Antimicrobial Effect of Concord Paper Containing with Lemongrass

Oil against Escherichia coli and Staphylococcus aureus

Bunda Amalia

, Retno Yunilawati

, Windri Handayani

, Agustina Arianita C.

and Cuk Imawan

Department of Physics, Faculty of Mathematics and Natural Sciences (FMIPA) Universitas Indonesia, 16424 Depok,

Indonesia

Department of Biology, Faculty of Mathematics and Natural Sciences (FMIPA) Universitas Indonesia, 16424 Depok,

Indonesia

Badan Penelitian dan Pengembangan Industri, BBKK, Kementerian Perindustrian, Indonesia

Keywords: Lemongrass Oils, Concord Paper, Antimicrobial Activity.

Abstract: The use of an antimicrobial label in food packaging as a form of active packaging is an interesting to

investigate. This label can be used to extend the shelf life of food. Lemongrass oil is one of essential oil that

is potential used as an antimicrobial agent. In this study, the antimicrobial effect of label made from concord

paper which incorporated with lemongrass oil was prepared and tested against the bacteria Escherichia coli

and Staphylococcus aureus using disk inhibition zone method. This antimicrobial label was tested using FTIR

to investigate the interaction between essential oil and the matrix. The lemongrass oil was tested using Gas

Chromatography-Mass Spectrometry to determine the levels and presence of compounds suspected of having

antimicrobial activity. The labels have antibacterial activity against E. coli with the diameter of inhibition

zone maximum about 47.85 mm but not active toward the S. aureus. From the results of the antibacterial test

can be seen that the use of antibacterial label is promising when used for food safety with a prolonged shelf

life.

1 INTRODUCTION

Contamination of food can occur during the process

of harvesting, food processing, packaging and

distribution. Packaging is one of the effective ways to

protect food from contamination from the outside

environment such as air, dust, physical, chemical and

biological impacts such as microbes that cause food

spoilage. Conventional packaging which is widely

used today, cannot actively control the reactions that

occur in food (Mousavi et al., 2018). One of the

packaging technologies that have been developed to

maintain the quality of food and extend the shelf life

of food is to use active packaging. The use of

antimicrobial labels on active packaging is now an

interesting technology for research.

With the aim to reduce the use of additional

chemical substances in food, one way is to use natural

ingredients to inhibit the growth of microbes that

cause food spoilage that does not have a negative

effect on human health (Chiralt and Atar, 2016).

Essential Oil is one of the antimicrobial agents

derived from plant extracts that have antimicrobial

properties. However, this essential has a strong

enough odour that it is rarely used to be added directly

to food because it will damage the taste of the food

itself. Because that reason, it is interested to combine

essential oils into a matrix to reduce the strong odour

as antimicrobial label.

Several studies have been carried out by

combining essential oil such as oregano with cassava

starch/chitosan with oregano essential oil (Pelissari et

al., 2009), alginate with lemongrass oil (Chiralt and

Atar, 2016), and coated paper with Cuminum

cyminum L. and Prubus mahaleb L. in terms of

improving antimicrobial properties (Ezel and Dal,

2018). In this research, lemongrass oil is combined

into a paper matrix. The paper used is Concord paper.

Concord paper which has another name namely

Japanese linen paper is textured paper. By combining

lemongrass oil into the concord, it is hoped that it can

make an effective antimicrobial label which effective

against Eschericha coli and Staphylococcus aureus

which can be used to extend the shelf life of food.

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

Antimicrobial Effect of Concord Paper Containing with Lemongrass Oil against Escherichia coli and Staphylococcus aureus.

DOI: 10.5220/0009956200540059

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 54-59

ISBN: 978-989-758-456-5

2 MATERIALS AND METHOD

2.1 Materials

Lemongrass oil was used in this experiment obtained

from Nusaroma, a local essential oils company in

Indonesia. The matrix used in this study is concord

paper with a gramatur of 220 gr / mm

produced by

PT. Parisindo Pratama.

2.2 Preparation of Antimicrobial

Labels

The antimicrobial label was prepared by dropping of

25 µL lemongrass oil using a micro pipette onto the

surface of the concord paper with a size of 1 cm x 3

cm, then allowed at room temperature for 5 minutes.

2.3 Characterization

2.3.1 Lemongrass Oil Characterization

Characterization Lemongrass Oil using GC- MS.

Lemongrass oil compounds were identified by gas

chromatography with a mass spectrometer detector

(GCMS) 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.

Antimicrobial Activities Assay of Lemongrass Oil:

Direct Contact Agar Diffusion Tests. 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.

