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

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).

Contents lists available atScienceDirect

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

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

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 L E I N F O

Keywords:

Orange essential oil

Starch films

Antibacterial activity

Mechanical properties

A B S T R A C T 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 starchfilms containing orange (Citrus sinensis var Valencia) essential oil (OEO) The corn starchfilms 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 thefilms increased the morphological heterogeneity and contributed to the reduction of the tensile strength and elongation of thefilms, and it increased the moisture content, water solubility, and water vapor permeability The water vapor permeability and partial or total solubility of afilm in water prior to consumption

of a product are of interest when thefilm is used as food coating or for encapsulation of specific molecules

1 Introduction

Because consumers are concerned about reducing the use of

syn-thetic 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)

Antimicrobialfilms have active compounds that are released into

the food when thefilms 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

prop-erties of thefilms (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 toMahato, 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 anti-oxidant potential of OEO, highlighting it as an alternative for the de-velopment of safer products accepted by consumers who prefer natural ingredients

The incorporation of active substances into starchfilms has been studied by several researchers (Acosta et al., 2016; Sapper, Wilcaso, Santamarina, Roselló, & Chiralt, 2018;Song, Zuo, & Chen, 2018) The production offilms with natural polymers offers an alternative to syn-thetic packaging (Romani, Prentice-Hernández, & Martins, 2018) Polysaccharides, proteins, and lipids used alone or in combination have the ability to form biodegradable and/or ediblefilms (Kim, Yang, Chun,

https://doi.org/10.1016/j.carbpol.2019.114981

Received 4 April 2019; Received in revised form 6 June 2019; Accepted 6 June 2019

⁎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)

Available online 10 June 2019

0144-8617/ © 2019 Elsevier Ltd All rights reserved

T

& Song, 2018) Among polysaccharides, starch has been widely used for

the production offilms because of the low cost of production from

re-newable sources (Khalid et al., 2018) and its properties that favor the

formation offilms (Luchese, Garrido, Spada, Tessaro, & La Caba, 2018)

The antimicrobial properties of several essential oils have been

widely studied as additives in biodegradablefilms, their effects on the

properties offilms is still less discussed in the literature Essential oils

have an oily and volatile nature which may affect the integrity or

de-gree of hydrophobicity of polymericfilms, 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 biodegradablefilms of corn starch

2 Material and methods

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 byMcGrane, Cornell and Rix (1998)), and

ge-latinization 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

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 ×0.25μm) The volume of the injected sample was

0.1μL 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−1until 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

2.4.2 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

disk-diffusion is an oft-employed method to determine the antimicrobial

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 10μL 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®)

2.4.2.2 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 per-formed in triplicate OEO was diluted in BHI broth with 3% Tween 20 (Vetec®), and concentrations of 166.7 to 0.3μL mL−1were obtained The bacteria (L monocytogenes, S aureus, B cereus, P aeruginosa, S dysenteriae, E coli, and S typhimurium) were added to obtain afinal concentration of 104UFC mL−1in 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 10μL aliquots inoculated on BHI agar plates and considering the lowest concentration at which no growth was observed as MBC

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 ofDiao,

Hu, Zhang, and Xu (2014)) OEO was added to BHI broth containing 3% Tween 20, and the MBC of OEO (5.208μL mL−1

) was obtained The pathogens were inoculated at 104CFU mL−1and 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

2.5 Production offilms Thefilms were produced using the casting technique, according to Souza, Goto, Mainardi, Coelho, and Tadin (2013) with some

mod-ifications The filmogenic solution was prepared with 3% (w/v) starch

in distilled water and 30% (w/w) glycerol (relative to dry starch mass) Thefilm-forming solutions were heated in a jacketed glass reactor, with water circulation at 90 °C for 10 min After cooling, OEO was added to thefilm-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, thefilms were conditioned at 16 °C and 58% relative humidity until

further use.

2.6 Characterization of thefilms

2.6.1 Morphology

The morphology of the surface and transverse sections of thefilms

was evaluated using scanning electron microscopy (SEM; JEOL,

JSM-6610LV, Japan) Samples of thefilms 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, thefilms were fractured with liquid nitrogen The surfaces and

cross-sections of thefilms were analyzed at 70× and 500×

magnifi-cations, respectively

2.6.2 Antibacterial activity

About 0.1 mL aliquots of the cell suspensions (103CFU·mL−1) of the

two OEO-sensitive bacteria (L monocytogenes and S aureus) were

in-oculated on the surface of BHI agar in petri dishes After absorption of

the inoculum, the entire surface of the agar was covered with

OEO-containingfilms (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

con-trols was used to express growth inhibition Three replicates were

performed for each tested bacterium

2.6.3 Film color and opacity

The color and opacity of thefilms were determined by averaging

five values, one in the center and the other in the perimeter, using a

colorimeter (MINOLTA, CR 400, Japan) Thefilms were placed on a

white plate defined as standard and illuminant D65 (daylight) for

de-termination 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)

