Study of Enzymatic Synthesis of Glycol Castor Oil Fatty Acid and
Glycol Palmitic Acid Esters as Emulsifier and Antimicrobial
Compounds Using Candida rugosa Lipase EC. 3.1.1.3
Adinda Azkia, Sumi Hudiyono
and Sri Handayani
Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Indonesia, West Java, Indonesia
Keywords: Antimicrobial.sal, castor oil, emulsifier, fatty acid glycol ester, lipase, palmitic acid.
Abstract: This study was conducted to synthesize glycol castor oil fatty acid and glycol palmitic acid esters using
Candida rugosa lipase as biocatalyst. The ester products was expected to have emulsifier and antimicrobial
properties. Esterification was conducted by reacting fatty acid and glycol at 37 C for 18 hours. The variation
of fatty acid mol ratio to glycol used were 1:1, 1:2, 1:3, and 1:4. The ester product was characterized using
FTIR and the conversion percentage was determined by titrimetric method. Emulsifier test also performed to
determine the ability of ester product as emulsifier. Antimicrobial assay were also conducted using disc
diffusion method against Propionibacterium acne and Staphylococcus epidermidis. FTIR spectra for glycol
castor oil fatty acid and glycol palmitic esters showed the absorption of C=O functional groups at wave
numbers 1732.27 and 1741.88 cm
-1
, respectively. The highest conversion percentage value for glycol castor
oil fatty acid and glycol palmitic ester were 85% and 82%, respectively. The emulsifier test showed both
glycol fatty acid ester have properties as emulsifiers. Antimicrobial assay showed that glycol castor oil
fatty acid ester has activity as antimicrobial against both bacteria. However, glycol palmitic ester has no
activity as an antimicrobial agent.
1 INTRODUCTION
Castor plant (Ricinus Communis L.) is a flowering
species in the spurge family; euphorbuaceae and
belong to the genus Ricinus. This plant contains 83
chemical compounds including alkaloid, terpenoid,
flavanoid, benzoic acid derivatives, coumarin,
tocopherol, and fatty acids such as risinoleic acid
(Ribeiro, 2016). In general, all parts of the castor
plant can be utilized. However, the most widely used
is the oil, because it has several functions as a drug
for skin rash, cosmetics, and biodiesel (Salihu, 2012).
Spesifically, most content of castor oil is ricinoleic
acid (87%) (Swern, 1979). Ricinoleic acid can be
obtained from castor oil by way of hydrolysis.
One of oleochemical production using fatty acid
is esterification reaction of glycol and fatty acid. The
reaction between carboxylic acid and alcohol with or
without the aid of catalyst, which will produce ester
compound known as esterification reaction. This
reaction is very slow and takes several times to reach
equilibrium when it is not using a catalyst. Therefore
a catalyst is needed to accelerate the rate of
esterification reaction (Mandake, 2013).
In previous study, esterification of glucose fatty
acid from coconut oil hydrolysis was performed using
Candida rugosa lipase as a catalyst (Hudiyono,
2012). In addition, the utilization of castor oil has
been made as a formulation of hand and body cream
products and cosmetic application (Perez, 2016).
Then, castor oil was also reported to be very effective
for the treatment of skin problems such as dry skin,
burnt skin and stretch marks (Salihu, 2012).
Furthermore, oil from castor seeds was reported to
have antibacterial activity against gram-positive and
negative bacteria (Rahmati, 2015).
In this study, the synthesis of emulsifier and
antibacterial compounds has been conducted through
esterification between glycol and fatty acid from
castor oil hydrolysis and palmitic acid. The
esterification was catalyzed by Candida rugosa
lipase. The emulsifier test and antimicrobial assay
were performed to observe the stability of the ester
product to have properties as emulsifier and able to
Azkia, A., Hudiyono, S. and Handayani, S.
Study of Enzymatic Synthesis of Glycol â
˘
A ¸S Castor Oil Fatty Acid and Glycol â
˘
A ¸S Palmitic Acid Esters as Emulsifier and Antimicrobial Compounds Using Candida rugosa Lipase EC. 3.1.1.3.
DOI: 10.5220/0008356800050011
In Proceedings of BROMO Conference (BROMO 2018), pages 5-11
ISBN: 978-989-758-347-6
Copyright
c
2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
5
inhibit the activity of Propionibacterium acne and
Staphylococcus epidermidis on human skin.