2.3.2 Label Characterization

Antimicrobial Activities Assay of Labels. The

antimicrobial activities of labels were tested in vapour

phase agar diffusion test, because in its application as

label antimicrobial will used vapour phase. The

vapour phase method follows the method used by

(Wang et.al, 2016).

Labels are cut in a circle with a diameter of 0.6 cm

and then placed in a petri dish to test antimicrobial

activity. The vapour 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 32° C for 24h. The diameter of the

inhibition zone was recorded.

Efficacy Test of Label on the Product. The efficacy

of the antimicrobial label was evaluated by placing

the label with a size of 1x3 cm above the surface of a

plastic package containing chicken meat (10 g)

purchased from the local market in Depok. Then the

chicken meat is kept at room temperature for 5 days

to see the visual changes found in the chicken meat.

Fourier Transform-Infra Red (FTIR) Analysis.

The spectra of the antimicrobial label (control paper

and paper that had been dropped with lemongrass oil)

were test using Fourier Transform Infrared (FTIR)

using a double beam spectrophotometer (Thermo

Nicolet iS5) to determine functional groups. FTIR

analysis was carried out on blank label before and

after it was used to store chicken breast filled.

UV-Vis Analysis. UV- vis spectrophotometer was

used to measure the reflectance of the antimicrobial

label (control paper and paper that had been dropped

with lemongrass oil) with a size of 1x3 cm. The brand

of UV-Vis apparatus is Shimadzu UV-2450.

3 RESULT AND DISCUSSION

3.1 Chemical Compounds of the

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

Antimicrobial Effect of Concord Paper Containing with Lemongrass Oil against Escherichia coli and Staphylococcus aureus

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).

Figure 1: GC-MS chromatogram of the lemongrass oil.

Table 1: Chemical compound identified of lemongrass oil

with GC-MS.

Retention

time

Identified

compound

Molecular

formula

Relative

percentage

area (%)

17.101

Neral (beta-

citral)

29.00

17.753

Geraniol

10.80

18.524

Geranial

(Alpha citral)

44.21

23.302

Geranyl

acetate

6.50

24.588

Beta-

caryophyllene

5.67

28.589

Gamma-

cadinene

3.83

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

unsaturated aldehyde, the most common flavour in

citrus oil and widely used in food and beverages.

Citral is the 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 vapour

form (Argyropoulou et al., 2007) It is revealed the

presence of C = O bond for aldehyde from indicates

the presence of citral compounds. Antimicrobial

activity of cinnamaldehyde was found against E. coli

and staphylococcus aureus. Citral that have aldehyde

function group plays a role in disrupting bacterial cell

membranes (Firmino et al., 2018)

3.2 Antimicrobial Activities of

Lemongrass Oil and the Label

The bacteria is one of indicator used for examination

of spoilage to meat products (Pranoto et al., 2005).

Meat and processed products that are perishable food

because they are very vulnerable to contamination by

microorganisms. Spoilage meat can contain

pathogenic bacteria such as S. Aureus and E. coli.

Therefore, in this research an Antimicrobial activity

of lemongrass oil against test was carried out against

E. coli and S. aureus (Figure 2). The inhibitory

activity is measured based on the clear zone that

occurs around the label. The measurement of the clear

zone diameter is calculated including the diameter of

the label. The diameter produced will be greater than

the diameter of the label if a clear zone is detected. If

no clear zone is formed around the label, then it is

assumed that there is no inhibitory region and the

diameter is declared zero.

Figure 2: Antimicrobial activities of lemongrass oil using

paper disk method against Gram-positive bacteria S. aureus

and Gram negative bacteria E. coli; A = negative control;

B=positive control; C=sample.

In Figure 2 it can be seen that the negative control

in the form of sterile distilled water does not form a

clear zone which means it does not show an inhibitory

effect on E. coli and S. aureus bacteria. Inhibition

diameter can be seen in Table 2. For positive control

in the form of antibiotic tetracycline, a clear zone with

a diameter of 1.9 cm can be seen for E. coli bacteria

and 3.1 cm for A. aureus bacteria. As for the

lemongrass oil, a clear zone with a diameter of 4.7 cm

is formed for E. coli bacteria and 2.5 cm for A. aureus

bacteria. The diameter of the clear zone formed in

lemongrass oil against E. coli bacteria (gram -) is

greater than that of S. aureus (gram +), which means

that lemongrass oil is more effective against E. coli

bacteria (gram -). This is because Gram positive has

5 . 0 0 1 0 . 0 0 1 5 . 0 0 2 0 . 0 0 2 5 . 0 0 3 0 . 0 0 3 5 . 0 0 4 0 . 0 0 4 5 . 0 0 5 0 . 0 0 5 5 . 0 0 6 0 . 0 0

