Antimicrobial Label from Lemongrass Oil Incorporated with

Chitosan/Ascorbic Acid

Retno Yunilawati

, Windri Handayani

, Agustina Arianita C.

, Bunda Amalia

and Cuk Imawan

Departemen Fisika, Fakultas Matematika dan Ilmu Pengetahuan Alam (FMIPA), Universitas Indonesia, Depok

16424, Indonesia

Departemen Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam (FMIPA), Universitas Indonesia, Depo

16424, Indonesia

Badan Penelitian dan Pengembangan Industri, Kementerian Perindustrian, Indonesia

Keywords: Antimicrobial Label, Lemongrass Oil, Chitosan, Ascorbic Acid

Abstract: Lemongrass oil is one of the essential oil which potential to be used as an antimicrobial agent in active

packaging. The aim of this research is to prepare antimicrobial labels and assess their activity. Antimicrobial

labels are made from a matrix of chitosan/ascorbic acid and lemongrass oil as active ingredients with various

concentrations ranging from 1% to 10%. Lemongrass oil was characterized using Gas Chromatography-Mass

Spectrometry (GC-MS) to determine compounds suspected of having antimicrobial activity. The GCMS

chromatograms have shown that lemongrass oil contains 73.21% citral compounds composed of 29% neral

(beta-citral) and 44.21% geranial (alpha-citral) as antimicrobial agents. Lemongrass oil was tested on Gram-

positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli using direct method and

vapor method. This test has shown that lemongrass oil has antimicrobial activity in both bacteria. The labels

provide optimum antimicrobial activity for the lemongrass oil concentration of 10%. These results conclude

that the lemongrass oil incorporated with chitosan/ascorbic acid has the potential to be an active packaging.

The abstract should summarize the contents of the paper and should contain at least 70 and at most 200 words.

It should be set in 9-point font size, justified and should have a hanging indent of 2-centimenter. There should

be a space before of 12-point and after of 30-point.

1 INTRODUCTION

Antimicrobial label is one form of active packaging

application, where the packaging made with the aim

to maintain the quality of the material it is packaged.

Antimicrobial labels are made by combining

antimicrobial materials into a polymer. Essential oil

has been widely used as an antimicrobial agent

considering its safe, natural, environmentally

friendly, and has a broad spectrum. One of the

essential oils is lemongrass oil. Lemongrass oil

contains several compounds such as neral and

geranial which can function as antimicrobials

(Argyropoulou et al., 2007).

In this research lemongrass oil is incorporated

with chitosan which is a biodegradable polymer

forming an antimicrobial label. Chitosan is a polymer

that insoluble in neutral pH, but soluble in acidic

environment, such as acetic acid, formic acid, and

hydrochloric acid. Acetic acid has an unpleasant and

pungent odor that can later affect food products in the

label application. Likewise, formic acid and

hydrochloric acid which has a pungent odor and can

penetrate (Ozdemir Kubra S, 2017). Therefore, this

research uses ascorbic acid as an alternative to

chitosan, which has safer than acetic acid and

hydrochloric acid.

This research aims to prepare antimicrobial label

using lemongrass oil incorporate with

chitosan/ascorbic acid and investigate their

antimicrobial activity

2 MATERIALS AND METHODS

2.1 Materials

Lemongrass oil was used in this experiment obtained

from Nusaroma, a local essential oils company in

Indonesia. The chemical materials used in this

Yunilawati, R., Handayani, W., Arianita C., A., Amalia, B. and Imawan, C.

Antimicrobial Label from Lemongrass Oil Incorporated with Chitosan/Ascorbic Acid.

DOI: 10.5220/0009968501470152

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 147-152

ISBN: 978-989-758-456-5

147

experiment were ascorbic acid (Merck), chitosan in

powder form (PT. Biokitosan Indonesia), and tween

80.

2.2 Methods

2.2.1 Lemongrass Oil Characterization

Lemongrass oil compounds were identified by gas

chromatography with a mass spectrometer detector

(GC-MS) Agilent 6890 series with capillary column

HP-5MS, 30 m x 0.25 mm id x 0.25 µm film

thickness. Helium gas was used as the carrier gas at

constant pressure of 65 kPa. The lemongrass oil was

injected with a volume of 1 µL in split ratio of 1:25.

