Bioactive Andean sweet potato starch-based foam incorporated with oregano or thyme essential oil

Food Packaging and Shelf Life 23 (2020) 100457

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Bioactive Andean sweet potato starch-based foam incorporated with oregano or thyme essential oil

T

J.P. Cruz-Tiradoa, Ramon Sousa Barros Ferreiraa, Edward Lizárragab, Delia R. Tapia-Blácidoc, N.C.C. Silvad, Luis Angelats-Silvae, Raúl Sicheb,* a

Department of Food Engineering, Faculty of Food Engineering (FEA), University of Campinas (UNICAMP), 13083-862, Campinas, Sao Paulo, Brazil Facultad De Ciencias Agropecuarias, Universidad Nacional De Trujillo, Av. Juan Pablo II s/n, Trujillo, Peru c Departamento De Química, Faculdade De Filosofia, Ciências e Letras, Universidade De São Paulo, Av. Bandeirantes, 3900, CEP 14040-901, Ribeirão Preto, SP, Brazil d Department of Food Science, Faculty of Food Engineering (FEA), University of Campinas (UNICAMP), 13083-862, Campinas, Sao Paulo, Brazil e Laboratorio De Investigación Multidisciplinaria, Universidad Privada Antenor Orrego, Av América Sur 3145, Trujillo, Peru b

A R T I C LE I N FO

A B S T R A C T

Keywords: Native starch Thermopressing Salmonella L. Monocytogenes Antimicrobial Biomaterial

In this research, sweet potato starch and oregano (OEO) or thyme (TEO) essential oil at two concentrations (7.5 and 10 %) were used to produce bioactive foams by thermopressing. The foams were characterized according to microstructure, mechanical properties, antimicrobial properties, and structural properties by X-ray diffraction, scanning electron microscopy, and Fourier-transform-infrared spectroscopy (FT-IR). In all cases, essential oil addition affected the foam color, yielding reddish/yellowish foams, but not the foam thickness. FT-IR spectrum and X-ray diffraction revealed starch-lipid interactions. According to the micrographs, the lipids were localized in the first layer. Thus, formation of amylose-essential oil complexes in the foam may have prevented the essential oil from degrading under the thermoforming temperature. Essential oil addition yielded starch foams with low water solubility and mechanical resistance, especially for 10 % OEO. Meanwhile, these foams were more effective against Salmonella (Gram-negative bacteria) and L. monocytogenes (Gram-positive bacteria). The antimicrobial activity of the foams containing essential oil makes them beneficial for application as bioactive materials. Therefore, bioactive sweet potato starch-based foams can be prepared by thermopressing and be applied as food container.

1. Introduction There has been growing interest in obtaining biopolymeric packaging consisting of biodegradable and renewable material (Gilfillan, Moghaddam, Bartley, & Doherty, 2016; Kaisangsri, Kerdchoechuen, & Laohakunjit, 2014) as opposed to petroleum-based packaging, whose accumulation is a real and worrisome problem for the environment. In addition, biopolymeric packaging with bioactive properties can be designed to offer protective effects against microbes. Active packaging is defined as a system where product, packaging, and free space interact, to result in improved product quality and safety (Suppakul, Miltz, Sonneveld, & Bigger, 2003; Vermeiren, Devlieghere, & Debevere, 2002). However, incorporating bioactive components into food products could alter the food product flavor and make the food product susceptible to fast degradation, not to mention that the bioactive component could interact negatively or positively with the other food matrix elements (Quirós-Sauceda, Ayala-Zavala, Olivas, &

⁎

González-Aguilar, 2014). In contrast, when appropriate concentrations of these components are added in a polymeric matrix (e.g., starch foam), they can generate active packaging that impact the food organoleptic properties to a lesser extent because the active components are gradually released. Biomaterials like starch allow incorporating of antimicrobial and antioxidant agents (Shen & Kamdem, 2015), such as essential oils (Atarés & Chiralt, 2016). Essential oils are classified as Generally Recognized as Safe (GRAS). They could be used to produce "bioactive packaging" with antimicrobial and antioxidant properties (Burt, 2004) and to help reduce the hydrophilic behavior. Several works have evaluated how diverse essential oils including thyme or oregano essential oil (Romani, Prentice-Hernández, & Martins, 2017; Yahyaoui, Gordobil, Herrera Díaz, Abderrabba, & Labidi, 2016), rosemary or clove (Mulla et al., 2017; Qin, Li, Liu, Yuan, & Li, 2017) essential oil, Thai essential oil (Klangmuang & Sothornvit, 2016), and cinnamon or ginger essential oil (Atarés, Bonilla, & Chiralt, 2010) affect film properties.

Corresponding author. E-mail address: [email protected] (R. Siche).

https://doi.org/10.1016/j.fpsl.2019.100457 Received 17 September 2019; Received in revised form 14 December 2019; Accepted 15 December 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.

