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 ⁰C 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 – Castor Oil Fatty Acid and Glycol – Palmitic Acid Esters as Emulsiﬁer and Antimicrobial Compounds Using Candida rugosa Lipase EC. 3.1.1.3.

DOI: 10.5220/0009841400002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

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 ⁰C. 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 ⁰C temperature. The

mixture was the heated at 80⁰C 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

Water (drops)

Oil (mL)

Var. 2

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

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 ⁰C. 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

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

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

. 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

-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 – Castor Oil Fatty Acid and Glycol – Palmitic Acid Esters as Emulsiﬁer and Antimicrobial

Compounds Using Candida rugosa Lipase EC. 3.1.1.3

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

After

Control

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

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

No Activity

Ineffective

No Activity

Ineffective

No Activity

Glycol – Castor oil

Fatty acid Ester

Weak

Ineffective

Weak

Ineffective

Weak

Ineffective

Palmitic Acid

No Activity

Castor oil fatty

acid

Weak

100

Weak

Castor oil

100

No Activity

Ethylene Glycol

100

No Activity

n-hexane

100

No Activity

(a)

(b)

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

Clindamycin

0.5

Weak

DMSO

100

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 – Castor Oil Fatty Acid and Glycol – Palmitic Acid Esters as Emulsiﬁer and Antimicrobial

Compounds Using Candida rugosa Lipase EC. 3.1.1.3