Carbohydrate Polymers 222 (2019) 114981

Contents lists available at ScienceDirect

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

Antibacterial activity, optical, mechanical, and barrier properties of corn T

starch films containing orange essential oil
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
Department of Agroindustrial Science and Technology, Federal University of Pelotas, Rio Grande do Sul, Pelotas, RS 96010-900, Brazil
b
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: The incorporation of antimicrobial compounds into natural polymers can promote increased shelf life and ensure
Orange essential oil food safety. The aim of this study was to evaluate the antibacterial activity, morphological, optical, mechanical,
Starch films and barrier properties of corn starch films containing orange (Citrus sinensis var. Valencia) essential oil (OEO).
Antibacterial activity The corn starch films were prepared using the casting method. OEO and the corn starch films incorporated with
Mechanical properties
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 the fruit exocarp, which is composed of the epidermis and a layer of
glandular cells. According to Mahato, Sharma, Sinha, and Cho (2018),
Because consumers are concerned about reducing the use of syn- large volumes of by-products are generated during the processing of
thetic additives, there is particular interest in the food industry for oranges, and they can be potentially used in the food industry for the
using natural preservatives that can maintain food freshness and quality extraction of essential oil. In a study on essential oils from plants that
and have no effects on human health (Atarés & Chiralt, 2016). New belong to the genus Citrus, including orange essential oil (OEO), against
technologies for active food packaging have been studied, and they can different food-borne pathogens, OEO exhibited antibacterial activity
protect and interact with the food, increasing its useful life (Adilah, against both gram-positive and gram-negative bacteria (Frassinetti,
Jamilah, Noranizan, & Hanani, 2018) and ensuring its safety Caltavuturo, Cini, Della Croce, & Maserti, 2011). Torrez-Alvarez et al.
(Dannenberg et al., 2017). (2017) also reported results that proved the antibacterial and anti-
Antimicrobial films have active compounds that are released into oxidant potential of OEO, highlighting it as an alternative for the de-
the food when the films touch the surface of the product (Guo, Yadav, & velopment of safer products accepted by consumers who prefer natural
Jin, 2017). Essential oils are active compounds that, in addition to ingredients.
providing antibacterial protection (Kumar, Narayani, Subanthini, & The incorporation of active substances into starch films has been
Jayakumar, 2011), can improve the functional and mechanical prop- studied by several researchers (Acosta et al., 2016; Sapper, Wilcaso,
erties of the films (Qin, Li, Liu, Yuan, & Li, 2017). These compounds can Santamarina, Roselló, & Chiralt, 2018; Song, Zuo, & Chen, 2018). The
have antifungal activities (Ribeiro-Santos, Andrade, & Sanches-Silva, production of films with natural polymers offers an alternative to syn-
2017) as well as antioxidant and anti-inflammatory effects (Liu, Xu, thetic packaging (Romani, Prentice-Hernández, & Martins, 2018).
Cheng, Yao, & Pan, 2012). Polysaccharides, proteins, and lipids used alone or in combination have
Orange (Citrus sinensis) is a source of essential oil concentrated in the ability to form biodegradable and/or edible films (Kim, Yang, Chun,

⁎
Corresponding author.
E-mail addresses: jarineamaral@hotmail.com (J.A. do Evangelho), gui.dannenberg@gmail.com (G. da Silva Dannenberg), babibiduski@hotmail.com (B. Biduski),
shanisemell@hotmail.com (S.L.M. el Halal), dianinikringel@hotmail.com (D.H. Kringel), marciagularte@hotmail.com (M.A. Gularte),
angefiore@gmail.com (A.M. Fiorentini), elessandrad@yahoo.com.br (E. da Rosa Zavareze).

https://doi.org/10.1016/j.carbpol.2019.114981
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.
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

