International Journal of Biological Macromolecules 52 (2013) 116–124 Contents lists available at SciVerse ScienceDirect International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac Characterization of antioxidant-antimicrobial ␬-carrageenan films containing Satureja hortensis essential oil Saeedeh Shojaee-Aliabadi a , Hedayat Hosseini a , Mohammad Amin Mohammadifar a , Abdorreza Mohammadi a , Mehran Ghasemlou b , Seyed Mahdi Ojagh c , Seyede Marzieh Hosseini a , Ramin Khaksar a,∗ a Department of Food Science and Technology, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran b Department of Food Science, Engineering and Technology, Faculty of Agricultural Engineering and Technology, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran c Department of Fisheries, Gorgan University of Agricultural Sciences and Natural Resources Golestan, Noor, Iran a r t i c l e i n f o Article history: Received 26 June 2012 Received in revised form 30 July 2012 Accepted 23 August 2012 Available online xxx Keywords: Kappa-carrageenan Biodegradable film Satureja hortensis Antimicrobial activity Vapor phase Antioxidant activity a b s t r a c t The present work was aimed at characterizing biodegradable composite kappa-carrageenan films incorporated with Satureja hortensis (SEO) in terms of their physical, optical, mechanical, barrier and antioxidant properties. Also, in a comparative study, we sought to evaluate the antimicrobial effectiveness of these films against five pathogens. The films’ water vapor barrier properties were found to improve considerably upon the addition of SEO. Carrageenan composite films were less resistant to breakage, more flexible and more opaque with lower gloss than the control film. These results can be explained by the film’s microstructure, which was analyzed by atomic force microscopy and scanning electron microscopy. The films incorporating SEO showed good antioxidant properties; this effect was greatly improved when the proportion of added SEO was 3%. Films with SEO effectively inhibited the five microorganisms tested. The results of the present study suggest that SEO as a natural antibacterial agent can potentially be used in packaging a wide range of food products, particularly those that are highly oxidative and microbial sensitive. © 2012 Elsevier B.V. All rights reserved. 1. Introduction For the past 50 years, synthetic petroleum polymers have been widely used in a variety of packaging materials; however, they have become a major source of waste-disposal problems due to their poor biodegradability. To solve this problem, much research has aimed to obtain an environmentally friendly packaging material [1,2]. Recent decades have seen extensive investigation into biodegradable coatings or films prepared from biopolymers, including proteins, polysaccharides and lipids or their combinations. Edible, biodegradable films, by acting as barriers to control the transfer of moisture, oxygen, lipids and flavors can prevent quality deterioration and increase the shelf life of food products [3]. Moreover, biodegradable film can be used to carry active ∗ Corresponding author at: Food Science and Technology Department, Faculty of Nutrition and Food Science, Shahid Beheshti University, P.O. Box 19395-4741, Tehran, Iran. Tel.: +98 21 22376480; fax: +98 21 22376480. E-mail address: r.khaksar@sbmu.ac.ir (R. Khaksar). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.026 ingredients, such as antioxidant and antimicrobial agents that provide an extra stress factor against foods’ oxidative and microbial deterioration [4,5]. Carrageenans are natural, water-soluble hydrocolloids composed of a linear chain of sulfated galactans and extracted from certain species of red seaweed. They are classified according to the number and position of a sulfated ester on 3,6-anhydro-d-galactose residues. Carrageenans have high potential as a film-forming material. Cooling a hot solution of carrageenan during film casting and drying leads to a transition of random coil to double helix, which results in the formation of a compact and structured film after the dehydration of the solution [6]. In one study, Park [7] reported that ␬-carrageenan can produce a clear film with good mechanical and structural properties, including a tensile strength higher than those of ␫ and ␥-carrageenan films. A number of hydrophobic compounds, such as lipids, are frequently incorporated into hydrocolloid-based films as depressors of water vapor permeability (WVP). The incorporation of plant essential oils into these films represents an interesting alternative to lipids. Their potential health benefits, as well as their S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 strong antioxidant-antimicrobial properties, make them possible substitutes for synthetic antioxidant-antimicrobial agents to achieve oxidative and microbial stability, as well as safer food products [8]. Satureja hortensis is an annual, aromatic and medicinal plant belonging to the Lamiaceae family, which mainly grows in the Mediterranean region. This plant is used as a seasoning agent and traditional herb in folk medicine [9]. Satureja