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Laurindo, 2008). Likewise, the plasticizer (glycerol) and the release agent (magnesium stearate) ratios had been established previously to obtain a complete foam. The essential oil (7.5 % or 10 %) (g of essential oil/g of starch), starch, water, glycerol (7.5 %), and magnesium stearate (3 %) were placed in a 500-mL beaker and mixed in a mechanical mixer at 1500 rpm for 10 min (Imaco, China). The time was defined so as to obtain a homogenous batter. Subsequently, the batter (50 g) was homogenized in Teflon molds (27 cm × 20 cm × 25 mm; thickness = 3.0 mm) in a mechanical thermopress (RELES, Lima, Peru) at 160 °C for 10 min and under 60 bar. The resulting foams were stored at 25 ± 1 °C and 60 % relative humidity for four days before the microbiological, physical, and mechanical analyses were conducted.

Debiagi, Kobayashi, Nakazato, Panagio and Mali (2014) incorporated essential oil as an additive in baked foams made from cassava bagasse and polyvinyl alcohol. The authors reported that the thermoforming temperatures volatilized the essential oil, and that the foams exerted no antimicrobial effect. Ketkaew et al. (2018) recently evaluated how oregano essential oil affected cassava starch foams and pointed out that bioactive foam foams could be obtained by thermopressing. Nevertheless, information on how starch and essential oils interact under high temperature (> 140 °C) and pressure to generate a bioactive foam is scarce. In fact, whether the essential oil is preserved in the foam layers after the thermoforming process has not been well established, so new information about this topic is necessary. In addition, the study of new starch sources as a basis for bioactive foams is relevant for industrial applications. In our previous work, we showed that native sweet potato starch can be a very useful source to generate resistant foams made by thermopressing, but it is necessary to improve its hydrophilicity and active property (Cruz-Tirado, Vejarano, Tapia-Blácido, Barraza-Jáuregui, & Siche, 2019). Therefore, here we hypothesize that addition of an essential oil can decrease the sweet potato starch hydrophilicity and improve the antimicrobial properties of sweet potato foams, yielding bioactive packaging. During the thermoforming process, microdrops of an essential oil can be encapsulated inside the gelatinized starch, preventing the oil from evaporating completely during foam drying. In this context, this work aims to evaluate how the addition and the concentration of thyme or oregano essential oil affect the physical and antimicrobial properties of foams based on native sweet potato starch.

2.3. Foam characterization 2.3.1. Foam visual aspect and color The foam color in terms of luminosity (L*) [black (0) to white (100)], redness (a*) [green (−80) to red (80)], and yellowness (b*) [blue (−80) to yellow (80)] was measured with a Z-300 colorimeter (Kingwell Shenzhen Co., China) according to Salgado et al. (2008). The measurements were taken on standard white backgrounds (L = 89.7, a = 1.9 y b = - 4.9). The total difference in color (ΔE) was calculated by using the following Eq. (1):

ΔE =

2.3.2. Water absorption index (WAI) and water solubility index (WSI) The water absorption index (WAI) and the water solubility index (WSI) were measured according to the AACC method 56-20 (1983). One gram of foam sample (piece of foam) was dispersed in 30 mL of distilled water in centrifuge tubes and placed in a water bath at 30 °C for 30 min. The tubes were centrifuged at 5000 rpm for 30 min in a centrifuge model NF 200 (Nüve, Germany). The supernatant was placed in Petri dishes and dried at 105 °C for 24 h, and the tube with the semi-solid sample was weighed. The WAI and WSI were calculated according to Eqs. (2) and (3):

2.1. Materials Sweet potato (Ipomoea batatas) starch (32.65 % amylose, 9.27 % moisture, and 0.30 protein) was provided by the Agroindustrial Processes Engineering Laboratory of the National University of Trujillo (Trujillo, Peru). Glycerol and magnesium stearate were acquired from Su Man (Pflücker e Hijos S.A., Lima, Peru). Thyme essential oil (Thymus vulgaris) and oregano essential oil (Origanum vulgaris) were purchased from Runcato (Runcato E.I.R.L, Lima, Peru). Oregano essential oil contained carvacrol (60–65 %) and thymol (1–5 %) and had specific density of 0.9232 and refractive index of 1.4774 at 20 °C. Thyme essential oil contained thymol (50–52 %) and had specific density of 0.9442 and refractive index of 1.5080 at 20 °C.

WAI =

WSI =

The foams were prepared by thermopressing sweet potato starch, water, and oregano essential oil (OEO) or thyme essential oil (TEO) according to the formulations listed in Table 1. The starch/water ratios for batter formulation had been optimized previously (data not shown). The starch concentration was set at 100 g for all the treatments, but the water ratio in the batter was decreased when the essential oil concentration was increased (Table 1). Reducing the water ratio was essential so as not to alter the batter viscosity and to obtain a complete foam (Salgado, Schmidt, Molina Ortiz, Mauri, &

Batter (g)

Control OEO OEO TEO TEO

0 7.5 10 7.5 10

75 70 62.5 70 62.5

50 50 50 50 50

Weight of plate with dry supernatant − plate weight x100 Sample weight

(3)