& Song, 2018). Among polysaccharides, starch has been widely used for susceptibility against bacterial and yeasts. This procedure is performed
the production of films because of the low cost of production from re- by agar plates inoculation containing a standardized inoculum of the
newable sources (Khalid et al., 2018) and its properties that favor the test microorganism and of the test compound. The antimicrobial agent
formation of films (Luchese, Garrido, Spada, Tessaro, & La Caba, 2018). inhibits germination and growth of the test microorganism, diffusing
The antimicrobial properties of several essential oils have been into the agar; the results are expressed by measurement of the
widely studied as additives in biodegradable films, their effects on the diameters of inhibition growth zones (Balouiri, Sadiki, & Ibnsouda,
properties of films is still less discussed in the literature. Essential oils 2016). Bacterial cultures (L. monocytogenes, S. aureus, B. cereus, P.
have an oily and volatile nature which may affect the integrity or de- aeruginosa, S. dysenteriae, E. coli, and S. typhimurium) were suspended in
gree of hydrophobicity of polymeric films, changing their mechanical peptone water (0.1%), and a concentration of 108 UFC/g (0.5
and barrier properties (Abdollahi, Damirchi, Shafafi, Rezaei, & Ariaii, McFarland) was achieved. The inoculum was seeded with sterile
2018; Atarés & Chiralt, 2016). Therefore, studies are needed to examine swabs on the surface of MH agar in petri dishes, on which sterile
the potential of each antibacterial agent as well as its interaction with paper disks (Laborclin®) were arranged. An aliquot of 10 μL of OEO
the material used to produce the active starch films. The aim of this was added to each disc (in triplicate; three discs per bacterium) and
study was to evaluate the antimicrobial activity of the OEO and its allowed to stand for 1 h for absorption; thereafter, the plates were
effect on the optical, microstructural, and mechanical and barrier incubated at 37 °C. After 24 h, the formation of inhibition halos was
properties of the biodegradable films of corn starch. 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
2.1. Material the lowest concentration of agent antimicrobial able to inhibit the
visible microbial growth and minimum bactericidal concentration
In this study, oranges (Citrus sinensis ‘Valencia’) harvested in 2017 in (MBC) is the lowest concentration of agent antimicrobial able to kill
the city of Pelotas, southern region of Rio Grande do Sul, Brazil, were 99.9% after incubation for determined time (24 h) (Balouiri et al.,
used. Brain heart infusion (BHI) broth (Acumedia®) and Mueller-Hinton 2016).
(MH) agar (Oxoid®) were used for the microbiological analyses. The minimum inhibitory concentration (MIC) was determined using
Commercially available corn starch (A-type crystallinity standard), 28% the plaque microdilution test (CLSI, 2015a). The analysis was per-
amylose (as described by McGrane, Cornell and Rix (1998)), and ge- formed in triplicate. OEO was diluted in BHI broth with 3% Tween 20
latinization peak of 69.9 °C (evaluated using a differential scanning (Vetec®), and concentrations of 166.7 to 0.3 μL mL−1 were obtained.
calorimeter; TA-60WS, Shimadzu, Kyoto, Japan). The bacteria (L. monocytogenes, S. aureus, B. cereus, P. aeruginosa, S.
dysenteriae, E. coli, and S. typhimurium) were added to obtain a final
2.2. Bacteria 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
Seven bacteria of relevance to food were used: three gram-positive spectrophotometer (Robonik® Readwel plate) at 625 nm, considering
bacteria, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC the highest dilution at which no cell growth was observed as MIC
6538, and Bacillus cereus ATCC 11778, and four gram-negative bacteria, (Ojeda-Sana, Baren, Elechosa, Juaréz, & Moreno, 2013).
Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 8739, The minimum bactericidal concentration (MBC) was determined
Shigella dysenteriae ATCC 13313, and Pseudomonas aeruginosa ATCC using 10 μL aliquots inoculated on BHI agar plates and considering the
15442. lowest concentration at which no growth was observed as MBC.

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.
OEO was extracted by hydrodistillation in a Clevenger apparatus monocytogenes and S. aureus), according to the methodology of Diao,
(Kringel et al., 2017). The fresh shells of the oranges were ground in Hu, Zhang, and Xu (2014)). OEO was added to BHI broth containing 3%
distilled water (ratio w/v = 1/10) and extracted for 3 h at 100 °C. The Tween 20, and the MBC of OEO (5.208 μL mL−1) was obtained. The
obtained essential oil was dehydrated with anhydrous sodium sulfate pathogens were inoculated at 104 CFU mL−1 and incubated at 37 °C
(Na2SO4; SYNTH®) and stored in an amber glass vial at −80 °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
2.4. Characterization of OEO 0.1 mL aliquots were plated on BHI agar. A control treatment was
performed under the same conditions, but without the addition of OEO.
2.4.1. Chemical composition of OEO The counts for each time were used to obtain the kinetics of action as
The chemical composition of OEO was determined using a gas well as the time required to promote bactericidal action on all the cells.
chromatograph coupled to a mass detector (GC/MS; QP 2010SE; The analysis was performed in triplicate.
Shimadzu®) equipped with an RTX-5MS (Restek®) capillary column
(30 m × 0.25 mm ×0.25 μm). The volume of the injected sample was 2.5. Production of films
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 The films were produced using the casting technique, according to
initially maintained at 60 °C for 2 min and gradually increased at a rate Souza, Goto, Mainardi, Coelho, and Tadin (2013) with some mod-
of 4 °C min−1 until it reached 220 °C. Identification of the compounds ifications. The filmogenic solution was prepared with 3% (w/v) starch
was based on the mass spectra (as compared with the Wiley 275 in distilled water and 30% (w/w) glycerol (relative to dry starch mass).
spectral library, 6th edition), and the concentrations were presented as The film-forming solutions were heated in a jacketed glass reactor, with
relative percentages of the area of each peak over the total area. 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
2.4.2. Antimicrobial activity of OEO and homogenized in an Ultraturrax at 14,000 rpm for 10 min. Then,
2.4.2.1. Agar diffusion. The determination of OEO action spectrum was 20 g of each solution was spread on acrylic plates (9 cm in diameter)
performed using the agar diffusion technique (CLSI, 2015b). Agar disk- and dried in an oven with air circulation at 30 °C for 16 h. After drying,
diffusion is an oft-employed method to determine the antimicrobial the films were conditioned at 16 °C and 58% relative humidity until

2
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

further use. Fm
TS=
A (3)

