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essential oil for bread: Release potential of the citral
Food Packaging and Shelf Life 23 (2020) 100431
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Packaging with cashew gum/gelatin/essential oil for bread: Release potential of the citral
T
Marília A. Oliveiraa, Maria L.C. Gonzagab, Maria S.R. Bastosa,*, Hilton C.R. Magalhãesa, Selene D. Benevidesa, Roselayne F. Furtadoa, Rafael A. Zambellib, Deborah S. Garrutia a b
Embrapa Agroindústria Tropical, Rua Dra. Sara Mesquita 2270, CEP 60511-110, Fortaleza, CE, Brazil Departamento de Engenharia de Alimentos, Universidade Federal do Ceará, CP 12200, 60455-760, Fortaleza, CE, Brazil
ARTICLE INFO
ABSTRACT
Keywords: Polysaccharide Cashew gum Gelatin Ferulic acid Active film
Cashew gum (CG) and gelatin (G) films were produced by incorporating ferulic acid (FA), as a cross-linking agent and Cymbopogon citratus essential oil (EO) for application bread packaging. The addition of FA and EO did not significantly affect water vapor permeability (WVP) or opacity. There was a significant decrease in the solubility of the films, possibly due to matrix cross-linking. Morphology revealed films with smooth surfaces and small cross-sections. Characteristic frequencies of CG and G demonstrated the cross-linking identified in infrared (IV). EO cross-linked films showed lower rupture stress than non-EO films. Elongation-at-break increased from 2% (cross-linked) to 140% (cross-linked/EO). CGG/EO packaging was more permeable than commercial packaging (polyethylene), giving bread more harshness. The experimental packaging provided six days of preservation to the bread compared with three days for the commercial packaging. Neral and geranial presented a higher proportion in the crust of the bread, suggesting antimicrobial action.
1. Introduction A wide variety of gums is produced in tropical and subtropical countries with attractive characteristics for commercial and industrial applications; a typical example is cashew gum, which has received attention because it is similar to gum arabic, which is widely used in the food industry. The Anacardium occidentale L. cashew tree is native to northeastern Brazil, and exudes a gum in response to the attack of pathogens and lesions. In Brazil, cashew trees produce an average of 700 g of cashew gum (CG) per year. Since there are more than 700 thousand hectares of planted cashew trees in Brazil, it is estimated that the production potential of the CG is around 50,000 tons per year (Ribeiro et al., 2016), and it is continuously studied aiming towards different applications. Cashew gum exhibits stabilizing, emulsifying and adhesion properties in water, and can be molded into a film with the addition of a suitable plasticizer such as glycerol. However, even with the use of glycerol in high concentrations, CG films are relatively fragile, inflexible and soluble, making it necessary to incorporate gelatin and a cross-linking agent to improve the mechanical and solubility properties. The need for cross-linking in packaging films is due to the hydrophilicity of the materials, and is therefore a way to improve the incorporation and adhesion of active substances such as antioxidants
⁎
and essential oil. Ferulic acid, a powerful antioxidant extracted from plants and grains, has been shown to be an excellent cross-linking agent for the gell-gelatin polyelectrolyte complex, it acts on the amine groups forming cross-links throughout the blended structure (Graf, 1992). Oxidation occurs employing 0.5% hydrogen peroxide solution. The acid forms a dimer as a side reaction or reacts with amine groups I and II which are free in the gelatin chain to form OeN covalent bonds. The latter response can be reoxidized and linked to another amine, resulting in cross-linking. Research on active food packaging has been developed by incorporating substances that can alter the food environment, affecting its metabolism, providing greater protection, consequently improving quality and increasing its useful life (Bastarrachea, Dhawan, & Sablani, 2011; Zhao & Saldaña, 2019). Packaging is the last stage of production, and its choice is based on promoting the intrinsic quality of the product and its effectiveness in preserving and communicating this quality (Licciardello et al., 2017). The most commonly used polymers in packagings are derivated from petroleum products and not biodegradable, generating solid waste from their final disposal and leading to serious environmental problems. This opens the way for the use of natural biodegradable polymers in manufacturing active food packaging (Arfat, Ejaz, Jacob, &
Corresponding author. E-mail address: [email protected] (M.S.R. Bastos).
https://doi.org/10.1016/j.fpsl.2019.100431 Received 13 August 2019; Received in revised form 14 October 2019; Accepted 25 October 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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Ahmed, 2017). Essential oils (EOs) extracted from plants and spices exhibit antimicrobial and antioxidant properties (Viuda-Martos et al., 2010), which makes them excellent additives in the food industry as a substitute for chemical additives (Alves-Silva et al., 2013). The composition of the oil and the specific interactions with the polymer determine its effectiveness as an active ingredient (Chiralt & Atar, 2016). Cymbopogon citratus essential oil (EO) contains 40–80% of Citral, being widely used in the cosmetic, food and pharmaceutical industries (Martinazzo, Corrêa, Melo, & Barbosa, 2007) for its antifungal (de Sá Silva et al., 2009) and antibacterial properties (Cimanga et al., 2002). Therefore, the combined effect ferulic acid as cross-linked cashew gum/gelatine films incorporated with commercial lemongrass (Cymbopogom citratus) and on their physicochemical, microscopic, spectrometric and mechanical characterizations and volatile compounds of essential oil release was analyzed in this work to verify the possibility of the an food packaging to bread.
of ± 0.01 mm. The thickness used was the arithmetic average of eight measurements performed randomly along each evaluated sample. 2.3.2. Opacity measurement Film samples were cut into a rectangle (8 x 40 mm) and placed on the internal side of a spectrophotometer test cell (Agilent technologies cary 60). The opacity was calculated using the following equation:
Opacity =
Abs750 X
Where: Abs750 is the absorbance value at 750 nm and X is the film thickness. The lower opacity values indicate higher sample transparency (Farhan & Hani, 2017). 2.3.3. Water vapor permeability (WPV) This was determined in accordance with ASTM method E96-05 (ASTM, 2005). Eight replicate samples (60-mm diameter each) were placed between the acrylic permeation cell fittings with a circular opening (diameter = 50 mm), corresponding to the film area exposed to migration. Each cell contained 5 mL of distilled water in its interior. The cells were placed in a desiccator (25 ± 5 °C temperature and 30 ± 5% relative humidity) containing silica gel. The weight of the cells was observed each hour on an analytical balance for 24 h.
2. Materials and methods 2.1. Cashew gum isolation Gum exudate was collected from cashew trees from Embrapa Experimental Station at Pacajus (Fortaleza-Ceará). It was ground to 100 mesh particle size, and 100 g resulting powder was solubilized in 300 mL water at the ratio of 1:3 (w/v) for 4 h, centrifuged at 10,000 rpm at 4 °C for 20 min, and filtered to remove the insoluble materials. 300 mL exudate solution was add in 1200 mL commercial ethanol at a 1:4 (v/v) ratio for 24 h to precipitate the polysaccharide. The precipitate was washed with acetone, then vacuum filtered on funnel with sintered glass disc nº 1, this process being repeated for five times and dried in a hot air circulation oven. This final powder is called “cashew gum” in this paper. Gelatin 225H B type (pH = 5.4) was kindly supplied by Rousselot® (São Paulo, Brazil).
2.3.4. Solubility in water The water solubility determination was carried out on 3 cm x 3 cm film pieces based on the method proposed by Gontard, Duchez, Cuq, and Guilbert (1994) with some modifications. Previously dried and weighed samples were immersed in 50 mL of distilled water for 24 h at 25 °C with stirring (75 rpm). The dry weight of the remaining film pieces was obtained after filtration on previously dried and weighed filter paper and used to calculate the insoluble matter as a percentage of the initial dry weight (g/100 g). All the dry weights (of the initial and final film pieces and the filter paper) were determined after drying at 105 °C for 24 h using a fan-assisted convection oven (Quimis model Q 31 4M22, Brazil).
