Journal of Food Engineering 81 (2007) 634–641 www.elsevier.com/locate/jfoodeng Effects of plant essential oils and oil compounds on mechanical, barrier and antimicrobial properties of alginate–apple puree edible films Maria A. Rojas-Graü a, Roberto J. Avena-Bustillos b,*, Carl Olsen b, Mendel Friedman b, Philip R. Henika b, Olga Martı́n-Belloso a, Zhongli Pan b, Tara H. McHugh b b a Department of Food Technology, UTPV-CeRTA, University of Lleida, Rovira Roure 191, 25198 Lleida, Spain Western Regional Research Center, US Department of Agriculture, Agricultural Research Service, 800 Buchanan Street, Albany, CA 94710, United States Received 18 September 2006; received in revised form 12 December 2006; accepted 12 January 2007 Available online 26 January 2007 Abstract Mechanical, barrier and antimicrobial properties of 0.1–0.5% suspensions of the following essential oils (EOs)/oil compounds (OCs) were evaluated against the foodborne pathogen Escherichia coli O157:H7 in alginate–apple puree edible film (AAPEF): oregano oil/carvacrol; cinnamon oil/cinnamaldehyde; and lemongrass oil/citral. The presence of plant essential oils did not significantly affect water vapor and oxygen permeabilities of the films, but did significantly modify tensile properties. Antimicrobial activities of solutions used to prepare edible films (AAPFFS) were also determined. The results obtained demonstrate that carvacrol exhibited the strongest antimicrobial activity against E. coli O157:H7. The data show that the antimicrobial activities were in the following order: carvacrol > oregano oil > citral > lemongrass oil > cinnamaldehyde > cinnamon oil. This study showed that plant-derived essential oils and their constituents could be used to prepare apple-based antimicrobial edible films for food applications. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Alginate film; Apple puree; Plant essential oils; Mechanical properties; Barrier properties; Antimicrobial activity; Escherichia coli O157:H7 1. Introduction Edible films can improve shelf life and food quality by serving as selective barriers to moisture transfer, oxygen uptake, lipid oxidation, and losses of volatile aromas and flavors (Kester & Fennema, 1986). Their use is gaining importance in food protection and preservation due to the fact that they provide advantages compared to films made from synthetic materials (Tharanathan, 2003; Weber, Haugaard, Festersen, & Bertelsen, 2002). Potential properties and applications of edible films and coatings have been extensively reviewed (Bravin, Peressini, & Sensidoni, 2006; Jagannath, Nanjappa, Das Gupta, & Bawa, 2006; Min, Harris, Han, & Krochta, 2005; Serrano et al., 2006). McHugh, Huxsoll, and Krochta (1996) developed the first edible films made from fruit purees. They found that * Corresponding author. Tel.: +1 530 559 5954; fax: +1 510 559 5851. E-mail address: ravena@pw.usda.gov (R.J. Avena-Bustillos). 0260-8774/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.01.007 apple-based edible films are excellent oxygen barriers, but not very good moisture barriers. Addition of hydrocolloids such as alginate may improve the barrier and tensile properties of fruit-based films (Mancini & McHugh, 2000). Novel films can be developed by combining fruit purees with various gelling agents. Alginate, a polysaccharide extracted from marine brown algae (Phaeophyceae), is a common type of gelling agent employed in the food industry (Mancini & McHugh, 2000; Yang & Paulson, 2000). This polysaccharide is of interest as a potential film or coating component because of its unique colloidal properties. These include thickening, stabilizing, suspending, film forming, gel producing, and emulsion stabilizing properties (King, 1982; Rhim, 2004). Interest in antimicrobial films has risen recently due to increased consumption of fresh-cut produce. Such consumption has resulted in occasional outbreaks of illness associated with contaminated fruits and vegetables (Brackett, 1999; Thunberg, Tran, Bennett, Matthews, & Belay, M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 2002). During minimal processing, spoilage and pathogenic microorganisms can contaminate fruits (Del Rosario & Beuchat, 1995; Thunberg et al., 2002). For