1 0 0 0 0 0 0

2 0 0 0 0 0 0

3 0 0 0 0 0 0

4 0 0 0 0 0 0

5 0 0 0 0 0 0

6 0 0 0 0 0 0

7 0 0 0 0 0 0

8 0 0 0 0 0 0

9 0 0 0 0 0 0

1 e + 0 7

1 . 1 e + 0 7

1 . 2 e + 0 7

1 . 3 e + 0 7

1 . 4 e + 0 7

T im e - - >

A b u n d a n c e

T I C : 1 3 0 3 . D \ d a t a . m s

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

a cell wall structure that is different from gram

negative bacteria. In addition, gram-positive bacteria

have cell walls composed of a thicker layer of

peptidoglycan (20 to 80 nanometres), while gram-

negative bacteria have a thinner layer of

peptidoglycan.

Table 2: Diameter of inhibition zone of lemongrass oil.

S. aureus(cm)

E.coli (cm)

Sample

Control

(-)

Contro

l (+)

Sample

Contro

l (-)

Control

(+)

2,5

3,1

4,7

1,9

Besides seeing the antimicrobial activity of

Lemongrass oil, it was also carry out an antimicrobial

test from the label. For the label antimicrobial test, the

vapour method is used to match the application used.

The antimicrobial activity of the label can be seen in

Figure 3.

Figure 3: Antimicrobial activities of antimicrobial labels

with lemongrass oil concentration 10% using vapour

method against Gram-positive bacteria S. aureus and Gram-

negative bacteria.

From Figure 3 it can be seen that the label

provides antimicrobial effect on E. coli (gram

negative bacteria) but not on S. aureus (gram positive

bacteria). It can be seen from a clear zone or the

diameter of the inhibition zone of E. coli is around

47.85 cm, while the S.aureus bacteria do not form a

clear zone, then it is assumed that there is no

inhibitory region and the diameter is zero, this is

possible because the antimicrobial testing of the label

was use the vapour method. The effectiveness of

lemongrass oil on E.coli is also similar as that of other

researchers (Faleiro, 2019) and (Naik et al., 2010).

Other research which states that lemongrass essential

oil also has antimicrobial properties against other

bacteria such as A. baumannii (Adukwu et al., 2016).

3.3 Efficacy Test of Label on the

Product

The efficacy of the antimicrobial label was evaluated

within 5 days using chicken breast filled. From Figure

4 it can be seen that there is a change in the colour,

texture and odour of the chicken breast filled. On the

fifth day, Figure 4 (A) is chicken breast filled without

using a label. Figure 4 (A) shows the colour of

chicken breast filled is paler compared to Figure 4

(B). In addition to colour observation, the texture of

chicken breast filled in Figure 4 (A) is also soggier

when compared to Figure 4 (B), this indicates that the

label application can maintain the freshness of

chicken breast filled. In addition to investigating the

colour and texture, an investigation was also

conducted on odours. In this experiment, the odour of

lemongrass oil still affected the odour of the food in

the packaging.

Figure 4: Label application on the chicken breast filled, (a)

label without application, (b) label with application.

3.4 FTIR Analysis of Label

Functional group analysis is performed to determine

changes in functional groups that occur during

efficacy tests on the labels. The performance test of

labels on chicken breast was carried out for five days.

During this time, functional group analyses are

carried out using FTIR. Figure 5 displays the spectra

of concord paper and label.

Figure 5: FT-IR Spectra.

500 1000 1500 2000 2500 3000 3500 4000

O=H

(3200-3600)

% Transmittance

Wave namber (cm-1)

Concord+EO H-5

Concord+EO H-4

Concord+EO H-3

Concord+EO H-2

Concord+EO H-1

Concord+EO H-0

Concord

C=O

(1690-1760)

Day 1

Day 5

Antimicrobial Effect of Concord Paper Containing with Lemongrass Oil against Escherichia coli and Staphylococcus aureus

Fingerprint for lemongrass oil is mostly in the

range of 1800-600 cm-1 (Li, 2013). In the IR spectra,

it is shown that the absorbance band at 1690-1760 cm

revealed the presence of C=O bond for aldehyde

from indicates the presence of citral compounds

(Adinew 2014). Besides that, it is shown that the

absorbance band at 3200-3600 cm-1 revealed the

presence of O=H, which indicates the presence of

compounds geraniol. From the Figure, the C=O

intensity of citral is decreasing. It is because citral has

to be released from the label and the presence of this

citral compound was strengthened by GC MS results

and the result of antimicrobial assay.