2.2. Formulation of cross-linked film
2.3.5. Contact angle The contact angles of coating surface were measured on GBX Digidrop MCAT (Modular Contact Angle Technology) system coupled to the CAN101 Tensiometer Optical Contact Angle Meter (KSV Instruments). Five drops of water were deformed on the surface of the films (FC and F1 to F4) individually. The angles were recorded by optical system immediately after droplet deposition, ∼ 2 s, after 1 hour of adhesion and after 2 hours with complete evaporation of water, all measurements being digitally determined. The assay was performed at room temperature (∼ 25 °C).
Cashew gum (CG) and gelatin (G) at a weight ratio of 5.0CG:5.0 G and 10% glycerol in relation to the total weight of the polymers were added in water and kept under stirring for 4 h at 60 °C. Then 1% oxidized ferulic acid (FA) was added relative to the total weight of the polymers. After this period, the surfactants span 80 (S) at 0.6% and tween 80 (T) at 0.5% were added, reaching an Hydrophile-Lipophile Balance (HLB) of 10.8 and then 10% lemon grass essential oil (EO) supplied by Embrapa was added. Homogenization occurred in an Ultra Turrax T25 (IKA, Wilmington, NC, USA) for 10 min. The films were produced by the casting method and dried at room temperature. Four films were prepared following the described procedure, called F1, F2, F3 and F4. Film F1 contains all of the above components, F2 does not contain lemon grass essential oil, F3 does not contain surfactants and F4 does not contain surfactants or essential oil. A non-crosslinked film without surfactants or essential oil was used as a control, called FC. The following were elaborated in order to facilitate identification of the films: F1(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S:1.0EO), F2(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S), F3(5.0CG:5.0 G:1.0 Gly:0.1FA:1.0EO), F4(5.0CG:5.0 G:1.0 Gly:0.1FA) and FC (5.0CG:5.0 G:1.0 Gly). The commercial polyethylene film was obtained in the local market.
2.3.6. Fourier transform infrared (FT-IR) The samples were analyzed as obtained by ATR/FT-IR in the medium infrared region, with wavelengths between 4000 and 400 cm−1 and 4 cm−1 resolution using the Perkin Elmer brand. 2.3.7. Scanning electron microscopy (SEM) Morphological characterization was performed by scanning electron microscopy (SEM, Zeiss DSM, model 940A). Samples were coated with platinum using a sputter coater (Electron Microscopy Sciences, Hatfield, PA, USA) and examined under SEM using an accelerating voltage of 15 kV and a magnification of 3,000–6,000×. 2.3.8. Mechanical properties Mechanical properties were tested according to ASTM Method D882-09 (ASTM, 2009) on a mechanical tester (EMIC DL 3000) using 12.8 cm film strips. The initial grip separation and cross-head speed were set to 10 cm/min. Both force (N) and deformation (mm) were recorded during extension. Tensile strength was calculated by dividing
2.3. Film characterization 2.3.1. Thickness Film thickness was measured using a digital micrometer (Digimatic model, Mitutoyo, Brazil) with a range of 0–25 mm and an accuracy 2
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the required force for film rupture by the cross-sectional area, and elongation-at-break was calculated as the percentage increase in sample length. The elastic modulus was calculated from the slope of the stress–strain curve in the elastic deformation region. The reported values are the averages of five measurements.
the films as barriers to water vapor bigger than for similar materials (Azeredo et al., 2012; Wu et al., 2016). The higher WVP values obtained for the FC, F3 and F4 films may be related to the absence of surfactants in these materials, resulting in lower compaction of the structure by decreasing of bonds. The higher thicknesses of these films may also contribute to higher WVP values. Ferulic acid action was observed by Ou, Wang, Tang, Huang, and Jackson (2005) and Silva, Santos, Coutinho, Mano, and Reis (2005), who reported no significant reduction in the vapor permeability of the soy protein films cross-linked with this acid. In addition to noting the low influence of ferulic acid on the WVP of gelatin films. Cao, Fu, and He (2007) suggest that its small effect is due to the possible combination of the hydroxyl groups of the acid with the water molecules, not favoring permeation. Edible films made from sodium caseinate crosslinked with feluric acid were less permeable to water vapor than αtocopherol cross-linked films (Fabra, Hambleton, Talens, Debeaufort, & Chiralt, 2011). The presence of gelatin in the composition of the films favors poor absorption of water since it can form organized structures, with it being possible to recover part of the triple helix structure of the collagen during the formation process of the film. The higher degree of organization optimizes molecular packaging, resulting in films with better gas barrier properties (Fan, Duquette, Dumont, & Simpson, 2018; Yun & Dong, 2017). Benavides, Villalobos-Carvajal, and Reyes (2012) formulated alginate films with oregano essential oil (EO) in varying proportions and observed a porous structure, whose increase in EO content resulted in greater packaging of the layers, accompanied by an improvement in water barrier properties. Ojagh, Rezaei, Razavi, and Hosseini (2010) incorporated cinnamon essential oil into chitosan films and observed final films with leaf-like structures stacked in thick layers, which presented better water barrier properties. The solubility of the films after cross-linking decreased from 53 to 3%, indicating good process efficiency. In a study by Cao et al. (2007), the effect of water on gelatin films cross-linked with ferulic acid was classified as slight. The presence or absence of essential oils and surfactants, associated or not, did not promote a change in solubility, as the data for F1 to F4 show. Due to the similarity in the results obtained individually for the physicochemical properties analyzed herein, it can be shown that EO was satisfactorily dispersed in the presence and absence of surfactants. The optical analysis of the films is essential to evaluate the acceptance of certain foods by the consumer. Transparency is essential for materials whose purpose as packaging is to promote good visualization of the packaged product. The developed films are promising, since the non-cross-linked film (FC) did not register opacity and the reticulated films presented small opacity values. It is important to note that such behavior was independent of the composition and thickness of the films. For thickness, the fluctuation in the values is due to the mold casting method, a fact reported by other authors (Cao et al., 2007; Henrique, Cereda, & Sarmento, 2008). The results (Table 1) show similar values for crosslinked films (F1 to F4), indicating that the presence or absence of surfactants, essential oil and glycerol did not influence the adhesive properties of the films structural composed by gelatin cashew gum. Non-crosslinking of the FC film, which gives it greater solubility, may account for its lower contact angle value. The low solubility of films (F1 to F4) may be related to the branched molecular structure of cashew gum (Ribeiro et al., 2016). This characteristic added to the presence of cross-linking may contribute to the non-exposure of hydroxyls present in glycosidic units, not favoring the formation of hydrogen bridges with water and, consequently, their solubility. Fig. 1 illustrates the behavior of sessile drop on the surface of the films. The interval recording of 2 h shows the efficient adhesion of water to film surfaces, regardless of their compositions. After 1 h of contact, it was observed that the drop deformed the films, without breaking them and spreading the droplets on the surfaces. This may be related to the ability of the material to absorb water by capillary infiltration or by reorienting hydrophilic groups towards the surface as a