hortensis essential oil’s (SEO) antioxidant and antimicrobial characteristics, which result from its high content of phenolic compounds, have been extensively demonstrated [10,11]. The addition of essential oils into a film matrix, instead of applying them directly on food products, could contribute to reducing required doses of essential oils, while keeping their antioxidant-antimicrobial activities [4]. Additionally, although carrageenan-based films have good mechanical and structural properties, they perform poorly as water-vapor barriers due to their hydrophilic nature; this limits their application. The presence in films of hydrophobic essential oils provides a practical solution by reducing its affinity for water [5]. Recently, several reports on the antimicrobial activity of various essential oils incorporated into biodegradable films using direct application (direct contact between microorganisms and antimicrobial agents) have been published [4,12]. Some authors reported that the vapor phase of essential oils (no direct contact between the essential oil and the medium surface) exhibits good inhibitive power against foodborne pathogens and spoilage bacteria, and is even more effective than direct application; this in turn can reduce the organoleptic alteration induced by essential oils [13,14]. To our knowledge, few studies have been carried out to evaluate the effectiveness of biodegradable films containing essential oils in the vapor phase versus direct contact. In addition, there are no reported data on the characteristics of carrageenan composite films containing SEO. This study aimed to develop a new biodegradable film based on carrageenan-SEO composite film through emulsification; to assess the film’s antimicrobial effect in both the vapor phase and direct contact, as well as its antioxidant activity; and to explore their impact on the relevant properties to evaluate the films’ suitability as food coatings: WVP, mechanical, optical and microstructural properties. These results, currently not present in the literature, but are very important for evaluating these films’ possible applications as packaging material. 2. Materials and methods 2.1. Materials Kappa-carrageenan (Rico Co, Philippine), Essential oil (SEO), supplied by Barij Company, (Kashan, Iran), Tween 80 and glycerol (Fluka, Sigma–Aldrich, MO, USA), were used to prepare film-forming dispersions (FFD). Mueller–Hinton agar (MHA) and Mueller–Hinton Broth (MHB) were bought from Merck Co (Darmstadt, Germany). Folin–Ciocalteu reagent, sodium carbonate, standard gallic acid and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co (St. Louis, MO). All other reagents used were of analytical grade. 2.2. Bacterial strains Staphylococcus aureus ATCC 25923; Bacillus cereus PTCC 1154, Escherichia coli ATCC 25922; Pseudomonas aeruginosa ATCC 27853; Salmonella typhimurium ATCC 14028 were provided by Iranian Research Organization for Science and Technology (Tehran, Iran). Stock cultures of the studied bacteria were grown in MHB at 30 ◦ C for 24 h before the tests. 117 2.3. Preparation of films Kappa-carrageenan based films were prepared by the method of Park [7] with some modifications. A series of preliminary experiments were conducted to determine the appropriate concentration of plasticizer (glycerol) for preparing films. Results showed that filmogenic solutions containing 50% (w/w) glycerol (based on carrageenan weight) were easily removed from the plate. Film solutions were prepared by dissolving ␬-carrageenan (1%, w/v), in distilled water under magnetic stirring. Following the addition of glycerol at constant concentration (50% (w/w) based on carrageenan weight), stirring was continued for a further 40 min at 82 ◦ C. The emulsions were obtained by adding SEO to the carrageenan solution to reach final concentrations of 1, 2 and 3% (v/v) and Tween 80 as an emulsifier in quantities proportional to the essential oils (0.1, 0.2 and 0.3%, v/v). FFDs without any essential oils were also prepared for later comparison. Homogenization was carried out using a rotor-stator homogenizer (IKA T25-Digital Ultra Turrax, Staufen, Germany) at 13,500 rpm for 3 min at 80 ◦ C, and then the emulsions were cooled to 65 ◦ C to remove any air bubbles incorporated during homogenization. The FFDs were casted on the center of a rimmed circular area (177 cm2 ) of clean and leveled glass plates, and then dried at 30 ◦ C for 30 h (casting and drying were carried out at 30 ◦ C, which is at temperature below the helix melting point reported for carrageenan polymer [6]). Dried films were peeled off the casting surfaces and stored inside desiccators at 25 ◦ C and 53% relative humidity (RH) until evaluation. Saturated magnesium nitrate solution was used to meet required RH. 2.4. Determination of physical properties of films 2.4.1. Thickness Film thickness was determined using a manual digital micrometer (Mituto, Tokyo, Japan) to the nearest 0.001 mm. Reported values were average of at least ten random locations for each film sheet. 