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).
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 starch films containing orange essential oil T Jarine Amaral 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 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 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: jarineamaral@hotmail.com (J.A 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 April 2019; Received in revised form June 2019; Accepted June 2019 Available online 10 June 2019 0144-8617/ © 2019 Elsevier Ltd All rights reserved Carbohydrate Polymers 222 (2019) 114981 J.A 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 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®) 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 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 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 (Restekđ) capillary column (30 m ì 0.25 mm ì0.25 m) 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 and gradually increased at a rate of °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 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 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 Carbohydrate Polymers 222 (2019) 114981 J.A 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 (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 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) 2.7 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 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 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 Carbohydrate Polymers 222 (2019) 114981 J.A Evangelho, et al the hydrophobic character of LPS hindered the penetration of the apolar components of OEO Table Chemical composition of orange essential oil (OEO) Peak number Retention time (min) Compound Peak area (%) 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 h of analysis, reaching 0.49 log CFU after 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 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 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 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 Carbohydrate Polymers 222 (2019) 114981 J.A Evangelho, et al Fig 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 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 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 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 Carbohydrate Polymers 222 (2019) 114981 J.A Evangelho, et al Table 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 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 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 Carbohydrate Polymers 222 (2019) 114981 J.A 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 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 Conclusion Table 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 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 References The moisture content, water solubility, and PWV of the films are presented in Table The starch films incorporated of 0.5 and 0.7 μL of OEO presented higher humidity in relation to the films without OEO and 0.3 μL 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 starch films with OEO facilitates the insertion of water molecules between the polymer chains On the other hand, Ghasemlou et al (2013) observed that the addition of essential oils from the plants Zataria multiflora Boiss and Mentha pulegium in starch films decreased the moisture content According 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 hydrophobicity indices Therefore, hydrophilic compounds tend to increase the solubility values, whereas hydrophobic compounds decrease these values (Caetano et al., 2017; 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Zuo, G., & Chen, F (2018) Effect of essential oil and surfactant on the physical and antimicrobial properties of corn and wheat starch films International Journal of Biological Macromolecules, 107,... same concentration of OEO Fig 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 Carbohydrate... with water and then lead to a decrease in the affinity of the film for water Fig 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 Conclusion