2.3.3. Thickness and density The foam thickness (mm) and density (g cm−3) were measured according to Shogren, Lawton, Doane and Tiefenbacher (1998). Ten samples (100 mm x 25 mm) were assayed for each formulation, and the average of the assays is presented herein. 2.3.4. Mechanical properties The foams were stored at 25 °C and 60 % RH for four days before the mechanical analysis was performed. The mechanical properties were determined by using a texturometer model TAHD Plus (Stable Micro System, Surrey, UK), with a 100-kg load cell. The tensile test (tensile strength (MPa) and elongation (%)) was carried out in accordance with the ASTM D828-97 standard test method (ASTM, 2002). Foams samples measuring 100 mm x 25 mm were used. The initial grip separation was 80 mm, and the crossing speed was 2 mm s−1. The breaking strength and elongation were calculated according to Eqs. (4) and (5):

Table 1 Batter composition used to prepare sweet potato starch-based foams incorporated with oregano or thyme essential oil. Water (g)

Weight of the tube with the semi-solid sample − tube weight Sample weight (2)

2.2. Foam preparation by thermopressing

Concentration (%)

(1)

Where, L*, a*, and b* are the values of the color parameters of the sample.

2. Materials and methods

Formulation

(L − L*)2 + (a − a*)2 + (b − b*)2

Tensilestrength= 2

F A

(4)

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Fig. 1. Appearance and microstructure of sweet potato starch-based foams incorporated with OEO or TEO at 7.5 % and 10 %. White arrows indicate defects in the foams such as cracks and holes.

Elongation=

L−L 0 L0

and compression), and the averages with their standard deviations were recorded.

(5)

Where, F is the maximum force; A is the cross-section of the calibration section; L0 is the initial length of the calibration; and L is the final length. The compression (hardness (N) and fracturability (mm)) analyses were conducted by using 50 mm x 50 mm squares according to CruzTirado et al. (2019). An HDP/CFS accessory and a P/0.50S spherical steel probe were employed. The test speed was 1.0 mm/s, and the probe was moved 25 mm to fracture the foam. Ten trials were accomplished for each treatment in each test (tensile

2.3.5. Fourier-transform-infrared spectroscopy (FT-IR) The FT-IR spectra were collected in a Fourier infrared transformation spectrometer Nicolet iS50 (Thermo Scientific, Germany) equipped with an attenuated total reflectance (ATR) Miracle diamond 3-rebound accessory (Thermo Scientific, Germany) in a frequency range of 4000–400 cm−1. FT-IR was performed on foams samples with thickness of 0.25 mm, width of 2.5 mm, and length of 2.5 mm; the resolution was 4 cm−1, and 32 scans were accumulated per spectrum in the reflectance mode. The spectra that were obtained with the ATR-FTIR accessory 3

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Table 2 presents the color parameters luminosity (L*), redness (a*), yellowness (b*), and total color difference (ΔE) for the foams. In general, essential oil addition significantly affected the foam color parameters (Tukey’s test, p < 0.05) as compared to the control foam. The lowest L * values were obtained when an essential oil was present in the foam, mainly for 10 % OEO. The phenolic compounds (carvacrol, thymol) that are present in essential oils absorb light at low wavelengths (Jouki, Mortazavi, Yazdi, & Koocheki, 2014), which might explain the decrease in L*. Because OEO has greater carvacrol and thymol contents than TEO, OEO addition affected the L* value to a larger extent. The a* and b* values increased with OEO or TEO addition (Table 2), which indicated the presence of yellow and red shades in the foams. Comparing both essential oils at the same concentration, OEO addition yielded more reddish and yellowish (> a* and b* values) foams especially at the higher concentration (10 %). This behavior was probably due to the greater presence of phenolic compounds in OEO as compared to TEO (see Section 2.1) (Klangmuang & Sothornvit, 2016). Essential oil incorporation elicited greater color variation (ΔE) (Tukey’s test, p < 0.05), but ΔE differences were not significant (p > 0.05) between OEO and TEO or between the two different essential oil concentrations (7.5 % or 10 %).

were corrected and analyzed with the OMNIC Spectra Material Characterization Advanced Analysis software (Thermo Scientific). 2.3.6. X-ray diffraction The foams were finely pulverized and sieved (particles < 300 μm), and the diffraction analysis was carried out in a Miniflex 600 model diffractor (Rigaku, Japan) using copper kα radiation (λ =1.5418 Å), voltage of 40 kV, and operating current of 15 mA. The analyses were conducted with a ramp of 1°/min for 2θ values between 10° and 60°. 2.3.7. Scanning electron microscopy (SEM) The SEM analyses were accomplished in a Tecsan VEGA 3 LM SEM microscope equipped with a gold coating system SPI 11430-AB (TESCAN, USA). The foams samples were mounted in bronze stubs for viewing of the cross-section. The surfaces were coated with gold (40−50 nm thickness). The acceleration voltage was 20 kV for all the samples. 2.3.8. Antimicrobial activity evaluation for the foams The E. Coli ATCC 25922, Salmonella ATCC 14028, S. aureus ATCC 25923, and L. Monocytogenes ATCC 35152 microbial strains were obtained from the culture collection of the Biological Chemistry Laboratory of the School of Microbiology and Parasitology of the National University of Trujillo (Trujillo, Peru). The disc inhibition zone method was used according to Pelissari, Grossmann, Yamashita and Pineda (2009), with some modifications. The inoculum was standardized at 108 CFU mL−1 on the McFarland scale, and 0.1 mL of culture was added to a plate containing MullerHinton agar (MHA). The control sample (0 % essential oil) and the foams with essential oil were cut into 10-mm circles and placed on the plate with MHA. The plates were incubated at 37 ± 0.5 °C for 18–24 h. The diameter of the inhibition zone was measured with a digital vernier (Stainless Hardened, 0–150 mm, China) with an accuracy of 0.001 mm. The analyses were conducted in triplicate for each treatment.