2.4.2. Moisture content The films moisture content was determined by drying in an oven at 110 ◦ C until a constant weight was reached (dry sample weight). Three replications of each film treatment were used for calculating the moisture content. 2.4.3. Film solubility in water The water solubility was determined in triplicate according to method of Ojagh et al. [5]. Briefly, pre-weighed film samples were immersed under constant agitation in 50 ml of distilled water for 6 h at 25 ◦ C. After filtration, undissolved film was dried at 110 ◦ C to constant weight. The initial dry weight was determined by drying at 110 ◦ C to constant weight. The water solubility (%) of the film was calculated according to the equation WS (%) = ((Wo − Wf )/Wo ) × 100, where Wo is the initial weight of the film expressed as dry matter and Wf is the weight of the desiccated undissolved film. 2.5. Mechanical properties Mechanical properties, including tensile strength (MPa) and elongation at break (%) of the film samples were measured at 25 ◦ C with a Testometric Machine M350-10CT (Testometric Co., Ltd., Rochdale, Lancs., England) according to ASTM standard method D882 [15]. All of the tested film strips (1.5 cm × 10 cm) equilibrated at 25 ◦ C and 53% RH in desiccators containing Mg(NO3 )2 saturated solutions for 48 h prior to testing. Equilibrated film strips were fixed between the grips with an initial separation of 50 mm, and the cross-head speed was set at 50 mm/min. Tensile strength was 118 S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 determined by dividing the peak load by the cross-sectional area of the initial film specimen. Elongation was calculated by percentile of a change in the length of the specimen to the original distance between the grips (50 mm). At least three replicates of each formulation were tested. FET × X3). Film samples were cryofractured by immersion in liquid nitrogen and then mounted on the specimen holder with a double-sided adhesive tape. After gold coating using a BAL-TEC SCD 005 sputter coater (BAL-TEC AG, Balzers, Liechtenstein), the images were captured using an accelerating voltage of 20 kV. 2.6. Water vapor permeability (WVP) 2.9. Atomic force microscopy (AFM) WVP of the film samples was determined at 25 ◦ C and 75% RH gradient according to the ASTM E96 gravimetric method [16]. The circular test cups containing anhydrous calcium chloride (0% RH, assay cup) or nothing (control cup) were sealed by the test films (0.00287 m2 film area). The cups were placed inside a desiccators maintained at 75% RH with a sodium-chloride-saturated solution (Merck, Darmstadt, Germany). The difference in RH corresponds to a driving force of 1753.55 Pa, expressed as water vapor partial pressure. Weight gain of the test cups along time was recorded periodically (with an accuracy of 0.0001 g). The slope of the weight gain vs. time plot was divided by exposed film area to obtain the water vapor transmission rate. This was multiplied by the thickness of the film and divided by the pressure difference between the inner outer surfaces to obtain the WVP. All tests were made in triplicate. The surface morphology of the films previously equilibrated at 53% RH, was studied by atomic force microscopy C26, DME, Denmark) with a (Dualscope/Rasterscope 200 ␮m × 200 ␮m scan size and a 6 ␮m vertical range. A sharpened cantilever was positioned over the sample, and 80 ␮m × 80 ␮m images were obtained. Three images of different zones were captured per formulation and analyzed offline with Dualscope/Rasterscope SPM software (Version 2.1.1.2) to transform into a three-dimensional image and to calculate the roughness values. Two statistical parameters related with sample roughness, were considered: Sa (average of the absolute value of the height deviations from a mean surface), and Sq (root-meansquare average of height deviations taken from the mean data plane). 2.7. Optical properties 2.10. Estimation of total phenolic (TP) content The lightness (L), redness (a), and yellowness (b) color system was used to evaluate the color of films by a colorimeter (Minolta CR 300 Series, Minolta Camera Co., Ltd., Osaka, Japan). The measurements were taken on white standard backgrounds (L* = 93.49, a* = −0.25 and b* = −0.09). All measurements were performed in triplicates. Total color difference (E) and whiteness index (WI) were calculated using following equations: The TP content of the films were estimated according to the previously reported method of Siripatrawan and Harte [19], involving the Folin–Ciocalteu reagent and gallic acid as standard with some modifications. Briefly, 25 mg of each film sample was dissolved in 5 ml of distilled water, then extract solution (0.1 ml), distilled water (7 ml), and Folin–Ciocalteu reagent (0.5 ml) were mixed and kept at room temperature for 8 min, after which 1.5 ml sodium carbonate (2%, w/v) and water were added to obtain a final volume of 10 ml. The mixture was stirred thoroughly and allowed to stand for 2 h at room temperature prior to an absorbance reading at 765 nm in a spectrophotometer (Shimadzu UV-VIS 1601, Japan). The results were expressed as mg gallic acid equivalents (GAE) per gram of dried film according to the following equation: E =  (L∗ − L)2 + (a∗ − a)2 + (b∗ − b) 2 (1) where