 mL up to 900 mL in each variable composition , while kaffir lime oil as essential
oil flavouring about 0,125 ml until 0,5 ml . Addition of kaffir lime oil is different from the
amount by water due if the excess addition of kaffir lime oil can make the drink become
embittered and bitter and are not realiable to drink[6]. Kaffir lime oil also had volatile
components such as limonene and sitronelal as main coumpond,[9] with a little the addition
been able to give the specific kaffir lime smell and fresh flavor that is directly can be be
acceptable to drink. The addition of fructose used to increase that to have an drink flavored
sweet and acceptable to drink.The addition of vitamins e [10] is also aimed at adding the
health or increase value of beverages emulsion [10]. We use RO-UV water ( reverse osmosis
with ultraviolet water ) because the process of filtration to a system of reverse osmosis can

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remove heavy mineral or inorganic substances like the iron and inorganic substances other
thus healthier and removing impurities . RO-UV also avoid disruption stability factors
because gum arabic is an emulsifier and working with the ion-exchange inside the droplet.
UV filtration process able to removing a microorgnism impurities so as to minimize the
growth of bacteria during the mixing process.

First of all Gum arabic and vitamin E must dissolved in hot water then mixed into the
water has been furctose added. Fructose has analysis to make sure his refractive index
showing 75 brix in accordance with the COA (certificate of analysis) from suppliers. Next
preparation is measure kaffir lime oil use a micropipet analytic and mass scale to weigh gum
arabic and vitamin E. The analitical scale should have the calibration to get an accurate
measurement, by heating RO-UV until the temperature reached then inserted fructose in
accordance the formulation. Mixing process water, fructose, gum arabic and vitamin e into
the water had been reached temperature 60⁰C observable visual change the color of the water
from the first clearly be cloudness. Cloudness is a gum arabic properties as a thickness agent
[11]

Gum arabic is widely used as an emulsifier in the beverage industry to stabilise cloud and
flavour emulsions. It is derived from the natural exudate of Acacia senegal and consists of at
least three high-molecular-weight biopolymer fractions [12]. The arabic gum surface has an
active side for colloid formation which can be suspended in solution. The surface-active
fraction is believed to consist of branched arabinogalactan blocks attached to a polypeptide
backbone [7] The hydrophobic polypeptide chain is believed to anchor the molecules to the
droplet surface, whereas the hydrophilic arabinogalactan blocks extend into solution. In
figure 1 can be seen depicting Arabic gum which binds air and oil [12]. Amino acids will
form bonds with the oil surface and more influence hydrophobically as an inhibitor will bind
more bonds with air molecules and more precisely hydrophilic [13].

Figure 1. Gum Arabic’s hydrophilic side and hydorphobic side (william)

Kaffir lime oil were then added a second after the addition of gum arab and vitamin e
accompanied with a round handblender with speed 1000 rpm for 3 minutes. At the time of
mixing it can be observed that the scent of kaffir lime appears from the mixture formulation.
We can smell an odor of kaffir lime is produced by the citronellal [6] compound which
evaporates at the time of mixing so that the aroma of lime can be received by the human
sense of nose sensor. Stirring for 3 minutes to avoid volatile components into the air, too long
then the heat received will continue to make volatile components and reveal the aroma in the
emulsion drink. Stirring time for 3 minutes aims to avoid volatile components released into
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the air, if too long then the heat received will continue to make the volatile components
continue to evaporate and cause the loss of aroma in the emulsion drink [2].

Table 2. RO-UV water quality measurement.