3.2. Foam microstructure Fig. 1 also shows the micrographs (surface and cross-section) of the sweet potato starch foams without (Control) and with essential oil (OEO and TEO). OEO or TEO addition modified the control foam microstructure. The control foam presented porosities on the surface. These porosities emerged during starch expansion and subsequent water evaporation by application of high heat and pressure. In contrast, the starchbased foams incorporated with essential oil displayed a more irregular but denser surface because starch-lipid complexes arose during the thermal process (Hafsa et al., 2016), causing the irregular surface. Regarding the essential oil type, the foams containing 7.5 % TEO had more cracks on its surface, whereas the foams with 7.5 % OEO presented a denser surface. In turn, the foams with 10 % OEO displayed discontinuous regions (marked with white arrow), whilst the foams with 10 % TEO exhibited a more compact surface. A greater discontinuity in the foams with 10 % OEO could be associated with the presence of more essential oil in the first layers of the foam. During the gelatinization process, starch and essential oil formed strong interactions, which promoted essential oil encapsulation within the gelatinized starch. Subsequently, steam displaced the essential oil to the first layers of the foam. Due to the essential oil hydrophobic nature, the water vapor that was trapped inside the foam caused deformations, but not cracks, as noticed in the control foam and in the foams with TEO. Therefore, OEO presented greater compatibility with sweet potato starch, which led to its greater encapsulation and presence in the first layers of the foam. Similar results have been reported for cassava starch foams with OEO (Ketkaew et al., 2018). The thermopressing process

2.4. Statistical analysis Analysis of variance (ANOVA) and Tukey's mean comparison test (p < 0.05) were performed with the Statistica software version 7.0 (Statsoft, Oklahoma, USA) to compare oil concentrations and types. 3. Results and discussion 3.1. Foam color and appearance Fig. 1 shows the appearance of the sweet potato starch foams without and with added essential oil (OEO or TEO). The thermoforming conditions provided complete foams. Foams with 7.5 % or 10 % OEO or TEO had small cracks, holes, and deformations because water vapor interacted with OEO or TEO during starch expansion. The foam color changed slightly to reddish/yellowish, which was consistent with the color results (Table 2).

Table 2 Color parameters of sweet potato starch-based foams incorporated with essential oil. Sample

Concentration (%)

L*

a*

b*

ΔE

Control OEO OEO TEO TEO

0 7.5 10 7.5 10

66.12 ± 1.33a1 59.71 ± 1.62bA 57.98 ± 1.41bB 61.93 ± 0.702A 60.93 ± 2.022A

1.46 ± 0.12c3 1.51 ± 0.43bcA 2.10 ± 0.38aA 1.59 ± 0.132A 1.60 ± 0.071B

9.16 ± 1.05c2 10.51 ± 1.04bcA 11.64 ± 1.50aA 9.30 ± 0.3412A 9.57 ± 0.511B

27.47 ± 1.57c2 33.74 ± 1.49abA 35.80 ± 1.60aA 31.19 ± 0.6912A 32.95 ± 1.841A

a−c, 1–3 Mean with different lowercase letter and numbers in the same column indicate significant difference between the control foam and the foams incorporated with oregano (letters) or thyme (numbers) essential oil (7.5 % and 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. A, B Mean with different uppercase letter in the same column indicates significant difference between the foams containing thyme essential oil (TEO) or oregano essential oil (OEO) at the same concentration (7.5 % or 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. 4

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involved temperatures above the sweet potato starch gelatinization temperatures (70–80 °C) (Cruz-Tirado et al., 2019). Therefore, during this process, small essential oil drops were probably trapped within the starch granules, to form starch-essential oil complexes in the foam layers. The oil droplets likely interacted with the starch bonds, were incorporated into the polymer matrix, and got trapped inside the foam structure, especially in the initial thick layer of the foam (sandwich structure) during drying. This entrapment could be advantageous because it could provide the foams with an antimicrobial effect. The cross-section micrograph obtained by SEM showed that all the foams presented a sandwich structure with two well-defined layers and the presence of air cells. Essential oil addition and type also affected the layer thickness and the air cell size between the foams. The foams with OEO showed homogenous air cell distribution of, smaller air cell size, and smaller layer thickness, while TEO addition caused a more packaged structure with heterogeneous air cell distribution and different air cell sizes. Additives generally interfere in starch expansion by forming the polymer matrix. TEO interfered less in starch expansion than OEO, thereby providing foams with larger air cell sizes. This would reflect that interactions between TEO and sweet potato starch were weaker, and that part of TEO was susceptible to volatilization (due high temperature) during TEO displacement toward the first layers of the foam. On the other hand, OEO interacted strongly with sweet potato starch even though this limited the interaction between the starch chains and possibly weakened the structure. However, this greater compatibility and strong interaction between OEO and sweet potato starch generated a more efficient bioactive packaging.

