Antibacterial activity, optical, mechanical, and barrier properties of corn starch films containing orange essential oil

Carbohydrate Polymers 222 (2019) 114981

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Antibacterial activity, optical, mechanical, and barrier properties of corn
starch films containing orange essential oil

T

Jarine Amaral do Evangelhoa, Guilherme da Silva Dannenberga, Barbara Biduskib,
Shanise Lisie Mello el Halala, Dianini Hüttner Kringela, Marcia Arocha Gulartea,
⁎
Angela Maria Fiorentinia, Elessandra da Rosa Zavarezea,
a
b

Department of Agroindustrial Science and Technology, Federal University of Pelotas, Rio Grande do Sul, Pelotas, RS 96010-900, Brazil
University of Passo Fundo (UPF), Faculty of Agronomy and Veterinary Medicine, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords:
Orange essential oil
Starch films
Antibacterial activity
Mechanical properties

The incorporation of antimicrobial compounds into natural polymers can promote increased shelf life and ensure
food safety. The aim of this study was to evaluate the antibacterial activity, morphological, optical, mechanical,
and barrier properties of corn starch films containing orange (Citrus sinensis var. Valencia) essential oil (OEO).
The corn starch films were prepared using the casting method. OEO and the corn starch films incorporated with
OEO showed higher antibacterial activity against Staphylococcus aureus and Listeria monocytogenes. The addition
of OEO to the films increased the morphological heterogeneity and contributed to the reduction of the tensile
strength and elongation of the films, and it increased the moisture content, water solubility, and water vapor
permeability. The water vapor permeability and partial or total solubility of a film in water prior to consumption
of a product are of interest when the film is used as food coating or for encapsulation of specific molecules.

1. Introduction
Because consumers are concerned about reducing the use of synthetic additives, there is particular interest in the food industry for
using natural preservatives that can maintain food freshness and quality
and have no effects on human health (Atarés & Chiralt, 2016). New
technologies for active food packaging have been studied, and they can
protect and interact with the food, increasing its useful life (Adilah,
Jamilah, Noranizan, & Hanani, 2018) and ensuring its safety
(Dannenberg et al., 2017).
Antimicrobial films have active compounds that are released into
the food when the films touch the surface of the product (Guo, Yadav, &
Jin, 2017). Essential oils are active compounds that, in addition to
providing antibacterial protection (Kumar, Narayani, Subanthini, &
Jayakumar, 2011), can improve the functional and mechanical properties of the films (Qin, Li, Liu, Yuan, & Li, 2017). These compounds can
have antifungal activities (Ribeiro-Santos, Andrade, & Sanches-Silva,
2017) as well as antioxidant and anti-inflammatory effects (Liu, Xu,
Cheng, Yao, & Pan, 2012).
Orange (Citrus sinensis) is a source of essential oil concentrated in

the fruit exocarp, which is composed of the epidermis and a layer of

glandular cells. According to Mahato, Sharma, Sinha, and Cho (2018),
large volumes of by-products are generated during the processing of
oranges, and they can be potentially used in the food industry for the
extraction of essential oil. In a study on essential oils from plants that
belong to the genus Citrus, including orange essential oil (OEO), against
different food-borne pathogens, OEO exhibited antibacterial activity
against both gram-positive and gram-negative bacteria (Frassinetti,
Caltavuturo, Cini, Della Croce, & Maserti, 2011). Torrez-Alvarez et al.
(2017) also reported results that proved the antibacterial and antioxidant potential of OEO, highlighting it as an alternative for the development of safer products accepted by consumers who prefer natural
ingredients.
The incorporation of active substances into starch films has been
studied by several researchers (Acosta et al., 2016; Sapper, Wilcaso,
Santamarina, Roselló, & Chiralt, 2018; Song, Zuo, & Chen, 2018). The
production of films with natural polymers offers an alternative to synthetic packaging (Romani, Prentice-Hernández, & Martins, 2018).
Polysaccharides, proteins, and lipids used alone or in combination have
the ability to form biodegradable and/or edible films (Kim, Yang, Chun,

⁎

Corresponding author.
E-mail addresses: (J.A. do Evangelho), (G. da Silva Dannenberg), (B. Biduski),
(S.L.M. el Halal), (D.H. Kringel), (M.A. Gularte),
angefi (A.M. Fiorentini), (E. da Rosa Zavareze).
/>Received 4 April 2019; Received in revised form 6 June 2019; Accepted 6 June 2019
Available online 10 June 2019
0144-8617/ © 2019 Elsevier Ltd. All rights reserved.


Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

& Song, 2018). Among polysaccharides, starch has been widely used for
the production of films because of the low cost of production from renewable sources (Khalid et al., 2018) and its properties that favor the
formation of films (Luchese, Garrido, Spada, Tessaro, & La Caba, 2018).
The antimicrobial properties of several essential oils have been
widely studied as additives in biodegradable films, their effects on the
properties of films is still less discussed in the literature. Essential oils
have an oily and volatile nature which may affect the integrity or degree of hydrophobicity of polymeric films, changing their mechanical
and barrier properties (Abdollahi, Damirchi, Shafafi, Rezaei, & Ariaii,
2018; Atarés & Chiralt, 2016). Therefore, studies are needed to examine
the potential of each antibacterial agent as well as its interaction with
the material used to produce the active starch films. The aim of this
study was to evaluate the antimicrobial activity of the OEO and its
effect on the optical, microstructural, and mechanical and barrier
properties of the biodegradable films of corn starch.

susceptibility against bacterial and yeasts. This procedure is performed
by agar plates inoculation containing a standardized inoculum of the
test microorganism and of the test compound. The antimicrobial agent
inhibits germination and growth of the test microorganism, diffusing
into the agar; the results are expressed by measurement of the
diameters of inhibition growth zones (Balouiri, Sadiki, & Ibnsouda,
2016). Bacterial cultures (L. monocytogenes, S. aureus, B. cereus, P.
aeruginosa, S. dysenteriae, E. coli, and S. typhimurium) were suspended in
peptone water (0.1%), and a concentration of 108 UFC/g (0.5
McFarland) was achieved. The inoculum was seeded with sterile
swabs on the surface of MH agar in petri dishes, on which sterile
paper disks (Laborclin®) were arranged. An aliquot of 10 μL of OEO
was added to each disc (in triplicate; three discs per bacterium) and

allowed to stand for 1 h for absorption; thereafter, the plates were
incubated at 37 °C. After 24 h, the formation of inhibition halos was
evaluated and quantified with a digital caliper (king.tools®).

2. Material and methods

2.4.2.2. Minimum inhibitory concentration and minimum bactericidal
concentration. Minimum inhibitory concentration (MIC) is defined as
the lowest concentration of agent antimicrobial able to inhibit the
visible microbial growth and minimum bactericidal concentration
(MBC) is the lowest concentration of agent antimicrobial able to kill
99.9% after incubation for determined time (24 h) (Balouiri et al.,
2016).
The minimum inhibitory concentration (MIC) was determined using
the plaque microdilution test (CLSI, 2015a). The analysis was performed in triplicate. OEO was diluted in BHI broth with 3% Tween 20
(Vetec®), and concentrations of 166.7 to 0.3 μL mL−1 were obtained.
The bacteria (L. monocytogenes, S. aureus, B. cereus, P. aeruginosa, S.
dysenteriae, E. coli, and S. typhimurium) were added to obtain a final
concentration of 104 UFC mL−1 in each well. The plates were incubated
at 37 °C for 24 h, and the reading was performed on a Robotic plate
spectrophotometer (Robonik® Readwel plate) at 625 nm, considering
the highest dilution at which no cell growth was observed as MIC
(Ojeda-Sana, Baren, Elechosa, Juaréz, & Moreno, 2013).
The minimum bactericidal concentration (MBC) was determined
using 10 μL aliquots inoculated on BHI agar plates and considering the
lowest concentration at which no growth was observed as MBC.

2.1. Material
In this study, oranges (Citrus sinensis ‘Valencia’) harvested in 2017 in
the city of Pelotas, southern region of Rio Grande do Sul, Brazil, were

used. Brain heart infusion (BHI) broth (Acumedia®) and Mueller-Hinton
(MH) agar (Oxoid®) were used for the microbiological analyses.
Commercially available corn starch (A-type crystallinity standard), 28%
amylose (as described by McGrane, Cornell and Rix (1998)), and gelatinization peak of 69.9 °C (evaluated using a differential scanning
calorimeter; TA-60WS, Shimadzu, Kyoto, Japan).
2.2. Bacteria
Seven bacteria of relevance to food were used: three gram-positive
bacteria, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC
6538, and Bacillus cereus ATCC 11778, and four gram-negative bacteria,
Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 8739,
Shigella dysenteriae ATCC 13313, and Pseudomonas aeruginosa ATCC
15442.
2.3. Extraction of OEO