L*, a*, and b* are the color parameter values of the standard and L, a, and b are the color parameter values of the sample. WI = 100 −  (100 − L)2 + a2 + b2 (2) The gloss of the films was measured at incidence angles of 60◦ with respect to the normal to the coating surface, according to the ASTM standard method D523 [17] using a flat surface gloss meter (Multi.Gloss 268, Minolta, Germany). Prior to optical measurements, films were conditioned in desiccators at 25 ◦ C and 53% RH. Gloss measurements were performed on the side of the film in contact with air during drying and over a black matte standard plate and six replicates were taken per formulation. Results were expressed as gloss units, relative to a highly polished surface of black glass standard with a value near to 100. The opacity of the film specimens was evaluated by measuring the absorbance at 600 nm using a spectrophotometer (Shimadzu UV-VIS 1601, Japan) according to the method of Gómez-Estaca et al. [18]. An empty test cell was used as the reference. The opacity was calculated using the following equation: Op = Abs600 x (3) where Abs600 is a value of absorbance at 600 nm and x is the film thickness (mm). According to this equation, the low values of Op demonstrate higher transparency and lower degree of opacity. Three replicates of each film were tested. 2.8. Scanning electron microscopy (SEM) Microstructure of the cross-sections of dried films was observed by scanning electron microscopy (Oxford Instruments INCA Penta T= C ·V M (4) where T is total content of phenolics compound (milligram per gram dried film, in GAE), C is the concentration of gallic acid obtained from the calibration curve (milligram per milliliter), V is the volume of film extract (milliliter) and M is the weight of dried film (gram). 2.11. DPPH radical-scavenging activity The hydrogen atom or electron donation abilities of the films were measured by the method of Brand-Williams et al. [20] on basis of bleaching of the bluish-red or purple-colored methanol solution of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a reagent. In its radical form, DPPH absorbs at 517 nm but, upon reduction by an antioxidant or a radical species, its absorption decreases. Decreasing the DPPH solution absorbance indicates an increase of the DPPH radical scavenging activity. Briefly, 25 mg of each film sample was dissolved in 5 ml of distilled water, and then a 0.1 ml of film extract solution were added to 3.9 ml of the DPPH solution (0.1 mM methanol solution) followed by 60 min incubation in the dark at ambient temperature. The absorbance was read against pure methanol at 517 nm and the percentage of DPPH radicalscavenging activity was calculated using following equation: DPPH scavenging activity (%) =  Ablank − Asample Ablank  × 100 (5) S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 where Ablank is the absorbance of the control, and Asample is the absorbance of the test compound. 2.12. Evaluation of antimicrobial activity of films 2.12.1. Disc diffusion method The antimicrobial activity of the films was qualitatively evaluated following an agar diffusion assay. The antimicrobial films were aseptically cut into 6 mm diameter discs and placed on plates containing MHA. The medium had been previously seeded with 100 ␮l of an overnight broth culture containing approximately 108 CFU/ml of the test bacteria. The plates were incubated at 30 ◦ C for 24 h. The diameter of the growth inhibition zones was measured using a caliper to the nearest 0.02 mm. The whole zone area was calculated then subtracted from the film disc area and this difference in area was reported as the “zone of inhibition” [5]. The tests were carried out in triplicate for each formulation. 2.12.2. Disc volatilization method Disc volatilization method was used to examine the antimicrobial activities of the films in vapor phase according to Lopez et al. [14]. Briefly, MHA medium was seeded with 100 ␮l of an overnight broth culture containing approximately 108 CFU/ml of the test bacteria. The antimicrobial films were aseptically cut into 6 mm diameter discs and laid on the inside surface of the upper lid, with no direct contact between it and the bacteria strains. The plate was then sealed using parafilm to prevent leakage of essential oil vapor then were incubated at 30 ◦ C for 24 h. The diameters of these zones were measured in millimeters and the whole zone area was reported as “zone of inhibition”. The tests were carried out in triplicate for each formulation. 2.13. Statistical analysis The statistical analysis of the data was performed using SPSS statistical software version 16 (SPSS Inc., Chicago, IL). Analysis of variance (ANOVA) followed by the Duncan’s multiple range test was used to determine any significant differences among the treatments at a 95% confidence level. 