2.6. Characterization of the films 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).
2.6.1. Morphology Eq. (4)
The morphology of the surface and transverse sections of the films dr
was evaluated using scanning electron microscopy (SEM; JEOL, JSM- E= × 100
di
6610LV, Japan). Samples of the films were coated onto the surface of
double-sided carbon tapes adhered to stubs and coated with a gold layer where: E is elongation (%); di is the initial separation distance (cm); and
by using a vacuum metallizer (Denton Desk V; Denton Vacuum, USA). dr is the distance at the moment of rupture (cm).
SEM was performed with a 10 kV electron beam. For the cross-section
analysis, the films were fractured with liquid nitrogen. The surfaces and 2.6.5. Moisture content and water solubility of the films
cross-sections of the films were analyzed at 70× and 500× magnifi- The moisture content of the films was determined using the AACC
cations, respectively. (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
2.6.2. Antibacterial activity
according to the method proposed by Gontard, Duchez, Cuq, and
About 0.1 mL aliquots of the cell suspensions (103 CFU·mL−1) of the
Guilbert, 1994). Disk samples with a diameter of 2.5 cm were used. The
two OEO-sensitive bacteria (L. monocytogenes and S. aureus) were in-
samples were dried in an oven at 105 °C until constant dry mass to
oculated on the surface of BHI agar in petri dishes. After absorption of
remove moisture. Then, they were immersed in a Falcon tube with
the inoculum, the entire surface of the agar was covered with OEO-
50 mL of distilled water. The tube was shaken (175 rpm) in a shaker for
containing films (0.3, 0.5, and 0.7 μL g−1). Control treatments for each
24 h at 25 °C. Then, the samples were oven-dried at 105 °C until con-
bacterium were performed similarly, but without the addition of the
stant weight to determine the final dry mass of the sample. The solu-
films. The plates were incubated at 37 °C for 24 h, and the percentage
bility was expressed in terms of the solubilized mass (SM) of the film,
difference between bacterial colony counts of the treatments and con-
according to Eq. (5).
trols was used to express growth inhibition. Three replicates were
performed for each tested bacterium. ( initial mass-final mass) × 100
SM (%) =
initial mass (g) (5)
2.6.3. Film color and opacity
The color and opacity of the films were determined by averaging 2.6.6. Water vapor permeability of the films
five values, one in the center and the other in the perimeter, using a The permeability to water vapor (PWV) was determined using the
colorimeter (MINOLTA, CR 400, Japan). The films were placed on a ASTM method E-96-95 (ASTM, 1995) at 25 °C. The films were sealed
white plate defined as standard and illuminant D65 (daylight) for de- with paraffin on aluminum permeation cells containing calcium
termination of color parameters. The parameter L* indicates clarity, chloride (0% relative humidity). The permeation cells were conditioned
which varies from 0 (black) to 100 (white); parameters a* and b* are in desiccators containing saline saturated with sodium chloride at room
the chromaticity coordinates, where a* varies from green (-) to red (+) temperature and 75% relative humidity. The mass gain of the system
and b* varies from (-) to yellow (+). The total color difference (ΔE) was was measured for 2 days. The evaluations were performed in triplicate,
calculated using Eq. (1). and PWV was calculated using Eq. (6).

ΔE= (ΔL2 + Δa2 + Δb)0.5 (1) ΔW X

PWV= ×
t AΔP (6)
where: ΔL = Lstandard – Lsample; Δa = astandard - asample; Δb = bstandard –
where: PWV is permeability to water vapor (g·mm/kPa·dia·m ); ΔW is 2
bsample.
mass gain (g); X is film thickness (mm); t is time (days); A = exposed
Opacity was calculated as the relation between the opacity of the
area (m2); and ΔP is the partial pressure difference (kPa).
film superimposed on the black standard (Sblack) and white standard
(Swhite), according to Eq. (2) (Hunterlab, 1997).
2.7. Statistical analysis
SBlack
Opacity= ×100
SWhite (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 3. Results and discussion
mean of eight random measurements of their surface by using a digital
micrometer (INSIZE model), and the results were expressed in mm. 3.1. Chemical composition of OEO
The mechanical properties (tensile strength and percentage of
elongation) of the films were determined using a texturometer GC-MS analysis identified the presence of seven components in OEO
(TA.XTplus, Stable Micro Systems, UK), according to the ATM D 882 (Table 1). The major compounds of OEO were 96% D-limonene and
method (ASTM, 1995) with initial grips separation at 50 mm and probe 2.6% β-myrcene. O’Bryan, Crandall, Chalova, and Ricke (2008) also
speed of 1 mm.s−1. Six to 10 samples of each film were trimmed reported the D-limonene (93.9%) and β-myrcene (2.1%) as the main
(85 mm × 25 mm) and fixed in the texturometer. The tensile strength constituents of OEO. Five other minor compounds were also identified
was calculated by dividing the maximum force at the breakage of the (in the decreasing order of concentration): octanal, α-pinene, β-linalool,
film by the cross-sectional area (Eq. (3)). The elongation was de- cyclohexene, and decanal (Table 1).
termined by dividing the final separation distance of the probe by the D-limonene usually exhibits antimicrobial and antiseptic activities
initial separation distance (50 mm) and multiplying by 100 (Equation (Hąc-Wydro, Flasiński, & Romańczuk, 2017; Umagiliyage, Becerra-
4). The mean thickness required for the sectional area calculation was Mora, Kohli, Fisher, & Choudhary, 2017; Zahi, El Hattab, Liang, &
determined using eight measurements obtained throughout the sample. Yuan, 2017). This compound has been reported to have applications in