2.4.2.3. Kinetics of action. The kinetics of OEO action were evaluated
for the two most sensitive bacteria in the previous tests (L.
monocytogenes and S. aureus), according to the methodology of Diao,
Hu, Zhang, and Xu (2014)). OEO was added to BHI broth containing 3%
Tween 20, and the MBC of OEO (5.208 μL mL−1) was obtained. The
pathogens were inoculated at 104 CFU mL−1 and incubated at 37 °C
under constant stirring (100 rpm). After 0, 3, 6, 9, 12, and 24 h, serial
dilutions of the samples were made in peptone water (0.1%), and
0.1 mL aliquots were plated on BHI agar. A control treatment was
performed under the same conditions, but without the addition of OEO.
The counts for each time were used to obtain the kinetics of action as
well as the time required to promote bactericidal action on all the cells.
The analysis was performed in triplicate.

OEO was extracted by hydrodistillation in a Clevenger apparatus
(Kringel et al., 2017). The fresh shells of the oranges were ground in

distilled water (ratio w/v = 1/10) and extracted for 3 h at 100 °C. The
obtained essential oil was dehydrated with anhydrous sodium sulfate
(Na2SO4; SYNTH®) and stored in an amber glass vial at −80 °C.
2.4. Characterization of OEO
2.4.1. Chemical composition of OEO
The chemical composition of OEO was determined using a gas
chromatograph coupled to a mass detector (GC/MS; QP 2010SE;
Shimadzu®) equipped with an RTX-5MS (Restek®) capillary column
(30 m × 0.25 mm ×0.25 μm). The volume of the injected sample was
0.1 μL. Helium was used as the entrainment gas at a flow of
1.2 mL·min−1. The total run time was 42 min; the temperature was
initially maintained at 60 °C for 2 min and gradually increased at a rate
of 4 °C min−1 until it reached 220 °C. Identification of the compounds
was based on the mass spectra (as compared with the Wiley 275
spectral library, 6th edition), and the concentrations were presented as
relative percentages of the area of each peak over the total area.

2.5. Production of films
The films were produced using the casting technique, according to
Souza, Goto, Mainardi, Coelho, and Tadin (2013) with some modifications. The filmogenic solution was prepared with 3% (w/v) starch
in distilled water and 30% (w/w) glycerol (relative to dry starch mass).
The film-forming solutions were heated in a jacketed glass reactor, with
water circulation at 90 °C for 10 min. After cooling, OEO was added to
the film-forming solution at concentrations of 0.3, 0.5, and 0.7 μL g−1
and homogenized in an Ultraturrax at 14,000 rpm for 10 min. Then,
20 g of each solution was spread on acrylic plates (9 cm in diameter)
and dried in an oven with air circulation at 30 °C for 16 h. After drying,
the films were conditioned at 16 °C and 58% relative humidity until

2.4.2. Antimicrobial activity of OEO

2.4.2.1. Agar diffusion. The determination of OEO action spectrum was
performed using the agar diffusion technique (CLSI, 2015b). Agar diskdiffusion is an oft-employed method to determine the antimicrobial
2


Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

further use.

TS=

2.6. Characterization of the films

Fm
A

(3)

where: TS is tensile strength (MPa); Fm is the maximum force at the
moment of film rupture (N); and A is the cross-sectional area (m2).
Eq. (4)

2.6.1. Morphology
The morphology of the surface and transverse sections of the films
was evaluated using scanning electron microscopy (SEM; JEOL, JSM6610LV, Japan). Samples of the films were coated onto the surface of
double-sided carbon tapes adhered to stubs and coated with a gold layer
by using a vacuum metallizer (Denton Desk V; Denton Vacuum, USA).
SEM was performed with a 10 kV electron beam. For the cross-section

analysis, the films were fractured with liquid nitrogen. The surfaces and
cross-sections of the films were analyzed at 70× and 500× magnifications, respectively.