example, the presence of Escherichia coli O157:H7 on fruit surfaces may adversely affect the safety of fresh and fresh-cut fruit. The use of edible films and coatings for food products, including fresh and minimally processed fruits and vegetables, is of interest because films and coatings can serve as carriers for a wide range of food additives, including antimicrobials (Pranoto, Salokhe, & Rakshit, 2005). Plant essential oils (EOs) and oil compounds (OCs) have been previously evaluated for their ability to protect food against pathogenic bacteria contaminating apple juice (Friedman, Henika, Levin, & Mandrell, 2004) and other foods (Burt, 2004; Seydim & Sarikus, 2006). However, little published data exist on the incorporation of EOs and OCs into edible films. EOs are also used as flavouring agents in baked goods, sweets, ice cream, beverages, and chewing gum (Fenaroli, 1995) and are designated as Generally Regarded as Safe (GRAS) (Burt, 2004). A complete analysis of both antimicrobial and physicochemical properties is important for predicting the behaviour of antimicrobial edible films in food systems (Cagri, Ustunol, & Ryser, 2001; Garcı́a, Martinó, & Zaritzky, 1998, 2000; Kester & Fennema, 1986; McHugh & Krochta, 1994a; Yang & Paulson, 2000). This is the first study to investigate the antimicrobial effects of alginate–apple puree edible films containing EOs or OCs against E. coli O157:H7. The objectives of this study were (a) to evaluate the effects of adding alginate and natural antimicrobials on the mechanical and barrier properties of the films, and (b) to determine antimicrobial activities against the E. coli O157:H7 of alginate–apple puree film forming solutions and alginate–apple puree films containing a variety of EOs and OCs. 635 solution, 10 g of N-acetylcysteine (1% w/w) and 15 g of glycerol (1.5% w/w). Samples were homogenized 1 min at 10,000 rpm and 2 min at 15,000 rpm using a Polytron 3000 homogenizer (Kinematica, Littau, Switzerland) according to McHugh and Senesi (2000). Natural antimicrobial EOs and OCs were then incorporated into the AAPFFS at the following concentrations: 0 (control), 0.1% w/w (oregano oil and carvacrol) and 0.5% w/w (lemongrass oil, citral, cinnamon oil and cinnamaldehyde). These solutions were homogenized for 3 min at 12,500 rpm using a Polytron 3000 homogenizer (Kinematica, Littau, Switzerland) and then used for the bactericidal studies and casting the films. 2.3. Preparation of alginate–apple puree edible film (AAPEF) AAPFFS was prepared as described previously and then, vacuum was applied to remove bubbles. Films were cast on level 29  29 cm square plates and dried at ambient conditions for 24 h. Dried films were cut and peeled from the casting surface. These film samples stored at 21 °C and 65% RH were used for determinations of barrier, mechanical and antimicrobial properties of the films. 2.4. Film thickness Film thickness was measured with a micrometer IP 65 (Mitutoyo Manufacturing, Tokyo, Japan) to the nearest 0.00254 mm (0.0001 in) at five random positions around the film. The mean value was used to calculate water vapor permeability (WVP), oxygen permeability (O2P), and tensile strength. 2.5. Water vapor permeability (WVP) of films 2. Materials and methods 2.1. Test compounds Food grade sodium alginate (KeltoneÒ LV, ISP, San Diego, CA., USA) and Golden Delicious apple puree (38° Brix) (Sabroso Co., Medford, OR) were the primary ingredients in all alginate/apple puree-based films. Glycerol (Fisher Scientific, Waukesha, WI) was added as a plasticizing agent and N-acetylcysteine (Sigma–Aldrich Chemical Co., Steinhein, Germany) was used as a browning inhibitor. The following EOs and OCs were obtained from Lhasa Kamash Herb Co. (Berkeley, CA): oregano, lemongrass, and cinnamon cassia. Citral and cinnamaldehyde were purchased from Sigma Chemical Co. (Milwaukee, WI). Carvacrol was donated by Millennium Chemical Co. (Jacksonville, FL). 