Quality **RO UV Water

measurement
pH 6,65
TDS 13,30
Total Hardness 2,79
Alkalynity 10.88
Cl- 5,22
Cl2 0
KmnO4 0,1580
Fe2+ 0
Silica 1,1240

The analitical scale should have the calibration to get an accurate measurement. The
process of mixing at a temperature of 60⁰C , by heating RO-UV until the temperature reached
then inserted fructose in accordance the formulation.The composition has a direct impact on
the selection of the ingredients needed to achieve the targeted texture and stability of the
beverage. Process parameters such as homogenisation, heat treatment (time, temperature) and
filling temperature also play a key role. The use of ingredients such as hydrocolloids and
emulsifiers are key to ensuring the stability and texture of the final product [14] . The concept
of stability of an emulsion system is described in a constant nature of the particle
characteristics, but even though it has been said to be stable sometimes there will still be a
decrease in stability or can be called relative stability [15]. The physical properties of
dispersed particles have always had a surface tension and electrostatic value on each droplet,
the physical properties of these particles that can change due to many external factors, such as
gravity, storage temperature, physical shock and others [8]. The concept of stability is applied
in measuring the physical character of the particle with a number of standard instruments
with the aim that it can be observed to what extent such stability during storage. The physical
characteristics of these particles will then be observed and become parameters for
determining the physiochemical stability of the emulsion drink. Physiochemical categories
observed included refractive index of dissolved solids (Brix), acidity (pH), Turbidity, Total
Dissolved Solids (TDS), conductivity

The refractive index value of dissolved solids can be measured because there are
sugar molecules in each formulation F1 to F10 of added fructose and from macromolecules
of dissolved sugar in water that binds to the amino acid chains of gum arabic that bind to
water molecules and are referred to as dissolved solids [16]. SNI 3719-2014 Standards on
SNI for fruit juice drinks have the terms and definitions of fruit juice drinks which are drinks
obtained by mixing drinking water, fruit juice or unfermented fruit juice mix, with other parts
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of one or more types of fruit, with or without sugar and other food additions allowed.
Standard brix for orange juice drink is a minimum of 11.2 brix, from 10 formulations that
have been made samples F2, F3, F5, F6, F7, F8, F9, and F10 have fulfilled SNI 3719-2014
standard criteria with values more than 11.2 (> 11.2). The physiochemical stability check of
the dissolved solid index was analyzed for 5 days of storage time. The analysis process was
carried out at the same time to observe the refractive index of dissolved solids in all samples
and is shown in table 3. All samples experienced a change in the value of brix ± 0.2. Shifting
the value of brix ± 0.2 is considered reasonable because the correction factor of the digital
refracto instrument is ±0.2 in accordance the specifications of the instrument used [17].

Table 3. Brix measurement of the samples F1 to F10 are stable with the brix value.

N Brix
O sta D D D D D
rt +1 +2 +3 +4 +5
F 10. 10 10 10 10 10
1 3 .2 .3 .1 .1 .2
F 14. 14 14 14 14 14
2 6 .5 .6 .6 .6 .3
F 14. 14 14 14 14 14
3 7 .7 .7 .7 .7 .6
F 10. 10 10 10 10 10
4 4 .2 .2 .3 .4 .5
F 15. 15 15 15 15 15
5 2 .2 .2 .2 .2 .3
F 19. 19 19 19 19 19
6 2 .2 .2 .3 .0 .0
F 15. 15 15 15 15 15
7 3 .2 .2 .2 .1 .1
F 15. 15 15 15 15 15
8 4 .2 .2 .2 .2 .3
F 19. 19 19 19 19 19
9 2 .2 .2 .1 .1 .1
F 14. 15 15 14 14 14
1 9 .0 .0 .8 .8 .9
0

pH parameter checking the degree of acidity is used to see if there are other reactions
that occur after mixing and storage process, pH decrease suspect such as the emergence of
microbes [18] that produce CO2 or Carbon dioxide (CO2) dissolved in water which causes an
increase in the concentration of hydrogen ions (H+) which can reduce the pH of water [19] .
Water quality parameter index that can be drunk has a limit between 6.2 to 8.5 to be drunk
[19]. Samples F1 to F10 on average have a pH value of not more than 8.0 and not less than
7.6 at the time of the initial analysis immediately after mixing which can be seen in Table 2.
However, for 5 days of storage time the pH value fell significantly with a decrease average of
0,3 per day can be seen on graph 1.