E=

dr
× 100
di

where: E is elongation (%); di is the initial separation distance (cm); and
dr is the distance at the moment of rupture (cm).
2.6.5. Moisture content and water solubility of the films
The moisture content of the films was determined using the AACC
(1995) in an oven at 105 °C with a natural air circulation to constant
mass; the results were expressed in g. (100 g)−1.
The water solubility was evaluated in triplicate and determined
according to the method proposed by Gontard, Duchez, Cuq, and
Guilbert, 1994). Disk samples with a diameter of 2.5 cm were used. The
samples were dried in an oven at 105 °C until constant dry mass to
remove moisture. Then, they were immersed in a Falcon tube with
50 mL of distilled water. The tube was shaken (175 rpm) in a shaker for
24 h at 25 °C. Then, the samples were oven-dried at 105 °C until constant weight to determine the final dry mass of the sample. The solubility was expressed in terms of the solubilized mass (SM) of the film,
according to Eq. (5).

2.6.2. Antibacterial activity
About 0.1 mL aliquots of the cell suspensions (103 CFU·mL−1) of the
two OEO-sensitive bacteria (L. monocytogenes and S. aureus) were inoculated on the surface of BHI agar in petri dishes. After absorption of
the inoculum, the entire surface of the agar was covered with OEOcontaining films (0.3, 0.5, and 0.7 μL g−1). Control treatments for each
bacterium were performed similarly, but without the addition of the
films. The plates were incubated at 37 °C for 24 h, and the percentage

difference between bacterial colony counts of the treatments and controls was used to express growth inhibition. Three replicates were
performed for each tested bacterium.

SM (%) =

( initial mass-final mass) × 100
initial mass (g)

(5)

2.6.3. Film color and opacity
The color and opacity of the films were determined by averaging
five values, one in the center and the other in the perimeter, using a
colorimeter (MINOLTA, CR 400, Japan). The films were placed on a
white plate defined as standard and illuminant D65 (daylight) for determination of color parameters. The parameter L* indicates clarity,
which varies from 0 (black) to 100 (white); parameters a* and b* are
the chromaticity coordinates, where a* varies from green (-) to red (+)
and b* varies from (-) to yellow (+). The total color difference (ΔE) was
calculated using Eq. (1).

2.6.6. Water vapor permeability of the films
The permeability to water vapor (PWV) was determined using the
ASTM method E-96-95 (ASTM, 1995) at 25 °C. The films were sealed
with paraffin on aluminum permeation cells containing calcium
chloride (0% relative humidity). The permeation cells were conditioned
in desiccators containing saline saturated with sodium chloride at room
temperature and 75% relative humidity. The mass gain of the system
was measured for 2 days. The evaluations were performed in triplicate,
and PWV was calculated using Eq. (6).

ΔE= (ΔL2 + Δa2 + Δb)0.5

PWV=

(1)

where: ΔL = Lstandard – Lsample; Δa = astandard - asample; Δb = bstandard –
bsample.
Opacity was calculated as the relation between the opacity of the
film superimposed on the black standard (Sblack) and white standard
(Swhite), according to Eq. (2) (Hunterlab, 1997).

SBlack
Opacity=
×100
SWhite

ΔW
X
×
t
AΔP

(6)

where: PWV is permeability to water vapor (g·mm/kPa·dia·m ); ΔW is
mass gain (g); X is film thickness (mm); t is time (days); A = exposed
area (m2); and ΔP is the partial pressure difference (kPa).
2

2.7. Statistical analysis

(2)

The results were statistically compared using one-way analysis of
variance and the Tukey test to detect significant differences (p ≤ 0.05).
Statistica software (StatSoft, France, version 6.1) was used.

2.6.4. Thickness and mechanical properties of the films
The thickness of the films was determined using the arithmetic
mean of eight random measurements of their surface by using a digital
micrometer (INSIZE model), and the results were expressed in mm.
The mechanical properties (tensile strength and percentage of
elongation) of the films were determined using a texturometer
(TA.XTplus, Stable Micro Systems, UK), according to the ATM D 882
method (ASTM, 1995) with initial grips separation at 50 mm and probe
speed of 1 mm.s−1. Six to 10 samples of each film were trimmed
(85 mm × 25 mm) and fixed in the texturometer. The tensile strength
was calculated by dividing the maximum force at the breakage of the
film by the cross-sectional area (Eq. (3)). The elongation was determined by dividing the final separation distance of the probe by the
initial separation distance (50 mm) and multiplying by 100 (Equation
4). The mean thickness required for the sectional area calculation was
determined using eight measurements obtained throughout the sample.