The increasing of oven temperature was programmed

from 60-240°C with step of 3°C per minute until

reaching 240°C.

2.2.2 Antimicrobial Assay of Lemongrass

Oil

Direct Contact Agar Diffusion Tests.

This

method used the method carried out by (Handayani et

al., 2019). The antimicrobial activities determined by

the paper disc diffusion method using type strain of

Staphylococcus aureus NBRC 100910 and

Escherichia coli NBRC 3301 in The Mueller Hinton

Agar. 10 ml of molten media poured into sterile Petri

plates (d=90 mm) and allowed to solidify for 5

minutes. After that, in a tube, 10 µl of bacteria culture

10-6 CFU/mL added with 10 ml of medium and

mixed gently with the inoculate before poured on the

top of molten media before and allowed to dry for 5

minutes. The negative control (sterile distilled

water), positive control (tetracycline 15 µg/mL),

lemongrass oil with concentration 1000 µg/mL

loaded on 6 mm disc, whereas the volume for each

disc was 10 µl. The loaded disc placed on the surface

of the medium then incubates at 35°C for 18 hours.

After the end of incubation, a clear zone formed

around the disc was measured.

Vapor Phase Agar Diffusion Test. This vapor

method used the method carried out by (Wang et al.,

2016). The vapor phase agar diffusion test was

technically similar to the direct contact diffusion test.

However, the filter discs were placed at the top in

centre of the inner side of the Petri dish cover. The

dishes were then sealed using laboratory parafilm to

avoid evaporation of the test compounds, followed by

incubation at 37°C for 24 h. The diameter of the

inhibition zone was recorded.

2.2.3 Antimicrobial Labels Preparation

The chitosan solution was prepared by dissolving 2 g

of chitosan powder into 100 mL of 1% (w/v) ascorbic

acid and stirring at 200 rpm for 2 h at 50 °C using a

magnetic stirrer. The antimicrobial label was

prepared by mixing lemongrass oil with 30 mL of the

chitosan solution in four different concentrations (1

% v/v, 3%v/v, 5% v/v and 10% v/v) with the added

of tween 80 as surfactant (0.2% v/v) and stirring the

resultant mixture for 10 min at room temperature

using a magnetic stirrer. The label solution was

poured onto a 10 × 15 cm acrylic board and left for

48 h at room temperature to form the film.

2.2.4 Antimicrobial Labels Characterization

A uv vis spectrophotometer (Shimadzu UV-2450)

was used to measure the reflectance of the chitosan

label and lemongrass-chitosan labels. A Fourier

Transform Infrared (FTIR) spectra were collected for

the chitosan label dan the lemongrass-chitosan labels

using a double-beam spectrophotometer (Thermo

Nicolet iS5) to determine the functional group

2.2.5 Antimicrobial Assay of Labels

The antimicrobial activities of labels were tested in

direct contact agar diffusion test and vapor phase agar

diffusion test. Labels are cut in a circle with a

diameter of 6 mm and then placed in a petri dish to

test antimicrobial activity with the technique as

described previously.

3 RESULTS AND DISCUSSION

3.1 Chemical Compounds of Lemongrass

Oil

Characterization using GC-MS showed the

chromatogram profile detected 6 peaks in lemongrass

oil (Figure 1) which indicated there were 6

compounds in lemongrass oil. The compounds were

identified based on comparison of mass spectrum

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Figure 1: GCMS chromatogram of lemongrass oil

Table 1: Chemical compound identified of lemongrass oil with GC-MS

No Retention

time

Identified compound Molecular formula Relative percentage

area (%)

1 17.101 Neral (beta-citral) C

O 29.00

2 17.753 Geraniol C

O 10.80

3 18.524 Geranial (Alpha citral) C

O 44.21

4 23.302 Geranyl acetate C

6.50

5 24.588 Beta-caryophyllene C

5.67

6 28.589 Gamma-cadinene C

3.83

with reference data from the database (Wiley 7).