3
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

Table 1 the hydrophobic character of LPS hindered the penetration of the

Chemical composition of orange essential oil (OEO). apolar components of OEO.
Peak number Retention time (min) Compound Peak area (%)
3.3. Action kinetics of OEO
1 5.14 α-pineno 0.53
2 6.23 ciclohexeno 0.29
The OEO action kinetics (Fig. 1) showed a similar behavior for S.
3 6.80 β-mirceno 2.35
4 7.15 octanal 0.55
aureus and L. monocytogenes, both of which showed a gradual reduction
5 8.20 d-limoneno 95.96 in viable cell count over OEO exposure time (MBC = 5.21 μL·mL−1); a
6 10.34 β-linalol 0.45 lethal effect was observed at 12 h of contact. L. monocytogenes was more
7 13.86 decanal 0.26 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 pharmaceutical and food industries (Chen et al., 2018; Li & Lu,
The kinetics of action of an antimicrobial depends on factors such as
2016). In humans, limonene is rapidly absorbed in the gastrointestinal
the cellular concentration of the bacterium under study and con-
tract and easily metabolized (Filipowicz, Kaminski, Kurlenda,
centration and mechanism of action of the component under study
Asztemborska, & Ochocka, 2003).
(Wang et al., 2011). Because essential oils are composed of different
β-Myrcene, the second major component of OEO, also has anti-
molecules, their mechanisms of action are attributable to both in-
microbial activity. Dannenberg et al. (2017) studied the essential oil
dividual action of each component on specific cellular targets and a
composition of pink pepper and found that β-myrcene (41%) was the
synergistic antimicrobial effect of all the compounds (Burt, 2004). An
major compound; cellulose acetate films containing this oil showed
OEO concentration of 5.21 μL mL−1 was able to cause the death of a
high antibacterial activity against S. aureus, L. monocytogenes, and B.
bacterium in 12 h of contact, and the initial concentrations of S. aureus
cereus.
and L. monocytogenes were more than 104 CFU·mL−1.

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 The morphology of the surfaces and cross-sections of the corn starch
three gram-positive bacteria, with inhibition halos of 10.59, 10.10, and films without and with different OEO concentrations are shown in
9.99 mm for L. monocytogenes, S. aureus, and B. cereus, respectively. The Fig. 2. The film without OEO presented a smooth and uniform surface
gram-negative bacteria P. aeruginosa and S. dysenteriae also showed (Fig. 2a). The addition of OEO in the films, regardless of the con-
sensitivity to OEO, presenting inhibition halos of 9.30 and 8.73 mm, centration, reduced the homogeneity of the cross-sections (Fig. 2f–h),
respectively. E. coli and S. typhimurium were not sensitive to OEO under with presence of more concentrated pores on the surface. The hydro-
the test conditions. phobicity of the oil and its density difference with the aqueous solution
MICs of up to 2.60 μL·mL−1 OEO were able to promote a bacterio- of starch can affect the stability of the filmogenic solution and conse-
static effect against L. monocytogenes and S. aureus, whereas the MIC for quently form heterogeneous structures because of the separation of
B. cereus was 5.21 μL·mL−1 ((Table 2). The MICs for gram-negative phases and presence of pores (Phan et al., 2002). These heterogeneities,
bacteria P. aeruginosa and S. dysenteriae were 10.42 and 41.67 μL·mL−1, such as the presence of preferential pathways (pores) shown in
respectively, and the values were higher than those found for the gram- Fig. 2f–h, may contribute to the antibacterial property of the films,
positive bacteria. considering that they facilitate the diffusion process of the essential oil
OEO concentrations up to 5.21 μL·mL−1 demonstrated a bactericidal from the interior of the polymer matrix to the surface to perform the
effect against the three gram-positive bacteria. For the gram-negative desired action.
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; 3.5. Antimicrobial activity of the OEO films
Table 2).
In the present study, it was possible to observe that the gram-ne- The starch films without OEO promoted a reduction of 16% and
gative bacteria were more resistant to OEO than the gram-negative 22% in the development of S. aureus and L. monocytogenes, respectively,
bacteria (Burt, 2004; Dannenberg et al., 2017; Silva et al., 2018). The when compared with the control (without film application; Fig. 3). This
gram-negative bacteria have a double outer phospholipid layer in their result indicates that the direct contact promoted by the coating of the
cell walls, which is composed of lipopolysaccharides (LPS); however, contaminated surface (agar) with the film promotes a physical im-
the gram-positive bacteria do not have this external layer, and their cell pediment to the development of the colonies, considering the inert
walls are mainly composed of peptidoglycan (90–95%) (Nazzaro, (non-antimicrobial) characters of starch and other components present
Fratianni, De Martino, Coppola, & De Feo, 2013). It is also possible that in the filmogenic solution.
The addition of OEO in the polymeric matrix of the film, at all
Table 2 evaluated concentrations, promoted the inhibition of both pathogens
Antimicrobial activity of orange essential oil (OEO). (Fig. 3). OEO concentrations of 0.3, 0.5, and 0.7 μL·g−1 reduced the
Bacteriaa ATCC Diffusion agar (mm) MIC (μL/mL) MBC (μL/mL) development of L. monocytogenes by 68, 80, and 83%, and the devel-
opment of S. aureus by 40, 51, and 66%, respectively. The increase in
Gram-positive OEO concentration resulted in a directly proportional increase in viable
L. monocytogenes 7644 10.59 ± 0.43 2.60 ± 0.00 5.21 ± 0.00
cell reduction in both pathogens. The lower concentration of OEO in the
S. aureus 6538 10.10 ± 0.88 2.60 ± 0.00 5.21 ± 0.00
B. cereus 11778 9.99 ± 0.18 5.21 ± 0.00 5.21 ± 0.00 films (0.3 μL g−1) was able to significantly reduce (p ≤ 0.05) the counts
Gram-negative of L. monocytogenes, when compared with the film without OEO. Only
P. aeruginosa 15442 9.30 ± 0.31 10.42 ± 0.00 20.83 ± 0.00 the highest OEO concentration (0.7 μL g−1) resulted in significant re-
S. dysenteriae 8739 8.73 ± 0.56 41.67 ± 0.00 41.67 ± 0.00 ductions (p ≤ 0.05) in the counts of S. aureus. These results demon-
E. coli 13313 ND ND ND
S. Typhimurium 14028 ND ND ND
strate that starch is a suitable polymer matrix for the incorporation of
antimicrobial agents such as OEO because it was able to store/en-
a
Values expressed as mean (n = 3) ± Standard deviation; ND = Not capsulate OEO and release it during direct contact with the con-
Detected. taminated surface of the medium (agar).