3. Results and discussion 3.1. Physical properties of films Preliminary experiments showed that the maximum amount of lipids that could be added to carrageenan films appeared to be 3% (v/v) of the FFD. Films containing higher percentages had an uneven lipid distribution, which most likely arose from the limited dispersion capability of the lipids, and from the poor stability of the emulsion systems at higher lipid concentrations. Table 1 shows the impact of incorporating SEO on the physical properties of carrageenan films. Film thickness varied from 0.031 to 0.068 mm. The films prepared with SEO showed lower moisture content and solubility than the control film; these decreased significantly (P < 0.05) as SEO content increased. Lower moisture content with minimum solubility (17.48% and 16.68%, respectively) was achieved for films formulated with 3% essential oil. These results could be attributed to a decrease in the hydrophilic nature of the films, as well as interaction between the components of SEO and the hydroxyl groups of carrageenan, which would reduce availability of hydroxyl groups for interaction with water molecules, consequently leading to a more water-resistant film. Similar results were found by Ghasemlou et al. [21] for kefiran films incorporating oleic acid. 119 3.2. Mechanical properties Analyzing the tensile strength (resistance to elongation) and elongation at break (capacity for stretching) of packaging materials gives better predictions about their mechanical properties in food applications [21]. Table 1 shows the influence of essential oil incorporation on the mechanical properties of carrageenan-based films. Film without essential oils had a tensile strength of 26.29 MPa, which agrees with that found by Park et al. [7] for carrageenanbased film (22–32 MPa). Tensile strengths were weaker for films containing SEO than for the control film, significantly (P < 0.05) decreasing as oil concentration increased. This coincides with the results reported by other authors when adding essential oil to a film-forming dispersion [4,22]. This effect could primarily be explained by the partial replacement of stronger polymer-polymer interactions by weaker polymer-oil interactions in the film network in the presence of the essential oil, which may weaken the network structure, and hence the tensile strength of the emulsified films [4]. As Table 1 shows, the incorporation of SEO caused a significant (P < 0.05) increase in the elongation of films. It appears that the aforementioned changes in the interaction balances had a plasticizing effect, even at small concentrations of essential oil, which made the film more stretchable (high elongation values at break); this coincides with the results observed for hydroxypropylmethylcellulose containing oleic acid [23]. 3.3. Water vapor permeability Table 1 shows the water vapor permeability (WVP) values of carrageenan films at different polysaccharide: essential oil ratios. The WVP was 2.38 g s−1 m−1 Pa−1 × 10−10 for the control sample (without essential oil). A significant increase was found in the moisture-barrier properties of films containing essential oil. When SEO concentration increased from 1 to 3%, WVP decreased markedly from 1.59 to 0.56 g s−1 m−1 Pa−1 × 10−10 (P < 0.05). For example, carrageenan films containing 3% SEO had a lower WVP (about 77%) than the essential oil-free films. It is generally accepted that tortuosity plays an important role in the water vapor transfer process [24]. The addition of SEO as a hydrophobic dispersed phase to hydrophilic ␬-carrageenan-based films tends to increase the tortuosity factor, leading to a decrease in the water vapor transmission rate. The same behavior has been observed by other authors [22,25]. For example, Sánchez-González et al. [4] found that the addition of 2% tea tree essential oil to a chitosan FFD reduced films’ WVP from 124 to 74.8 g s−1 m−1 Pa−1 × 10−11 . In contrast, Bonilla et al. [26] reported an increase in the WVP of chitosan films with increasing concentrations of thyme and basil essential oils. They suggested that water-molecule diffusivity increased because the interruption of the film network provoked by the essential oils overcame the overall hydrophobic nature of the film matrix. In the present study, although the presence of essential oils reduced film strength (Table 1), it did not contribute to a loss of matrix cohesion and, thus increasing WVP, even at the highest concentration. 3.4. Optical properties of films Consumer acceptability of biodegradable films as a food coating could be affected by their optical properties. To better understand the optical properties of SEO-carrageenan films, Hunter Lab color values (L, a, b), total color difference (E), gloss and opacity values were analyzed (Table 2). Carrageenan films without essential oil appeared clear and transparent. However, emulsified films had a slightly yellow appearance, as indicated by a significant (P < 0.05) increase in the b value and E but a decrease in the L, a, and WI values as a function of SEO concentration. These results agreed with visual observations. This phenomenon is probably due to the 120 S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 Table 1 Physical, WVP and mechanical properties of carrageenan films formulated with different concentrations of SEO.a Film Thickness (mm) Control SEO1 SEO2 SEO3 a 0.031 0.038 0.043 0.068 ± ± ± ± a 0.006 0.005ab 0.008b 0.005c Moisture content (%) 20.15 20.01 