Journal of Food Engineering 110 (2012) 232239

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Journal of Food Engineering

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Physical, mechanical and antibacterial properties of alginate lm: Effect of the crosslinking degree and oregano essential oil concentration
Sergio Benavides a, R. Villalobos-Carvajal b,, J.E. Reyes b
a b

Universidad Adventista de Chile, Facultad de Ingeniera y Negocios, P.O. Box 7-D, Chilln, Chile Universidad del Bio Bio, Departamento de Ingeniera en Alimentos, P.O. Box 447, Chilln, Chile

a r t i c l e

i n f o

a b s t r a c t
Alginate lms with different degrees of crosslinking obtained by internal gelation, and alginate lms incorporated with oregano essential oil (OEO) were prepared. The impact of the degree of crosslinking caused by the use of calcium carbonate as crosslinking agent and the incorporation of OEO into the alginate lms on their antibacterial, optical, mechanical, microstructural and water vapour barrier properties was evaluated. An increase in the degree of crosslinking produced alginate lms that were signicantly thicker (0.031 0.038 mm) and stronger (51.952.9 MPa) but less elastic (2.3%) than those non-crosslinked lms (0.029 mm; 39.7 MPa; 4.4%). The water vapour permeability (WVP) of the lms decreased signicantly only with the highest level of crosslinking. The incorporation of OEO in alginate lms affected signicantly their physical properties. Thickness and percent elongation at break of the lms were increased by the addition of OEO (0.0360.042 mm and 2.73.7%), while the tensile strength and water vapour permeability decreased (31.155.5 MPa and 2.73.0 109 g/m s Pa). Films incorporated with OEO were more effective against Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) than Gram-negative bacteria ( Escherichia coli and Salmonella Enteritidis). A minimum concentration of 1.0% of OEO was necessary to ensure their antibacterial efcacy. 2011 Elsevier Ltd. All rights reserved.

Article history: Available online 31 May 2011 Keywords: Oregano essential oil Alginate lm Crosslinking Antibacterial activity Mechanical properties Microstructure

1. Introduction Microbial contamination of ready-to-eat products such as refrigerated meats and intermediate moisture foods is a serious concern to human health (Crdenas et al., 2008; Cutter, 2000; Devlieghere et al., 2004). A traditional method used to control the growth of microorganism has been the application of antimicrobial dips or sprays on the surface of products. However, this has had limited success because the antimicrobial substances may interact with food components by evaporating or diffusing into bulk food (Kerry et al., 2006; Quintavalla and Vincini, 2002; Siragusa and Dickson, 1992). One new approach to overcome these limitations could be the use of antimicrobial packaging techniques (Appendini and Hotchkiss, 2002), or the application of antimicrobial edible coatings (Zhou et al., 2010). To control undesirable microorganisms on food surfaces, volatile and non-volatile antimicrobial agents can be incorporated into packaging materials or coatings. Through this approach, it is possible to achieve greater efciency if the antimi-

Corresponding author. Tel.: +56 42 253095; fax: +56 42 253066.

E-mail address: rivillal@ubiobio.cl (R. Villalobos-Carvajal). 0260-8774/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2011.05.023

crobial agents are released slowly to the surface of food and remaining at effective concentrations for extended periods of time (Coma et al., 2001; Ouattara et al., 2000). In order to meet consumer demands for more natural products and for packaging materials with low environmental impact, many researchers have focused on the incorporation of plant extracts into lms, edible coatings and bio-based packaging materials (Del Nobile et al., 2008; Norajit et al., 2010; Oussalah et al., 2006; Rojas-Gra et al., 2007). Essential plant oils and their constituents have been widely used as avouring agents in food since early recorded history (Tiwari et al., 2009) and are categorised as Generally Recognised as Safe (GRAS) (Lpez et al., 2007). Essential oils rich in phenolic compounds have been reported to have a wide spectrum of antimicrobial activity. Among these, clove, oregano, rosemary, thyme, sage and vanillin oils have been found to be the most effective (Holley and Patel, 2005). Carvacrol, thymol, c-therpinene and p-cymene are the principal constituents of oregano essential oil (Burt, 2004; Lamber et al., 2001). Its antimicrobial properties have been demonstrated in numerous studies (Avila-Sosa et al., 2010; Emiroglu et al., 2010; Ozkalp et al., 2010; Zivanovic et al., 2005; Seydim and Sarikus, 2006; Zinoviadou et al., 2009).