Based on this, lemongrass oil was known contain 6

compounds, namely neral (beta-citral), geraniol,

geranial (alpha-citral), geranyl acetate, beta-

caryophyllene and gamma cadinene (Table 1) with

the main compounds being citral and geraniol. These

results appropriated with previous finding reported in

literature, citral and geraniol has been described as the

main compounds of lemongrass oil (Ganjewala,

2009). Citral as the major component of lemongrass

oil present at level of approximately 65%-85%

(Saddiq and Khayyat, 2010). The content of citral in

this research was 73.21%.

Citral (3,7 dimethyl-2-6-octadienal) is mixture of

two isomers geometric, neral (beta-citral) and

geranial (alpha-citral) which are monoterpene

aldehyde. Citral has an activity antibacterial against

Gram-positive bacteria and Gram-negative bacteria,

both on oil form and vapor form (Argyropoulou et al.,

2007) Geraniol (3,7-dimethyl-octa-trans-2,6-dien-1-

ol) is an acyclic monoterpene alcohol with the

chemical formula C

O (Ternus ZR, 2015).

Geraniol is reported to have activity against several

pathogenic bacteria (Ternus ZR, 2015). The aldehyde

groups in citral and alcohol groups in geraniol that

play a role in antibacterial activity. Aldehydes,

phenols, esters, oxygenated terpenoids, ketones, and

amines are the principle components responsible for

the antimicrobial activity of essential oil (Ju et al.,

2019).

3.2 Antimicrobial Activity of

Lemongrass Oil

The result of antimicrobial assay showed that the

clear zone was formed in positive control and sample

(lemongrass oil) both in Gram-positive Bacteria S.

aureus and Gram-negative bacteria E. coli (Figure 2).

The diameter of clear zone/inhibition zone in S.

aureus is lower than in E. coli (Table 2). Generally,

essential oils are more active in Gram-positive

bacteria than in Gram-negative bacteria

(Bhavaniramya et al., 2019) (Huang et al., 2014).

Gram-negative bacteria have a rigid outer membrane,

composed of a double layer of phospholipids

(lipopolysaccharide),

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0 10203040506070

Abundance

Time(minutes)

Antimicrobial Label from Lemongrass Oil Incorporated with Chitosan/Ascorbic Acid

149

Figure 2: Antimicrobial activities of lemongrass oil against Gram-positive bacteria S. aureus and Gram-negative

bacteria E. coli; A = negative control; B=positive control; C=sample

Tabel 2: Diameter of Inhibition zone of lemongrass oil

Samples S. aureus (mm) E. coli (mm)

Lemongrass

oil

25 47

Lemongrass

oil (vapor)

22 36

thereby limiting the diffusion of hydrophobic

compounds through it. In this experiment, lemongrass

oil is more active in Gram-negative bacteria E. coli,

contrary to that statement. The antimicrobial activity

of essential oil is influenced by many factors, such as

the respective composition of the essential oils, the

structural conﬁguration of the constituent

components, their functional groups and possible

synergistic interactions between components

(Dorman and Deans, 2000). The lemongrass oil has

two functional groups (aldehydes and alcohol) which

expected have synergistic interactions in

antimicrobial activity.

The antimicrobial activity of lemongrass oil in

vapor form was lower compare with in oil form.

Some experiments have indicate that lemongrass oil

in vapor phase is more effective than in the liquid

phase (Tyagi and Malik, 2010) (Hyun et al., 2015),

contrary with this experiment. It can be explained that

the antimicrobial activity in vapor contact was

influence by the concentration of vapor, and the major

constituent (Inouye, Takizawa and Yamaguchi,

2001).

3.3 Labels Characterization

The antimicrobial labels made of lemongrass oil and

chitosan/ascorbic acid were shown at Figure 3. The

colour of control label (chitosan/ascorbic acid

without lemongrass oil) was yellowish and the label

was transparent. When lemongrass incorporated in

matrix, the more lemongrass oil was added, the label

was opaquer and less transparent. The transparency of

the label was optically expressed as a reflectance and

determined using a UV spectrophotometer. The

reflectance of each label was shown in Figure 4. The

reflectance value decreases with increasing

concentration of lemongrass oil.

FTIR spectroscopy was performed to explore the

intermolecular interaction between lemongrass oil

and chitosan. The FTIR spectra of the control

(chitosan/ascorbic acid matrix) along with the

lemongrass oil incorporated chitosan/ascorbic acid

are shown in Fig.5.