19.11 17.48 ± ± ± ± Solubility in water (%) a 2.53 1.44a 2.06b 1.88c 26.32 24.53 22.47 16.68 ± ± ± ± WVP (g s−1 m−1 Pa−1 × 10−10 ) a 1.03 0.75b 0.49c 1.66d 2.383 1.591 0.840 0.556 ± ± ± ± Elongation at break (%) 0.044a 0.112b 0.093c 0.032d 36.46 35.82 41.46 44.77 ± ± ± ± Tensile strength (MPa) 1.04c 2.02c 1.77b 3.49a 26.29 19.88 11.44 9.52 ± ± ± ± 2.93a 2.37b 1.53c 0.94d Data reported are average values ± standard deviations. Values within each column with different letters are significantly different (P < 0.05). Table 2 Effect of different concentrations of SEO on optical properties of carrageenan films.a Film L Control SEO1 SEO2 SEO3 a 88.41 76.52 74.65 72.79 a ± ± ± ± a 1.04 0.85b 0.74c 1.36d b −0.27 −1.32 −1.60 −2.00 ± ± ± ± d 0.07 0.55c 1.08b 0.09a 0.86 2.86 5.23 7.28 WI E ± ± ± ± d 1.15 0.24c 0.86b 1.64a 5.26 17.23 19.58 21.98 ± ± ± ± d 0.41 0.96c 1.03b 0.87a 88.27 76.33 74.11 71.80 Opacity ± ± ± ± a 0.52 0.68b 2.14c 1.30d 0.81 4.12 5.76 7.35 ± ± ± ± 0.02d 0.36c 0.58b 0.84a Data reported are average values ± standard deviations. Values within each column with different letters are significantly different (P < 0.05). phenolic compounds of SEO, which might have light absorption at low wavelengths. An increase in SEO concentration significantly (P < 0.05) reduced the transparency of carrageenan films, giving more-opaque films. Opacity values were also significantly (P < 0.05) higher in films incorporating the highest amounts of SEO. It has been previously explained that addition of a dispersed, less-miscible phase into a FFD makes a film less transparent than its pure form [27]. This phenomenon is related to the light-scattering provoked by the distribution of lipid droplets (with a different refractive index from the continuous phase) throughout the film network, as well as initial emulsion properties (volume fraction and concentration of lipid phase) [28]. The gloss of films is linked to the surface roughness: generally, the rougher the surface, the lower the gloss [25]. Fig. 1 shows the gloss values of the films measured at an incidence angle of 60◦ . The addition of SEO to the FFD significantly (P < 0.05) affected the gloss of the films. A linear decrease of glossiness as a function of the SEO concentration in the film matrix was observed. Film incorporated with 3% SEO had the lowest gloss value (14.1%). The composite films were not as glossy as the pure carrageenan films, suggesting an increase in the surface roughness of the composite films. This roughness appears to be the consequence of the migration of SEO droplets to the top of the film during drying, decreasing the specular reflectance in the air-film interface, and thus contributing to reduced gloss. Gloss reduction in composite films containing lipids has also been observed by other authors [25,26]. 60 50 Scanning electron microscopy (SEM) can provide a better understanding of the relationships of water vapor transmission, mechanical and optical properties with the films’ structural characteristics. Fig. 2 shows the cross-section micrographs for the control and the SEO-containing films at 1 and 3% (v/v). The pure carrageenan film displayed a compact, smooth and continuous microstructure with no irregularities. However, adding essential oil caused a heterogeneous structure in which oil droplets were entrapped in the continuous polysaccharide network. The oil droplets (Fig. 2c) were not exactly spherical, as is usual for oil/water emulsions; this may be due to the upright forces induced by the retraction of the carrageenan network during solvent evaporation. No creaming was observed in the emulsified films even at the highest concentration, probably because of the reduction of oil droplet mobility induced by the higher viscosity of the carrageenan dispersion, which increases during the film dehydration. Nevertheless, the size of the oil droplets in the film increased as the SEO concentration increased. The oil droplets were big enough to be visible at the magnification level used in films prepared with the highest SEO content. This could be explained by the fact that in the oil/water emulsions, a higher lipid content increases the collision frequency between droplets, which in turn increases flocculation and coalescence rate [29]. Moreover, in this study, although the amount of emulsifier present in each case was proportional to the SEO percentage, it was assumed that it was not high enough to prevent aggregation, particularly at 3%. 