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Alginates are natural substances extracted from brown seaweed and are composed of 1-4b-D-mannuronic acid (M) and a-L-guluronic acid (G). In polymer chains, monomers are arranged alternately in GG and MM blocks, together with MG blocks (King, 1983; Grant et al., 1973). The most interesting property of alginates is their ability to react with polyvalent metal cations, specifically calcium ions. The ions establish the cooperative association between M and G blocks, resulting in a tridimensional network where they may pack and be coordinated. This arrangement is pictured as the eggbox model (Grant et al., 1973). Because alginate lms are hydrophilic matrices, the crosslinking process with polyvalent cations has been used to improve their water barrier properties, mechanical resistance, cohesiveness and rigidity (Rhim et al., 2003; Rhim, 2004) and to delay the release of some drugs (Al-Musa et al., 1999; Chan et al., 2006; RemuanLpez and Bodmeier, 1997). Due to the fast crosslinking process between alginate and calcium ions, localised gelling areas are produced, compromising the uniformity and quality of lms. Draget et al. (1991) have proposed a technique (internal gelation) to form homogeneous matrices through the slow release of calcium ions from insoluble calcium salt in an acidied medium. Antimicrobial lms containing volatile antimicrobial compounds can be considered controlled release systems, and their effectiveness depends on the diffusion of volatile compounds through the polymer as well as their vapour partial pressure at saturation. Therefore, control of the release rates and migration of antimicrobial compounds from lms, either by regulating the degree of crosslinking in the lm or by using multilayer structures, could be an interesting strategy (Buonocore et al., 2003; Han, 2000). According to Cagri et al. (2001), a complete analysis of both antimicrobial and physicochemical properties is important for predicting the behaviour of antimicrobial edible lms in food systems. The objectives of this work were to evaluate the effects of the degree of internal crosslinking and the addition of oregano essential oil on the antimicrobial, optical, mechanical, microstructural and barrier properties of alginate lms. The antimicrobial activity of alginate lms containing different amounts of oregano essential oil was tested against Gram-negative bacteria (Escherichia coli and Salmonella Enteritidis) and Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes). 2. Materials and methods 2.1. Materials The material used for lm formation included food-grade sodium alginate purchased from SigmaAldrich Chemical Co., (St. Louis, MO, USA). Glycerol (used as a plasticising agent) and Tween 80 (used as a surfactant) were obtained from Merck Co., (Darmstadt, Germany). The analytical grade glucono-d-lactone (GDL), calcium chloride and calcium carbonate used in the crosslinking process were purchased from Merck Co., (Darmstadt, Germany). Oregano essential oil (OEO), obtained from leaves and the aerial part of oregano (Origanum vulgare) by steam-distillation was provided by Oilife (Rancagua, Chile). 2.2. Organism and cultures The bacteria used in this study were E. coli (ATCC 25922), S. Enteritidis (ATCC 13076), S. aureus (ATCC 6538) and L. monocytogenes (ATCC 7644). They were obtained from the culture collection of the Food Microbiology Laboratory the Food Engineering Department of the Universidad del Bio Bio, Chile.

