Chapter 4: Result and discussion

4.1 Introduction

In this chapter, all the result and data recorded from this research were clearly discussed.
The results were specifically discussed about the effectiveness of essential oil as
antimicrobial agent in edible film, effect of essential oil towards the properties of film and
analysed the result of FTIR. Besides, the discussion in this chapter was also compared and
strong supported with the relevant theories of the previous research.

4.2 Raw data of the experiment

Some research was done before this experiment was conducted to determine the
potential of essential oil in bio-edible film as food packaging, in order, to prevent the
contamination of food product. The core ingredient in making edible film was essential oil
that extracted from cinnamon bark because it acted as antimicrobial agent. In this research,
the essential oil was extracted by using Soxhlet extraction because it was low cost and good
for plant that yield low amount of oil. There were no parameters involved in the extraction of
essential. This is because the taken to get the essential oil, the temperature, amount of
solvent and the mass of sample were constant until this method were finished. Extraction for
30g of cinnamon powder was took 6 hours to get the essential oil at 80˚C by using 250ml of
ethanol as a solvent. The time taken of material to be extracted is depend on the type of
plant material. This is to avoid from components in essential oil volatile when too long
heating. Moreover, the temperature on this process depends on the volatility of solvent used.
Thus, the correct solvent used is important in order to get the good yield of product. To get
the purity of essential oil, it was separated by using rotary evaporator for about 20minutes
and then, keep in fridge before the analysis. This is because to avoid the changes
composition in essential oil. Hence, the pure cinnamon essential oil extract that collected
was 6.10 ml.
Besides, there were two parameters involved in edible film which is amount of essential oil
and different type of packaging. The amount of essential oil that used in this research is 1ml,
1.5ml and 2ml. These three amounts of essential oils were used to determine the
effectiveness as antimicrobial agent. So, the edible film was produced by mixing 25g of
starch, 175g of water, 6.8ml of glycerol and three different amounts of essential oil.
Moreover, the different amount of essential oil added into edible films were not affect the
heating time which is 20 minutes until gelatinization. Next, different type of packaging that
used in this experiment was edible film and plastic wrapper. The used of different type of
packaging was to compare the potential of edible film added with essential oil with other food
packaging. Furthermore, bread was used in antimicrobial test because it was easy to
determine the growth of microbial. The bread was cut into 2 x 2 cm size and wrapped with
edible film with different amount of essential oil and polyethylene plastic wrapper. There was
one uncovered bread as a control. The parameter was shown in table 4.1 below.

Table 4.1 Type of packaging use

Types of packaging Days of bread contamination

Edible film 1ml 9
1.5ml 10
2ml 14
Plastic wrapper 5
Control 5

4.3 Analysis cinnamon essential oil by using FTIR

The major active components in cinnamon essential oil is cinnamaldehyde and

eugenol. Cinnamaldehyde is an aromatic compound that highly found in cinnamon essential
oil that extracted from cinnamon bark. Moreover, cinnamon bark also composed the eugenol
component. In this research, Fourier transform Infrared spectroscopy (FTIR) was used to
determine the active components in CEO by its functional groups and types of bond in the
materials in this product.

From figure 4.1, it shows that the IR spectrum of the cinnamon essential oil has 24
wavenumbers in which were used in determine the possible components in the sample. The
functional groups were determined by referring their wavenumbers in IR spectrum to the
Correlation chart in order to interpret the spectral graph obtained. The interpretation results
were listed on the table 4.1 below.

Table 4.1: Interpretation results of IR spectrum wavenumbers.

Wavenumbers in IR Spectrum (cm-1) Functional group

 3409.51 Alcohol (O-H)
2922.41 Alkane (C-H)
2853.20 Aldehyde (=C-H)
2741.31 Aldehyde (=C-H)
1966.93 Aromatic compound (C-H)
1722.71 Aldehyde (C=O)
1688.54 α,β-unsaturated Ketones (C=O)
1623.89 Alkene (C=C)
1451.52 Aromatic compound (C=C)
1378.88 Phenol (O-H)
1253.61 Ether (C-O)
1124.21 Secondary alcohol (C-O)
973.06 Alkene disubstituted (C=C)
829.23 Alkene trans-substituted (C=C)

Based on the interpretation result on the table 4.1, the functional group of active
components in CEO were detected in the extracted sample of cinnamon essential oil by
using Soxhlet extraction which cinnamaldehyde and eugenol. The functional groups in the
cinnamaldehyde were aldehyde, aromatic compound, ketones and carbonyl groups. While
for eugenol were alcohol, aromatic compound and ether.

Both active components were function as antimicrobial and have a main

bacteriostatic effect on bacteria cell walls. Cinnamaldehyde is the major active components
of the CEO has shown as good antimicrobial spectrum against bacteria, yeast and moulds.
This was supported by Zhang (1995), who did the inhibition experiment of cinnamaldehyde
against fungi which the cellular ultrastructure of fungal was significantly changes including
the disappearance of organelles and degradation of cell wall and cytoplasm. In addition,
eugenol component is usually used in perfumes, flavouring and in medical as antiseptic and
anaesthetic. According to Gill et al. (2004), eugenol can be used in food as antimicrobial
because it can reduce the presence of Listeria monocytogenes and Lactobacillus sakei. It
was also act as antioxidant for plastic and rubbers.