2 MATERIALS AND METHODS
2.1 Materials
Materials used in this study were Candida rugosa
Lipase (2.45 U/mg) that obtained from Sigma-
Aldrich, castor oil, ethylene glycol, palmitic acid,
ethanol, potassium hydroxide, hydrogen chloride, ,
aquades, n-hexane, sodium hydroxide phosphate
buffer pH 8, phenolphthalein, eosin, clindamycin,
DMSO 10%, nutrient agar, nutrient broth,
Propionibacterium acne and Staphylococcus
epidermidis.
2.2 Methods
2.2.1 Hydrolysis of Castor Oil
Hydrolysis of castor oil was performed by mixing 100
g of castor oil and 100 mL potassium hydroxide in
ethanol and then heated for 1 hour at 70±2 C. After
heated, 55 mL of Hydrogen Chloride was added while
stirring for 1 hour. Then, the product must be waited
for 24 hours. After 24 hours it will form 2 phase.
2.2.2 Esterification
Esterification was started by mixing ethylene glycol,
castor oil fatty acid, n-hexane, and Candida rugosa
lipase as a catalyst. The variation of mol ratio for
glycol to fatty acid that used were 1:1, 1:2, 1:3, and
1:4 (mol/mol). The ratio of solvent and substrate used
was 1:1 (v/v substrate), while the enzyme used was
5% of the total substrate (w/w substrate). The mixture
then was incubated on a horizontal incubator shaker
at 200 rpm, for 18 hours at 37 C temperature. The
mixture was the heated at 80C to terminated the
reaction. After that, the mixture was centrifuged at
3400 rpm for 15 minutes. The same process was
conducted for palmitic acid.
2.2.3 Conversion Percentage Value
Determination
The value of determination conversion was
determined using titration method. The remaining
fatty acids in the organic phase was titrated by 0.1 N
sodium hydroxide and indicator that use was
phenolphthalein.
2.2.4 FTIR Characterization for
Esterification Product
FTIR Characterization was conducted for
esterification product, glycol, fatty acids obtained
from hydrolysis and palmitic acid.
2.2.5 Emulsifier Test and Determination of
Emulsion Type
The emulsifier test was performed by mixing 0.1 g of
ester product, water, and oil according to Table 1.
Then, the mixture was shaken by vortex for 30 second
and the emulsion stability was observed.
Table 1: Variation of water and oil for emulsifier test.
Water (mL)
Oil (drops)
Var. 1
1
1
1
1
1
4
6
8
10
Water (drops)
Oil (mL)
Var. 2
2
4
6
8
10
1
1
1
1
Determination of emulsion type test was
performed by mixing 1 drop of emulsion with eosin
in preparation glass. Then, the mixture was observed
under a microscope to determine the type of
emulsion; oil in water or water in oil emulsion.
2.2.6 Antimicrobial Assay Using Disc
Diffusion Method
Antimicrobial assay were performed using disc
diffusion method. Suspension of 200 μL P. acnes
bacteria with a cell density of 1 x 10
8
cell/m L was
aseptically mixed with 20 mL nutrient agar in a sterile
petri dishes. The media then was left to harden. The
sterile disc paper (6 mm in diameter) was placed on
each section of media and dropped by 4 μL of sample.
After that, the media was incubated for 24 hours at
37 C. Clindamycin 0.5% was used as positive
control while DMSO as negative control. The clear
zone around the disc paper was measured. The same
test was performed using S. epidermidis.
3. RESULT AND DISCUSSION
3.1 Hydrolysis of Castor Oil
The catalyst used in this reaction is a strong base;
potassium hydroxide, because the reaction that occur
can take place quickly and commercially. In addition,
the use of a strong base aims to break ester bonds in
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
6
castor oil. The castor oil fatty acids that obtained from
this process were used for esterification. Afterwards,
the yield percentage obtained from castor oil
hydrolysis was 83.6%.
3.2 Determination of Conversion
Percentage
Esterification product formed emulsion system. To
break the emulsion, the mixture is needed to be
centrifuged. Thereafter, three phases were formed
and the upper phase was used for determination of
conversion percentage.
