Simple Antimicrobial Labels from Cinnamon Oil Added to

Recycled Paper

Agustina Arianita Cahyaningtyas

, Retno Yunilawati

, Bunda Amalia

, Windri Handayani

and Cuk

Imawan

Badan Penelitian dan Pengembangan Industri, Kementerian Perindustrian

Departemen Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Indonesia,

Kampus Depok, Indonesia 16424

Departemen Fisika, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Indonesia,

Kampus Depok, Indonesia 16424

Keywords: Essential Oils, Cinnamon Oil, Antimicrobial Label, Recycled Paper.

Abstract: Essential oils are one of the antimicrobial agents that are safe for food, and thus can be used as an

antimicrobial label to extend the shelf life of food products. This study aims to prepare antimicrobial labels

and to investigate their activities in shrimp. Antimicrobial labels are made using cinnamon oil in the

recycled paper as a simple matrix. Cinnamon oil was tested on Gram-positive Staphylococcus aureus and

Gram-negative bacteria Escherichia coli using the paper disk diffusion method. From the results obtained,

cinnamon oil has both antimicrobial activities. Cinnamon oil is also characterized using Gas

Chromatography-Mass Spectrometry (GC-MS) to determine the level and presence of compounds suspected

of having antimicrobial activity. Cinnamon oil has interactions with recycled paper functional groups as

measured by Fourier Transform Infrared Spectroscopy (FTIR). Testing of antimicrobial labels on shrimp

shows that the Total Volatile Basic Nitrogen (TVB-N) value is better than without the label. From the

results of antimicrobial activity, can be seen that cinnamon oil applied to recycled paper has the potential to

be used as a simple antimicrobial label.

1 INTRODUCTION

Fresh shrimp is very easy to damage. Many methods

have been carried out to maintain the freshness and

shelf life of shrimp. The use of synthetic

preservatives to maintain the freshness and quality

of shrimp can endanger health. At present natural

preservatives with excellent antimicrobial properties

have been searched and implemented as safe

alternatives in seafood processing to extend shelf

life. Natural preservatives commonly used include

plant extracts, bacteriocins, bioactive peptides,

chitosan and chitooligosaccharide, and essential oils

(Olatunde and Benjakul, 2018). Essential oils from

aromatic plants have antimicrobial properties and

are safe to add to food or food packaging (Santos et

al., 2017).

Currently, some researchers are developing the

addition of essential oil as an antimicrobial to the

paper matrix. Researches on adding essential oil as

an antimicrobial and antifungal to paper matrix that

have been carried out are carvacrol (Mascheroni et

al., 2011) and cinnamon essential oil (Echegoyen

and Nerin, 2014). From the research that has been

done, the addition of essential oil to the paper matrix

mostly uses a coating method that requires

applicator coating equipment. Therefore, necessary

to develop a preparation method that is simple,

practical, can be used as an antimicrobial, and

integrated with product packaging.

This research aims to develop a simple

antimicrobial label by using cinnamon oil. As the

matrix of this simple antimicrobial label is recycled

paper. From studies on active paper packaging that

have been done, no one has ever used a matrix of

recycling paper. The use of recycled paper can

increase the added value of the recycled paper. Also,

the recycled paper easily absorbs essential oils

compared to other types of paper. The preparation

method of the label is simple, by dropping cinnamon

oil on the circular shape of the recycled paper. The

simple antimicrobial label is then tested to detect the

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

Simple Antimicrobial Labels from Cinnamon Oil Added to Recycled Paper.

DOI: 10.5220/0009956300600066

In Proceedings of the 2nd International Conference of Essential Oils (ICEO 2019), pages 60-66

ISBN: 978-989-758-456-5

characterization, antimicrobial activity, and TVB-N

of the shrimp after applied by a label.

2 MATERIALS AND METHOD

2.1 Materials

The simple antimicrobial label was made using

materials recycle paper purchased from local

stationery shop and cinnamon oil purchased from

Nusa Aroma local essential oils company in

Indonesia.

2.2 Antimicrobial Label Preparation

The simple antimicrobial labels were composed of

the cinnamon oil and the matrix made from recycled

paper. The labels were prepared by dropping of

50 µL cinnamon oil on circular shape cutting of the

recycle paper with a diameter of 6 mm. The label

then dried in room temperature for 5 minutes ready

for use.

2.3 Characterization

2.3.1 Cinnamon Oil Characterization using

GCMS

Cinnamon oil was characterized using a mass

spectrometer detector (GCMS), to find out the

chemical compounds contained in cinnamon oil. The

tools used are GC / MS with Agilent 6890 series

specifications with capillary column HP-5MS, 30

m x 0.25 mm id x 0.25 µm film thickness. As the

carrier gas was used helium gas at constant pressure.