2.3. Preparation of lms 2.3.1. Sodium alginate lms Alginate lms were prepared by a modication of the method used by Rhim (2004). A solution of sodium alginate (1.5% w/v) was prepared by dissolving 3.0 g of sodium alginate in 200 mL of sterile deionised water at 70 C under mechanical stirring for 30 min. After complete dissolution, glycerol (0.243 g/g of alginate) was added as a plasticizer to enhance lm exibility, decrease brittleness and to facilitate their detachment from the petri dishes. The lm-forming solution was cast onto petri dishes 9 cm in diameter and allowed to dry in an oven at 30 C for approximately 24 h. The dried lms were then removed from the petri dishes and stored in desiccators until being used. 2.3.2. Calcium alginate lms (internal crosslinking) The calcium alginate lms were prepared based on the methods proposed by Draget et al. (1991) and Rhim (2004). A specic amount of CaCO3 (0.00; 0.01; 0.02 and 0.03 g/g alginate) with GDL (5.4 g/g CaCO3) was dispersed in 50 mL of distilled water with magnetic stirring. Then this dispersion was added to a 150 mL of sodium alginate solution to provide 1.5% w/v alginate. The mixture was homogenised at 13,500 rpm for 3 min at room temperature, using a homogeniser (UltraTurrax IKA, T25, Werke, Germany), and nally was left gently stirring overnight to promote complete gelation of the alginate. Films were prepared and stored as previously described. 2.3.3. Oregano essential oilcalcium alginate lms Oregano essential oil was previously mixed with Tween 80 (0.250 g/g of essential oil) to help create a uniform and stable distribution in the alginate matrix; it was then incorporated into a sodium alginate solution at several nal concentrations (0.0, 0.5, 1.0 and 1.5% w/v). The mixture was homogenised at 13,500 rpm for 3 min at room temperature, using a homogeniser (UltraTurrax IKA, T25, Werke, Germany). The homogenised mixture was ultrasonicated for 20 min to remove air bubbles formed during agitation (Zivanovic et al., 2005). In order to obtain a lm with a high level of internal crosslinking and evaluate its effect on antimicrobial efciency, 50 mL of a dispersion composed of CaCO3 (0.03 g/g alginate) and GDL (5.4 g/g CaCO3) was added to 150 mL of essential oilsodium alginate solution to provide 1.5% w/v alginate, and the emulsion was slowly stirred overnight to promote a slow internal crosslinking. Films were prepared and stored as previously described. 2.4. Properties of lms 2.4.1. Appearance, thickness and colour of the lms Manageability and exibility of all the lms were qualitatively evaluated at the time of detachment from the petri dish. The surface homogeneity and the presence of cracks and bubbles in the lms were analyzed by observation under an optical microscope (ECLIPSE E200, Nikon, Japan) whit a magnication of 40. The visual assessment of transparency and the apparent colour of the lms were evaluated qualitatively by four untrained students considering the recommendations outlined in ASTM D1729. The lms illuminated at 45 with a uorescent white light source, were examined directly from above (0 angle) on a white background and black background. Film thickness was measured using a digital micrometre (Fowler, USA). Measurements were taken at ve different locations of each lm sample (Moreno-Osorio et al., 2010). Average values were used in the WVP and tensile tests. Film colour was measured using a CR-200 Minolta Chroma Meter (Minolta Camera Co., Osaka, Japan). The instrument was

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calibrated with a standard white plate, and the (CIE) Lab colour space was used (CIE, 1986). Lightness (L) and chromaticity parameters a (redgreen) and b (yellowblue) were obtained using illuminant D65 at an observer angle of 2 (Moreno-Osorio et al., 2010). Five measurements were taken from each sample, and three samples of each lm were measured. Total colour differences (DE) were calculated as follows (1):