2.2. Preparation of alginate–apple puree film forming solution (AAPFFS) A 26% w/w AAPFFS was formed by combining 260 g of 38° Brix apple puree with 715 g of 2% w/w alginate The gravimetric Modified Cup Method (McHugh, Avena-Bustillos, & Krochta, 1993) based on standard method E96-80 (ASTM, 1989) was used to determine WVP. A cabinet with a variable speed fan was used to test film WVP. Cabinet temperature of 25 ± 1 °C was maintained in a Forma Scientific reach-in incubator (Thermo Electron Corp., Waltham, MA). Fan speeds were set to achieve air velocities of 152 m/min to ensure uniform relative humidity throughout the cabinets. Cabinets were preequilibrated to 0% room humidity (RH) using anhydrous calcium sulphate (W.A. Hammond Drierite, Xenia, OH). Circular test cups made from polymethylmethacrylate (PlexiglasTM) were used. A film was sealed to the cup base with a ring containing a 19.6 cm2 opening using 4 screws symmetrically located around the cup circumference. Both sides of the cup contacting the film were coated with silicon sealant. Distilled water (6 mL) was placed in the bottom of the test cups to expose the film to a high percentage RH inside the test cups. Average stagnant air gap heights between the water surface and the film were measured. Test cups holding films were then inserted into the pre-equilibrated 0% RH 636 M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 desiccator cabinets. Steady state of water vapor transmission rate was achieved within 2 h. Each cup was weighed 8 times at 2 h intervals. Eight replicates of each film were tested. Room humidities at the film undersides and WVPs were calculated using the WVP Correction Method (McHugh et al., 1993). The WVP of the films was calculated by multiplying the steady state water vapor transmission rate by the average film thickness determined as described above and dividing by the water vapor partial pressure difference across the films: WVP ¼ ðWVTRÞðthicknessÞ ðpA1  pA2 Þ ð1Þ where WVTR = water vapor transmission rate and pA1 and pA2 = water vapor partial pressure inside and outside the cup, respectively. Units for WVP were g mm/kPa h m2. 2.6. Oxygen permeability (O2P) of films An Ox-Tran 2/20 ML modular system (Modern Controls Inc., Minneapolis, MN) was utilized to measure oxygen transmission rates through the films according to standard method D3985 (ASTM, 1995). Oxygen transmission rates were determined at 23 °C and 50 ± 1% RH. Each film was placed on a stainless steel mask with an open testing area of 5 cm2. Masked films were placed into the test cell and exposed to 98% N2 + 2% H2 flow on one side and pure oxygen flow on the other. The system was programmed to have a 10 h waiting period to allow the films to achieve equilibrium. Oxygen permeability was calculated by dividing O2 transmission rate by the difference in O2 partial pressure between both sides of the film (1 atm) and multiplying by the average film thickness measured at 5 random places. Four replicates of each film were evaluated. Units for O2P were cm3 lm/m2 d kPa. 2.7. Tensile properties of films Standard method D882-97 (ASTM, 1997) was used to measure tensile properties of films. Films were cut into strips with a test dimension of 165 mm  19 mm according to standard method D638-02a (ASTM, 2002). All films were conditioned for 48 h at 23 ± 2 °C and 50% ± 2% RH before testing using a saturated salt solution of magnesium nitrate (Fisher Scientific, Fair Lawn, NJ). The ends of the equilibrated strips were mounted and clamped with pneumatic grips on an Instron Model 55R4502 Universal Testing Machine (Instron, Canton, MA) with a 100 N load cell. The initial gauge length was set to 100 mm and films were stretched using a crosshead speed of 7.5 mm/min. Tensile properties were calculated from the plot of stress (tensile force/initial cross-sectional area) vs. strain (extension as a fraction of original length), using Series IX Automated Materials Testing System Software (Instron, Canton, MA). Fifteen specimens of each type of film were evaluated. 2.8. Source of bacteria The Food and Drug Administration (FDA) provided E. coli O157:H7 (strain SEA18B88; our file, strain RM1484). This strain was isolated from apple juice associated with an outbreak of human infection (Friedman, Henika, & Mandrell, 2002). 2.9. Test buffers Phosphate-buffered saline (PBS, pH 7.0) was prepared by mixing dibasic sodium phosphate (100 mM) and monobasic sodium phosphate (100 mM) at 2:1 ratio, diluting by half with H2O (v/v), and adding NaCl (150 mM). For lower pH buffers, 2 mM citric acid–150 mM NaCl was adjusted to pH 3.3–3.7 (saline solutions). 