4
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

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 com-
pared 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 sen-
sorial 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 Fig. 3. Antimicrobial activity of the films with OEO on the growth of S. aureus
films with or without OEO are listed in Table 3. The brightness (L) of ATCC 6538 and L. monocytogenes ATCC 7644. Results expressed as means
the films ranged from 96.38 to 96.80 (Table 2). The films with 0.3 and (n = 3) ± standard deviation; Different lowercase letters indicate significant
0.7 μL of OEO showed higher values of a* and b* (coordinates re- difference between OEO concentrations for the same bacterium; Different up-
sponsible for chromaticity), indicating a tendency to green and yellow. percase letters indicate significant difference between the bacteria for the same
OEO addition increased the opacity of the films with higher OEO concentration of 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. 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
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

Table 3 Table 4
Color parameters (L*, a* and b*) and opacity of the corn starch films with and Thickness, tensile strength and percent elongation of starch films with and
without orange essential oil (OEO). without orange essential oil (OEO).
OEO (μL/ L* a* b* Opacity (%) OEO (μL/g)a Thickness (mm) Tensile strength (MPa) Elongation (%)
g)a
0.0 0.084 ± 0.008c 5.11 ± 0.57a 64.58 ± 8.95a
0.0 96.38 ± 0.06b −0.16 ± 0.00b 2.63 ± 0.02b 10.86 ± 0.37b 0.3 0.112 ± 0.016b 4.08 ± 0.40b 9.94 ± 0.46b
0.3 96.41 ± 0.11b −0.32 ± 0.03a 2.76 ± 0.11ab 12.02 ± 1.16b 0.5 0.142 ± 0.024a 2.73 ± 0.20c 12.64 ± 3.45b
0.5 96.55 ± 0.03b −0.15 ± 0.02b 2.51 ± 0.06b 13.07 ± 1.52ab 0.7 0.131 ± 0.020a 2.40 ± 0.46c 15.25 ± 2.85b
0.7 96.80 ± 0.02ª −0.33 ± 0.06a 2.97 ± 0.16a 16.24 ± 1.87a
a
The results are the average of three determinations. Values with different
a
The results are the average of three determinations. Values with different letters in the same column are significantly different (p < 0.05).
letters in the same column are significantly different (p < 0.05).
an increase in film thickness with the addition of licorice essential oil
attributed to the essential oil droplets (refractive index of 1.472) dis- (Glycyrrhiza glabra L.) and attributed this behavior to the entrapment of
tributed throughout the polymer matrix (refractive index of 1.450), essential oil microdroplets into the polymeric matrix, thereby in-
promoting light scattering. Essential oils dispersed in the polymeric creasing the compactness of the starch matrix structure.
matrix promotes an increase of light scattering and consequently, in the The mechanical characteristics of films are important because they
opacity of the films. This behavior is due to change in the film refractive are related to the end-use characteristics of these materials, such as
index at the polymer interface promotes promoted by essential oils strength and elongation (Bastos et al., 2016). The tensile strength and
addition (Atarés & Chiralt, 2016; Valencia-Sullca, Vargas, Atarés, & elongation of the films ranged from 2.40 MPa to 5.11 MPa and from
Chiralt, 2018). 9.94% to 64.5%, respectively (Table 4).
Opacity is an important property because the amount of light that In our study, as in the majority reported research, decreases in
affects food and the appearance of packaged products is relevant to strength upon essential oil incorporation are evidenced (Li, Ye, Lei, &
consumer acceptance (Villalobos, Chanona, Hernandez, & Gutierrez, Zhao, 2018; Sánchez-González, Cháfer, Hernández, Chiralt, & González-
2005). Martínez, 2011). This may be explained by the heterogeneous film
structure featuring discontinuities in presence of essential oil (Fig. 2).
3.7. Mechanical properties of films with OEO Furthermore, stronger intermolecular polysaccharide interactions can
be partially replaced by the weaker polysaccharide-essential oil inter-
The incorporation of OEO increased the thickness of the films actions, generating more flexible domains within the film (Li et al.,
(Table 4). Luís, Pereira, Domingues, and Ramos (2019)) also reported 2018; Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015). On the

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
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

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 ben-
eficial 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 inter-
actions 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
Fig. 5. Stress vs strain curves of the corn starch films with 0.0, 0.3, 0.5 and
affinity of the film for water.
0.7 μL g-1 of orange essential oil.