2.4. Film application as bread package 2.4.1. Packaging preparation The package (Fig. 4c) was prepared using the F1 film, selected for its mechanical resistance. Three 45 cm x 30 cm films each were closed by vacuum-sealed Selovac model 200B. The basic formulation bread (wheat flour, margarine, baking powder, sugar and salt) was prepared at the Laboratório de Cereais of Departamento de Tecnologia de Alimentos UFC - Brazil, cut into 1.25 cm thick x 15 cm in length, packed as shown in Fig. 4b and c and conditioned at room temperature (25 °C) for 8 days, during which the analyzes were performed. 2.4.2. Volatile compounds release in the bread Extraction of volatiles in the F1 films (the package) and packed bread (Fig. 4c) was performed by Headspace Solid-Phase Microextraction (SPME) using a carboxen/divinylbenzene/polydimethylsiloxane 50/30 μm fiber. First, 0.5 g of sample film and bread separately were cut in small pieces and weighed in 20 mL vials. The fiber was then exposed into the headspace above the sample for 40 min, maintaining the temperature at 40 °C. The volatiles were thermally desorbed into the splitless injection mode of a gas chromatograph (GC Shimadzu GC2010 Plus, Japan) fitted with a 5% phenyl stationary phase capillary column (30 m length, 0.25 mm id, 0.25 mm film thickness). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL min−1. The oven temperature was programmed from 70 °C to 180 °C at 4.0 °C min−1, and then raised to 250 °C at 8 °C min−1, held for 5 min. The injector temperature was 250 °C and the FID detector was 260 °C. Gas Chromatography-Mass Spectrometry (GC–MS SHIMADZU QP2010, Japan) and Retention indices were used to identify neral and geranial. Analyses were performed in triplicate. 2.4.3. Texture The texture profile was obtained by the double compression test of the bread slices packed in a TA-XT2i texture analyzer (Stable Micro Systems). A texture profile analysis (TPA) with velocity of 1.0 mm/s, compression distance of 40 mm, equivalent to 50% of compression, contact force of 5.0 g was performed using a SMS Aluminum Cylinder probe (P35). Texture Expert for Windows 1.20 program (Stable Micro Systems) collected the data. The hardness parameter was evaluated in this test. 2.5. Statistical analyses The volatile compounds data were evaluated by regression analysis, while the quality of the models (linear or quadratic) was evaluated by the coefficient of determination (R2) and by the lack of fit test. The film characterization data were submitted to analysis of variance (ANOVA) and Tukey test with a level of 5% of significance for comparison of means. All analyses were performed using using the XLSTAT v. 18.1 statistical software program (Addinsoft). 3. Results and discussion 3.1. Characterization of films 3.1.1. Thickness, opacity, WVP, solubility and contact angle Table 1 presents the properties of thickness, opacity, water vapor permeability, solubility and contact angle of films obtained under different conditions. As shown in Table 1, cross-linking through ferulic acid did not influence the WVP of the films (F1 to F4). The results show 3
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Table 1 Thickness, water vapor permeability, solubility and opacity for F1, F2, F3, F4 and FC films. Film
Thickness§ (μm)
FC F1 F2 F3 F4
59.67 58.94 49.90 95.10 68.90
± ± ± ± ±
1.50bc 3.93 bc 6.25 c 8.42 a 5.61 b
WVP (g.mm.k. Pa−1. h−1. m-2) 1.42 1.78 1.72 2.60 2.10
± ± ± ± ±
0.20 0.07 0.40 0.30 0.22
c bc bc a ab
Solubility (%)
Thickness† (μm)
53.40 ± 2.50a 3.04 ± 0.31 b 2.39 ± 0.42 b 3.00 ± 0.48 b 3.03 ± 0.35 b
76.59 47.21 51.10 58.13 46.35
± ± ± ± ±
3.27b 5.94 c 7.10 c 10.66 c 6.27 c
Opacity 0.00 0.11 0.10 0.10 0.11
± ± ± ± ±
Contact angle 0.00 0.00 0.00 0.00 0.00
d a c bc ab
50.98 61.66 61.78 62.72 60.68
± ± ± ± ±
2.23 1.30 1.78 2.91 1.25
a b b b b
§
Thickness for WVP (mean of eight replicate samples, each measured eight times). Opacity thickness (average of six replicate samples, each measured eight times). Mean of three values with standard deviation, the same letter in the row indicates that there is no significant difference between the means by the Tukey test (p < 0.05). F1(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S:1.0EO), F2(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S), F3(5.0CG:5.0 G:1.0 Gly:0.1FA:1.0EO), F4 (5.0CG:5.0 G:1.0 Gly:0.1FA) and FC (5.0CG:5.0 G:1.0 Gly).
†
function of hydration generated by interaction with water (Gugliuzza & Drioli, 2004). The non-rupture of the films was better recorded after 2 h of application, with drop evaporation.
stretches of the C–H bonds of CH3 and C]O of the aldehyde, are added to those recorded at 2923 cm−1 and 1633-1638 cm−1, respectively. As proposed in this work, the bond that structures the cross-linking is likely made between the amino group (NH2) of the gelatin and the hydroxyl group (OH) of ferulic acid, the registration of which can be identified by symmetrical stretching at 1546 cm−1 for NO (Silverstein et al., 2007), present in all cross-linked films.
3.1.2. Fourier transform infrared (FT-IR) The IR spectra of the films, ferulic acid, cashew gum, and gelatin are shown in Fig. 2. Characteristic frequencies of the present polymers, CG and G and FA on the films are reported at 3295-3304 cm−1 for OH, and NH stretches, 2923 cm−1 for asymmetric stretches CH of CH2, 1150 cm−1 for CO (anomeric carbon), 1072-1077 cm−1 for COC (glycosidic bonding), 1031-1036 cm−1 for alcohol CO, with the latter three being specific for CG. C]O acid stretches, characteristic of gelatin and ferulic acid, are recorded at 1650 and 1679 cm−1, respectively. The record of this group in the films can be observed displaced in the range of 1633-1638 cm−1. The presence of ferulic acid on the films is also recorded in the stretches at 1454 cm−1, the characteristic frequency of C]C aromatic ring bonds (Wang, Sun, Liu, Li, & Ma, 2011; Yeung, Eskici, & Axelsen, 2013; Silverstein, Webster & Kiemle, (2007)). Three intense bands at 1618, 1598 and 1518 cm-1 observed in the aromatic ring of fungal acid appear in some regions between 1630 to 1556 cm-1. EO-specific frequencies added in F1 and F3, such as the asymmetric
3.1.3. Scanning electron microscopy (SEM) Fig. 3 micrographs were obtained for the control-FC and F3-sample films. The F1, F2 and F4 images were suppressed by F3 similarity, despite differences in compositions. The FCa and F3a films show homogeneity in the surfaces with no irregularities, as reported by Cao et al. (2007). The recording of air bubbles is due to the casting method used in the molding of films (Mathew & Abraham, 2008). The transverse sessions (FCb and F3b) show higher compression in F3b, with few pores without depth. The bumps in some points are due to the blade action used in the cut. The compact aspect observed may be related to the degree of interaction between the gelatin molecules, one of the structural polymers of the film, provided by the cross-linker action. Mathew and Abraham (2008)) observed a similar compact aspect for composite chitosan and starch films cross-linked with ferulic acid.
Fig. 1. Wettability behavior of FC and F1-F4 films after 2 s, 1 h and 2 h. 4
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Fig. 2. Infrared spectra of the films samples (F1, F2, F3, and F4) and control (FC), Ferulic acid (FA), Gelatin (G) and Cashew gum (CG).
Considering the similarity in the compositions of FC and F4, the absence of ferulic acid (the only non-common component) may respond to the aggregates visualized in FCb. The presence of the essential oil did not promote morphological changes in the F1 and F3 films when compared to the other cross-linked F2 and F4 films.
on the concentration of the cross-linking agent, the literature indicates that the cross-linking process makes the polymer matrix structure more rigid, leaving the films less flexible (Dammak, Bittante, Lourenço, & do Amaral Sobral, 2017). The obtained results reveal a good cross-linking/ matrix ratio able to flexibilization the polymeric chain. The E in the cross-linked films decreases and the ε increases considerably, because in the case of ferulic acid, the cross-linker can preserve the matrix structure in its action, herein formed by CG:G, resulting in a more flexible film with lower tensile strength, especially in F1 and F3, which present EO. A decrease in tensile strength caused by adding EO in films based on polysaccharides was repeatedly observed (Atarés, Pérez-Masiá, & Chiralt, 2011; Ma, Hu, Wang, & Wang, 2016; Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010; Sánchez-González, Chiralt, González-Martínez, & Cháfer, 2011). Tongnuanchan, Benjakul, and Prodpran (2012) and Limpisophon, Tanaka, and Osako (2010) observed
3.1.4. Mechanical properties The mechanical properties of the film indicate the expected integrity under stress conditions that would occur during processing, handling, and storage. The stiffness, strength, and flexibility of the film are described by Young’s modulus (E), tensile strength (σ) and elongation-at-break (ε). Table 2 shows the modulus, tensile and elongation values for the ferulic acid cross-linked films with and without the surfactants, lemongrass essential oil, as well as the non-cross-linked (FC) film. It is observed that FC showed to be more rigid than the crosslinked films. However, its elongation-at-break was very low. Depending
Fig. 3. Micrographs of the surfaces (a) and cross-sections (b) of the films: FCa and FCb (5.0CG: 5.0 G: 1.0 Gly); F3a and F3b (5.0CG: 5.0 G: 1.0 Gly: 0.1FA: 1.0EO). 5
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Table 2 Mechanical properties for the F1, F2, F3, F4 and control (FC) films. Films FC F1 F2 F3 F4
Thickness(μm)
E(Mpa) b
74.00 ± 5.00 51.53 ± 0.79 cd 46.12 ± 0.92 d 140.60 ± 5.16 a 56.46 ± 1.49 c
3.54 0.59 0.09 0.27 0.14
± ± ± ± ±
σ(MPa) 1.34 0.25 0.00 0.09 0.67
a b c c c
58.36 19.16 46.60 19.40 50.51
± ± ± ± ±
ε(%) 0.03 4.18 1.25 0.65 2.18
a c b c ab
2.97 ± 0.98 b 141.57 ± 46.75 105.50 ± 65.42 121.69 ± 41.75 137.13 ± 34.89
a a a a
Mean of three values with standard deviation, same letter in the line indicates that there is no significant difference between the means by Tukey test (p < 0.05). Fig. 4. (a) Neral and geranial determination in percentage of area and their respective equations. Bread crust: neral (□): y = 176.37x² – 731.18x + 2057.9 R² = 0.9309; geranial (●): y = 212.11x² – 929.76x + 2278.2 R² = 0.9451 and bread crumbs: neral (Δ) : y = 28.111x² – 93.522x + 340.27 R² = 0.7308; geranial (▼) : y = 22.114x² – 73.734x + 233.45 R² = 0.6725 (b) Bread in commercial packaging (c) Bread in CGG / EO film and (d) aspect of bread wrapped in CGG / EO film over days.