2.10. Preparation of samples for bactericidal assays of AAPFFS To facilitate pipetting, the 26% AAPFFS solution was further diluted by 1/2 with pH 3.3 saline solution, v/v. This AAPFFS sample was used to prepare suspensions for the assay. Oregano oil or carvacrol (10 lL) was added to 9.99 mL of diluted AAPFFS. Lemongrass oil, citral, cinnamon oil or cinnamaldehyde (50 lL) was added to 9.95 mL diluted AAPFFS in 50 mL tubes. The tubes were warmed in a microwave oven for 10 s and then shaken to form uniform suspensions. The content of the tubes were then diluted as follows: saline solution (500 lL) was added to five sterile 1.9 mL tubes. Serial dilutions were made starting with 1 mL of each original test solution, using 500 lL for each transfer for a total of five dilutions. Microtiter plates with 96 wells (Nalge, Rochester, NY) were prepared with saline pH 3.3 negative controls (100 lL each in 6 wells) and three test substances with five dilutions plus the test solution (100 lL each dilution per well, 6 wells). These 24 wells were sampled at three time intervals: 3, 30 and 60 min at 21 °C. 2.11. Bactericidal assays of AAPFFS A previously described assay (Friedman et al., 2004, 2002) was used with some modifications. E. coli O157:H7 bacteria streaked on Luria-Bertani (LB) agar plates (Difco Inc., Sparks, MD) were subcultured and incubated for 16– 18 h at 37 °C. LB broth cultures were prepared by harvesting a few isolated colonies from the plates with a sterile loop and suspending them into 5 mL LB broth in 15 mL sterile plastic tubes. The capped tubes were incubated with agitation at 37 °C for 18 h. Bacterial suspensions were prepared for growth of 100–200 CFU per lane on the square plates with grids used for counting. A sample (1 mL) of an 18 h LB broth culture of E. coli O157:H7 was added to a 1.9 mL microfuge tube. The bacteria were pelleted by centrifugation in a microfuge (15,800g) for 1 min. After the supernatant was removed, 1 mL of sterile PBS (phosphate-buffered 637 M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 saline, pH 7.0) was added to the pellet. The pellet was resuspended by gentle aspiration in and out of a transfer pipette. The sample’s optical density at 620 nm was adjusted by 1/4 dilution with PBS to ca. 0.4. The suspension (20 lL) was added to PBS (980 lL). The resulting suspension (1000 lL) was then added to 5 mL saline solution pH 3.3, vortexed, and poured into a sterile, plastic Petri dish. The suspensions (50 lL) were drawn with a multichannel Eppendorf pipette and added to six microtiter plate wells. This was repeated until all of the 24 prepared wells were inoculated. The inoculated microtiter plates were sampled three times (3, 30, and 60 min) at 21 °C without agitation. At the end of each incubation time, aliquots (10 lL) from each of six wells were drawn with an Eppendorf multichannel pipette for spotting of six 10-lL drops at the top of a square LB agar Petri plate. The plates were tilted before spotting to avoid coalescence of drops and tapped gently to facilitate movement of the liquid to the bottom. They were then placed uncovered for 10 min in a biological safety hood until dry, recovered, and incubated overnight at 37 °C. Each well with test solution (150 lL) plus bacteria contained 1500–3000 cells. Experiments were done in duplicate. 3. Results and discussion 2.12. Bactericidal activities (BA50 values) 3.1. Barrier and mechanical properties Bactericidal activities, defined as the % of test compound that kills 50% of the bacteria under the test conditions, were determined as follows. Each compound was tested at a series of dilutions. The control pH 3.3 saline diluent was matched with pH of AAPFFS. The CFU values from all experiments were transferred to a Microsoft Excel 8.0 Spreadsheet. The number of CFU from each dilution was matched with the average control value to determine the percent of bacteria killed per well. Each of the dose–response profiles (% test compound versus % bactericidal activity) was examined graphically and the BA50 values were estimated by a linear regression. The lower the BA50, the higher the bactericidal activity was observed. 