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pH
8.5
8
7.5
7
6.5
6
5.5
1 2 3 4 5 6 7

F1 F2 F4

Figure 2. Graph F1, F2, F3 pH decrease for 5 days

pH decrease is due to the intensity of sunlight penetrating through the clear cup. light
intensity can increase the surface temperature of emulsion drinks [20]. The second possibility
can also be from changes in room temperature during storage. Research on water cooperation
conditions (pH) previously stated that relatively lower water temperatures increase the
solubility of CO2 which can reduce water pH and low temperature conditions will also cause
high DIC (dissolved inorganic carbon) concentrations, so that the water pH will drop [21].
The decrease in pH can be seen in graph 1 in F1, F2 and F4 samples.

Turbidity can be used as a reference parameter for determining stability in a book on

emulsion stability discussed about the characterization of emulsion microemulsions with
sonification techniques[13]. Then the stability determination is determined by the turbidity
value where the more stable the turbidity value measured in a different time interval, the
better the stability value. Turbidity values can also be used in reference to determine the size
of droplets distributed in an emulsion system [13] Turbidity can also be used as a reference to
determine relative stability. the more non-uniform droplet size results in high turbidity values
[8]. Modern analysis to measure Turbidity is usually stated in the Nephelometric Turbidity
Unit (NTU). This unit is also recommended by USEPA drinking water regulation. Analysis
can be done quickly and easily with the Nephelometric method [22]..

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Figure 3. Ion exchenge mechanism inside a droplet

Conductivity value can measure the potential value of energy. Each emulsion droplet
basically has a surface tension and has a different polar group because the surface of the
emulsion is usually covered by a layer of organic matter[13]. The book explained that the
ions from the diffused layer are partially distributed outside the organic matter layer and
partly into the layer [8]. The phenomenon of ion exchange also occurs in the adsorption layer
and electrostatic interactions occur so as to produce information about ion exchange outside
the adsorption layer which can be measured in conductivity values [8]. It can be observed in
the illustration in Figure 2 that there is an ion exchange between the inside of the water
droplet and most of the outside [13].

TURBIDITY
170
165
160 F1
F2
155
NTU

F4
150
145
140
1 2 3 4 5 6 7

Figure 4. Graph F1, F2, F3 Turnidity stable for 5 days

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CONDUCTIVITY AT 25⁰
c
250 F1
200 F2
150 F4

μS/ cm
100
50
0
1 2 3 4 5 6 7

Figure 5. Graph F1, F2, F3 Conductivity stable for 5 days

Turbidity is different from total Dissolved Solids. Turbidity states how much light is
dispersed by the water sample, while TDS expresses the weight of the suspended material in
the sample. Turbidity figures greater than 5 Nephelometric Turbidity Unit (NTU) can show a
quite clear turbidity. In samples F1 to F10 the value of the stability of the conductivity and
turbidity are positively correlate.. Where when the turbidity value rises the conductivity value
will also be increase linearly [23].

All natural water contains minerals and dissolved substances. These minerals in water
can begenerally measured as total dissolved solids (TDS). TDS is mainly comprised of
naturally occurring minerals, mostly inorganic salts (calcium, magnesium, potassium,
sodium, bicarbonates, chlorides and sulfates) [24] . It can also include contaminants such as
heavy metals, but usually at low concentrations that represent a very small fraction of the
TDS. The presence of contaminants (such as heavy metals) can not be determined by
measuring TDS. In drinking water [25], TDS is measured as parts per million (ppm) or mg/L.
Because TDS does not present a risk to human health, the U.S. EPA has not set a mandatory
TDS limit, but suggests a maximum level of 500 ppm. At or above this level, consumers may
notice deposits, colored water, staining and/or a salty taste. These are not harmful, but rather
noticeable aesthetic (such as smell, taste or color) effects when the TDS is above 500 ppm.
Meanwhile TDS value under 300 mg/L (<300 mg/L) is a good criteria and can be drunk
(Broséus et al., 2009) [25].

Table 3. TDS measurement of the samples F1 to F10 are stable with the brix value.