4. Conclusion
Table 5
Moisture content, water solubility and water vapor permeability (WVP) of corn The major component of OEO was D-limonene, and it showed higher
starch films with and without orange essential oil (OEO). antimicrobial activity against S. aureus and L. monocytogenes. The starch
OEO (μL/g)a Moisture (%) Water solubility (%) WVP (g.mm/m2.day.kPa) films with OEO were effective against L. monocytogenes and S. aureus,
and the antimicrobial activity was higher against L. monocytogenes than
0.0 18.81 ± 0.80b 15.25 ± 0.70b 2.82 ± 0.69b
S. aureus. The starch films with OEO, regardless of the OEO con-
0.3 18.39 ± 1.77b 18.27 ± 1.11a 3.90 ± 0.62ab
0.5 21.67 ± 0.44a 18.38 ± 0.52a 4.44 ± 0.61ab centration used, showed porosity in their morphological structure.
0.7 21.93 ± 0.90a 18.67 ± 0.66a 4.53 ± 0.28a Addition of OEO reduced the tensile strength and elongation of the
films and increased the moisture, water solubility, and PWV. The results
a
The results are the average of three determinations. Values with different of this study suggest that starch films incorporated with OEO have
letters in the same column are significantly different (p < 0.05). potential for use as bioactive films. However, the applications of these
films need to be evaluated further to analyze their efficiency in food
other hand, essential oils increase the elongation due to its plasticizing bioconservation.
effect (Lee, Garcia, Chin, & Kim, 2019; Song et al., 2018). Nevertheless,
the elongation of the films with OEO was substantially lower than na- Acknowledgements
tive film.
Stress vs strain curves of the films can be visualized in Fig. 5. Briefly, This study was financed in part by the Coordenação de
the results shown in this study indicate that increase of essential oil Aperfeiçoamento de Pessoal de Nível Superior—Brasil
decrease the strength of the films but enhance their flexibility. These (CAPES)—Finance Code 001, CNPq, FAPERGS and the Center of
characteristics may contribute to predicting their possible applications Southern Electron Microscopy (CEME-SUL) of the Federal University of
as a packaging material. Rio Grande (FURG).

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

The moisture content, water solubility, and PWV of the films are AACC- AMERICAN ASSOCIATION OF CEREAL CHEMISTS (1995). (9ª ed.). Approved
presented in Table 5. The starch films incorporated of 0.5 and 0.7 μL of methods of the american association of cereal chemistsVol. 1 and 2 St. Paul.
Abdollahi, M., Damirchi, S., Shafafi, M., Rezaei, M., & Ariaii, P. (2018). Carboxymethyl
OEO presented higher humidity in relation to the films without OEO cellulose-agar biocomposite film activated with summer savory essential oil as an
and 0.3 μL of OEO (Table 5). The increase in the moisture content of the antimicrobial agent. International Journal of Biological Macromolecules.
films with OEO may be related to the rupture of the films (Fig. 2). The Acosta, S., Chiralt, A., Santamarina, P., Rosello, J., Gonzalez-Martínez, C., & Cháfer, M.
(2016). Antifungal films based on starch-gelatin blend, containing essential oils. Food
formation of a porous structure in the starch films with OEO facilitates Hydrocolloids, 61, 233–240.
the insertion of water molecules between the polymer chains. Adilah, A. N., Jamilah, B., Noranizan, M. A., & Hanani, Z. A. N. (2018). Utilization of
On the other hand, Ghasemlou et al. (2013) observed that the ad- mango peel extracts on the biodegradable films for active packaging. Food Packaging
and Shelf Life, 16, 1–7.
dition of essential oils from the plants Zataria multiflora Boiss and ASTM (1995). Standard test methods of water vapor trans- mission of materials. Annual book
Mentha pulegium in starch films decreased the moisture content. Ac- of ASTM standards. Philadelphia, E: American Society for Testing and Materials 96-
cording to these authors, the incorporation of hydrophobic essential oils 95, p.
Atarés, L., & Chiralt, A. (2016). Essential oils as additives in biodegradable films and
may affect the film’s ability to retain water and consequently decrease
coatings for active food packaging- review. Trends in Food Science & Technology, 48,
its moisture. 51–62.
Solubility is a factor that directs the application of the film as Balouiri, M., Sadiki, M., & Ibnsouda, S. K. (2016). Methods for in vitro evaluating anti-
packaging for food products. The films with OEO, regardless of OEO microbial activity: A review. Journal of Pharmaceutical Analysis, 6(2), 71–79.
Bastos, M. S. R., Laurentino, L. S., Canuto, K. M., Mendes, L. G., Martins, C. M., Silva, S. M.
concentration, showed higher solubility (Table 5) than the films F., et al. (2016). Physical and mechanical testing of essential oil-embedded celulose
without OEO. Overall, the solubility of films depend the type and ester films. Polymer Testing, 49, 156–161.
concentration of the compounds as well their hydrophilicity and hy- Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in
foods: A review. International Journal of Food Microbiology, 94, 223–253.
drophobicity indices. Therefore, hydrophilic compounds tend to in- Caetano, K., dos, S., Hessel, C. T., Tondo, E. C., Flôres, S. H., & Cladera-Olivera, F. (2017).
crease the solubility values, whereas hydrophobic compounds decrease Application of active cassava starch films incorporated with oregano essential oil and
these values (Caetano et al., 2017; Ghasemlou et al., 2013). The pumpkin residue extract on ground beef. Journal of Food Safety, 37(4), 12355.