3.1.1. Water vapor permeability In the present study, WVP properties were not affected by the incorporation of EOs and OCs into the film, presumably because these EOs consist mostly of terpene-like compounds, not lipids. However, a slight decrease in WVP was observed after incorporation of 0.5% w/w cinnamaldehyde (Table 1). Hernandez (1994) indicated that water vapor transfer generally occurs through the hydrophilic portion of the film and depends on the hydrophilic–hydrophobic ratio of the film components. 2.13. Antimicrobial activity of alginate–apple puree edible films (AAPEF) Disc inhibition zone assays were performed as a qualitative test for antimicrobial activity of the films. AAPEF with and without EOs and OCs were aseptically cut into 12 mm diameter discs and then placed on MacConkey-Sorbitol agar (Biokar Diagnostics, Beauvais, France) plates for E. coli O157:H7, which had been previously spread with 0.1 mL of inoculum containing 105 CFU/mL of tested bacterium. Plates were incubated at 37 °C for 48 h. The thickness (mm) of the inhibition zone around the film disc (colony free perimeter) was then measured and the growth below the film discs (the contact area of edible film with agar surface) was examined visually. Tests were done in duplicate. 2.14. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) using Minitab version 13.31 software (Minitab Inc., State College, PA). Tukey test was used to determine the difference at 5% significance level (SAS, 1999). 3.1.2. Oxygen permeability Oxygen permeability of the AAPEF with and without EOs and OCs are summarized in Table 1. The O2P of the Table 1 Effect of concentration (% w/w) and type of plant essential oils/oil compounds on water vapor permeability (WVP) and oxygen permeability (O2P) properties of alginate–apple puree edible films Essential oil and oil compounds (% w/w) ThicknessA (mm) RH inside cupAB (%) WVPAB (g mm/kPa h m2) Oxygen permeabilityA (cm3 lm/m2 d kPa) Control (0) Oregano oil (0.1) Carvacrol (0.1) Lemongrass oil (0.5) Citral (0.5) Cinnamon oil (0.5) Cinnamaldehyde (0.5) 0.119 ± 0.004NS 0.118 ± 0.007 0.117 ± 0.008 0.122 ± 0.006 0.118 ± 0.004 0.117 ± 0.008 0.118 ± 0.009 65.03 ± 1.81a 63.46 ± 0.65a 64.11 ± 0.86a 65.70 ± 1.77ab 63.87 ± 0.89a 64.77 ± 0.79a 67.10 ± 0.80b 4.95 ± 0.43a 5.25 ± 0.33a 5.02 ± 0.22a 4.91 ± 0.40a 5.12 ± 0.13a 4.90 ± 0.27a 4.37 ± 0.54b 10.20 ± 0.91a 11.00 ± 0.92a 10. 89 ± 0.76a 9.38 ± 0.32b 9.94 ± 0.15ab 10.50 ± 0.62a 11.03 ± 0.70a A Thickness and RH data are mean values. WVP (N = 8) and O2P (N = 4) data are mean values ± standard deviations. Relative humidity at the inner surface and WVP values were corrected for stagnant air effects using the WVP Correction Method (McHugh et al., 1993). NS Not significantly different. a,b Means in same column with different letters are significantly different (p < 0.05). B 638 M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 alginate–apple puree film was 10.20 ± 0.91 cm3lm/ m2 d kPa indicating that this film is a good oxygen barrier. This value is two times lower than that of an apple pureepectin film (22.64 ± 1.28 cm3lm/m2 d kPa) as it was observed in a previous study (McHugh et al., 1996; Rojas-Graü et al., 2006). This difference is hypothesized to be caused by the effect of the type of carbohydrate used in the formulation (McHugh et al., 1996). Addition of antimicrobial agents did not affect the oxygen permeability of the films. Compared to the control films, a slight decrease in O2P of the films was observed with lemongrass oil and citral (0.5% w/w) (Table 1). 3.1.3. Tensile properties Tensile strength, elongation, and elastic modulus are parameters that relate mechanical properties of films to their chemical structures (McHugh & Krochta, 1994b). Tensile strength expresses the maximum stress developed in a film during tensile testing (Gennadios, Brandenburg, Park, Weller, & Testin, 1994). Incorporation of EOs and OCs caused a significant reduction (p < 0.05) in tensile strength of the films (Table 2). This effect was more pronounced in films containing oregano oil and carvacrol, which displayed lower values of tensile strength 2.47 ± 0.37 and 2.58 ± 0.37 MPa, respectively. Elongation at break is a measure of the film’s stretch ability prior to breakage (Krochta & DeMulder-Johnston, 1997). The percent elongation of control AAPEF was 51.06% and increased in all films containing EOs and OCs, reaching a maximum value of 58.33% with carvacrol (Table 2). The elastic modulus of AAPEF (7.07 ± 1.09 MPa) was significantly greater than most of the films containing antimicrobial agents (Table 2). No significant differences were observed in the elastic modulus between films with and without cinnamon oil or cinnamaldehyde (p < 0.05). 