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)

Opacity SBlack

2.6.4 Thickness and mechanical properties of thefilms

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) andfixed 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

de-termined by dividing thefinal 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

=

TS F A

m

(3) where: TS is tensile strength (MPa); Fm is the maximum force at the moment offilm rupture (N); and A is the cross-sectional area (m2)

Eq (4)

E d

r i

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 thefilms The moisture content of thefilms was determined using theAACC (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 con-stant weight to determine thefinal dry mass of the sample The solu-bility was expressed in terms of the solubilized mass (SM) of thefilm, according to Eq.(5)

SM (%) ( initial mass-final mass) 100

2.6.6 Water vapor permeability of thefilms The permeability to water vapor (PWV) was determined using the ASTM method E-96-95 (ASTM, 1995) at 25 °C Thefilms 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)

t

X

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

2.7 Statistical analysis 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

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 theD-limonene (93.9%) andβ-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, Becerra-Mora, Kohli, Fisher, & Choudhary, 2017; Zahi, El Hattab, Liang, & Yuan, 2017) This compound has been reported to have applications in

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

anti-microbial activity Dannenberg et al (2017)studied the essential oil

composition of pink pepper and found thatβ-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

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 2.60μL·mL−1

OEO were able to promote a bacterio-static effect against L monocytogenes and S aureus, whereas the MIC for

B cereus was 5.21μL·mL−1 ((Table 2) The MICs for gram-negative

bacteria P aeruginosa and S dysenteriae were 10.42 and 41.67μL·mL−1,

respectively, and the values were higher than those found for the

gram-positive bacteria

OEO concentrations up to 5.21μL·mL−1demonstrated 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 41.67μL·mL−1for P aeruginosa and S dysenteriae, respectively;

Table 2)

In the present study, it was possible to observe that the

gram-ne-gative bacteria were more resistant to OEO than the gram-negram-ne-gative

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

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 = 5.21μL·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 con-centration 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 in-dividual action of each component on specific cellular targets and a synergistic antimicrobial effect of all the compounds (Burt, 2004) An OEO concentration of 5.21μL mL−1was 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 104CFU·mL−1

3.4 Morphology of thefilms with OEO The morphology of the surfaces and cross-sections of the corn starch films without and with different OEO concentrations are shown in Fig 2 Thefilm without OEO presented a smooth and uniform surface (Fig 2a) The addition of OEO in the films, regardless of the con-centration, reduced the homogeneity of the cross-sections (Fig 2f–h), with presence of more concentrated pores on the surface The hydro-phobicity of the oil and its density difference with the aqueous solution

of starch can affect the stability of the filmogenic solution and conse-quently 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 OEOfilms The starchfilms without OEO promoted a reduction of 16% and 22% in the development of S aureus and L monocytogenes, respectively, when compared with the control (withoutfilm 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 im-pediment to the development of the colonies, considering the inert (non-antimicrobial) characters of starch and other components present

in thefilmogenic solution

The addition of OEO in the polymeric matrix of thefilm, at all evaluated concentrations, promoted the inhibition of both pathogens (Fig 3) OEO concentrations of 0.3, 0.5, and 0.7μL·g−1

reduced the 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 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 thefilm without OEO Only the highest OEO concentration (0.7μL g−1) resulted in significant re-ductions (p≤ 0.05) in the counts of S aureus These results demon-strate that starch is a suitable polymer matrix for the incorporation of antimicrobial agents such as OEO because it was able to store/en-capsulate OEO and release it during direct contact with the con-taminated surface of the medium (agar)

Table 1

Chemical composition of orange essential oil (OEO)

Peak number Retention time (min) Compound Peak area (%)

Table 2

Antimicrobial activity of orange essential oil (OEO)

Bacteria a ATCC Diffusion agar (mm) MIC (μL/mL) MBC (μL/mL)