DE

q 2 2 2 L 0 L a0 a b0 b

where, L0, a0 and b0 are the standard values of the white plate, and L, a and b are the measured values of the samples. 2.4.2. Mechanical properties Tensile strength (TS) is the maximum stress supported by the lm before break (Gennadios et al., 1997), and percent elongation at break (%E) is the maximum elongation of the lm before rupture (Krochta and De Mulder-Johnston, 1997). TS and %E were measured with a texture analyser (TA-XT2, TA Instruments, UK) according to the ASTM standard method D882 (ASTM, 2001). Sample lms were cut into strips 20 mm wide strips and 90 mm long. The samples were conditioned for 72 h at 25 C and 50% RH in a dessicator containing Mg (NO3)2 saturated solutions. Equilibrated lm specimens were mounted between the grips with an initial separation of 50 mm, and the cross-head speed was set to 1 mm/s. TS (MPa) was calculated by dividing the maximum load required to break the lm by the cross-sectional area, and %E was calculated by dividing the lm elongation at rupture by its initial length and multiplying by 100. At least eight samples from each type of lm were evaluated. 2.4.3. Water vapour permeability The water vapour permeability (WVP) of the lm samples was measured gravimetrically using a modication of the ASTM E96-95 (ASTM, 1995) proposed by McHugh et al. (1993). Film samples were mounted on permeation cups (Fisher Scientic, USA) to expose the coated face to the outside of the permeation cups lled with 10 ml of distilled water. The cups were placed inside a dessicator containing a sodium chloride saturated solution (75% RH) to maintain a 75/100% RH gradient across the lm. The air over the surface of the lms was homogenised constantly by a fan operated within the dessicator. The desiccators were stored at 25 C. The cups were weighed eight times at 2-h intervals. For each type of lms, WVP measurements were replicated three times and WVP was calculated following Villalobos et al. (2006). 2.4.4. Scanning electron microscopy The microstructural characteristics of the surface and cross-sections of all lms were examined using a scanning electron microscope (JEOL JSM-6380LV). Dried samples (5 3 mm) were mounted on specimen stubs. Samples were freeze-dried, goldcoated and observed using an accelerating voltage of 15 kV. For cross-section observation, the samples were previously fractured using liquid nitrogen.

2.4.5. Antibacterial activity The antibacterial activity of the lms with OEO was determined using the agar diffusion method, according to Pellissari et al. (2009). The edible lms were aseptically cut into 2-mm discs and placed on Muller Hinton agar plates, which had been previously spread with 0.1 mL of inocula, each containing 105106 CFU/mL of bacterial culture, previously standardised using the McFarland scale. The plates were incubated at 37 1 C for 24 h. The diameter of the growth inhibition zones around the discs was measured using a digital calliper (VWR, USA). The growth under the lm discs (area of contact with the agar surface) was visually examined. The measurements were made in triplicate for each lm. 2.5. Statistical analysis Studies conducted at different CaCO3 and OEO concentrations were carried out and considered independent experiments. Oneway analysis of variance (ANOVA) was conducted to analyse the data from each experiment, using Statgraphics plus for Windows 5.1 (Manugistics Corp., Rockville, Md). Differences between the mean values of the lms properties were determined using least signicant difference (LSD) test for multiple comparisons. The signicance of the difference was determined at a 95% condence level (a = 0.5). 3. Results and discussion 3.1. Properties of alginate lm with internal gelation The effect of the internal crosslinking of the alginate lms, promoted by the slow release of calcium ions from CaCO3 in an acidied medium with GDL, on functional properties is shown in Table 1. 3.1.1. Appearance, thickness and colour of the lms Alginate lms were visually homogeneous with no brittle areas or bubbles and were easily manageable and exible. The addition of CaCO3 increased the thickness of the lms (3138 lm), depending on the CaCO3 content. This effect was statistically signicant (p < 0.05) from 0.02 g CaCO3/g alginate content as compared to control lm (29 lm). According to Han and Krochta (1999), lm thickness is inuenced by the solid content of the lm-forming solution. The incorporation of CaCO3 did not signicantly (p < 0.05) affect the total colour difference (DE) of the alginate lms. It was observed that the low concentration of calcium ions (0.010.03 g CaCO3/g alginate) used in the internal gelation of the alginate lms was not sufcient to change the light scattering in the polymeric matrix. 3.1.2. Mechanical properties The tensile strength (TS) and elongation at break (%E) are parameters representing the mechanical properties of the lms, which depend on their microstructural characteristics (Krochta

Table 1 Effect of CaCO3 concentration on the physical properties of alginate lmsa. CaCO3 (g/g alginate) 0.00 0.01 0.02 0.03 Thickness (mm) 0.029 0.0025 0.031 0.0019a 0.037 0.0028b 0.038 0.0032b
a

DE
2.76 0.49 2.85 0.93 3.55 0.84 3.04 0.67
a a a a

TS (MPa) 39.7 5.3 52.6 6.1b 51.9 5.8b 52.9 5.5b

a

%E 4.4 0.5 4.5 0.8a 2.3 1.2b 2.3 0.9b

a

WVP (g/m s Pa) 109 3.9 0.12a 3.8 0.18a 3.8 0.15a 3.0 0.21b

DE, total colour differences; TS, tensile strength; %E, percent elongation; WVP, water vapour permeability. a Data reported are mean values standard deviations. Mean values with different letters in the same column are signicantly different (p < 0.05).