3.2. Antimicrobial properties 3.2.1. Antimicrobial activity of plant essential oils and oil compounds in AAPFFS The experimental BA50 values for EOs and OCs at three time periods, 3, 30, and 60 min are shown in Table 3. All compounds inhibited the growth of E. coli O157:H7. AAPFFS in saline pH 3.3 without EOs or OCs and containing N-acetylcysteine as an antibrowning agent was not effective against the pathogen. The pH of the AAPFFS oscillated between 4.2 and 4.7. At pH values near 5, the alginate chains repel each other and provide stable solutions without significant change in viscosity between pH values of 5.5 to 11 (King, 1982). Table 3 shows that carvacrol at a concentration of 0.1% w/w in the AAPFFS was effective at 3 min with a BA50 value of 0.020 (0.020% of carvacrol inhibited 50% of the E. coli O157:H7 after 3 min). The activity at 30 min was twice as great (BA50 = 0.011%) than at 3 min; at 60 min, it was the same (BA50 = 0.011%) as at 30 min. Carvacrol appears to exhibit high antimicrobial effects against E. coli O157:H7. Similar behaviour was observed with Table 2 Effect of concentration (% w/w) and type of plant essential oils/oil compounds on the tensile properties of alginate–apple puree edible films Essential oil and oil compounds (% w/w) Control (0) Oregano oil (0.1) Carvacrol (0.1) Lemongrass oil (0.5) Citral (0.5) Cinnamon oil (0.5) Cinnamaldehyde (0.5) A a,b Tensile strengthA (MPa) a 2.90 ± 0.52 2.47 ± 0.37b 2.58 ± 0.37b 2.56 ± 0.46b 2.52 ± 0.44b 2.84 ± 0.48ab 2.75 ± 0.42ab ElongationA (%) a 51.06 ± 3.89 56.96 ± 3.86b 58.33 ± 4.66b 55.95 ± 5.55ab 57.38 ± 5.71b 57.88 ± 5.37b 55.50 ± 7.40ab Elastic modulusA (MPa) 7.07 ± 1.09a 5.75 ± 0.96b 5.96 ± 1.12b 6.02 ± 1.07b 6.46 ± 1.27ab 6.86 ± 1.16a 6.77 ± 0.87a Tensile strength, elongation, and elastic modulus data (N = 10) are mean values ± standard deviations. Means in same column with different letters are significantly different at p < 0.05. Table 3 Bactericidal activities (BA50 values) of plant essential oils/oil compounds against E. coli O157:H7 in alginate–apple puree film forming solution (AAPFFS)a incubated for 3, 30, and 60 min at 21 °C Oil/oil compound (% w/w) in 50% AAPFFSa Oregano oil (0.1) Carvacrol (0.1) Lemongrass oil (0.5) Citral (0.5) Cinnamon oil (0.5) Cinnamaldehyde (0.5) BA50 value for E. coli O157:H7b 3 min 30 min 60 min 0.025nd 0.020 ± 0.0007 >0.34c >0.34c >0.34c >0.34c 0.010 ± 0 0.011 ± 0.001 0.066 ± 0.01 0.093 ± 0.02 0.16 ± 0.08 0.11 ± 0 0.012 ± 0 0.011 ± 0.0007 0.059 ± 0.006 0.057 ± 0.0007 0.087 ± 0.05 0.086 ± 0.03 nd: not detected. a AAPFFS is 50% apple puree film formula suspension in saline pH 3.7 buffer. b BA50 = Average values and standard deviations of two replicates of BA50 values. c >: less than 50% of bacteria were killed at the highest dose used. 639 M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 Table 4 Antibacterial activity of plant essential oils/oil compounds incorporated into alginate–apple puree edible films against E. coli O157:H7 Essential oil and oil compounds Control Oregano oil Carvacrol Lemongrass oil Citral Cinnamon oil Cinnamaldehyde a b Concentration (% w/w) 0 0.1 0.1 0.5 0.5 0.5 0.5 E. coli O157:H7 Inhibitory zone (mm2)a Inhibitory effect under filmb 0.0 49.8 68.4 40.8 49.8 19.6 40.8  + + + + + + Values (N = 3) are measurements of area (mm2) of inhibitory growth zone on agar around film. Not growth (+) or growth () on Petri dish agar directly underneath film pieces. oregano oil. The BA50 values of oregano oil against E. coli O157:H7 at 3, 30, and 60 min were 0.025%, 0.010%, and 0.012%, respectively, only slightly higher (the activity was lower) than those mentioned for carvacrol. The antimicrobial activity of oregano oil can be accounted for by its content of carvacrol. The antibacterial properties of carvacrol are associated with its