The relation between mol variations of fatty acid
with conversion percentage found on Figure 1. As the
mol of glycol increase, the conversion percentage
increased. In accordance with Le Chatelier principle
which states that the addition of one excess reactants
in equilibrium reaction will cause a reaction shift
leading to the formation of the product. The highest
value of conversion percentage was obtained at ratio
1:4 with conversion percentage were 84.7% for
castor oil fatty acid and 81.9% for palmitic acid.
Figure 1: Relation between mol variations of fatty acid
versus conversion percentage value.
3.3 Characterization of Esterification
Product using FTIR
The IR spectra of fatty acid from castor oil hydrolysis,
palmitic acid, glycol castor oil fatty acid and glycol
- palmitic esters are shown in Figure 2 and Figure 3.
Figure 2: FTIR spectra (a) fatty acids from castor oil
hydrolysis, (b) palmitic acid
Figure 2(a) shows the FTIR spectra of castor oil
fatty acid. There are several typical functional groups
appeared in the spectra. The absorption peak at wave
number 3009.73 cm
-1
for CH sp
2
which indicates that
the compound have double bonds. Then, at the wave
number 1712.86 cm
-1
there is an absorption of C=O
carboxylate in the range 1700-1725 cm
-1
(Silverstein,
2005). Furthermore, Figure 2(b) shows FTIR
spectrum of palmitic acid. The absorption at the wave
numbers 1701.28 cm
-1
for C=O carboxylic acid and
1471.75 cm
-1
for CH sp
2
. Both spectrum indicate that
absorption of typical functional groups for fatty acid,
i.e. C=O carboxylic acids.
Figure 3 (a) and (b) shows FTIR spectra for glycol
castor oil fatty acid and glycol palmitic acid ester,
respectively. Both spectrum showed the absorption of
C=O ester functional groups at the range 1735-1750
cm
-1
. The wave number 1735.64 cm
-1
is the
absorption of C=O ester functional group for glycol -
castor oil fatty acid ester. While the wave number
1738.16 cm
-1
for the glycol palmitic ester.
72,3
77,7
81,5
84,7
77,9
78,5
81,7
81,9
1:1 1:2 1:3 1:4
Castor oil fatty acid Palmitic acid
(a)
(b)
Study of Enzymatic Synthesis of Glycol â
˘
A¸S Castor Oil Fatty Acid and Glycol â
˘
A¸S Palmitic Acid Esters as Emulsifier and Antimicrobial
Compounds Using Candida rugosa Lipase EC. 3.1.1.3
7
Figure 3: FTIR spectra (a) glycol castor oil fatty acid ester, (b) glycol palmitic acid ester.
3.4 Simple Emulsifier Test
The result of the emulsion test on the ester product
showed that glycol castor oil fatty acid emulsion
was better than glycol palmitate ester. After 36
hours observation glycol castor oil fatty acid
emulsion still looks turbid, indicating that the ester
product has properties as emulsifier. Similarly for
glycol palmitic acid ester emulsion, after 24 hours
the mixture still looks turbid. However, the ability of
glycol-palmitic acid ester was not good as glycol
castor oil fatty acid that can last for 36 hours. In
comparison, the emulsion properties of glycol
castor oil fatty acid ester more stable than glycol
palmitic acid ester (Figure 4).
Figure 4: Emulsion formed using (a) glycol castor oil fatty acid ester (after 36 hours), (b) glycol palmitic ester (after 24
hours).
Microscopic observations for the determination
type of emulsions are shown in Fig. 5. Both glycol
castor oil fatty acid and glycol palmitic esters show
water in oil emulsion type because red droplets are
produced in yellow medium.
The emulsion type produced depends on the
emulsifier properties and the Hydrophillic to
Liophillic Balance (HLB) value. Emulsifiers with low
HLB values (3-6) can produce water in oil emulsion.
Whereas high HLB vallue (8-18) produces an oil in
water emulsions (Luna et al, 2013). However, in this
study HLB value were not determined.
(a)
(b)
(a)
(b)
Before
Before
After
After
Control
BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity
8
Figure 5: The observe emulsion type using microscope (a) glycol castor oil fatty acid, (b) glycol palmitic
esters.
3.5 Antimicrobial Assay
Propionibacterium acne and Staphylococcus
epidermidis bacteria were used in this study. The
principle of the disc diffusion method is to measure
the zone of bacterial growth resistance that occurs due
to the diffusion of antimicrobial substances in solid
media. The inhibition zone is a clear region around
the disc. The area of the inhibition zone is directly
proportional to the antimicrobial activity. The
stronger antimicrobial power the more prevelant the
inhibition area (Chouhan et al, 2017).