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and De Mulder-Johnston, 1997). TS and %E of the internal crosslinked alginate lms are summarised in Table 1. The crosslinked lms were signicantly stronger than the control lm (p < 0.05), as indicated by high values of TS. This suggests that the extent of crosslinking increased the mechanical strength of the lms, making them more resistant and requiring a greater force to rupture them. This phenomenon is explained by the development of a more rigid polymer structure as a result of interactions between carboxyl groups of the alginate chain and calcium ions (Pavlath et al., 1999). The incorporation of calcium ions into the lms may partially replace the weak polymerpolymer interactions by the formation of ionic polymercationpolymer complexes (Zactiti and Kieckbusch, 2004). However, no signicant differences in crosslinked lms with different amounts of CaCO3 were observed. According to Chan et al. (2006), in the internal gelation method, the liberation of calcium ions from insoluble CaCO3 salt promoted by the dissolution of GDL is accompanied by CO2 formation. Hence, the presence of cavities due to the liberation of CO2 may have disrupted the alginate matrix, thereby compromising its TS. Consistent with the highest TS shown by the crosslinked lm, %E decreased from 4.5% to 2.3% with increasing concentration of calcium ions. This effect was statistically signicant (p < 0.05) from 0.02 g CaCO3/g alginate compared with the control lm. However, lms crosslinked with the addition of 0.01 g CaCO3/g alginate did not show a signicant decrease in its %E compared with the control lm. This result can be attributed to the microstructural features presented by the crosslinked lms. The degree of crosslinking produced, although signicantly increased the TS of the lm, was not sufcient to affect its plasticity. Also, the porous microstructure and the formation of a dense structure in the lmair interface, observed in the crosslinked lms, which are described in Section 3.1.4, may also explain the lower effect of calcium on the %E of this lm. The loss of exibility or rigidity observed in the other crosslinked lms may be a consequence of the reduced mobility of the polymer chains, promoted by the highest level of crosslinking achieved. The increase in the TS and decrease in %E due to the incorporation of calcium ions in alginate lms has been noted by other authors (Chan et al., 2006; Rhim, 2004; Zactiti and Kieckbusch, 2006). However, the TS and %E values found in this study are lower than those reported by these same authors. The differences in these mechanical properties can be related to the non-incorporation of plasticizer, highest concentration and different sizes of particles of CaCO3 used (Chan et al., 2006), different calcium salt (CaCl2), concentration of plasticizers used and thicker lms (Rhim, 2004) and could also be explained by the different conditions used in the measurement of mechanical properties, such as cross-head speed (8.3 mm/s, Rhim, 2004; 0.16 mm/s, Chan et al., 2006). 3.1.3. Water vapour permeability The WVP of the crosslinked lms (Table 1) was not signicantly affected (p < 0.05) by the internal gelation process, except at the highest calcium concentration studied (0.03 g CaCO3/g alginate), which revealed a signicant decrease in WVP (p < 0.05). The degree of crosslinking obtained by increasing the calcium concentration in alginate lms was not sufcient to reduce water vapour diffusion through it. Only at a concentration of 0.03 g CaCO3 was it possible to obtain a degree of crosslinking that reduced the mobility of polymeric chains, thus reducing signicantly the WVP through the lm matrix. This behaviour has also been reported in alginate lms with higher concentrations of calcium (Rhim, 2004). On the other hand, the formation of a porous matrix, generated by the evolution of CO2 from the reaction between H+ and CaCO3, may also have screened the effect of the crosslinking process on the WVP. The magnitudes of the WVP values of alginate lms obtained