3. Results and discussion
3.1. Chemical composition of OEO
GC-MS analysis identified the presence of seven components in OEO
(Table 1). The major compounds of OEO were 96% D-limonene and
2.6% β-myrcene. O’Bryan, Crandall, Chalova, and Ricke (2008) also
reported the D-limonene (93.9%) and β-myrcene (2.1%) as the main

constituents of OEO. Five other minor compounds were also identified
(in the decreasing order of concentration): octanal, α-pinene, β-linalool,
cyclohexene, and decanal (Table 1).
D-limonene usually exhibits antimicrobial and antiseptic activities
(Hąc-Wydro, Flasiński, & Romańczuk, 2017; Umagiliyage, BecerraMora, Kohli, Fisher, & Choudhary, 2017; Zahi, El Hattab, Liang, &
Yuan, 2017). This compound has been reported to have applications in
3


Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

the hydrophobic character of LPS hindered the penetration of the
apolar components of OEO.

Table 1
Chemical composition of orange essential oil (OEO).
Peak number

Retention time (min)

Compound

Peak area (%)

1
2
3
4

5
6
7

5.14
6.23
6.80
7.15
8.20
10.34
13.86

α-pineno
ciclohexeno
β-mirceno
octanal
d-limoneno
β-linalol
decanal

0.53
0.29
2.35
0.55
95.96
0.45
0.26

3.3. Action kinetics of OEO
The OEO action kinetics (Fig. 1) showed a similar behavior for S.

aureus and L. monocytogenes, both of which showed a gradual reduction
in viable cell count over OEO exposure time (MBC = 5.21 μL·mL−1); a
lethal effect was observed at 12 h of contact. L. monocytogenes was more
sensitive and showed statistically significant reductions (p ≤ 0.5) than
S. aureus at all times after 3 h of analysis, reaching 0.49 log CFU after
9 h of contact.
The kinetics of action of an antimicrobial depends on factors such as
the cellular concentration of the bacterium under study and concentration and mechanism of action of the component under study
(Wang et al., 2011). Because essential oils are composed of different
molecules, their mechanisms of action are attributable to both individual action of each component on specific cellular targets and a
synergistic antimicrobial effect of all the compounds (Burt, 2004). An
OEO concentration of 5.21 μL mL−1 was able to cause the death of a
bacterium in 12 h of contact, and the initial concentrations of S. aureus
and L. monocytogenes were more than 104 CFU·mL−1.

the pharmaceutical and food industries (Chen et al., 2018; Li & Lu,
2016). In humans, limonene is rapidly absorbed in the gastrointestinal
tract and easily metabolized (Filipowicz, Kaminski, Kurlenda,
Asztemborska, & Ochocka, 2003).
β-Myrcene, the second major component of OEO, also has antimicrobial activity. Dannenberg et al. (2017) studied the essential oil
composition of pink pepper and found that β-myrcene (41%) was the
major compound; cellulose acetate films containing this oil showed
high antibacterial activity against S. aureus, L. monocytogenes, and B.
cereus.
3.2. Antimicrobial activity of OEO

3.4. Morphology of the films with OEO

In the agar diffusion test (Table 2), OEO showed activity against the
three gram-positive bacteria, with inhibition halos of 10.59, 10.10, and

9.99 mm for L. monocytogenes, S. aureus, and B. cereus, respectively. The
gram-negative bacteria P. aeruginosa and S. dysenteriae also showed
sensitivity to OEO, presenting inhibition halos of 9.30 and 8.73 mm,
respectively. E. coli and S. typhimurium were not sensitive to OEO under
the test conditions.
MICs of up to 2.60 μL·mL−1 OEO were able to promote a bacteriostatic effect against L. monocytogenes and S. aureus, whereas the MIC for
B. cereus was 5.21 μL·mL−1 ((Table 2). The MICs for gram-negative
bacteria P. aeruginosa and S. dysenteriae were 10.42 and 41.67 μL·mL−1,
respectively, and the values were higher than those found for the grampositive bacteria.
OEO concentrations up to 5.21 μL·mL−1 demonstrated a bactericidal
effect against the three gram-positive bacteria. For the gram-negative
bacteria, higher MBCs were required to produce a lethal effect (20.83
and 41.67 μL·mL−1 for P. aeruginosa and S. dysenteriae, respectively;
Table 2).
In the present study, it was possible to observe that the gram-negative bacteria were more resistant to OEO than the gram-negative
bacteria (Burt, 2004; Dannenberg et al., 2017; Silva et al., 2018). The
gram-negative bacteria have a double outer phospholipid layer in their
cell walls, which is composed of lipopolysaccharides (LPS); however,
the gram-positive bacteria do not have this external layer, and their cell
walls are mainly composed of peptidoglycan (90–95%) (Nazzaro,
Fratianni, De Martino, Coppola, & De Feo, 2013). It is also possible that