that the incorporation of citric oil resulted in a reduction in the tensile strength of edible films of fish skin gelatine, which was attributed to the lower interaction between the gelatin molecule and provides flexible domains within the film. The effects of destabilization phenomena during the film drying were considered by Hosseini, Rezaei, Zandi, and
Farahmandghavi (2015) to justify the reduction of tensile strength in gelatine-chitosan fish films added with Origanum vulgare essential oil. Therefore, in the films studied herein greater flexibility and lower stiffness of the cross-linked films is a probable consequence of the presence of the essential oil, favoring the behavior of the glycerol in 6
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study (Fig. 4d). The efficiency of active packaging by the action of essential oils was verified by Priyadarshi, Sauraj Kumar, Deeba, Kulshreshtha, and Negi (2018) by incorporating apricot kernel oil into chitosan films. These authors not only observed better antioxidant activity, but also substantial antibacterial activity against Gram-negative E. coli, as well as Gram-positive B. subtilis bacteria. The films were also effective in inhibiting mold growth on bread for ten days, thus increasing its shelf life. Antioxidant action was also reported by the authors, similar to that disclosed for chitosan films added with green tea extract as presented by Siripatrawan and Harte (2010). Similarly, by adding cinnamaldehyde essential oils to wheat glycine films, Balaguer, Lopez-carballo, Catala, Gavara, and Hernandez-munoz (2013) obtained a material with antibacterial and antifungal action, which promoted a drastic reduction of fungi, and consequently extended the shelf life of the bread and packaged cheese. An important issue nowadays is the selection of packaging systems which are not only efficient, meaning capable of maintaining quality characteristics, but also useful in containing the environmental impact and costs generated by the production and disposal of packaging (Licciardello et al., 2017). In this aspect, developed packaging is favorable, since all the components are biodegradable (Biswas et al., 2018) and of affirmative action in the biochemical processes of the environment.
Fig. 5. Bread hardness packed in CGG / EO film and commercial packaging.
leaving spaces in the matrix structure. The action of the essential oil was also observed in films based on synthetic polymers of cellulose acetate, with reduction of the tension at rupture by the action of Cymbopogon citratus (Oliveira et al., 2017). The action of EO on elongation-at-break of films was also reported. Pranoto, Salokhe, and Rakshit (2005) observed that the proportion of EO determines its effect on elongation-at-break. Shojaee-aliabadi, Hosseini, and Amin (2013) found an increase in the elongation of κcarrageenan film caused by adding Satureja hortensis EO. Zinoviadou, Koutsoumanis, and Biliaderis (2009) incorporated oregano EO into serum protein films, and observed an increase in the elongation properties at an oil concentration of up to 1%; a fact explained by the development of composite film with discontinuities. Different behaviors have also been observed. Increased tensile strength by adding EO in films was observed by Atarés, Bonilla, and Chiralt (2010) in cinnamon EO soy protein films, and by Ojagh et al. (2010) who incorporated the same oil into chitosan films. These studies justified such behavior by the strong interaction between the EO and the polymer matrix, promoting chain cross-linking. Ojagh et al. (2010) also found a decrease in elongation-at-break in films, similar to Moradi et al. (2012) when incorporating Zataria multiflora EO into chitosan films, where the latter attributed this to an increase in the pore size of the films, creating possible rupture points.
3.1.6. Texture Fig. 5 shows hardness data for bread packed with CGG/EO film and polyethylene (PE) film (commercial packaging). It is observed that the hardness is inversely proportional to the water levels of the bread. Bread packed in CGG/EO film is more stringent than food wrapped in commercial packaging of PE over the course of days because the WVP of the CGG/EO film (1.78 g.mm.k.pa−1. h−1. m-2) is high enough to let the bread lose its moisture, making it harder as the days go by compared to polyethylene packaging (0.008 g.mm.k.pa−1. h−1. m-2), already widely used in the market. Mudgil, Barak, and Khatkar (2016) observed that the bread hardness remained almost unchanged up to 60% water level, but the bread hardness decreased with the increasing water level beyond 60%. The increase in permeability to water vapor (WVP) and water absorption is one of the main factors that may contribute to the degradation of packaging made with CGG film (Oliveira et al., 2018). From the characteristics presented, the packaging under study proved to be efficient in protecting the bread from microbial action for at least six days, due the less water available for the microbial growth as demonstrated in Fig. 4d
3.1.5. Application of the film as bread packaging Several studies have demonstrated the efficacy of packaging using various materials and techniques in maintaining bread quality characteristics, delaying moisture loss and fungus growth (Latou, Mexis, Badeka, & Kontominas, 2010; Licciardello, Cipri, & Muratore, 2014; Pagani, Lucisano, Mariotti, & Limbo, 2006; Piergiovanni & Fava, 1997; Cozmuta et al., 2015). These results imply in an increase in the shelf life of the bread. Research of the active components identified neral and geranial in higher proportions in the bread crust, and increasing throughout the days (Fig. 4a), with data indicating significant differences by the value of P < 0.0001. Considering the arrangement of the slices inside the package (Fig. 4b Commercial packaging and 4c CGG/EO packaging) and the observed shelf life, the antimicrobial barrier action do film was observed mainly in the bread crust. This fact can be suggested by retaining the active compounds of the Cymbopogon citratus essential oil (EO) incorporated in the experimental film. The presence of the assets in the core is added to the bread’s stability. Such behavior is relevant due to the absence of preservatives in the food used. Assay research was completed after the sixth day of storage due to fungus presence (visual observation) (Fig. 4c). This fact reveals oxygen permeability of the packaging under study, however without compromising its effectiveness given that the satisfactory storage period promoted six days in function of the commercial package evaluated in this
4. Conclusion Ferulic acid was efficient in cross-linking the GC:G matrix, providing films with low solubility and opacity, and without significant change in water vapor permeability when compared to FC. The IR spectrum confirmed the cross-linking at 1546 cm−1 relative to the N–O covalent bond. The cross-linking provided more compact films. The cross-linked films were shown to be less rigid according to the Young’s modulus and having higher elongation-at-break by more than 100%, which gives them a plastic material characteristic. The essential oil favored the mechanical properties, with a 38% decrease in the tensile strength in the films. In general, the presence and absence associated or not of the surfactants and essential oil did not affect the investigated characteristics. The essential oil promoted bread preservation. The results present the films as an alternative to food packaging. Declaration of Competing Interest The authors declare that they have no conflict of interest regarding this manuscript. 7
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Acknowledgements
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Packaging with cashew gum/gelatin/essential oil for bread: Release potential of the citral
T
Marília A. Oliveiraa, Maria L.C. Gonzagab, Maria S.R. Bastosa,*, Hilton C.R. Magalhãesa, Selene D. Benevidesa, Roselayne F. Furtadoa, Rafael A. Zambellib, Deborah S. Garrutia a b
Embrapa Agroindústria Tropical, Rua Dra. Sara Mesquita 2270, CEP 60511-110, Fortaleza, CE, Brazil Departamento de Engenharia de Alimentos, Universidade Federal do Ceará, CP 12200, 60455-760, Fortaleza, CE, Brazil
ARTICLE INFO
ABSTRACT
Keywords: Polysaccharide Cashew gum Gelatin Ferulic acid Active film
Cashew gum (CG) and gelatin (G) films were produced by incorporating ferulic acid (FA), as a cross-linking agent and Cymbopogon citratus essential oil (EO) for application bread packaging. The addition of FA and EO did not significantly affect water vapor permeability (WVP) or opacity. There was a significant decrease in the solubility of the films, possibly due to matrix cross-linking. Morphology revealed films with smooth surfaces and small cross-sections. Characteristic frequencies of CG and G demonstrated the cross-linking identified in infrared (IV). EO cross-linked films showed lower rupture stress than non-EO films. Elongation-at-break increased from 2% (cross-linked) to 140% (cross-linked/EO). CGG/EO packaging was more permeable than commercial packaging (polyethylene), giving bread more harshness. The experimental packaging provided six days of preservation to the bread compared with three days for the commercial packaging. Neral and geranial presented a higher proportion in the crust of the bread, suggesting antimicrobial action.