in the present study are higher than those reported by Rhim (2004) for similar lms. These differences may be explained by the higher relative humidity gradient used in this study (10075). Several authors have reported that when the relative humidity increases, the capacity of water vapour barrier of lms decreases (Gontard et al., 1996; Hagenmaier and Shaw, 1990; Olivas and BarbosaCnovas, 2008). Water acts as a plasticiser in the polymer matrix, reducing the number of intermolecular bonds between the polymer chains (free volume increase), thereby facilitating the transfer of water vapour through the lm (Cisnero-Zeballos and Krochta, 2002). 3.1.4. Microstructural characteristics The effect of internal gelation on the surface and cross-sectional microstructure is shown in Fig. 1. The surface of the lm without addition of CaCO3 was smooth and homogenous, typical of a soft polymer (Avella et al., 2007), with some irregularities spread on the surface, probably belonging to foreign materials and without pores or cracks. SEM observations of the cross-section of the control lm showed a brous structure, similar to those reported by Norajit et al. (2010), with a more compact region at the lm air interface. The incorporation of CaCO3 generated a heterogeneous surface with gelled areas that increased proportionally with the amount of calcium added. The cross-section of the crosslinked alginate lms showed a sponge-like structure, with pores distributed throughout the alginate matrix. The pores in the matrix increased with the concentration of calcium added, as a result of the formation of CO2 during the release of calcium ions from CaCO3 salt during the gelation process, as previously mentioned. Similar results have been found by Choi et al. (2002) to evaluate the effect of CaCO3 content in the internal microstructure of alginate beads. A dense structure at the lmair interface was also observed in these lms, though its thickness decreased as the calcium content increased in the matrix lm. 3.2. Properties of alginate lms with incorporation of OEO 3.2.1. Appearance, thickness and colour of the lms The effects of the incorporation of OEO on the properties of the alginate lms are presented in Table 2. The appearance of the alginate lms was affected by the addition of OEO. The transparency of the lms, evaluated visually, decreased with the addition of OEO, showing the development of a yellowish colour. The addition of OEO to the lm-forming emulsion led to an increase in the thickness of the lms, which ranged between 0.036 mm and 0.042 mm. (Table 2). However, this effect was only signicant at the highest level of OEO used (1.5%). Norajit et al. (2010) noted that the addition of ginseng extract in alginate lm increased signicantly their thickness. However, the concentration of ginseng extract (0.5 g/ml of lm-forming solution) added was much higher than the OEO concentrations used in this work. Alginate lms incorporated with a concentration greater than 0.5% of OEO showed a total colour difference (DE) signicantly greater (p < 0.05) than the control lm (Table 2). This behaviour is attributed to the decrease in brightness (L) and the increase observed in the colourimetric coordinate b. Similar results have been reported by Pranoto et al. (2005), who studied the incorporation of garlic oil into alginate lms. 3.2.2. Mechanical properties Contrary to the increase in TS and decrease in %E observed by adding CaCO3 in the alginate lms, a signicant reduction in TS and increase in %E was observed with the addition of oregano essential oil (p < 0.05). According to Table 2, a 56% reduction in the TS values (71.031.1 MPa) was obtained when adding 1.5% of

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Fig. 1. SEM micrographs of the surface and cross-section of the alginate lms. (a) 0.00 g CaCO3/g alginate, (b) 0.01 g CaCO3/g alginate, (c) 0.02 g CaCO3/g alginate, (d) 0.03 g CaCO3/g alginate.