3.6. Surface morphology a 40 % gloss 60º 3.5. Film microstructure b 30 c 20 d 10 0 0% 1% 2% 3% Essential oil concentration (%v/v based on carageenan content) Fig. 1. Gloss values of carrageenan films with different concentrations of SEO measured at 60◦ . The presence of SEO not only altered the internal structure of film, as observed by SEM, but also promoted changes in the film surface as analyzed through AFM. Fig. 3 shows typical 3D-plots and the corresponding results for roughness parameters obtained for the control and emulsified carrageenan films. The oil-free film presents a smooth surface, with Sa and Sq values of 103 and 137 nm, respectively. The addition of SEO into the FFD produced films with much rougher surfaces, as indicated by higher Sa and Sq values (1220 and 1470 nm, respectively, for 3% essential oil) compared to the control film. These results are in line with those of Fabra et al. [27] for sodium caseinate-based films containing oleic acid-beeswax. The reliefs were accentuated when oil content increased, in according to SEM micrographs. These results also confirm those observed by gloss analyses. As mentioned above, this roughness is probably a result of irregularities in the surface induced by the presence of oil droplets and their aggregation during drying. S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 121 Fig. 2. SEM micrographs of the cross-sections of the films (scale bars: 50 ␮m). (a) Control, (b) SEO1, and (c) SEO3. 3.7. Total phenolic (TP) content and antioxidant activity Fig. 4a shows the TP content for different carrageenan films. A low TP content for pure carrageenan (1.12 mg gallic acid/g film) was measured. Our results showed that the films’ TP content significantly increased with increasing SEO concentration (Fig. 4a). The highest TP content (20.56 mg gallic acid/g film) was observed in films incorporating 3% of SEO. A DPPH-scavenging assay was used to determined films’ antioxidant activity. Fig. 4b shows the antioxidant activity of different SEO-formulated carrageenan films. The control film showed low antioxidant activities, probably because of its naturally occurring polyphenols. The results showed that the DPPH-scavenging activity of the films significantly increased (P < 0.05) with increasing SEO concentrations, as shown in Fig. 4b. The effect of essential oil concentration on the films’ antioxidant activity was expected and has been previously discussed in the literature [18,19,30]. GómezEstaca et al. [31] reported that the degree of antioxidant power of biodegradable film is generally proportional to the amount of added antioxidant additives. Our observation corroborated this and showed that SEO-containing films at a level of 3% had the highest antioxidant activity (78.99%). Similar results were found for gelatin film formulated with oregano and rosemary extracts [31] and chitosan film incorporated with Zataria multiflora essential oil [30]. The antioxidant power of essential oils, caused mainly by their phenolic compounds, has been reviewed by Dimitrios [32]. It has been reported that the phenolic compounds of SEO are carvacrol, ␥-terpinene and p-cymene, which are known to be capable of quenching free radicals [9,32]. It is interesting to note that for all the SEO concentrations studied, there was a linear correlation between TP content and antioxidant activity, as reported by Shan et al. [33]. The results suggested that incorporating SEO into carrageenan film improved the film’s antioxidant activity. 3.8. Antimicrobial activity The antimicrobial activity of carrageenan films incorporated with SEO, either by direct contact or through vapor phase, against five selected bacteria was assessed by the presence or absence of an inhibition zone (Table 3). An SEO-free carrageenan film served as a control to determine any potential antibacterial activities of the films with no additives. The control films showed no inhibition against any test microorganisms. Using the direct contact method, films containing 1% of SEO were not effective against either S. typhimurium or P. aeruginosa, but exhibited a weak inhibitory effect on the growth of S. aureus, E. coli and B. cereus, as evidenced by minimal growth around the film cuts. When SEO concentration was higher than 1%, the films showed a clear antibacterial effect against all studied bacteria directly proportional to the concentration: as expected, the films containing the highest oil content (3%) presented the greatest zone of inhibition (P < 0.05). Among the bacteria examined, P. aeruginosa showed the highest resistance, while S. aureus was the most sensitive to SEO-containing films, with an inhibition zone of 319.97 mm2 . Because of the lack of information in the literature about the antimicrobial effects of films incorporating SEO, our results were compared with those found for free SEO. For example, in accordance with this study’s results, Oussalah et al. [10] showed that S. aureus 122 S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 Fig. 3. Typical AFM image of emulsified carrageenan films: (a) Control, (b) SEO1, and (c) SEO3 (mean values with different superscript in small letters for Sa and in capital letters for Sq are significantly different (P < 0.05)). was more susceptible to SEO than E. coli 0157:H7, S. thyphimarium and L. monocytogenes. As has been reported by other researchers, the inhibitory effect of SEO essential oil is due to its relatively high concentration of carvacrol, ␥-terpinene and p-cymene [9]. These compounds can disintegrate the outer membrane of gram-negative bacteria, and thus increasing the cytoplasmic membrane permeability [34]. In the vapor-phase test, the inhibitory effects of composite films were determined by circular inhibition areas, rather than the clear inhibition zones around the film cuts in the direct-contact test. At an