The morphology of the surfaces and cross-sections of the corn starch
films without and with different OEO concentrations are shown in
Fig. 2. The film without OEO presented a smooth and uniform surface
(Fig. 2a). The addition of OEO in the films, regardless of the concentration, reduced the homogeneity of the cross-sections (Fig. 2f–h),
with presence of more concentrated pores on the surface. The hydrophobicity of the oil and its density difference with the aqueous solution
of starch can affect the stability of the filmogenic solution and consequently form heterogeneous structures because of the separation of
phases and presence of pores (Phan et al., 2002). These heterogeneities,
such as the presence of preferential pathways (pores) shown in

Fig. 2f–h, may contribute to the antibacterial property of the films,
considering that they facilitate the diffusion process of the essential oil
from the interior of the polymer matrix to the surface to perform the
desired action.
3.5. Antimicrobial activity of the OEO films
The starch films without OEO promoted a reduction of 16% and
22% in the development of S. aureus and L. monocytogenes, respectively,
when compared with the control (without film application; Fig. 3). This
result indicates that the direct contact promoted by the coating of the
contaminated surface (agar) with the film promotes a physical impediment to the development of the colonies, considering the inert
(non-antimicrobial) characters of starch and other components present
in the filmogenic solution.
The addition of OEO in the polymeric matrix of the film, at all
evaluated concentrations, promoted the inhibition of both pathogens
(Fig. 3). OEO concentrations of 0.3, 0.5, and 0.7 μL·g−1 reduced the
development of L. monocytogenes by 68, 80, and 83%, and the development of S. aureus by 40, 51, and 66%, respectively. The increase in
OEO concentration resulted in a directly proportional increase in viable
cell reduction in both pathogens. The lower concentration of OEO in the
films (0.3 μL g−1) was able to significantly reduce (p ≤ 0.05) the counts
of L. monocytogenes, when compared with the film without OEO. Only
the highest OEO concentration (0.7 μL g−1) resulted in significant reductions (p ≤ 0.05) in the counts of S. aureus. These results demonstrate that starch is a suitable polymer matrix for the incorporation of
antimicrobial agents such as OEO because it was able to store/encapsulate OEO and release it during direct contact with the contaminated surface of the medium (agar).

Table 2
Antimicrobial activity of orange essential oil (OEO).
Bacteriaa
Gram-positive
L. monocytogenes
S. aureus
B. cereus

Gram-negative
P. aeruginosa
S. dysenteriae
E. coli
S. Typhimurium

ATCC

Diffusion agar (mm)

MIC (μL/mL)

MBC (μL/mL)

7644
6538
11778

10.59 ± 0.43
10.10 ± 0.88
9.99 ± 0.18

2.60 ± 0.00
2.60 ± 0.00
5.21 ± 0.00

5.21 ± 0.00
5.21 ± 0.00
5.21 ± 0.00

15442
8739
13313
14028

9.30 ± 0.31
8.73 ± 0.56
ND
ND

10.42 ± 0.00
41.67 ± 0.00
ND
ND

20.83 ± 0.00
41.67 ± 0.00
ND
ND

a
Values expressed as mean (n = 3) ± Standard deviation; ND = Not
Detected.

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Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

Fig. 1. Kinetics of action of the OEO for S. aureus ATCC 6538 (A) and L. monocytogenes ATCC 7644 (B). Results expressed as means (n = 3) ± standard deviation.