Gram-positive

L monocytogenes 7644 10.59 ± 0.43 2.60 ± 0.00 5.21 ± 0.00

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

Gram-negative

P aeruginosa 15442 9.30 ± 0.31 10.42 ± 0.00 20.83 ± 0.00

S dysenteriae 8739 8.73 ± 0.56 41.67 ± 0.00 41.67 ± 0.00

S Typhimurium 14028 ND ND ND

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

Detected

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 OEOfilms

The color parameters (L*, a*, and b*) and opacity of the corn-starch

films with or without OEO are listed inTable 3 The brightness (L) of

thefilms ranged from 96.38 to 96.80 (Table 2) Thefilms with 0.3 and

0.7μL of OEO showed higher values of a* and b* (coordinates

re-sponsible for chromaticity), indicating a tendency to green and yellow

OEO addition increased the opacity of thefilms with higher OEO

concentrations (Table 3) However, this behavior only is visually noted

in thefilm with 0.7 μL of OEO (Fig 4) This increase in opacity can be

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

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

Fig 3 Antimicrobial activity of thefilms 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 up-percase letters indicate significant difference between the bacteria for the same concentration of OEO

attributed to the essential oil droplets (refractive index of 1.472)

dis-tributed 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 thefilms 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 offilms with OEO

The incorporation of OEO increased the thickness of the films

(Table 4).Luís, Pereira, Domingues, and Ramos (2019))also reported

an increase infilm 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 in-creasing the compactness of the starch matrix structure

The mechanical characteristics offilms 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, & González-Martínez, 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 inter-actions, generating more flexible domains within the film (Li et al.,

2018;Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015) On the

Table 3

Color parameters (L*, a* and b*) and opacity of the corn starchfilms with and

without orange essential oil (OEO)

OEO (μL/

g) a

0.0 96.38 ± 0.06 b −0.16 ± 0.00 b 2.63 ± 0.02 b 10.86 ± 0.37 b

0.3 96.41 ± 0.11 b −0.32 ± 0.03 a 2.76 ± 0.11 ab 12.02 ± 1.16 b

0.5 96.55 ± 0.03 b −0.15 ± 0.02 b 2.51 ± 0.06 b 13.07 ± 1.52 ab

0.7 96.80 ± 0.02ª −0.33 ± 0.06 a 2.97 ± 0.16 a 16.24 ± 1.87 a

a The results are the average of three determinations Values with different

letters in the same column are significantly different (p < 0.05)

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

Table 4 Thickness, tensile strength and percent elongation of starchfilms with and without orange essential oil (OEO)

OEO (μL/g) a Thickness (mm) Tensile strength (MPa) Elongation (%) 0.0 0.084 ± 0.008 c 5.11 ± 0.57 a 64.58 ± 8.95 a

0.3 0.112 ± 0.016 b 4.08 ± 0.40 b 9.94 ± 0.46 b

0.5 0.142 ± 0.024 a 2.73 ± 0.20 c 12.64 ± 3.45 b

0.7 0.131 ± 0.020 a 2.40 ± 0.46 c 15.25 ± 2.85 b

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 thefilms with OEO was substantially lower than

na-tivefilm

Stress vs strain curves of thefilms can be visualized inFig 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

3.8 Moisture, water solubility, and PWV of thefilms with OEO

The moisture content, water solubility, and PWV of thefilms are

presented inTable 5 The starchfilms incorporated of 0.5 and 0.7 μL of

OEO presented higher humidity in relation to thefilms without OEO

and 0.3μL of OEO (Table 5) The increase in the moisture content of the

films with OEO may be related to the rupture of the films (Fig 2) The

formation of a porous structure in the starchfilms with OEO facilitates

the insertion of water molecules between the polymer chains

On the other hand,Ghasemlou et al (2013)observed that the

ad-dition of essential oils from the plants Zataria multiflora Boiss and

Mentha pulegium in starch films decreased the moisture content

Ac-cording to these authors, the incorporation of hydrophobic essential oils

may affect the film’s ability to retain water and consequently decrease

its moisture

Solubility is a factor that directs the application of the film as

packaging for food products The films with OEO, regardless of OEO

concentration, showed higher solubility (Table 5) than the films

without OEO Overall, the solubility of films depend the type and

concentration of the compounds as well their hydrophilicity and

hy-drophobicity indices Therefore, hydrophilic compounds tend to

in-crease the solubility values, whereas hydrophobic compounds dein-crease

these values (Caetano et al., 2017; Ghasemlou et al., 2013) The

increase in solubility may due the rupture of thefilms, easing the water insertion in the polymeric matrix and also with increase of thickness and irregular surface structures of thefilms, increasing the contact area

offilm 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 starchfilms 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 thefilms 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

affinity of the film for water

4 Conclusion The major component of OEO wasD-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 con-centration 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 bioactivefilms However, the applications of these films need to be evaluated further to analyze their efficiency in food bioconservation

Acknowledgements This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brasil (CAPES)—Finance Code 001, CNPq, FAPERGS and the Center of Southern Electron Microscopy (CEME-SUL) of the Federal University of Rio Grande (FURG)

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