1. Introduction A wide variety of gums is produced in tropical and subtropical countries with attractive characteristics for commercial and industrial applications; a typical example is cashew gum, which has received attention because it is similar to gum arabic, which is widely used in the food industry. The Anacardium occidentale L. cashew tree is native to northeastern Brazil, and exudes a gum in response to the attack of pathogens and lesions. In Brazil, cashew trees produce an average of 700 g of cashew gum (CG) per year. Since there are more than 700 thousand hectares of planted cashew trees in Brazil, it is estimated that the production potential of the CG is around 50,000 tons per year (Ribeiro et al., 2016), and it is continuously studied aiming towards different applications. Cashew gum exhibits stabilizing, emulsifying and adhesion properties in water, and can be molded into a film with the addition of a suitable plasticizer such as glycerol. However, even with the use of glycerol in high concentrations, CG films are relatively fragile, inflexible and soluble, making it necessary to incorporate gelatin and a cross-linking agent to improve the mechanical and solubility properties. The need for cross-linking in packaging films is due to the hydrophilicity of the materials, and is therefore a way to improve the incorporation and adhesion of active substances such as antioxidants
⁎
and essential oil. Ferulic acid, a powerful antioxidant extracted from plants and grains, has been shown to be an excellent cross-linking agent for the gell-gelatin polyelectrolyte complex, it acts on the amine groups forming cross-links throughout the blended structure (Graf, 1992). Oxidation occurs employing 0.5% hydrogen peroxide solution. The acid forms a dimer as a side reaction or reacts with amine groups I and II which are free in the gelatin chain to form OeN covalent bonds. The latter response can be reoxidized and linked to another amine, resulting in cross-linking. Research on active food packaging has been developed by incorporating substances that can alter the food environment, affecting its metabolism, providing greater protection, consequently improving quality and increasing its useful life (Bastarrachea, Dhawan, & Sablani, 2011; Zhao & Saldaña, 2019). Packaging is the last stage of production, and its choice is based on promoting the intrinsic quality of the product and its effectiveness in preserving and communicating this quality (Licciardello et al., 2017). The most commonly used polymers in packagings are derivated from petroleum products and not biodegradable, generating solid waste from their final disposal and leading to serious environmental problems. This opens the way for the use of natural biodegradable polymers in manufacturing active food packaging (Arfat, Ejaz, Jacob, &
Corresponding author. E-mail address: [email protected] (M.S.R. Bastos).
https://doi.org/10.1016/j.fpsl.2019.100431 Received 13 August 2019; Received in revised form 14 October 2019; Accepted 25 October 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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M.A. Oliveira, et al.
Ahmed, 2017). Essential oils (EOs) extracted from plants and spices exhibit antimicrobial and antioxidant properties (Viuda-Martos et al., 2010), which makes them excellent additives in the food industry as a substitute for chemical additives (Alves-Silva et al., 2013). The composition of the oil and the specific interactions with the polymer determine its effectiveness as an active ingredient (Chiralt & Atar, 2016). Cymbopogon citratus essential oil (EO) contains 40–80% of Citral, being widely used in the cosmetic, food and pharmaceutical industries (Martinazzo, Corrêa, Melo, & Barbosa, 2007) for its antifungal (de Sá Silva et al., 2009) and antibacterial properties (Cimanga et al., 2002). Therefore, the combined effect ferulic acid as cross-linked cashew gum/gelatine films incorporated with commercial lemongrass (Cymbopogom citratus) and on their physicochemical, microscopic, spectrometric and mechanical characterizations and volatile compounds of essential oil release was analyzed in this work to verify the possibility of the an food packaging to bread.
of ± 0.01 mm. The thickness used was the arithmetic average of eight measurements performed randomly along each evaluated sample. 2.3.2. Opacity measurement Film samples were cut into a rectangle (8 x 40 mm) and placed on the internal side of a spectrophotometer test cell (Agilent technologies cary 60). The opacity was calculated using the following equation:
Opacity =
Abs750 X
Where: Abs750 is the absorbance value at 750 nm and X is the film thickness. The lower opacity values indicate higher sample transparency (Farhan & Hani, 2017). 2.3.3. Water vapor permeability (WPV) This was determined in accordance with ASTM method E96-05 (ASTM, 2005). Eight replicate samples (60-mm diameter each) were placed between the acrylic permeation cell fittings with a circular opening (diameter = 50 mm), corresponding to the film area exposed to migration. Each cell contained 5 mL of distilled water in its interior. The cells were placed in a desiccator (25 ± 5 °C temperature and 30 ± 5% relative humidity) containing silica gel. The weight of the cells was observed each hour on an analytical balance for 24 h.
2. Materials and methods 2.1. Cashew gum isolation Gum exudate was collected from cashew trees from Embrapa Experimental Station at Pacajus (Fortaleza-Ceará). It was ground to 100 mesh particle size, and 100 g resulting powder was solubilized in 300 mL water at the ratio of 1:3 (w/v) for 4 h, centrifuged at 10,000 rpm at 4 °C for 20 min, and filtered to remove the insoluble materials. 300 mL exudate solution was add in 1200 mL commercial ethanol at a 1:4 (v/v) ratio for 24 h to precipitate the polysaccharide. The precipitate was washed with acetone, then vacuum filtered on funnel with sintered glass disc nº 1, this process being repeated for five times and dried in a hot air circulation oven. This final powder is called “cashew gum” in this paper. Gelatin 225H B type (pH = 5.4) was kindly supplied by Rousselot® (São Paulo, Brazil).
2.3.4. Solubility in water The water solubility determination was carried out on 3 cm x 3 cm film pieces based on the method proposed by Gontard, Duchez, Cuq, and Guilbert (1994) with some modifications. Previously dried and weighed samples were immersed in 50 mL of distilled water for 24 h at 25 °C with stirring (75 rpm). The dry weight of the remaining film pieces was obtained after filtration on previously dried and weighed filter paper and used to calculate the insoluble matter as a percentage of the initial dry weight (g/100 g). All the dry weights (of the initial and final film pieces and the filter paper) were determined after drying at 105 °C for 24 h using a fan-assisted convection oven (Quimis model Q 31 4M22, Brazil).
2.2. Formulation of cross-linked film
2.3.5. Contact angle The contact angles of coating surface were measured on GBX Digidrop MCAT (Modular Contact Angle Technology) system coupled to the CAN101 Tensiometer Optical Contact Angle Meter (KSV Instruments). Five drops of water were deformed on the surface of the films (FC and F1 to F4) individually. The angles were recorded by optical system immediately after droplet deposition, ∼ 2 s, after 1 hour of adhesion and after 2 hours with complete evaporation of water, all measurements being digitally determined. The assay was performed at room temperature (∼ 25 °C).