solubility (WSI) reduced significantly (Tukey’s test, p < 0.05) upon TEO or OEO addition as compared to the control, especially for 10 % essential oil. Furthermore, the foams incorporated with OEO had lower WSI values as compared to foams incorporated with TEO (p < 0.05). This probably resulted from the greater OEO homogeneity in the polymeric matrix as compared to TEO. Also, reduced foam solubility could be related to the essential oil hydrophobic characteristic and to ester and/or amide group formation in the starch matrix (Möller, Grelier, Pardon, & Coma, 2004). A similar behavior has been detected in wheat flour-based films incorporated with oregano or thyme essential oil (Pagno, Klug, Costa, de Oliveira Rios, & Flôres, 2016). The foam water absorption (WAI) decreased slightly with TEO or OEO addition, but there were no significant differences between TEO or OEO. The porosities present in the control foam may have led to greater water absorption as compared to the foam with essential oil. In contrast, the reduction in the WAI values of the foams with essential oil (OEO or TEO) was probably related to the essential oil hydrophobic character and to the possible formation of covalent bonds between the functional groups of starch and the essential oils, which reduced the availability of hydroxyl and amino groups and limited the polysaccharide-water molecular interactions through hydrogen bonds (Park & Zhao, 2004). A similar behavior has been reported for films containing clove, cinnamon, or thyme essential oil (Hosseini, Razavi, & Mousavi, 2009). Besides, as seen in Fig. 1, the foams with essential oil (TEO or OEO) presented more compact structures with fewer porosities (transversal section), which may have decreased water absorption, especially at the surface.

3.3. Foam physical properties

3.4. Mechanical properties

Table 3 summarizes the thickness, density, water solubility index (WSI), and water absorption index (WAI) values for sweet potato starchbased foams incorporated with essential oil. Thickness varied from 0.24 to 0.25 mm, without significant differences (Tukey’s test, p > 0.05) between the control foam and the foams incorporated with essential oil. Moreover, the essential oil type and concentration did not affect the foam thickness significantly. Similar results have been found for basil seed rubber films with oregano essential oil (Hashemi & Mousavi Khaneghah, 2017). As for the density values, there was a slight but significant increase (Tukey’s test, p < 0.05) in the foam with 10 % TEO (Table 3) as compared to the control foam. Interaction between essential oil and starch chains (lipid-carbohydrate) seemed to reduce the rate at which vapor bubbles expanded, which increased the sweet potato starch foam density (Shogren et al., 1998). This agreed with this foam transversal structure (Fig. 1): a more compact sandwich structure was observed for the foam with 10 % TEO (Kaewtatip, Tanrattanakul, & Phetrat, 2013). The solubility index (WSI) and the water absorption index (WAI) indicate the interaction of polymers with water molecules and its hydrophilic characteristic (Xu, Dzenis, & Hanna, 2005). The foam

The mechanical properties of foams are one of the most important for their practical application. Table 4 depicts the hardness (N), fracturability (mm), tensile strength (MPa), and elongation (%) values obtained for the starch-based foams incorporated with oregano (OEO) or thyme (TEO) essential oil. In general, essential oil (TEO and OEO) addition significantly decreased the foam mechanical resistance and elongation (Tukey’s test, p < 0.05), which could indicate that the starch matrix destabilized due to interruption of amylose-amylose, amylopectin-amylopectin, or amylose-amylopectin interactions in the presence of an essential oil. The compression tests helped to assess the strength of the foams and to measure their stiffness, which allowed them to absorb any energy impact (Fang & Hanna, 2001). The decrease in the hardness and fracturability of the sweet potato starch foams containing essential oil may be related to internal structure destabilization (Fig. 1) due to interactions between starch chains and lipids (Shogren, Lawton, & Tiefenbacher, 2002). The lowest hardness and fracturability values were recorded for the foams with 10 % essential oil, especially OEO. However, a significant difference (Tukey’s test, p > 0.05) emerged only between OEO and TEO for fracturability. Increased fracturability could

Table 3 Physical properties of sweet potato starch-based foams incorporated with essential oil. Sample Control OEO OEO TEO TEO

Concentration (%) 0 7.5 10 7.5 10

Density (g cm−3)