SEO concentration of 1% (v/v), the clear zone of inhibition was not observed for the test bacteria. As the concentration increased to 2%, the zone of inhibition increased significantly (P < 0.05) for S. aureus, B. cereus, E. coli and S. typhimurium, whereas P. aeruginosa was sensitive only to the highest concentration. As with the direct-contact method, the most sensitive bacteria were S. aureus and E. coli. Comparatively, all tested bacteria were more inhibited by direct contact with the antimicrobial films than by the SEO vapors. However, there is a close relationship between the results of the Table 3 Antimicrobial activities of different concentrations of SEO incorporated in carrageenan edible based films in direct contact and in vapor phase.a Film Inhibition zone (mm2 ) S. aureus B. cereus E. coli S. typhimurium P. aeruginosa Direct contact Control SEO1 SEO2 SEO3 0.00d 35.88 ± 7.18c 127.91 ± 15.13b 319.97 ± 20.71a 0.00d 11.55 ± 2.27c 113.46 ± 9.25b 174.62 ± 25.31a 0.00d 38.76 ± 4.83c 105.22 ± 14.43b 256.76 ± 28.41a 0.00c 0.00c 54.29 ± 9.09b 128.10 ± 17.88a 0.00c 0.00c 37.17 ± 0.64b 110.28 ± 13.05a Vapor phase Control SEO1 SEO2 SEO3 0.00c 0.00c 78.69 ± 10.27b 283.74 ± 34.46a 0.00c 0.00c 38.29 ± 9.15b 114.71 ± 15.03a 0.00c 0.00c 37.68 ± 3.25b 201.12 ± 23.53a 0.00c 0.00c 30.78 ± 8.38b 95.22 ± 11.47a a Data reported are average values ± standard deviations. Values within each column with different letters are significantly different (P < 0.05). 0.00b 0.00b 0.00b 49.72 ± 4.68a S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 mg gallic acid/g film a 24 22 20 18 16 14 12 10 8 6 4 2 0 a b c d Control SEO1 SEO2 SEO3 123 antioxidant-antimicrobial films and coatings for various food applications. Films containing SEO exhibited a large inhibitory effect against five selected bacteria not only in direct contact, but also in vapor phase, which could have additional benefits such as efficacy without direct application to the food, ease of application and no alteration in foods’ organoleptic properties. The films incorporating SEO showed good antioxidant properties; this effect was greatly improved when 3% (v/v) SEO was added. The films’ microstructure, which was analyzed through SEM and AFM, revealed that emulsified films had a homogenous matrix in which SEO droplets embedded in a continuous polymer network. Our findings demonstrated that carrageenan films incorporating SEO have a good potential to be used as an active biodegradable packaging material that controls food pathogens. Further investigations are needed to test the effectiveness of these films on selected food systems. Film type b Acknowledgment 90 DPPH Scavenging activity (%) a 80 Authors acknowledge financial support from National Nutrition and Food Technology Research Institute (NNFTRI) of Iran. b 70 60 c 50 References 40 [1] F.G. Torres, O.P. Troncoso, C. Torres, D.A. Díaz, E. Amaya, International Journal of Biological Macromolecules 48 (2011) 603–606. [2] M. Ghasemlou, F. Khodaiyan, A. Oromiehie, M.S. Yarmand, Food Chemistry 127 (2011) 1496–1502. [3] F. Debeaufort, J.A. Quezada-Gallo, A. Voilley, Critical Reviews in Food Science and Nutrition 38 (1998) 299–313. [4] L. Sánchez-González, C. González-Martínez, A. Chiralt, M. Cháfer, Journal of Food Engineering 98 (2010) 443–452. [5] S.M. Ojagh, M. Rezaei, S.H. Razavi, S.M.H. 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[14] for basil and rosemary oil, which did not show inhibitory effects in vapor phase despite their effectiveness in direct contact. However, the results of this study were in contrast with those obtained by Goñi et al. [13], who stated that essential oils were more effective against most bacteria in vapor phase than in direct contact. This discrepancy can be attributed to the fact that they added essential oil to a filter disc rather than producing a film. Evaporation and diffusion may occur with much more difficulty through the film because the essential oil droplets are trapped in the polymer matrix. However, in the direct-contact test, films are directly exposed to the agar surface, which has a high moisture content. Because of the carrageenan polymer’s hydrophilic nature, the interpenetration of water molecules into the film matrix results in swelling, thus gradually widening the meshes of the polymer network and leading to more release of essential oils into the surroundings; this results in higher antimicrobial activity compared to the vapor-phase test, where availability of water is limited. 4. Conclusion Carrageenan-SEO composite films with improved WVP were obtained by emulsification. The properties of the composite films were greatly affected by SEO incorporation. The composite films were less rigid, more flexible, and less transparent than the control film. The WVP of the pure film was significantly reduced by SEO incorporation. Moreover, the results of this study showed that SEO has a good potential for use with carrageenan to make 124 S. Shojaee-Aliabadi et al. / International Journal of Biological Macromolecules 52 (2013) 116–124 [30] M. Moradi, H. Tajik, S.M. Razavi Rohani, A.R. 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