The essential oil was injected with a volume of 1 µL

(split ratio of 25: 1). The oven temperature was

programmed from 60 °C - 240 °C for an increase of

3 °C per minute until reaching 250 °C.

2.3.2 Characterization using FTIR

FTIR characterization is used to monitor label

activity. Tests are carried out on blank paper and

labels before and after storing shrimp and carried out

every day. The blank and the label were measured

on the Seri Nicolet iS5 FTIR spectrometer. All

spectra were taken in the spectral range of 4000 cm

-1

until 500 cm

-1

2.4 Antimicrobial Activity Assay

2.4.1 Direct Contact Agar Diffusion Tests

Direct contact agar diffusion tests determined by the

paper disk diffusion method using strain type of

Staphylococcus aureus NBRC 100910 and

Escherichia coli NBRC 3301 in The Mueller Hinton

Agar. 10 mL of molten media was put into a sterile

petri dish (d = 90 mm) until it became solid for

5 minutes. 10 µL bacterial culture 10

-6

CFU / mL is

added with 10 mL medium into the tube and mixed

slowly with inoculating before pouring on the top

surface of the molten media and allowed to dry for 5

minutes. The negative control (sterile distilled

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

and cinnamon oil with a concentration of 1000 µg /

mL are poured on 6 mm discs, where the volume for

each disc is 10 µL. The disc is then placed on the

surface of the medium then incubated at 35 °C for

18 hours. After completion of incubation, a clear

zone is formed around the disc measured.

2.4.2 Vapour Phase Agar Diffusion Tests

This vapour phase agar diffusion uses the method

used by (Wang et al., 2016). The vapour phase agar

diffusion test technically has the same method as the

direct contact method. The test uses a 6 cm diameter

petri dish, bacterial culture, filter disc size, and

cinnamon oil adding. The disk filter is placed in the

middle of the lid of the petri dish. The dishes are

then sealed using a paraffin laboratory to prevent

evaporation of the test compound. Incubation ran at

32 °C for 24 hours. The clear zone diameter was

measured.

2.5 Total Volatile Basic Nitrogen

(TVB-N)

The shrimp used for this experiment were fresh

obtained from the local market. Shrimp after being

bought directly delivered to the laboratory and

prepared as soon as possible for observation. The

shrimp weighed as much as 10 g and then put into a

PVC square packaging. Then a simple antimicrobial

label was attached to the top of PVC square

packaging containing the shrimp, placed indirectly

in contact with the shrimp. The distance between the

label and shrimp is about 1 cm. The PVC square

packaging is then tightly closed. Observation of

shrimp freshness was carried out at room

temperature for three days. During this time, TVB-N

levels were measured every day, as a control used

Simple Antimicrobial Labels from Cinnamon Oil Added to Recycled Paper

shrimp that are packaged without using simple

antimicrobial label. Measurement of TVB-N levels

in shrimp according to the Total Volatile Basic

Nitrogen (TVB-N) method based on Commission

Regulation (EC) No 2074/2005 (EC, 2005).

3 RESULT AND DISCUSSION

3.1 Chemical Compounds of the

Cinnamon Oil

The method used to analyse volatile oils for many

years is using gas chromatography. GC-MS is the

most appropriate technique used to identify the

compounds contained in essential oils. The

chromatogram profile of cinnamon oil is shown in

Figure 1, and the results of the characterization of

the chemical compounds listed in cinnamon are

shown in Table 1.

A total of four different components, with

different retention times, were indicated by the

chromatogram in Figure 1. Based on Table 1, GC-

MS analysis revealed that different chemical

compositions were identified in cinnamon oils,

including cinnamaldehyde, iso-bornyl acetate,

cinnamaldehyde dimethyl acetyl, and cynamil

alcohol. The main component of cinnamon oil is

cinnamaldehyde (83.87%). This result is the same as

some of the results of previous studies conducted by

Figure 1: GCMS chromatogram of the cinnamon oil.

Table 1: Chemical component identified of cinnamon oil

with GCMS.

Retenti

on time

Identified

compound

Molecular

formula

Relative

percentage

area (%)

18.501

Cinnamaldehy

83.87

18.913

Iso-bornyl

acetate

4.71

23.817

Cinnamaldehy

de dimethyl

acetal

7.31

25.788

Cynamil

alcohol

4.10

researchers that cinnamaldehyde is a major

component of cinnamon oil (Gotmare and Tambe,

2019; Dwijatmoko, 2016; Li, Kong and Wu, 2013).