Thickness (mm)

b2

a1

0.25 ± 0.00 0.25 ± 0.00aA 0.24 ± 0.00aA 0.25 ± 0.011A 0.24 ± 0.021A

0.18 ± 0.02 0.18 ± 0.02bA 0.19 ± 0.01abA 0.18 ± 0.012A 0.20 ± 0.021A

WSI

WAI a1

2.11 ± 0.02 1.08 ± 0.02bB 1.06 ± 0.01bB 1.20 ± 0.022A 1.24 ± 0.032A

13.78 ± 0.83a1 12.24 ± 0.93abA 11.94 ± 0.80bA 12.57 ± 0.8512A 12.00 ± 0.802A

a−c, 1–3 Mean with different lowercase letter and numbers in the same column indicates a significant difference between the control foam and the foams incorporated with oregano (letters) or thyme (numbers) essential oil (7.5 % and 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. A, BMean with different upper case letter in the same column indicates a significant difference between the foams containing thyme essential oil (TEO) or oregano essential oil (OEO) at the same concentration (7.5 % or 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. WSI: water solubility index. WAI: water absorption index. 5

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Table 4 Mechanical properties of sweet potato starch-based foams incorporated with essential oil. Sample Control OEO OEO TEO TEO

Concentration (%) 0 7.5 10 7.5 10

Hardness (N) a1

2.62 ± 0.20 1.74 ± 0.12bA 1.43 ± 0.11cA 1.63 ± 0.142A 1.54 ± 0.173A

Fracturability (mm) a1

7.39 ± 0.33 3.09 ± 0.14bB 2.59 ± 0.17cB 3.63 ± 0.282A 3.45 ± 0.152A

Tensile strength (MPa) a1

1.08 ± 0.16 0.54 ± 0.03bA 0.44 ± 0.06cA 0.48 ± 0.052A 0.47 ± 0.062A

Elongation (%) 1.65 ± 0.20a1 1.06 ± 0.08bA 0.92 ± 0.18bcA 1.07 ± 0.052A 0.96 ± 0.1123A

a−d, 1–3 Mean with different lowercase letter and numbers in the same column indicates a significant difference between the control foam and the foams incorporated with oregano (letters) or thyme (numbers) essential oil (7.5 % and 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. A, B Mean with different uppercase letter in the same column indicates a significant difference between the foams containing thyme essential oil (TEO) or oregano essential oil (OEO) at the same concentration (7.5 % or 10 %) for each analyzed parameter according to Tukey's test, p < 0.05.

be related to the lower ability of the foams with essential oil to absorb water (plasticizer) (see Table 3). Therefore, the foams deformed more easily under compression forces (Cruz‐Tirado, Vejarano, Tapia‐Blácido, Angelats‐Silva, & Siche, 2019). In addition, increased fracturability could be related to the irregular/heterogeneous structure of foams with essential oil (Fig. 1). The tension tests showed that OEO or TEO addition reduced the foam strength and elongation (Table 4). The foams with 10 % essential oil exhibited the lowest values for tensile properties without significant difference between TEO and OEO (Tukey’s test, p > 0.05). This reduction in foam resistance could be associated with the essential oil plasticizing effect, which destabilized the biopolymer matrix (Pelissari et al., 2009), and with the essential oil cross-linker effect, which reduced the polymer free volume and molecular mobility (Hosseini et al., 2009). A similar behavior has been reported in cassava bagasse-based foams with OEO (Debiagi et al., 2014) and cassava starch-based foams with OEO (Ketkaew et al., 2018). About elongation, the foam irregular structure of contributed to reducing its elasticity (as seen in Fig. 1) (Ramos, Jiménez, Peltzer, & Garrigós, 2014). Moreover, the reduction in the foam capacity to absorb water (Table 3) may have contributed to decreasing elasticity-water functions as a plasticizer and can modify natural polymer structure (Martelli-Tosi et al., 2017). 3.5. Fourier-transform infrared spectroscopy (FT-IR) The FT-IR analyses were carried out to identify possible molecular interactions between the starch and essential oil functional groups (Fig. 2). The spectra of all the starch foams displayed a wide absorption band between 3440 and 3400 cm−1, due to OeH bond stretching. This band indicates intermolecular hydrogen interactions between starch/glycerol/essential oil generated during thermoforming (Matsuda, Verceheze, Carvalho, Yamashita, & Mali, 2013). This intermolecular hydrogen interaction could also be associated with the angular flexion of water molecules reflected in absorption bands between 1656 and 1640 cm−1. Additionally, the band at 1640 cm−1 indicates an ester bond (Matsuda et al., 2013), which could be attributed to a more intense interaction between starch and the essential oil. The foams presented absorption peaks at 1035, 894, 1022, and 1060 and in the region between 1200 and 1220 cm−1, corresponding to CeO and CeOeC bond stretching (glycosidic linkage) in starch (Vercelheze et al., 2012), respectively. Absorption bands emerged in the region between 2920 and 2870 cm−1 and referred to CeH stretching in the eCH2 and eCH3 groups (Abdollahi, Rezaei, & Farzi, 2012) and to HeCeH and CeOeH vibrations, which appeared in all the spectra. These peaks became less intense when the essential oil (OEO or TEO) concentration increased from 7.5–10%. This behavior was probably associated with the presence of hydrogen bonds between the eOH groups in the essential oil and the eNH and eOH groups in starch. Also, the band at 1257 cm−1, related to the flexural vibration of eOH of the phenol groups present in the structure of oregano and thyme essential oils (Salarbashi et al., 2013).