7
J.A. do Evangelho, et al. Carbohydrate Polymers 222 (2019) 114981

Calo, J. R., Crandall, P. G., O’Bryan, C. A., & Ricke, S. C. (2015). Essential oils as anti- growth of foodborne pathogens. LWT- Food Science and Technology, 111, 218–225.
microbials in food systems - A review. Food Control, 54, 111–119. Mahato, N., Sharma, K., Sinha, M., & Cho, M. H. (2018). Citrus waste derived nutra-/
Chen, Y., Shu, M., Yao, X., Wu, K., Zhang, K., He, Y., et al. (2018). Effect of zein-based pharmaceuticals for health benefits: Current trends and future perspectives. Journal
microencapsules on the release and oxidation of loaded limonene. Food Hydrocolloids, of Functional Foods, 40, 307–316.
84, 330–336. Malhotra, B., Keshwani, A., & Kharkwal, H. (2015). Antimicrobial food packaging:
CLSI (2015b). M02-A12: Performance standards for antimicrobial disk susceptibility tests; Potential and pitfalls. Frontiers in Microbiology, 6. https://doi.org/10.3389/fmicb.
approved standard (twelfth edition). CLSI (Clinical and Laboratory Standards 2015.00611.
Institute)1 35. Mcgrane, S. J., Cornell, H. J., & Rix, C. J. (1998). A simple and rapid colourimetric
CLSI (2015a). M07-A10: Methods for dilution antimicrobial susceptibility tests for Bacteria method for determination of amylose in starch products. Starch/Stärke, 50, 158–163.
That grow aerobically; approved standard (tenth edition). CLSI (Clinical and Laboratory Nazzaro, F., Fratianni, F., De Martino, L., Coppola, R., & De Feo, V. (2013). Effect of
Standards Institute)2 35. essential oils on pathogenic Bacteria. Pharmaceuticals, 6, 1451–1474.
Dannenberg, G. S., Funk, G. D., Cruxen, C. E. S., Marques, J. L., Silva, W. P., & Fiorentini, O’Bryan, C. A., Crandall, P. G., Chalova, V. I., & Ricke, S. C. (2008). Orange essential oils
A. M. (2017). Essential oil from pink pepper as an antimicrobial component in cel- antimicrobial activities against Salmonella spp. Journal of Food Science, 73, 264–267.
lulose acetate film: Potential for application as active packaging for sliced cheese. Ojeda-Sana, A. M., Baren, C. M., Elechosa, M. A., Juaréz, M. A., & Moreno, S. (2013). New
LWT- Food Science and Technology, 81, 314–318. insights into antibacterial and antioxidant activities of rosemary essential oils and
Diao, W.-R., Hu, Q.-P., Zhang, H., & Xu, J.-G. (2014). Chemical composition, antibacterial their main components. Food Control, 31, 189–195.
activity and mechanism of action of essential oil from seeds of fennel (Foeniculum Phan, T. D., Péroval, C., Debeaufort, F., Despré, D., Courthaudon, J. L., & Voilley, A.
vulgare Mill.). Food Control, 35, 109–116. (2002). Arabinoxylan-lipid-based edible films and coatings. Influence of drying
Filipowicz, N., Kaminski, M., Kurlenda, J., Asztemborska, M., & Ochocka, R. J. (2003). temperature on film structure and functional properties. Journal of Agricultural and
Antibacterial and antifungal activity of juniper berry oil and its selected components. Food Chemistry, 50, 2423–2428.
Phytotherapy Research, 17, 227–231. Qin, Y., Li, W., Liu, D., Yuan, M., & Li, L. (2017). Development of active packaging film
Frassinetti, S., Caltavuturo, L., Cini, M., Della Croce, C. M., & Maserti, B. E. (2011). made from poly (lactic acid) incorporated essential oil. Progress in Organic Coatings,
Antibacterial and antioxidant activity of essential oils from Citrus spp. Journal of 103, 76–82.
Essential Oil Research, 23, 27–31. Ribeiro-Santos, R., Andrade, M., & Sanches-Silva, A. (2017). Application of encapsulated
Ghasemlou, M., Aliheidari, N., Fahmi, R., Shojaee-Aliabadi, S., Keshavarz, B., Cran, M. J., essential oils as antimicrobial agents in food packaging. Current Opinion in Food
et al. (2013). Physical, mechanical and barrier properties of corn starch films in- Science, 14, 78–84.
corporated with plant essential oils. Carbohydrate Polymers, 98, 1117–1126. Romani, V. P., Prentice-Hernández, C., & Martins, V. G. (2018). Pink pepper phenolic
Gontard, N., Duchez, C., Cuq, J. L., & Guilbert, S. (1994). Edible composite flms of wheat compounds incorporation in starch/protein blends and its potential to inhibit apple
gluten and lipids, water vapor permeability and other physical properties. browning. Food Packaging and Shelf Life, 15, 151–158.
International Journal of Food Science & Technology, 29, 39–50. Sánchez-González, L., Cháfer, M., Hernández, M., Chiralt, A., & González-Martínez, C.
Guo, M., Yadav, M. P., & Jin, T. Z. (2017). Antimicrobial edible coatings and films from (2011). Antimicrobial activity of polysaccharide films containing essential oils. Food
micro-emulsions and their food applications. International Journal of Food Control, 22(8), 1302–1310.
Microbiology, 263, 9–16. Sapper, M., Wilcaso, P., Santamarina, M. P., Roselló, J., & Chiralt, A. (2018). Antifungal
Hąc-Wydro, K., Flasiński, M., & Romańczuk, K. (2017). Essential oils as food eco-pre- and functional properties of starch-gellan films containing thyme (Thymus zygis)