Figure 3: Antimicrobial label from lemongrass oil

incorporated with chitosan/ascorbic acid

Figure 4: Reflectance spectra of antimicrobial labels

control LO LO 3% LO 5%

LO 10%

S.aureus E.coli

S.aureus

E.coli

direc

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4000 3500 3000 2500 2000 1500 1000 500

Wavenumber (cm-1)

Chit + LO 10%

Chit + LO 5%

Chit + LO 3%

Chit + LO 1%

Control

Figure 5: FTIR spectra of chitosan and all of the labels

FTIR spectroscopy was performed to

explore the intermolecular interaction between

lemongrass oil and chitosan. The FTIR spectra of the

control (chitosan/ascorbic acid matrix) along with the

lemongrass oil incorporated chitosan/ascorbic acid

are shown in Fig.5. The FTIR spectra of chitosan and

all of the labels gave a broad peak in the range of

3200–3500 cm

-1

indicate the stretching vibration of

hydroxyl group (O-H) (Zhang et al., 2018). When the

lemongrass oil was incorporated into

chitosan/ascorbic acid, the major peak of the infrared

spectrum did not change very much, suggested that

there was no signiﬁcant change in the

chitosan/ascorbic acid. There were no significant

changes was due the lemongrass oil didn’t form

covalent bonding with chitosan. These results were

appropriate with several previous studies that used

chitosan as a matrix for essential oils (Gursoy et al.,

2018) (Li et al., 2019). However, there was the peak

in 1722 cm

-1

in the labels indicating the presence of

citral, the major component of lemongrass oil

(Natrajan et al., 2015). The intensity of this peak was

greater when more lemongrass oil was added.

3.4 Antimicrobial Activity of the

Labels

The antimicrobial test results from the label showed

that the label has antimicrobial activity on the

lemongrass oil concentration was 10% as

summarized in the diameter of inhibition zone were

shown in Tabel 5. The clear zone/inhibition zone was

formed both in Gram-positive bacteria S. aureus and

Gram-negative bacteria E. coli (Figure 6).

Figure 6: Antimicrobial activities of antimicrobial labels

with lemongrass oil concentration 10% against Gram-

positive bacteria S. aureus (a) and Gram-negative bacteria

E. coli (b)

Tabel 3: Diameter of inhibition zone of the antimicrobial labels

No. % lemongrass oil (v/v) Direct contact test Vapor test

S. aureus (cm) E. coli (cm) S. aureus(cm) E. coli (cm)

1. 1 - - - -

2. 3 - - - -

3. 5 - - - -

4. 10 2.6 3.0 2.5 2.7

Labels with lemongrass oil concentrations above 10%

have been tried in this experiment but the results show

that the labels are not compatible. There was a

separation between lemongrass oil and chitosan

matrix when the concentration of lemongrass was

above 10%. Therefore, the optimal concentration of

lemongrass oil on the label is 10%. Previous studies

that have been conducted have shown that lemongrass

concentrations below 10% have had antimicrobial

activity (Ali, Noh and Mustafa, 2014) but in

difference of the solvent of chitosan and difference

of microbe.

4 CONCLUSIONS

The lemongrass oil was used in this study contained

73.21% of citral as the major compound which is an

antimicrobial agent. The lemongrass oil has the

antimicrobial activity in Gram-positive bacteria S.

aureus) and Gram-negative bacteria E. coli. The

labels from lemongrass oil incorporated with

chitosan/lemongrass oil shown the antimicrobial

activity in Gram-positive bacteria S. aureus and

Gram-negative bacteria E. coli with the optimal

lemongrass oil concentration of 10% (v/v).

(

)

(

)

Antimicrobial Label from Lemongrass Oil Incorporated with Chitosan/Ascorbic Acid

151

ACKNOWLEDGEMENT

This research is supported by PSNI (Penelitian

Strategis Nasional Institusi) from Kementerian Riset,

Teknologi, dan Perguruan Tinggi Republik Indonesia

No NKB-1798/UN2.R3.1/HKP.05.00/2019. We also

thank the Center of Excellence Biology Resources

Genome Study (CoE IBR-GS) FMIPA UI and the

Center for Chemical and Packaging (CCP) for the

facilities and equipment to support this research.

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