Fig. 2. FT-IR spectra of sweet potato starch-based composite foam incorporated with essential oil.

A new peak that was not observed in the spectrum of the control foam appeared at 1560 cm−1 in the spectra of the foams with essential oil. Hence, this absorption band could be attributed to the interaction between starch and the essential oil under thermoforming conditions. Furthermore, this peak could be ascribed to benzene ring unsaturation, which would correspond to the aromatic hydrocarbons of p-cymene, thymol, γ-terpene, and carvacrol present in both essential oils (OEO and TEO) (Pelissari et al., 2009). In fact, the benzene ring presented abnormal stability during the thermoforming process (160 °C), which was a consequence of ring resonance (Solomons & Fryhle, 2001), as previously reported for extruded chitosan films incorporated with OEO (Pelissari et al., 2009). On the other hand, the peak at 1022 cm−1, which appeared in the spectrum of the control foam, no longer appeared in the spectra of the foams with essential oil, which could be attributed to hydroxyl and ether bond C]O stretching. Therefore, the FTeIR spectra peaks indicated that intermolecular interactions existed

6

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this type of structure are desirable because this structure indicates that the materials are more hydrophobic and consequently present lower WSI and WAI values as demonstrated in Table 3 (Corradini, Carvalho, Curvelo, Agnelli, & Mattoso, 2007). In addition, the essential oil plasticizing effect increased chain mobility due to weakening of the intermolecular interactions (amorphous area > crystalline area) (Benavides, Villalobos-Carvajal, & Reyes, 2012). This effect reduced the starch chain rigidity, which consequently lowered the foam mechanical strength (Liu, Adhikari, Guo, & Adhikari, 2013). This was consistent with the results found for the mechanical properties of the foams with essential oil (Table 4). Besides evidencing the matrix structure weakening, the amorphous peaks that emerged in the diffractograms suggested molecular miscibility and interaction between the essential oil and starch. This behavior has also been observed in rice starch, fish protein, and oregano essential oil films (Romani et al., 2017). 3.7. Antimicrobial analysis Table 5 summarizes the antibacterial activities (inhibitory zone) of the sweet potato starch-based foams incorporated with oregano or thyme essential oil against E. coli and Salmonella (Gram -) bacteria and S. aureus and L. monocytogenes (Gram +). Fig. 4 shows an example of the antimicrobial activity of the control foam and the foams with essential oil against S. aureus. The control foam (0 % essential oil) had no antimicrobial effect against the studied bacteria, while the foams with essential oil displayed activity against the microorganisms tested in this work. Migration of the phenolic compounds from the inner layers to the surface of the polymeric matrix could be related to the starch hydrophilic nature, water vapor penetration, and polymer structure swelling, as previously reported for other hydrophilic polymers (Hashemi & Mousavi Khaneghah, 2017; Jouki et al., 2014; Pelissari et al., 2009). The foam structure could also influence essential oil diffusion from inside the foams to the surface strongly. The surface deformations shown in the micrographs revealed that the essential oil was in the first layer of the foam and was later displaced by water vapor during thermoforming. Therefore, the foams with 10 % essential oil exerted a greater antimicrobial effect due to a greater amount of essential oil that diffused to the environment. The foams with 10 % OEO exhibited greater zones of inhibition probably due to a greater presence of phenolic compounds such as carvacrol, thymol, therpinene, and p-cymene in the foam, as previously reported by Hashemi and Mousavi Khaneghah (2017), Jouki et al. (2014) and Seydim and Sarikus (2006). Table 5 shows that a higher concentration of essential oil provided greater zones of inhibition (p < 0.05), especially for Salmonella and L. monocytogenes. All the strains were sensitive to the foams with OEO and TEO, and the foams with 10 % OEO and 10 % TEO promoted significantly different L. monocytogenes inhibition Pelissari et al. (2009) reported higher inhibition halos for Gram positive bacteria (B. cereus, S. aureus, and L. monocytogenes) in starch-chitosan films incorporated with OEO. The antimicrobial effect on Gram-positive bacteria may result

Fig. 3. X-ray diffractogram of sweet potato starch-based composite foam incorporated with essential oil.