The ability to release antimicrobial components through direct
contact is an important feature because, normally, the highest microbial
contamination occurs on the surface (Malhotra, Keshwani, & Kharkwal,
2015). Therefore, antimicrobial films would act directly at the most
critical point.
These interactions result in a gradual release of the antimicrobial
compounds and guarantee their action for a longer period when compared with direct application (Atarés & Chiralt, 2016). In addition, the
incorporation of essential oils into packages is interesting because it is
an indirect method of using this natural extract in foods without the
need for adding them as an ingredient, thus reducing undesirable sensorial interferences (Calo, Crandall, O’Bryan, & Ricke, 2015).
3.6. Color and opacity of the OEO films
The color parameters (L*, a*, and b*) and opacity of the corn-starch
films with or without OEO are listed in Table 3. The brightness (L) of
the films ranged from 96.38 to 96.80 (Table 2). The films with 0.3 and
0.7 μL of OEO showed higher values of a* and b* (coordinates responsible for chromaticity), indicating a tendency to green and yellow.
OEO addition increased the opacity of the films with higher OEO
concentrations (Table 3). However, this behavior only is visually noted
in the film with 0.7 μL of OEO (Fig. 4). This increase in opacity can be

Fig. 3. Antimicrobial activity of the films with OEO on the growth of S. aureus
ATCC 6538 and L. monocytogenes ATCC 7644. Results expressed as means
(n = 3) ± standard deviation; Different lowercase letters indicate significant
difference between OEO concentrations for the same bacterium; Different uppercase letters indicate significant difference between the bacteria for the same
concentration of OEO.

Fig. 2. Surface micrographs (a, b, c, d) and cross-sections (e, f, g, h) of the corn starch films with 0.0, 0.3, 0.5 and 0.7 μL g−1 of orange essential oil, respectively.
5

Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

Table 3
Color parameters (L*, a* and b*) and opacity of the corn starch films with and
without orange essential oil (OEO).
OEO (μL/
g)a

L*

0.0
0.3
0.5
0.7

96.38
96.41
96.55
96.80

a*

±
±
±
±

0.06b
0.11b
0.03b
0.02ª

−0.16
−0.32
−0.15
−0.33

b*

±
±
±
±

0.00b
0.03a
0.02b
0.06a

2.63
2.76
2.51
2.97

Table 4
Thickness, tensile strength and percent elongation of starch films with and

without orange essential oil (OEO).

Opacity (%)

±
±
±
±

0.02b
0.11ab
0.06b
0.16a

10.86
12.02
13.07
16.24

±
±
±
±

0.37b
1.16b
1.52ab
1.87a

OEO (μL/g)a

Thickness (mm)

0.0
0.3
0.5
0.7

0.084
0.112
0.142
0.131

±
±
±
±

0.008c
0.016b
0.024a
0.020a

Tensile strength (MPa)

Elongation (%)

0.57a
0.40b
0.20c

0.46c

64.58 ± 8.95a
9.94 ± 0.46b
12.64 ± 3.45b
15.25 ± 2.85b

5.11
4.08
2.73
2.40

±
±
±
±

a

The results are the average of three determinations. Values with different
letters in the same column are significantly different (p < 0.05).

a

The results are the average of three determinations. Values with different
letters in the same column are significantly different (p < 0.05).

an increase in film thickness with the addition of licorice essential oil
(Glycyrrhiza glabra L.) and attributed this behavior to the entrapment of
essential oil microdroplets into the polymeric matrix, thereby increasing the compactness of the starch matrix structure.

The mechanical characteristics of films are important because they
are related to the end-use characteristics of these materials, such as
strength and elongation (Bastos et al., 2016). The tensile strength and
elongation of the films ranged from 2.40 MPa to 5.11 MPa and from
9.94% to 64.5%, respectively (Table 4).
In our study, as in the majority reported research, decreases in
strength upon essential oil incorporation are evidenced (Li, Ye, Lei, &
Zhao, 2018; Sánchez-González, Cháfer, Hernández, Chiralt, & GonzálezMartínez, 2011). This may be explained by the heterogeneous film
structure featuring discontinuities in presence of essential oil (Fig. 2).
Furthermore, stronger intermolecular polysaccharide interactions can
be partially replaced by the weaker polysaccharide-essential oil interactions, generating more flexible domains within the film (Li et al.,
2018; Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015). On the

attributed to the essential oil droplets (refractive index of 1.472) distributed throughout the polymer matrix (refractive index of 1.450),
promoting light scattering. Essential oils dispersed in the polymeric
matrix promotes an increase of light scattering and consequently, in the
opacity of the films. This behavior is due to change in the film refractive
index at the polymer interface promotes promoted by essential oils
addition (Atarés & Chiralt, 2016; Valencia-Sullca, Vargas, Atarés, &
Chiralt, 2018).
Opacity is an important property because the amount of light that
affects food and the appearance of packaged products is relevant to
consumer acceptance (Villalobos, Chanona, Hernandez, & Gutierrez,
2005).
3.7. Mechanical properties of films with OEO
The incorporation of OEO increased the thickness of the films
(Table 4). Luís, Pereira, Domingues, and Ramos (2019)) also reported