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TDS mg/L
star D+1 D+2 D+3 D+4 D+5
t
F1 167. 167. 167. 167. 167. 167.
5 5 5 5 5 5
F2 99 99 99 99 99 99
Physiochemical
stability test F3 137 137 137 137 137 137 parameters as long as
the TDS value does F4 98 98 98 98 98 98 not change, it can be
said that there is no F5 143 143 143 143 143 143 change in the solute
in an emulsion F6 183 183 183 183 183 183 system and it can be
said that there is a F7 137. 137. 137. 137. 137. 137. hydrophilic and
hydrophobic balance 5 5 5 5 5 5 [8]. Initial analysis of
TDS in F1 and F10 F8 135 135 135 135 135 135 samples varies in
value because the F9 156 156 156 156 156 156 formulation of solids
in emulsion drinks is F1 197. 197. 197. 197. 197. 197. also different, the
more solids are 0 5 5 5 5 5 5 dissolved [23].

In the organoleptic test selected 30 panelists who have the ability and high sensitivity
to product quality specifications and have knowledge and experience on how to assess
organoleptic and sensory and pass the selection of standard panelists. Panelists are employees
of PT CS2 Pola Sehat from the Manager level to the operational level who have passed the
organoleptic test. The assessment using this sensory instrument includes the appearance
quality specifications, texture, odor, taste and several other factors needed to assess the
product. The panel is asked to choose 3 out of 10 samples based on their general appearance
and taste. And an assessment is made by ranking between 1 and 3. Value 1 is given to the
sample with the most preferred rating, value 2 is given to the sample with a good rating, then
value 3 is given to the sample with a moderate rating. The rank test results are summed with
the values listed in table 4.

Table 4.. Scoring test result summary sample F1 ; F10

F F2 F3 F4 F5 F6 F7 F8 F9 F10
1

39 36 11 41 20 12 8 8 0 5

Then the 3 highest scores were taken. Next step we use a hedonic test was performed
to see based on the specific level of preference which was divided into 4 groups, namely by
evaluating the visual visual, odor, taste and fresh sensation for each sample. Rating
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specifications are assessed based on numbers 1 to 9 to see ratings of preference. The

assessment specifications are set out in Table 5. And make a sensory value interval with the
interval formulas.

Table 5.. Hedonic rating specification with number

Most like 9
Really like 8
line 7
Rather like 6
Neutral 5
rather not like 4
Not like 3
Really not like 2
Most not like 1

The formula for calculating the preferred index interval with the following formula listed in
table 6

Table 6. Preferred index interval F1 F2 F4

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Sampl Observatio
e n Interval

P(8,00 ≤ μ ≤8,00)
Visual

P(6,89 ≤ μ ≤ 7,46)
Odor
F1
P(6,89 ≤ μ ≤ 7,46)
Taste
Fresh P(7,28 ≤ μ ≤ 8,04)
sensation
Visual  

P(8,00 ≤ μ ≤8,00)
Odor  

P(7,53 ≤ μ ≤ 8,13)
F2
Taste  

P(8,00 ≤ μ ≤8,00)
Fresh  
sensation
P(7,09 ≤ μ ≤ 7,90)
Visual  

P(8,0 ≤ μ ≤8,0)
Odor  

F4 P(6,95 ≤ μ ≤ 7,71)
Taste  

P(7,09 ≤ μ ≤ 7,90)
Fresh P(7,71 ≤ μ ≤ 9,88)
sensation

CONCLUSION
The stability of the products can be seen from pH values, Turbidity and Conductivity in
accordance ISO standart product storage before the distribution process and no deviations
organoleptik and visual products during storages. The sensory value interval of the
appearance of F1 samples is P (8) ≤μ≤ (8) and for the final value of appearance the smallest
value is 8 (very like). and obtained values for odor, tastes and fresh sensations in table 6 from
which conclusions can be drawn that the F1, F2, and F4 samples from the calculation of the
Design Expert 10 program can have good preferences and can be accepted by consumers
through orgnoleptic sensory tests. A good interval has a distance that is not too far away,
meaning all panelists have almost the same sensory ratings when tasting a sample.

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