Table 2 Effect of oregano essential oil on physical properties of the alginate lmsa. Essential oil (% p/v) 0.0 0.5 1.0 1.5 Thickness (mm) 0.033 0.0028 0.036 0.0025ab 0.038 0.0023ab 0.042 0.0019b
a

DE
2.28 0.23 3.05 0.63ab 3.19 0.53b 3.35 0.38b
a

TS (MPa) 71.0 6.3 55.5 5.7b 46.5 5.4b 31.1 6.0c

a

%E 2.2 0.5 2.7 0.7ab 3.3 0.7bc 3.7 0.9c

a

WVP (g/m s Pa) 109 3.1 0.12a 3.0 0.08a 2.8 0.06b 2.7 0.11b

DE, total colour differences; TS, tensile strength; %E, percent elongation; WVP, water vapour permeability. a Data reported are mean values standard deviations. Mean values with different letters in the same column are signicantly different (p < 0.05).

OEO to the alginate lms. Consequently, the %E of alginate lms increased signicantly, ranging from 2.2% (control) to 3.7% for lms containing 1.5% of OEO (p < 0.05). This trend is consistent with those obtained by Cagri et al. (2001), Pranoto et al. (2005) and Rojas-Gra et al. (2007). These studies concluded that the incorporation of essential oils or other extra-polymeric components usually reduce the TS as a result of the development of a heterogeneous lm structure featuring discontinuities. Because

OEO is liquid at room temperature, it will be present in the lm in the form of oil droplets that can easily be deformed, enhancing the lms extensibility (Fabra et al., 2008). Alginate lms incorporated with oregano essential oil were designed to have a high internal crosslinking by addition of 0.03 g CaCO3/g alginate. The rigid and less exible structure characteristic of these crosslinked lms was affected by the addition of OEO. The presence of OEO in the lm-forming emulsion may have interfered

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with ionic interactions between the alginate and calcium ions, reducing the intermolecular forces along polymer chains, improving the exibility and chain mobility. Thus, OEO can act as a plasticizer, reducing TS and increasing %E of the lms.

(Gontard et al., 1996; Hagenmaier and Shaw, 1990; Olivas and Barbosa-Cnovas, 2008). 3.2.4. Microstructural characteristics Scanning electron micrographs of cross-sections of alginate lms incorporated with OEO are shown in Fig. 2. The nal microstructure of the lms results from the internal gelation process of the alginate and the interactions between the components as well as the destabilising phenomena that place during the drying step (Atars et al., 2010a). Alginate lms incorporated with OEO showed a loose texture with sponge-like structure, with pores distributed throughout the alginate matrix. However, this structure transformed into one that featured the more compact stacking of sheet-like layers with increasing OEO content. These latter structures could explain the observation of lower WVP in alginate lms with higher oil content. 3.2.5. Antibacterial activity The antibacterial activities of alginate lms incorporated with OEO against Gram-negative and Gram-positive bacteria are shown in Table 3. An alginate lm without OEO was used as a control to determine any possible antibacterial effect of the lm without additives. The control lm did not inhibit the growth of the four pathogenic bacteria. Alginate lms showed antibacterial effect against all bacteria studied after the incorporation of 1.0% OEO. As the concentration of OEO increased in the alginate lms, the zone of inhibition also increased signicantly (p < 0.05). The minimum concentration of OEO at which the lms begin to express antibacterial activity coincides with that found by Du et al. (2009), who studied the antibacterial activity of tomato lms incorporated with OEO against E. coli 0157:H7, S. enterica and L. monocytogenes, and by Zivanovic et al. (2005), who studied the antibacterial activity of chitosan lms enriched with OEO against E. coli 0157:H7 and L. monocytogenes. However, Seydim and Sarikus (2006) reported that wheyprotein isolate lms containing OEO had a higher minimum inhibitory concentration against the same bacteria. The antibacterial efciency of OEO can be related to the concentration and proportion of phenolic compounds, which in turn depend on the variety of plant, origin, time of harvest, and processing, as well as storage conditions (Zivanovic et al., 2005).