3.5 UV-VIS Analysis

UV-Vis analysis was carried out to see the Reflect

ants from the label before and after the addition of

essential oil. From Figure 6 there is a change in the %

reflectance intensity of the label. The color change

occurred from blue to green can be seen in Figure 7.

The green color change occurred after the addition of

lemongrass oil followed by a decrease in the intensity

value of % Reflectance at wavelength around 600-

650 nm.

400 450 500 550 600 650 700

0.0

0.1

0.2

0.3

0.4

0.5

% Reflectance

wavelenght (nm)

Blanko Concord

Concord+EO H0

Concord+EO H2

Concord+EO H5

Figure 6: Reflectance spectra of antimicrobial labels.

Figure 7: Discoloration of label.

4 CONCLUSIONS

In this study, it can be concluded that labels made

from Concorde paper added with lemongrass oil have

the potential to become antimicrobial label. The

labels have antibacterial activity against E. coli with

the diameter of inhibition zone maximum about 47,85

mm but not active toward the S. aureus. However, the

application will depend on the type of food where

flavour is not a problem.

ACKNOWLEDGEMENTS

This research 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.

REFERENCES

Adukwu, E. C., Bowles, M., Jones, V, W., Bone, H., 2016.

Antimicrobial Activity , Cytotoxicity and Chemical

Analysis of Lemongrass Essential Oil (Cymbopogon

flexuosus) and Pure Citral, Applied Microbiology and

Biotechnology. Applied Microbiology and

Biotechnology, 9619–9627.

Argyropoulou, C., Daferera, D., Tarantilis, P, A., Fesseas,

C., Polissiou, M., 2007. Chemical Composition of the

Essential Oil from Leaves of Lippia citriodora H.B.K.

(Verbenaceae) at Two Developmental Stages,

Biochemical Systematics and Ecology, 35(12), 831–

837.

Chiralt, A., Atar, L., 2016. Trends in Food Science &

Technology Essential Oils as additives in

Biodegradable Films and Coatings for Active Food

Packaging, 48.

Ezel, A., Dal, B., 2018. Strength Properties of Coated Paper

with Cuminum cyminum L. and Prunus mahaleb L,

14(2), 247–249.

Gago, C, M, L., Artigas, M, A., Antunes, M, D, C., Faleiro,

M, L., Miguel, M, G., Belloso, O, M., 2019.

Effectiveness of Nanoemulsions of Clove and

Lemongrass Essential Oils and Their Major

Components Against Escherichia coli and Botrytis

cinerea’, 56, 2721–2736.

Firmino, D, F., Cavalcante, T, T, A., Gomes, G, A.,

Firmino, N, C, S., Rosa, L, D., de Carvalho, M, G.,

Junior, F, E, A, C., 2018. Antibacterial and Antibiofilm

Activities of Cinnamomum Sp . Essential Oil and

Cinnamaldehyde : Antimicrobial Activities, 2018.

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

Ganjewala, D., 2009. Cymbopogon Essential Oils :

Chemical Compositions and Bioactivities,

International Journal of Essential Oil Therapeutics, 3,

56–65.

Khaneghah, A, M., Hashemi, S, M, G., Limbo, S., 2018.

Food and Bioproducts Processing Antimicrobial

Agents and Packaging Systems in Antimicrobial Active

Food Packaging : An Overview of Approaches and

Interactions, Food and Bioproducts Processing.

Institution of Chemical Engineers, 111, 1–19.

Naik, M. I., Fomda, B, A., Jaykumar, E., Bhat, J, A., 2010.

Antibacterial Activity of Lemongrass (Cymbopogon

citratus) Oil Against some Selected Pathogenic

Bacterias, Asian Pacific Journal of Tropical Medicine,

3(7), 535–538.

Pelissari, F. M., Grossmann, M, V, E., Yamashita, F.,

Pineda, E, A, G., 2009. Antimicrobial, Mechanical, and

Barrier Properties of Cassava Starch-Chitosan Films

Incorporated with Oregano Essential Oil, Journal of

agricultural and food chemistry, 7499–7504.

Pranoto, Y., Rakshit, S, K, A., Salokhe, V, M., 2005.

Enhancing Antimicrobial Activity of Chitosan Films by

incorporating garlic oil , Potassium Sorbate and Nisin,

38, 859–865.

Antimicrobial Effect of Concord Paper Containing with Lemongrass Oil against Escherichia coli and Staphylococcus aureus