servatives: Model system studies on the effect of temperature on limonene anti- essential oil. Food Control, 92, 505–515.
bacterial activity. Food Chemistry, 235, 127–135. Silva, F. T. da, Cunha, K. F. da, Fonseca, L. M., Antunes, M. D., Halal, S. L. M. E.,
Hunterlab (1997). The color management company. Reston: Universal software, ver- Fiorentini, Â. M., et al. (2018). Action of ginger essential oil (Zingiber officinale)
sion 3.2. encapsulated in proteins ultrafine fibers on the antimicrobial control in situ.
Khalid, S., Yu, L., Feng, M., Meng, L., Bai, Y., Ali, A., et al. (2018). Development and International Journal of Biological Macromolecules, 118, 107–115.
characterization of biodegradable antimicrobial packaging films based on poly- Song, K., Zuo, G., & Chen, F. (2018). Effect of essential oil and surfactant on the physical
caprolactone, starch and pomegranate rind hybrids. Food Packaging and Shelf Life, 18, and antimicrobial properties of corn and wheat starch films. International Journal of
71–79. Biological Macromolecules, 107, 1302–1309.
Kim, S., Yang, S.-Y., Chun, H. H., & Song, K. B. (2018). High hydrostatic pressure pro- Souza, A. C., Goto, G. E. O., Mainardi, J. A., Coelho, A. C. V., & Tadin, C. C. (2013).
cessing for the preparation of buckwheat and tapioca starch films. Food Hydrocolloids, Cassava starch composite films incorporated with cinnamon essential oil:
81, 71–76. Antimicrobial activity, microstructure, mechanical and barrier properties. LWT - Food
Kringel, D. H., Antunes, M. D., Klein, B., Crizel, R. L., Wagner, R., Oliveira, R. P., et al. Science and Technology, 54, 346–352.
(2017). Production, characterization, and stability of orange or Eucalyptus Essential Tongnuanchan, P., Benjakul, S., Prodpran, T., & Nilsuwan, K. (2015). Emulsion film based
oil/β-Cyclodextrin inclusion complex. Journal of Food Science, 82, 2598–2605. on fish skin gelatin and palm oil: Physical, structural and thermal properties. Food
Kumar, A., Narayani, M., Subanthini, A., & Jayakumar, M. (2011). Antimicrobial activity Hydrocolloids, 48, 248–259.
and phytochemical analysis of citrus fruit peels – Utilization of fruit waste. Torrez-Alvarez, C., Núñez González, A., Rodríguez, J., Castillo, S., LeosRivas, C., & Báez-
International Journal of Engineering, 3, 5414–5421. González, J. G. (2017). Chemical composition, antimicrobial, and antioxidant ac-
Lee, J. Y., Garcia, C. V., Chin, G. H., & Kim, J. T. (2019). Antibacterial and antioxidant tivities of orange essential oil and its concentrated oils. CyTA - Journal of Food, 15,
properties of hydroxypropyl methylcellulose-based active composite films in- 129–135.
corporating oregano essential oil nanoemulsions. LWT- Food Science and Technology, Umagiliyage, A. L., Becerra-Mora, N., Kohli, P., Fisher, D. J., & Choudhary, R. (2017).
106, 164–171. Antimicrobial efficacy of liposomes containing d -limonene and its effect on the
Li, J., Ye, F., Lei, L., & Zhao, G. (2018). Combined effects of octenyl succination and storage life of blueberries. Postharvest Biology and Technology, 128, 130–137.
oregano essential oil on sweet potato starch films with an emphasis on water re- Valencia-Sullca, C., Vargas, M., Atarés, L., & Chiralt, A. (2018). Thermoplastic cassava
sistance. International Journal of Biological Macromolecules, 115, 547–553. starch-chitosan bilayer films containing essential oils. Food Hydrocolloids, 75,
Li, P. H., & Lu, W. C. (2016). Effects of storage conditions on the physical stability of D- 107–115.
limonene nanoemulsion. Food Hydrocolloids, 53, 218–224. Villalobos, R., Chanona, J., Hernandez, P., & Gutierrez, G. A. (2005). Gloss and trans-
Liu, L., Xu, X., Cheng, D., Yao, X., & Pan, S. (2012). Structure-Activity relationship of parency of hydroxypropyl methylcellulose films containing surfactants as affected by
Citrus polymethoxylated flavones and their inhibitory effects on Aspergillus niger. their microstructure. Food Hydrocolloids, 19, 53–61.
Journal of Agricultural and Food Chemistry, 60, 4336–4341. Wang, L., Liu, F., Jiang, Y., Chai, Z., Li, P., Cheng, Y., et al. (2011). Synergistic anti-
Luchese, C. L., Garrido, T., Spada, J. C., Tessaro, I. C., & La Caba, K. (2018). Development microbial activities of natural essential oils with chitosan films. Journal of Agricultural
and characterization of cassava starch films incorporated with blueberry pomace. and Food Chemistry, 59, 12411–12419.
International Journal of Biological Macromolecules, 106, 834–839. Zahi, M. R., El Hattab, M., Liang, H., & Yuan, Q. (2017). Enhancing the antimicrobial
Luís, Â., Pereira, L., Domingues, F., & Ramos, A. (2019). Development of a carboxymethyl activity of d -limonene nanoemulsion with the inclusion of ε-polylysine. Food
xylan film containing licorice essential oil with antioxidant properties to inhibit the Chemistry, 221, 18–23.