Cinnamaldehyde is a compound containing aldehyde

groups and conjugated double bonds outside the ring

(Sachdeva et al., 2017). Cinnamaldehyde is an

organic mixture that gives wood a sweet taste and

smell (also known as cinnamic aldehyde). This

organic compound is significant to inhibit bacterial

growth (Ashakirin et al., 2017). Antimicrobial

activity of cinnamaldehyde was found against E. coli

and staphylococcus aureus. Cinnamaldehyde plays a

role in disrupting bacterial cell membranes (Firmino

et al., 2018).

3.2 Antimicrobial Activity of

Cinnamon Oil

The antimicrobial activity of cinnamon oil was

analysed for gram-positive bacteria (S. aureus) and

gram-negative bacteria (E. coli). The results of the

analysis of antimicrobial activity are shown in

Figure 2 and Table 2.

Antimicrobial ability is shown from the diameter

of the inhibition zone (measured the clear area) as

shown in Figure 2. Based on Table 2, cinnamon oil

has antimicrobial activity against Escherichia coli

and Staphylococcus aureus. In the paper disc

diffusion method, the area of inhibition depends on

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

Figure 2: The inhibition zone cinnamon oil by the paper

disc diffusion method (a : positive control, tetracycline; b :

sample, cinnamon oil; c : negative control (sterile distilled

water)).

Table 2: Antimicrobial activity of cinnamon oil.

Essential

Oil

E. coli (mm)

S.aureus (mm)

Sample

Control

(-)

Control

(+)

Sample

Control

(-)

Control

(+)

Cinnamon

oil

the ability of the essential oil to diffuse evenly to

medium and also releases volatile compounds from

essential oil. Inhibition zone of cinnamon oil against

E. coli is 34 mm, and against a S. aureus is 35 mm.

These results are similar with previous research

conducted by (Adinew, 2014) reported that

cinnamon oil shows an inhibitory effect against the

gram-positive bacteria (Bacillus cereus,

Micrococcus luteus, Staphylococcus aureus, and

Enterococcus faecalis) and gram-negative bacteria

(Alcaligenes faecalis, Enterobacter cloacae, and

Escherichia coli).

3.3 Antimicrobial Activity of the

Simple Antimicrobial Label

Cinnamon oil that has been added to the recycle

paper is then analysed for its antimicrobial activity

compared to the blank, to determine the

antimicrobial ability of the label. Analysis of

antimicrobial activity on labels is done by paper disk

(direct contact) and vapour phase diffusion test

because when applied to shrimp analysed using the

phase diffusion vapour method. The analysis results

are shown in Figure 3 and Table 3.

Based on Figure 3, the blank (only recycled

paper) does not show the inhibition zone. The

absence of the inhibition zone indicates that recycle

paper does not have the antimicrobial ability.

Figure 3: The inhibition zone simple antimicrobial label

by the paper disc and vapour phase agar diffusion method.

Table 3: Antimicrobial activity of simple antimicrobial

label.

Direct contact

Vapor

E. coli

(mm)

S.aureus

(mm)

E. coli

(mm)

S.aureus

(mm)

Recycle

Paper

cinnamon oil

36.98

50.31

28.75

44.54

Meanwhile, after adding cinnamon oil to the

recycle paper, the inhibition zone was seen, which

stated that the label has the antimicrobial ability. The

antimicrobial label has an antimicrobial ability

against the gram-positive bacteria (Staphylococcus

aureus) and gram-negative bacteria (Escherichia

coli). It can be seen from Table 3 that a clear zone or

diameter of the inhibition zone of 36.98 cm (E. coli)

and 50.31 cm (S. aureus) for the direct contact

method, while the vapour diffusion method is 28.75

cm (E. coli) and 44.54 cm (S. aureus). Antimicrobial

labels have a better antimicrobial ability against S.

aureus than E. coli, both from the test results using

vapour or direct contact. This result is the same as

the results of research conducted by (Zhang et al.,

2016), which states that E. coli is more resistant to

cinnamon oil than S. aureus. This phenomenon

probably due to differences in the structure of the

bacterial outer membrane. E. coli has a thick layer of

lipopolysaccharide on its outer membrane that

covers the cell wall, whereas S. aureus has only a

single peptidoglycan layer structure. Therefore E.

coli is more resistant to essential oils (hydrophobic

substance) compared with S. aureus.