Fig. 4. Photographs of the corn starch films with 0.0 (a) 0.3 (b) 0.7 (c) and 0.9 μL g−1 (d) of orange essential oil.
6

Carbohydrate Polymers 222 (2019) 114981

J.A. do Evangelho, et al.

increase in solubility may due the rupture of the films, easing the water
insertion in the polymeric matrix and also with increase of thickness
and irregular surface structures of the films, increasing the contact area
of film and water (Song et al., 2018). The high solubility may be beneficial for the application of the films in fruits and vegetables, for later
removal of the same (Wang et al., 2011).
The PWV of the starch films with and without OEO increased from
2.82 to 4.53 g.mm/m2·day·kPa, with the OEO films showing higher
PWV than the control. The increase in the PWV of the films is related to
the formation of cavities (Fig. 2) that caused changes in the structural
integrity of the films, increasing the amount of free spaces in the
polymer network and facilitating the passage of water vapor.
Ghasemlou et al. (2013) observed a reduction in the PWV of corn-starch
films incorporated with essential oils of Z. multiflora and M. pulegium.
These authors related this behavior to the presence of hydrogen interactions between the starch network and polyphenolic compounds of the
oils. These interactions may limit the availability of hydrogen groups to
form hydrophilic bonds with water and then lead to a decrease in the
affinity of the film for water.

Fig. 5. Stress vs strain curves of the corn starch films with 0.0, 0.3, 0.5 and
0.7 μL g-1 of orange essential oil.

4. Conclusion
Table 5
Moisture content, water solubility and water vapor permeability (WVP) of corn

starch films with and without orange essential oil (OEO).
OEO (μL/g)a

Moisture (%)

0.0
0.3
0.5
0.7

18.81
18.39
21.67
21.93

±
±
±
±

0.80b
1.77b
0.44a
0.90a

Water solubility (%)
15.25
18.27
18.38
18.67

±
±
±
±

0.70b
1.11a
0.52a
0.66a

The major component of OEO was D-limonene, and it showed higher
antimicrobial activity against S. aureus and L. monocytogenes. The starch
films with OEO were effective against L. monocytogenes and S. aureus,
and the antimicrobial activity was higher against L. monocytogenes than
S. aureus. The starch films with OEO, regardless of the OEO concentration used, showed porosity in their morphological structure.
Addition of OEO reduced the tensile strength and elongation of the
films and increased the moisture, water solubility, and PWV. The results
of this study suggest that starch films incorporated with OEO have
potential for use as bioactive films. However, the applications of these
films need to be evaluated further to analyze their efficiency in food
bioconservation.

WVP (g.mm/m2.day.kPa)
2.82
3.90
4.44
4.53

±

±
±
±

0.69b
0.62ab
0.61ab
0.28a

a
The results are the average of three determinations. Values with different
letters in the same column are significantly different (p < 0.05).

other hand, essential oils increase the elongation due to its plasticizing
effect (Lee, Garcia, Chin, & Kim, 2019; Song et al., 2018). Nevertheless,
the elongation of the films with OEO was substantially lower than native film.
Stress vs strain curves of the films can be visualized in Fig. 5. Briefly,
the results shown in this study indicate that increase of essential oil
decrease the strength of the films but enhance their flexibility. These
characteristics may contribute to predicting their possible applications
as a packaging material.

Acknowledgements
This study was financed in part by the Coordenaỗóo de
Aperfeiỗoamento
de
Pessoal
de
Nớvel
SuperiorBrasil

(CAPES)Finance Code 001, CNPq, FAPERGS and the Center of
Southern Electron Microscopy (CEME-SUL) of the Federal University of
Rio Grande (FURG).

3.8. Moisture, water solubility, and PWV of the films with OEO

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8