between the functional groups of starch, glycerol, and the essential oils. This could explain the weak mechanical behavior and the low solubility in water of the foams incorporated with TEO and OEO. 3.6. X-ray diffraction Fig. 3 illustrates the X-ray diffraction patterns of sweet potato starch, control foam (without essential oil), and foams incorporated with OEO and TEO. Native sweet potato starch presents characteristic peaks at 2θ = 12°, 14.94°, 16.8°, and 22.8, known as the C-type pattern (Fig. 3) (Huang, Zhou, Jin, Xu, & Chen, 2015). These peaks disappeared in the diffractograms of the foams due to the cooking process, when starch gelatinization occurs and predominantly amorphous materials emerge (Machado, Benelli, & Tessaro, 2017). The foams with essential oil (OEO or TEO) showed a peak at 21.5°, attributed to the formation of a lipidamylose linkage (Zobel, 1988), which became more intense upon increasing essential oil concentration (10 %) in the foam. Baked corn foams (Shogren et al., 1998) displayed a similar behavior although this peak was located at 19.58°. The foams with essential oil had type V structure, which stemmed from amylose crystallization in individual helices involving glycerol and lipids (Bastioli, 1998). Structure V can be divided into two regions: Vh (hydrated) for the peaks at 12.6 and 19.4°, and Va (anhydrous) for the region from 13.2° to 20.6°. Materials with

Table 5 Diameter of the inhibitory zone (mm) of the bioactive foams against the analyzed microorganisms. Samples

Control OEO OEO TEO TEO

Concentration (%)

0 7.5 10 7.5 10

S. aureus

Microorganisms Salmonella

E. coli

L. monocitogenes

0 ± 0c3 38.2 ± 1.4bA 46.2 ± 0.9aA 14.7 ± 1.72B 37.7 ± 2.41B

0 ± 0b2 65.7 ± 2.4aA Total Inhibition 0 ± 02B 11.8 ± 0.41B

0 ± 0c2 29.2 ± 1.5bA 40.4 ± 0.9aA 0 ± 02B 13.7 ± 0.91B

0 ± 0c2 44.1 ± 1.4bA 48.8 ± 2.9aA 0 ± 02B 41.0 ± 1.61A

a−d, 1–3 Mean with different lowercase letter and numbers in the same column indicates a significant difference between the control foam and the foams incorporated with oregano (letters) or thyme (numbers) essential oil (7.5 % and 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. A, B Mean with different uppercase letter in the same column indicates a significant difference between the foams containing thyme essential oil (TEO) or oregano essential oil (OEO) in the same concentration (7.5 % or 10 %) for each analyzed parameter according to Tukey's test, p < 0.05. 7

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for the first time in this investigation. Finally, a bioactive and moderately resistant packaging has been created and can be a potential substitute for petroleum-based packaging. Funding This work was supported by Universidad Nacional Trujillo Contract CanonPIC 2-2013. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgement J. P. Cruz-Tirado acknowledges scholarship funding from FAPESP, grant n° 2018/02500- 4. Ramon Sousa Barros Ferreira acknowledges scholarship funding from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnologico), grant n° 132428/2018-0. The authors acknowledge the Innovation Program for Competitiveness and Productivity (INNÓVATE PERÚ) Contract 407-PNICP-PIAP-2014. Appendix A. Supplementary data Fig. 4. Inhibition zones of sweet potato starch-based foams containing TEO or OEO on S. aureus.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fpsl.2019.100457.

from the absence of the outer membrane around the cell wall, which facilitates the diffusion of hydrophobic compounds such as essential oils. On the other hand, here we have found a significant OEO effect on Salmonella (Gram-negative bacteria) and E. coli. The antimicrobial effect on Gram-negative bacteria could result from the action of carvacrol as well as a system for electron delocalization. The antimicrobial activity of carvacrol is associated with its hydroxyl group and is reinforced by the presence of the benzene ring (Ben Arfa, Combes, Preziosi-Belloy, Gontard, & Chalier, 2006). Also, the hydroxyl group of phenolic compounds inactivates microbial enzymes such as ATPase, histidine decarboxylase, amylase, and protease (Swamy, Akhtar, & Sinniah, 2016). These components disintegrate the cellular outer membrane and mitochondria of gram-negative bacteria, releasing lipopolysaccharides and increasing the cytoplasmic membrane permeability to ATP (Burt, 2004). A similar behavior has been reported for OEO in whey protein-based films toward Gram-negative bacteria (Salmonella enteritidis and E. coli) (Seydim & Sarikus, 2006). In addition, the relative position of the hydroxyl group on the phenolic ring affects the antimicrobial activity (Rao, Chen, & McClements, 2019). Carvacrol is more effective than thymol and eugenol against E. coli, S. aureus, and Bacillus cereus (Gallucci et al., 2009), which agrees with our data for foams with OEO (high carvacrol content).

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4. Conclusion Bioactive sweet potato starch-based foams with oregano or thyme essential oil were prepared by thermopressing. The foams with OEO showed a greater antimicrobial effect than the foams with TEO. Besides, the foams with 10 % OEO completely inhibited Salmonella (Gram-negative bacteria). The results suggested that small essential oil drops were trapped in the first layers of the foams, which caused an effective antimicrobial effect. However, the drops altered the polymer matrix structure and interfered in the proximity of the starch chains, decreasing their mechanical strength. The presence of the essential oils within the starch matrix reduced their solubility and water absorption, which is highly beneficial for the practical application of the packaging. The FT-IR and X-ray diffraction results evidenced starch-lipid interactions in the biodegradable foams. These interactions may have been established during the thermoforming process and have been reported 8

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