RP-CO : Vapor

RP-CO : Direct contact

Blank : Direct contact

Simple Antimicrobial Labels from Cinnamon Oil Added to Recycled Paper

3.4 Total Volatile Basic Nitrogen

(TVB-N)

The enzymatic and bacteriological activity can

quickly reduce the protein content and quality of

stale seafood, some ammonia, trimethylamine,

dimethylamine, and other volatile basic nitrogen

compounds are produced, which together are called

TVB-N (Fallah et al., 2016). Total volatile basic

nitrogen (TVB-N) is one method that is often used to

measure seafood quality and, most commonly, as an

indicator of chemical decay in marine products

(Altissimi et al., 2017). TVB-N analysis was

performed to find out the freshness of shrimp stored

without or using simple antimicrobial labels. TVB-N

analysis results are shown in Figure 4.

Based on the graph in Figure 4 shows that the

value of TVB-N is increasing. It is consistent with

the results of previous research conducted by

(Chakrabortty et al., 2017), which states that the

value of TVB-N increases with storage time. The

low value of TVB-N is an indication of the quality

of fresh shrimp, while the high value of TVB-N may

be due to the action of the enzyme autolysis and

spoilage bacteria. TVB-N values for “high quality”

quality up to 25 mg / 100 g, “good quality” up to

30 mg / 100 g, “limit of acceptability” up to

35 mg / 100 g, and “spoiled” above 35 mg / 100 g

(Jinadasa, 2014). From the graph in Figure 4 also

shows that the value of TVB-N for shrimp stored

using simple antimicrobial labels is lower than

shrimp stored without using simple antimicrobial

labels. It shows that simple antimicrobial labels can

be used to maintain the freshness of shrimps,

however further research is needed to determine the

optimization of the addition of cinnamon oil to

recycled paper.

3.5 Fourier Transform Infrared

Spectroscopy

The functional group of the label was analysed using

FTIR for three days to determine changes in

functional groups that occur during that day. Figure

5 displays the spectra of the simple antimicrobial

labels.

Figure 4: Total volatile base nitrogen (TVB-N) of shrimp.

Figure 5: FTIR spectra of recycling paper and simple

antimicrobial label.

Based on Figure 5, the IR characteristic

fingerprint for cinnamon oil is mostly in the range of

1800 cm

-1

- 600 cm

-1

(Li et al., 2013). In the IR

spectra, it is shown that the absorbance band at 1690

-1

- 1760 cm

-1

revealed the presence of C = O

bond for aldehyde from cinnamaldehyde (Adinew,

2014). These results are consistent with the results of

the analysis using GCMS, which shows that the

main component of cinnamon oil is cinnamaldehyde.

The IR spectroscopy spectrum display characteristic

bands corresponding to aromatic CH bonds

(between 3000 cm

-1

and 3100 cm

-1

), CH alquenes

(between 3020 cm

-1

and 3080 cm

-1

), and C = C

(between 1640 cm

-1

- 1680 cm

-1

) (Singh et al.,

2011). From the Figure, the C = O intensity of

cinnamaldehyde is decreasing. It is because

cinnamaldehyde has to be released from the label.

0 1 2

100

120

Total volatile base nitrogen (mg/100 g)

Days

Without label

With label

4000 3500 3000 2500 2000 1500 1000 500

Transmittance

Wavenumber (1/cm)

RP-Cinnamon day 2

RP-Cinnamon day 1

RP-Cinnamon day 0

C=O aldehyde

(1690-1760 cm

-1

)

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia

4 CONCLUSION

In this experiment, cinnamon oil has antimicrobial

ability against the gram-positive bacteria

(Staphylococcus aureus) and gram-negative bacteria

(Escherichia coli). Simple antimicrobial labels from

cinnamon oil added to the recycled paper also have

antimicrobial ability against the gram-positive

bacteria (Staphylococcus aureus) and gram-negative

bacteria (Escherichia coli). The results obtained

from this experiment indicated that this simple

antimicrobial label could be used to maintain the

freshness of shrimps. Further research is needed to

determine the optimization of the addition of

cinnamon oil to recycled paper.

ACKNOWLEDGEMENT

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

Adinew, B., 2014. GC-MS and FT-IR Analysis of

Constituents of Essential Oil From Cinnamon Bark

Growing in South-west of Ethiopia, International

Journal of Herbal Medicine, 1(6), 22–31.

Altissimi, S., Mercuri, M. L., Framboas, M., Tommasino,

M., Pelli, S., Benedetti, F., Di Bella, S., Haouet, N.,

2017. Indicators of Protein Spoilage in Fresh and

Defrosted Crustaceans and Cephalopods Stored in

Domestic Condition, Italian Journal of Food Safety,

6(4), 217–221.