Cashew gum (CG) and gelatin (G) at a weight ratio of 5.0CG:5.0 G and 10% glycerol in relation to the total weight of the polymers were added in water and kept under stirring for 4 h at 60 °C. Then 1% oxidized ferulic acid (FA) was added relative to the total weight of the polymers. After this period, the surfactants span 80 (S) at 0.6% and tween 80 (T) at 0.5% were added, reaching an Hydrophile-Lipophile Balance (HLB) of 10.8 and then 10% lemon grass essential oil (EO) supplied by Embrapa was added. Homogenization occurred in an Ultra Turrax T25 (IKA, Wilmington, NC, USA) for 10 min. The films were produced by the casting method and dried at room temperature. Four films were prepared following the described procedure, called F1, F2, F3 and F4. Film F1 contains all of the above components, F2 does not contain lemon grass essential oil, F3 does not contain surfactants and F4 does not contain surfactants or essential oil. A non-crosslinked film without surfactants or essential oil was used as a control, called FC. The following were elaborated in order to facilitate identification of the films: F1(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S:1.0EO), F2(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S), F3(5.0CG:5.0 G:1.0 Gly:0.1FA:1.0EO), F4(5.0CG:5.0 G:1.0 Gly:0.1FA) and FC (5.0CG:5.0 G:1.0 Gly). The commercial polyethylene film was obtained in the local market.
2.3.6. Fourier transform infrared (FT-IR) The samples were analyzed as obtained by ATR/FT-IR in the medium infrared region, with wavelengths between 4000 and 400 cm−1 and 4 cm−1 resolution using the Perkin Elmer brand. 2.3.7. Scanning electron microscopy (SEM) Morphological characterization was performed by scanning electron microscopy (SEM, Zeiss DSM, model 940A). Samples were coated with platinum using a sputter coater (Electron Microscopy Sciences, Hatfield, PA, USA) and examined under SEM using an accelerating voltage of 15 kV and a magnification of 3,000–6,000×. 2.3.8. Mechanical properties Mechanical properties were tested according to ASTM Method D882-09 (ASTM, 2009) on a mechanical tester (EMIC DL 3000) using 12.8 cm film strips. The initial grip separation and cross-head speed were set to 10 cm/min. Both force (N) and deformation (mm) were recorded during extension. Tensile strength was calculated by dividing
2.3. Film characterization 2.3.1. Thickness Film thickness was measured using a digital micrometer (Digimatic model, Mitutoyo, Brazil) with a range of 0–25 mm and an accuracy 2
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the required force for film rupture by the cross-sectional area, and elongation-at-break was calculated as the percentage increase in sample length. The elastic modulus was calculated from the slope of the stress–strain curve in the elastic deformation region. The reported values are the averages of five measurements.
the films as barriers to water vapor bigger than for similar materials (Azeredo et al., 2012; Wu et al., 2016). The higher WVP values obtained for the FC, F3 and F4 films may be related to the absence of surfactants in these materials, resulting in lower compaction of the structure by decreasing of bonds. The higher thicknesses of these films may also contribute to higher WVP values. Ferulic acid action was observed by Ou, Wang, Tang, Huang, and Jackson (2005) and Silva, Santos, Coutinho, Mano, and Reis (2005), who reported no significant reduction in the vapor permeability of the soy protein films cross-linked with this acid. In addition to noting the low influence of ferulic acid on the WVP of gelatin films. Cao, Fu, and He (2007) suggest that its small effect is due to the possible combination of the hydroxyl groups of the acid with the water molecules, not favoring permeation. Edible films made from sodium caseinate crosslinked with feluric acid were less permeable to water vapor than αtocopherol cross-linked films (Fabra, Hambleton, Talens, Debeaufort, & Chiralt, 2011). The presence of gelatin in the composition of the films favors poor absorption of water since it can form organized structures, with it being possible to recover part of the triple helix structure of the collagen during the formation process of the film. The higher degree of organization optimizes molecular packaging, resulting in films with better gas barrier properties (Fan, Duquette, Dumont, & Simpson, 2018; Yun & Dong, 2017). Benavides, Villalobos-Carvajal, and Reyes (2012) formulated alginate films with oregano essential oil (EO) in varying proportions and observed a porous structure, whose increase in EO content resulted in greater packaging of the layers, accompanied by an improvement in water barrier properties. Ojagh, Rezaei, Razavi, and Hosseini (2010) incorporated cinnamon essential oil into chitosan films and observed final films with leaf-like structures stacked in thick layers, which presented better water barrier properties. The solubility of the films after cross-linking decreased from 53 to 3%, indicating good process efficiency. In a study by Cao et al. (2007), the effect of water on gelatin films cross-linked with ferulic acid was classified as slight. The presence or absence of essential oils and surfactants, associated or not, did not promote a change in solubility, as the data for F1 to F4 show. Due to the similarity in the results obtained individually for the physicochemical properties analyzed herein, it can be shown that EO was satisfactorily dispersed in the presence and absence of surfactants. The optical analysis of the films is essential to evaluate the acceptance of certain foods by the consumer. Transparency is essential for materials whose purpose as packaging is to promote good visualization of the packaged product. The developed films are promising, since the non-cross-linked film (FC) did not register opacity and the reticulated films presented small opacity values. It is important to note that such behavior was independent of the composition and thickness of the films. For thickness, the fluctuation in the values is due to the mold casting method, a fact reported by other authors (Cao et al., 2007; Henrique, Cereda, & Sarmento, 2008). The results (Table 1) show similar values for crosslinked films (F1 to F4), indicating that the presence or absence of surfactants, essential oil and glycerol did not influence the adhesive properties of the films structural composed by gelatin cashew gum. Non-crosslinking of the FC film, which gives it greater solubility, may account for its lower contact angle value. The low solubility of films (F1 to F4) may be related to the branched molecular structure of cashew gum (Ribeiro et al., 2016). This characteristic added to the presence of cross-linking may contribute to the non-exposure of hydroxyls present in glycosidic units, not favoring the formation of hydrogen bridges with water and, consequently, their solubility. Fig. 1 illustrates the behavior of sessile drop on the surface of the films. The interval recording of 2 h shows the efficient adhesion of water to film surfaces, regardless of their compositions. After 1 h of contact, it was observed that the drop deformed the films, without breaking them and spreading the droplets on the surfaces. This may be related to the ability of the material to absorb water by capillary infiltration or by reorienting hydrophilic groups towards the surface as a
2.4. Film application as bread package 2.4.1. Packaging preparation The package (Fig. 4c) was prepared using the F1 film, selected for its mechanical resistance. Three 45 cm x 30 cm films each were closed by vacuum-sealed Selovac model 200B. The basic formulation bread (wheat flour, margarine, baking powder, sugar and salt) was prepared at the Laboratório de Cereais of Departamento de Tecnologia de Alimentos UFC - Brazil, cut into 1.25 cm thick x 15 cm in length, packed as shown in Fig. 4b and c and conditioned at room temperature (25 °C) for 8 days, during which the analyzes were performed. 2.4.2. Volatile compounds release in the bread Extraction of volatiles in the F1 films (the package) and packed bread (Fig. 4c) was performed by Headspace Solid-Phase Microextraction (SPME) using a carboxen/divinylbenzene/polydimethylsiloxane 50/30 μm fiber. First, 0.5 g of sample film and bread separately were cut in small pieces and weighed in 20 mL vials. The fiber was then exposed into the headspace above the sample for 40 min, maintaining the temperature at 40 °C. The volatiles were thermally desorbed into the splitless injection mode of a gas chromatograph (GC Shimadzu GC2010 Plus, Japan) fitted with a 5% phenyl stationary phase capillary column (30 m length, 0.25 mm id, 0.25 mm film thickness). Hydrogen was used as the carrier gas at a flow rate of 1.0 mL min−1. The oven temperature was programmed from 70 °C to 180 °C at 4.0 °C min−1, and then raised to 250 °C at 8 °C min−1, held for 5 min. The injector temperature was 250 °C and the FID detector was 260 °C. Gas Chromatography-Mass Spectrometry (GC–MS SHIMADZU QP2010, Japan) and Retention indices were used to identify neral and geranial. Analyses were performed in triplicate. 