lipophilic character, leading to change in membrane potential and increase in permeability of the cytoplasm membrane for protons and potassium ions, including depletion of the intracellular ATP pool (Friedman, 2006; Sikkema, De Bont, & Poolman, 1995). Previously it was shown by HPLC that oregano oil contains about 86% carvacrol (Friedman et al., 2004). Carvacrol, the major component of oregano oil, is designated as Generally Regarded as Safe (GRAS) (Dingman, 2000). The activity of lemongrass oil at concentrations of 0.5% in the AAPFFS against E. coli O157:H7 were similar to those of citral (Table 3). Compared to carvacrol and oregano oil, it took about five times more of citral and lemongrass oil to achieve the same activity against E. coli O157:H7. On the other hand, cinnamaldehyde at a concentration of 0.5% w/w in the AAPFFS was only effective at 30 and 60 min with BA50 values of 0.11 and 0.086%, respectively (Table 3). These results indicate that cinnamaldehyde at a fivefold greater concentration was less effective than carvacrol. The activity of cinnamon oil against E. coli O157:H7 at a concentration of 0.5% in the AAPFFS was of the same order as that observed with cinnamaldehyde (Table 3). These results were expected in view of the fact that cinnamon oil contains about 85% of cinnamaldehyde (Friedman et al., 2004). 3.2.2. Antimicrobial activity of plant essential oils and oil compounds in AAPEF Table 4 shows the results of antimicrobial activities of the films containing the essential oils and oil compounds. The listed inhibitory activities were estimated from measurement of clear inhibition zones surrounding the film disks. If a surrounding clear zone was not present, it was assumed that the compound was not inhibitory and the area was assigned as zero. AAPEF without EOs and OCs served as a control to determine any possible antimicrobial effect of the film without additives. The control film did not inhibit the E. coli O157:H7. The results show that all films containing added essential oils and oil compounds significantly inhibited the growth of E. coli O157:H7. As expected, AAPEF containing carvacrol was the most effective (greater surrounding clear zone) against E. coli O157:H7 (Table 4). Inhibition of E. coli O157:H7 by lemongrass oil/citral and cinnamon oil/cinnamaldehyde at 0.5% w/w in the films was lower than that observed with oregano oil/carvacrol (Table 4). Compared to carvacrol, it took about five times more cinnamaldehyde and citral to achieve the same activity against E. coli O157:H7. Because cinnamon oil is present in numerous commercial foods (Friedman, Kozukue, & Harden, 2000), has a pleasant taste, and is GRAS-listed (Adams et al., 2004), the compound merits use as an antimicrobial in edible films. 4. Conclusions There was no adverse effect of the additives on water vapor and oxygen permeabilities. Tensile properties, however, were significantly affected by addition of EOs and OCs. The antimicrobial activity of oregano essential oil and of carvacrol in alginate–apple puree edible films and film forming solutions against E. coli O157:H7 was significantly greater than the activities of lemongrass oil, citral, cinnamon oil, and cinnamaldehyde. The antimicrobial data obtained with the alginate–apple puree forming solution can serve as a guide for selection of appropriate levels of plant compounds for incorporation into antimicrobial edible films. Incorporating EOs and OCs into edible films provides a novel way to enhance the safety and shelf-life in food systems. Acknowledgements This work was supported by the Ministry of Science and Technology (AGL2003-09208-C03-01), the European Social Fund and by the Departament d’Universitats, Recerca i Societat de la Informació of the Generalitat de Catalunya (Spain), that also awarded author Rojas-Graü with a predoctoral grant and by NRI grant 2006-35201-17409 provided by the USDA, CSREES. We also thank Dr. J. M. 640 M.A. Rojas-Graü et al. / Journal of Food Engineering 81 (2007) 634–641 Krochta and his colleagues of the University of California, Davis, for support for oxygen permeability evaluation. References Adams, T. B., Cohen, S. M., Doull, J., Feron, V. J., Goodman, J. 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