The antimicrobial assay showed that glycol
palmitic acid were all ineffective as antimicrobials.
While for the glycol castor oil fatty acid ester
showed the antimicrobial activity. The highest
inhibition zone was obtained at 80% concentration
against P. acnes bacteria with clear zone diameter 12
mm and classified as weak activity. Then for
Staphylococcus epidermidis, the highest inhibition
zone was obtained at 60% with clear zone diameter
10 mm and classified as weak activity. Table 2 shows
the antimicrobial activity of esterification product,
ethylene glycol, n-hexane, and DMSO against P.
acne and S. epidermidis bacteria. While Table 3
shows the classification of effectiveness
antimicrobial compounds.
Table 2: Antimicrobial activity against P. acnes and S. epidermidis
Sample
Concentration
(%)
Inhibition Zone
P. acnes
Classification
S.epidermidis
Classification
Glycol palmitic
ester
20
-
No Activity
9
Ineffective
40
-
No Activity
9
Ineffective
60
-
No Activity
-
No Activity
80
-
No Activity
-
No Activity
Glycol Castor oil
Fatty acid Ester
20
11
Weak
8
Ineffective
40
10
Weak
9
Ineffective
60
8
Ineffective
10
Weak
80
12
Weak
9
Ineffective
Palmitic Acid
50
-
No Activity
-
No Activity
Castor oil fatty
acid
50
12
Weak
14
Weak
100
13
Weak
15
Weak
Castor oil
100
-
No Activity
-
No Activity
Ethylene Glycol
100
-
No Activity
-
No Activity
n-hexane
100
-
No Activity
-
No Activity
(a)
(b)
Study of Enzymatic Synthesis of Glycol â
˘
A¸S Castor Oil Fatty Acid and Glycol â
˘
A¸S Palmitic Acid Esters as Emulsifier and Antimicrobial
Compounds Using Candida rugosa Lipase EC. 3.1.1.3
9
Clindamycin
0.5
12
Weak
14
Weak
DMSO
100
-
No Activity
-
No Activity
Table 3. Antibacterial effectiveness classification
Inhibit zone diameter
Response of growth barrier
>20 mm
Strong
16-20 mm
Medium
10-15 mm
Weak
<10 mm
Ineffective
[Source: Greenwood, 1996]
From Table 2, it can be seen that 500 ppm
clindamycin has inhibitory activity against both
bacteria; P. acnes and S. epidermidis for 12 mm and
14 mm, respectively. Negative control that used in
this research was DMSO which is dropped on sterile
disc paper. The purpose of using DMSO as negative
control was to compare that the solvent used did not
affect the results of antibacterial test. Therefore, the
negative control used was 100% DMSO. It can be
seen from Table 2 that the inhibitory zone results in
negative control for both bacteria were 0 mm. these
results indicate that the use of DMSO as solvent did
not affect the antibacterial test results. Furthermore,
the result of inhibition zone for both bacteria goes into
the weak classification. So it can be concluded that
glycol castor oil fatty acid has the same inhibitory
power as clindamycin. As known that clindamycin is
an antibiotic usually used for drug acne (Handayani,
et al, 2015). This indicates that glycol castor oil
fatty acid ester can potentially be an antimicrobial
agent.
4 CONCLUSIONS
The glycol castor oil fatty acid and glycol palmitic
acid esters were successfully synthesized
enzymatically using Candida rugosa lipase that
indicated by characteristic of C=O ester group at
FTIR spectra. Both ester products have properties as
emulsifiers for water in oil (w/o) emulsion type, but
only glycol-castor oil fatty acid ester has potential to
be an antimicrobial agent.
ACKNOWLEDGEMENTS
This work was funded by Hibah Kompetensi
Publikasi Internasional Terindeks Untuk Tugas Akhir
Mahasiswa (PITTA), Universitas Indonesia 2018.
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Study of Enzymatic Synthesis of Glycol â
˘
A¸S Castor Oil Fatty Acid and Glycol â
˘
A¸S Palmitic Acid Esters as Emulsifier and Antimicrobial
Compounds Using Candida rugosa Lipase EC. 3.1.1.3
11