2.4.3. Texture The texture profile was obtained by the double compression test of the bread slices packed in a TA-XT2i texture analyzer (Stable Micro Systems). A texture profile analysis (TPA) with velocity of 1.0 mm/s, compression distance of 40 mm, equivalent to 50% of compression, contact force of 5.0 g was performed using a SMS Aluminum Cylinder probe (P35). Texture Expert for Windows 1.20 program (Stable Micro Systems) collected the data. The hardness parameter was evaluated in this test. 2.5. Statistical analyses The volatile compounds data were evaluated by regression analysis, while the quality of the models (linear or quadratic) was evaluated by the coefficient of determination (R2) and by the lack of fit test. The film characterization data were submitted to analysis of variance (ANOVA) and Tukey test with a level of 5% of significance for comparison of means. All analyses were performed using using the XLSTAT v. 18.1 statistical software program (Addinsoft). 3. Results and discussion 3.1. Characterization of films 3.1.1. Thickness, opacity, WVP, solubility and contact angle Table 1 presents the properties of thickness, opacity, water vapor permeability, solubility and contact angle of films obtained under different conditions. As shown in Table 1, cross-linking through ferulic acid did not influence the WVP of the films (F1 to F4). The results show 3
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Table 1 Thickness, water vapor permeability, solubility and opacity for F1, F2, F3, F4 and FC films. Film
Thickness§ (μm)
FC F1 F2 F3 F4
59.67 58.94 49.90 95.10 68.90
± ± ± ± ±
1.50bc 3.93 bc 6.25 c 8.42 a 5.61 b
WVP (g.mm.k. Pa−1. h−1. m-2) 1.42 1.78 1.72 2.60 2.10
± ± ± ± ±
0.20 0.07 0.40 0.30 0.22
c bc bc a ab
Solubility (%)
Thickness† (μm)
53.40 ± 2.50a 3.04 ± 0.31 b 2.39 ± 0.42 b 3.00 ± 0.48 b 3.03 ± 0.35 b
76.59 47.21 51.10 58.13 46.35
± ± ± ± ±
3.27b 5.94 c 7.10 c 10.66 c 6.27 c
Opacity 0.00 0.11 0.10 0.10 0.11
± ± ± ± ±
Contact angle 0.00 0.00 0.00 0.00 0.00
d a c bc ab
50.98 61.66 61.78 62.72 60.68
± ± ± ± ±
2.23 1.30 1.78 2.91 1.25
a b b b b
§
Thickness for WVP (mean of eight replicate samples, each measured eight times). Opacity thickness (average of six replicate samples, each measured eight times). Mean of three values with standard deviation, the same letter in the row indicates that there is no significant difference between the means by the Tukey test (p < 0.05). F1(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S:1.0EO), F2(5.0CG:5.0 G:1.0 Gly:0.1FA:0.05T:0.06S), F3(5.0CG:5.0 G:1.0 Gly:0.1FA:1.0EO), F4 (5.0CG:5.0 G:1.0 Gly:0.1FA) and FC (5.0CG:5.0 G:1.0 Gly).
†
function of hydration generated by interaction with water (Gugliuzza & Drioli, 2004). The non-rupture of the films was better recorded after 2 h of application, with drop evaporation.
stretches of the C–H bonds of CH3 and C]O of the aldehyde, are added to those recorded at 2923 cm−1 and 1633-1638 cm−1, respectively. As proposed in this work, the bond that structures the cross-linking is likely made between the amino group (NH2) of the gelatin and the hydroxyl group (OH) of ferulic acid, the registration of which can be identified by symmetrical stretching at 1546 cm−1 for NO (Silverstein et al., 2007), present in all cross-linked films.
3.1.2. Fourier transform infrared (FT-IR) The IR spectra of the films, ferulic acid, cashew gum, and gelatin are shown in Fig. 2. Characteristic frequencies of the present polymers, CG and G and FA on the films are reported at 3295-3304 cm−1 for OH, and NH stretches, 2923 cm−1 for asymmetric stretches CH of CH2, 1150 cm−1 for CO (anomeric carbon), 1072-1077 cm−1 for COC (glycosidic bonding), 1031-1036 cm−1 for alcohol CO, with the latter three being specific for CG. C]O acid stretches, characteristic of gelatin and ferulic acid, are recorded at 1650 and 1679 cm−1, respectively. The record of this group in the films can be observed displaced in the range of 1633-1638 cm−1. The presence of ferulic acid on the films is also recorded in the stretches at 1454 cm−1, the characteristic frequency of C]C aromatic ring bonds (Wang, Sun, Liu, Li, & Ma, 2011; Yeung, Eskici, & Axelsen, 2013; Silverstein, Webster & Kiemle, (2007)). Three intense bands at 1618, 1598 and 1518 cm-1 observed in the aromatic ring of fungal acid appear in some regions between 1630 to 1556 cm-1. EO-specific frequencies added in F1 and F3, such as the asymmetric
3.1.3. Scanning electron microscopy (SEM) Fig. 3 micrographs were obtained for the control-FC and F3-sample films. The F1, F2 and F4 images were suppressed by F3 similarity, despite differences in compositions. The FCa and F3a films show homogeneity in the surfaces with no irregularities, as reported by Cao et al. (2007). The recording of air bubbles is due to the casting method used in the molding of films (Mathew & Abraham, 2008). The transverse sessions (FCb and F3b) show higher compression in F3b, with few pores without depth. The bumps in some points are due to the blade action used in the cut. The compact aspect observed may be related to the degree of interaction between the gelatin molecules, one of the structural polymers of the film, provided by the cross-linker action. Mathew and Abraham (2008)) observed a similar compact aspect for composite chitosan and starch films cross-linked with ferulic acid.
Fig. 1. Wettability behavior of FC and F1-F4 films after 2 s, 1 h and 2 h. 4
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Fig. 2. Infrared spectra of the films samples (F1, F2, F3, and F4) and control (FC), Ferulic acid (FA), Gelatin (G) and Cashew gum (CG).
Considering the similarity in the compositions of FC and F4, the absence of ferulic acid (the only non-common component) may respond to the aggregates visualized in FCb. The presence of the essential oil did not promote morphological changes in the F1 and F3 films when compared to the other cross-linked F2 and F4 films.
on the concentration of the cross-linking agent, the literature indicates that the cross-linking process makes the polymer matrix structure more rigid, leaving the films less flexible (Dammak, Bittante, Lourenço, & do Amaral Sobral, 2017). The obtained results reveal a good cross-linking/ matrix ratio able to flexibilization the polymeric chain. The E in the cross-linked films decreases and the ε increases considerably, because in the case of ferulic acid, the cross-linker can preserve the matrix structure in its action, herein formed by CG:G, resulting in a more flexible film with lower tensile strength, especially in F1 and F3, which present EO. A decrease in tensile strength caused by adding EO in films based on polysaccharides was repeatedly observed (Atarés, Pérez-Masiá, & Chiralt, 2011; Ma, Hu, Wang, & Wang, 2016; Sánchez-González, Cháfer, Chiralt, & González-Martínez, 2010; Sánchez-González, Chiralt, González-Martínez, & Cháfer, 2011). Tongnuanchan, Benjakul, and Prodpran (2012) and Limpisophon, Tanaka, and Osako (2010) observed
3.1.4. Mechanical properties The mechanical properties of the film indicate the expected integrity under stress conditions that would occur during processing, handling, and storage. The stiffness, strength, and flexibility of the film are described by Young’s modulus (E), tensile strength (σ) and elongation-at-break (ε). Table 2 shows the modulus, tensile and elongation values for the ferulic acid cross-linked films with and without the surfactants, lemongrass essential oil, as well as the non-cross-linked (FC) film. It is observed that FC showed to be more rigid than the crosslinked films. However, its elongation-at-break was very low. Depending
Fig. 3. Micrographs of the surfaces (a) and cross-sections (b) of the films: FCa and FCb (5.0CG: 5.0 G: 1.0 Gly); F3a and F3b (5.0CG: 5.0 G: 1.0 Gly: 0.1FA: 1.0EO). 5
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Table 2 Mechanical properties for the F1, F2, F3, F4 and control (FC) films. Films FC F1 F2 F3 F4
Thickness(μm)
E(Mpa) b
74.00 ± 5.00 51.53 ± 0.79 cd 46.12 ± 0.92 d 140.60 ± 5.16 a 56.46 ± 1.49 c
3.54 0.59 0.09 0.27 0.14
± ± ± ± ±
σ(MPa) 1.34 0.25 0.00 0.09 0.67
a b c c c
58.36 19.16 46.60 19.40 50.51
± ± ± ± ±
ε(%) 0.03 4.18 1.25 0.65 2.18
a c b c ab
2.97 ± 0.98 b 141.57 ± 46.75 105.50 ± 65.42 121.69 ± 41.75 137.13 ± 34.89
a a a a
Mean of three values with standard deviation, same letter in the line indicates that there is no significant difference between the means by Tukey test (p < 0.05). Fig. 4. (a) Neral and geranial determination in percentage of area and their respective equations. Bread crust: neral (□): y = 176.37x² – 731.18x + 2057.9 R² = 0.9309; geranial (●): y = 212.11x² – 929.76x + 2278.2 R² = 0.9451 and bread crumbs: neral (Δ) : y = 28.111x² – 93.522x + 340.27 R² = 0.7308; geranial (▼) : y = 22.114x² – 73.734x + 233.45 R² = 0.6725 (b) Bread in commercial packaging (c) Bread in CGG / EO film and (d) aspect of bread wrapped in CGG / EO film over days.