Ashakirin, S. N., Tripathy, M., Patil, U, K., Majeed, A, B,

A., 2017. Chemistry and Bioactivity of

Cinnamaldehyde : A Natural Molecule of Medicinal

Importance, International Journal of Pharmaceutical

Sciences and Research, 8(6), 2333–2340.

Chakrabortty, A., Aziz, M, A., Rashad, M, A., Quality, F.,

2017. Study on Protein Losses and Total Volatile Base

Nitrogen (TVBN) of Fresh Water Prawn (Galda) in

the Shrimp Value Chain of Bagerhat Region,

Bangladesh for the Development of Sea Food Quality,

Journal of Engineering Science, 8(1), 79–82.

Dwijatmoko, M, I., 2016. Effect of Cinnamon Essential

Oils Addition in the Sensory Attributes of Dark

Chocolate, Nusantara Bioscience, 8(2), 301–305.

EC., 2005. Commission regulation (EC) No 2074/2005,

Official Journal of the European Union.

Echegoyen, Y., Nerin, C., 2014. Performance of An

Active Paper Based on Cinnamon Essential Oil in

Mushrooms Quality, Food Chemistry, 1–28.

Fallah, F., Ebrahimnezhad, Y., Maheri-Sis, N., Ghasemi-

Sadabadi, M., 2016, The Effect of Different Levels of

Diet Total Volatile Nitrogen on Performance, Carcass

Characteristics and Meat Total Volatile Nitrogen in

Broiler Chickens, Archives Animal Breeding, 59(2),

191–199.

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

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

Catunda, F, E, A., 2018. Antibacterial and Antibiofilm

Activities of Cinnamomum Sp. Essential Oil and

Cinnamaldehyde: Antimicrobial Activities, The

Scientific World Journal, 2018, 1–9.

Gotmare, B., Tambe, E., 2019. Identification of Chemical

Constituents of Cinnamon Bark Oil by GCMS and

Comparative Study Garnered from Five Different

Countries, 19(1).

Jinadasa, B, K, K, K., 2014. Determination of Quality of

Marine Fishes Based on Total Volatile Base Nitrogen

Test (TVB-N), Nature and Science, 12(5), 106–111.

Li, Y, Q., Kong, D, X., Wu, H., 2013. Analysis and

Evaluation of Essential Oil Components of Cinnamon

Barks using GC-MS and FTIR Spectroscopy,

Industrial Crops and Products, 41(1), 269–278.

Mascheroni, E., Guillard, V., Gastaldi, E., Gontard, N.,

Chalier, P., 2011. Anti-Microbial Effectiveness of

Relative Humidity-Controlled Carvacrol Release from

Wheat Gluten/Montmorillonite Coated Papers, Food

Control, 22(10), 1582–1591.

Olatunde, O, O., Benjakul, S., 2018. Natural Preservatives

for Extending the Shelf-Life of Seafood: A Revisit,

Comprehensive Reviews in Food Science and Food

Safety, 17(6), 1595–1612.

Sachdeva, A., Vashist, S., Chopra, R., Puri, D., 2017.

Antimicrobial Activity of Active Packaging Film to

Prevent Bread Spoilage, International Journal of Food

Science and Nutrition, 2(4), 29–37.

Santos, R, R., Andrade, M., Melo, N, R., Sanches-Silva,

A., 2017. Use of Essential Oils in Active Food

Packaging: Recent Advances and Future Trends,

Trends in Food Science and Technology, 61, 132–140.

Singh, P., Andola, H, C., Rawat, M, S, M., Pant, G, J, N,

P., Purohit, V K., 2011. Fourier Transform Infrared

(FT-IR) Spectroscopy in An-Overview, Research

Journal of Medicinal Plant, 5(2), 127–135.

Simple Antimicrobial Labels from Cinnamon Oil Added to Recycled Paper

Wang, T, H., Hsia, S, M., Wu, C, H., Ko, S, Y., Chen, M,

Y., Shih, Y, H., Shieh, T, M., Chuang, L, C., Wu, C,

Y., 2016. Evaluation of the Antibacterial Potential of

Liquid and Vapor Phase Phenolic Essential Oil

Compounds Against Oral Microorganisms, Plos One,

11(9), 1–17.

Zhang, Y., Liu, X., Wang, Y., Jiang, P., Quek, S, Y., 2016.

Antibacterial Activity and Mechanism of Cinnamon

Essential Oil Against Escherichia Coli and

Staphylococcus aureus, Food Control, 59, 282–289.

ICEO 2019 - 2nd International Conference of Essential Oil Indonesia