4.4 Effectiveness of essential oil as antimicrobial in edible film

 Days of microbial growth vs type of packaging
16

14

12
Days of contamination

10

0
Edible film (1%) Edible film Edible film (2%) Plastic Wrapper Non-Packaging
(1.5%) (control)

Type of packaging

Figure 4.2: Graph of Days of Microbial Growth vs Types of Packaging

Based on the result obtained from Graph 4.2, the bread that had been wrapped with
edible films can last longer compare to the plastic wrapper and control sample. The edible
film with different amount of essential oil took longer time to observe the mould growth which
is 9 to 14 days. Edible films with 2% essential oil took longer time which is 14days to bread
spoiled with mould. It shows that the increasing amount of essential oil added into edible film
will affect the times for bread to contaminate with microbial. While for plastic wrapper and
control sample, it took 5 days to observe the growth of microbial on the bread. It shows that
the plastic wrapper was the faster packaging for mould growth. This is because the plastic
wrapper was not an antimicrobial packaging and just a normal plastic bag for food wrapper
purpose only. Moreover, the control sample took shortest time for microbial to grow due to
the exposure to air of environment without packaging wrappers.

Based on recent research, essential oil in edible film helps to degrade in cell wall of
microbial and weakening their membrane. It was also reacted with the cell membrane
proteins of microbial in order to inhibit its function. Thus, it was leads to death of microbial
cells. This is show that the essential oil can control the microbial spoilage in food. In addition,
Cristani (2007) was reported that the aromatic component in essential oil modify the active
sites via hydrogen bonding to amino acid residue of active sites. Therefore, cinnamon
essential oil which is have bacteriostatic effect to fungi give good impact to edible film in
order to produce antimicrobial bio-degradable food packaging.
4.5 Tensile strength

Tensile strength vs Edible Film

0.78

0.76
Tensile strength (kg/mm2)

0.74

0.72

0.7

0.68

0.66

0.64
0 1 1.5 2
Edible Film with CEO (%)

Figure 4.3: Graph Tensile strength vs edible film

Tensile strength is one of the film properties in edible film which reflects the
maximum stress that the film sustains before it eventually breaks. It shows the resisting of
mechanical damage in film strength. The content in CEO might give an effect on the tensile
strength of edible film.

According to the result, the addition of CEO in edible film give a reduction towards
the film strength. Edible film with 2% CEO has the lowest tensile strength which is _____.
However, edible film with 1.5% CEO has a constant result with the edible film without CEO
which is a control sample. The difference was explained by Warkoyo et al. (2014), it was due
to the loss of intermolecular interaction among starch molecules. The arrangement in the
protein network have some degree of changes due to the addition of CEO in edible film,
thus, it decreased the film resistance to elongation. Similar results were also reported by
Ahmad et al. (2012), who found that the addition of 5-20% of bergamot oil or lemongrass oil
in gelatin film can caused the reduction of film tensile strength.

Besides, edible film without CEO has the higher tensile strength compared to edible
film with CEO. This was because the addition of lipid or oil in protein-based or
polysaccharide-based films, it will give an effect in the partial replacement of stronger
polymer-polymer interactions by weaker polymer-oil interaction in the film network. The low
interaction between gelatin monomers interfere the polymer chain-to-chain interactions
(Atares & Chiralt, 2016).In addition, Wang et al. (2011), was explained the leading of
declination in film strength was due to the existence of poles apart substances in the film
which make the film microstructure become discontinuous, hence, the external force
distributed on each matrix bond becomes uneven. Therefore, the addition of CEO in edible
film resulting in decrease of film tensile strength.

4.6 Water Absorption

Percentage of water absorption vs edible films

100
87.5
90
80
water absorption (%)

70
65.27
60
50 43.18
40
30 32.74
20
10
0
0 0.5 1 1.5 2 2.5
Edible film with CEO (ml)

Figure 4.4: Graph of percentage of water absorption vs edible film incorporated with CEOs

Water absorption test is important in film properties because to determine the

absorption of water in film when exposed to water. Based on the graph, percentage of water
absorption was reduced as the amount of CEO in edible film was increased. Edible film
without CEO was control sample and it was showed the highest water absorption compared
to edible film with CEO. This is because edible film was made from cassava starch which is
polysaccharide films that has limitation towards water barrier properties due to high
hydrophilicity. Film with high hydrophilic compound will increase the water absorption.

Therefore, edible film with CEO can decrease the absorption of water for the film
which give a positive result towards film properties. Based on the theory, essential oil is a
concentrated hydrophobic liquid that contains volatile aromatic compounds from plants.
Because of that, the EOs help to increase the hydrophobicity of film by interact with the
hydroxyl group and thus, the polysaccharide-water interaction will be reduced.
 Gill, A. O.; Holley, R. A. (2004). "Mechanisms of Bactericidal Action of Cinnamaldehyde against Listeria
monocytogenes and of Eugenol against L. Monocytogenes and Lactobacillus sakei".  Applied and
Environmental Microbiology. 70 (10): 5750–5. doi:10.1128/AEM.70.10.5750-
5755.2004. PMC  522076.  PMID  15466510.