that the incorporation of citric oil resulted in a reduction in the tensile strength of edible films of fish skin gelatine, which was attributed to the lower interaction between the gelatin molecule and provides flexible domains within the film. The effects of destabilization phenomena during the film drying were considered by Hosseini, Rezaei, Zandi, and
Farahmandghavi (2015) to justify the reduction of tensile strength in gelatine-chitosan fish films added with Origanum vulgare essential oil. Therefore, in the films studied herein greater flexibility and lower stiffness of the cross-linked films is a probable consequence of the presence of the essential oil, favoring the behavior of the glycerol in 6
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study (Fig. 4d). The efficiency of active packaging by the action of essential oils was verified by Priyadarshi, Sauraj Kumar, Deeba, Kulshreshtha, and Negi (2018) by incorporating apricot kernel oil into chitosan films. These authors not only observed better antioxidant activity, but also substantial antibacterial activity against Gram-negative E. coli, as well as Gram-positive B. subtilis bacteria. The films were also effective in inhibiting mold growth on bread for ten days, thus increasing its shelf life. Antioxidant action was also reported by the authors, similar to that disclosed for chitosan films added with green tea extract as presented by Siripatrawan and Harte (2010). Similarly, by adding cinnamaldehyde essential oils to wheat glycine films, Balaguer, Lopez-carballo, Catala, Gavara, and Hernandez-munoz (2013) obtained a material with antibacterial and antifungal action, which promoted a drastic reduction of fungi, and consequently extended the shelf life of the bread and packaged cheese. An important issue nowadays is the selection of packaging systems which are not only efficient, meaning capable of maintaining quality characteristics, but also useful in containing the environmental impact and costs generated by the production and disposal of packaging (Licciardello et al., 2017). In this aspect, developed packaging is favorable, since all the components are biodegradable (Biswas et al., 2018) and of affirmative action in the biochemical processes of the environment.
Fig. 5. Bread hardness packed in CGG / EO film and commercial packaging.
leaving spaces in the matrix structure. The action of the essential oil was also observed in films based on synthetic polymers of cellulose acetate, with reduction of the tension at rupture by the action of Cymbopogon citratus (Oliveira et al., 2017). The action of EO on elongation-at-break of films was also reported. Pranoto, Salokhe, and Rakshit (2005) observed that the proportion of EO determines its effect on elongation-at-break. Shojaee-aliabadi, Hosseini, and Amin (2013) found an increase in the elongation of κcarrageenan film caused by adding Satureja hortensis EO. Zinoviadou, Koutsoumanis, and Biliaderis (2009) incorporated oregano EO into serum protein films, and observed an increase in the elongation properties at an oil concentration of up to 1%; a fact explained by the development of composite film with discontinuities. Different behaviors have also been observed. Increased tensile strength by adding EO in films was observed by Atarés, Bonilla, and Chiralt (2010) in cinnamon EO soy protein films, and by Ojagh et al. (2010) who incorporated the same oil into chitosan films. These studies justified such behavior by the strong interaction between the EO and the polymer matrix, promoting chain cross-linking. Ojagh et al. (2010) also found a decrease in elongation-at-break in films, similar to Moradi et al. (2012) when incorporating Zataria multiflora EO into chitosan films, where the latter attributed this to an increase in the pore size of the films, creating possible rupture points.
3.1.6. Texture Fig. 5 shows hardness data for bread packed with CGG/EO film and polyethylene (PE) film (commercial packaging). It is observed that the hardness is inversely proportional to the water levels of the bread. Bread packed in CGG/EO film is more stringent than food wrapped in commercial packaging of PE over the course of days because the WVP of the CGG/EO film (1.78 g.mm.k.pa−1. h−1. m-2) is high enough to let the bread lose its moisture, making it harder as the days go by compared to polyethylene packaging (0.008 g.mm.k.pa−1. h−1. m-2), already widely used in the market. Mudgil, Barak, and Khatkar (2016) observed that the bread hardness remained almost unchanged up to 60% water level, but the bread hardness decreased with the increasing water level beyond 60%. The increase in permeability to water vapor (WVP) and water absorption is one of the main factors that may contribute to the degradation of packaging made with CGG film (Oliveira et al., 2018). From the characteristics presented, the packaging under study proved to be efficient in protecting the bread from microbial action for at least six days, due the less water available for the microbial growth as demonstrated in Fig. 4d
3.1.5. Application of the film as bread packaging Several studies have demonstrated the efficacy of packaging using various materials and techniques in maintaining bread quality characteristics, delaying moisture loss and fungus growth (Latou, Mexis, Badeka, & Kontominas, 2010; Licciardello, Cipri, & Muratore, 2014; Pagani, Lucisano, Mariotti, & Limbo, 2006; Piergiovanni & Fava, 1997; Cozmuta et al., 2015). These results imply in an increase in the shelf life of the bread. Research of the active components identified neral and geranial in higher proportions in the bread crust, and increasing throughout the days (Fig. 4a), with data indicating significant differences by the value of P < 0.0001. Considering the arrangement of the slices inside the package (Fig. 4b Commercial packaging and 4c CGG/EO packaging) and the observed shelf life, the antimicrobial barrier action do film was observed mainly in the bread crust. This fact can be suggested by retaining the active compounds of the Cymbopogon citratus essential oil (EO) incorporated in the experimental film. The presence of the assets in the core is added to the bread’s stability. Such behavior is relevant due to the absence of preservatives in the food used. Assay research was completed after the sixth day of storage due to fungus presence (visual observation) (Fig. 4c). This fact reveals oxygen permeability of the packaging under study, however without compromising its effectiveness given that the satisfactory storage period promoted six days in function of the commercial package evaluated in this
4. Conclusion Ferulic acid was efficient in cross-linking the GC:G matrix, providing films with low solubility and opacity, and without significant change in water vapor permeability when compared to FC. The IR spectrum confirmed the cross-linking at 1546 cm−1 relative to the N–O covalent bond. The cross-linking provided more compact films. The cross-linked films were shown to be less rigid according to the Young’s modulus and having higher elongation-at-break by more than 100%, which gives them a plastic material characteristic. The essential oil favored the mechanical properties, with a 38% decrease in the tensile strength in the films. In general, the presence and absence associated or not of the surfactants and essential oil did not affect the investigated characteristics. The essential oil promoted bread preservation. The results present the films as an alternative to food packaging. Declaration of Competing Interest The authors declare that they have no conflict of interest regarding this manuscript. 7
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Acknowledgements
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