3.2.3. Water vapour permeability WVP of alginate lms incorporated with OEO are shown in Table 2. In general, incorporation of OEO into alginate lms leads to a decrease in WVP values from 3.1 to 2.7 109 g m1 s1 Pa1. However, a signicant decrease (p < 0.05) was observed only after the incorporation of 1.0% of OEO. This behavior may be due to the porous microstructure produced by the internal crosslinking process and the addition of OEO in the lms of alginate, as described in Sections 3.1.4 and 3.2.4, respectively. In the latter case, a more compact structure was observed to increase essential oil content, which could explain the decrease in the WVP. Hernandez (1994) suggested that water vapour transfer generally occurs through the hydrophilic portion of the lm and depends on the hydrophilichydrophobic ratio of the lm components. Thus, it is generally accepted that the addition of hydrophobic lipids to hydrophilic polymer lms improves water vapour barrier properties. Previous studies on the WVP of chitosan lms (Hosseini et al., 2009; Zivanovic et al., 2005), and starchchitosan lms (Pellissari et al., 2009) containing essential oils, have shown similar trends. However, others studies on the incorporation of essential oils and natural extracts have not shown improvements in WVP (Atars et al., 2010b; Pranoto et al., 2005; Zinoviadou et al., 2009). According to Atars et al. (2010b), it cannot be assumed that the WVP of edible lms is reduced simply by adding a hydrophobic component to the formulation, though the impact of lipid addition on the microstructure of emulsied lms is a determining factor of water vapour barrier efciency. In general, the WVP values of all alginate lms tested (Table 2) were higher than those reported by the authors previously mentioned. This difference may be due to the hydrophilic character of alginate and the high RH gradient used in this study (100:75), which was selected to simulate the surface conditions of moist food. Under these conditions of high relative humidity, the ability of lms to act as a barrier to water vapour and gases decreases

Fig. 2. SEM micrographs showing the effect of the addition of oregano essential oil on the cross-sectional microstructure of alginate lms. (a) 0.0%, (b) 0.5%, (c) 1.0%, (d) 1.5%.

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Table 3 Antibacterial activity of alginate lms incorporated with oregano essential oil. Bacteria Escherichia coli Oregano essential oil (w/v) 0.0 (control) 0.5 1.0 1.5 0.0 (control) 0.5 1.0 1.5 0.0 (control) 0.5 1.0 1.5 0.0 (control) 0.5 1.0 1.5 Diameter of the zone of inhibitiona 0aA 0aA 21.89 0.12bB 22.13 0.02bCD 0aA 0aA 21.83 0.04bB 22.09 0.07cC 0aA 0aA 22.19 0.05bD 22.69 0.03cE 0 0aA 22.62 0.03bE 22.86 0.06bF
aA

Salmonella Enteritidis

Staphylococcus aureus

OEO exhibited signicant antibacterial activity against four bacteria studied. A minimum concentration of 1.0% of OEO was necessary to ensure their antibacterial efcacy. The lms were more effective against Gram-positive bacteria (S. aureus and L. monocytogenes) than Gram-negative bacteria (E. coli and S. Enteritidis). These results suggest that physical, mechanical and antibacterial properties of alginate lms can be modied by controlling the level of internal crosslinking produced by the addition of CaCO3. Therefore, it would be possible to obtain an antimicrobial alginate lm, with a degree of crosslinking to allow a slow migration of essential oil of oregano from the alginate matrix to the surface of the food. Acknowledgments The authors thank the nancial support of the Direccin de Investigacin de la Universidad del Bio Bio (DIUBB) through Project 085922 3/R. References
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Listeria monocytogenes

a Diameter of the zone of inhibition in mm. (n = 3). Mean values with different superscript letters in the same column for OEO are signicantly different (p < 0.05). Mean values with different superscript in capital letters in the same column for bacteria are signicantly different (p < 0.05).

Hosseini et al. (2009) also noted that the nature and structural characteristics of the matrix in which the essential oil is dispersed along with the method of preparation of the lms has a crucial effect on antimicrobial activity. In general, alginate lms with OEO were signicantly more effective against Gram-positive bacteria (S. aureus and L. monocytogenes) than against Gram-negative bacteria ( E. coli and S. Enteritidis). According to the values of the inhibition zone diameter, the relative resistance to OEO had the following order: E. coli P S. Enteritidis > S. aureus > L. monocytogenes. Several studies that have investigated the activity of essential oils against food spoilage organisms and food-borne pathogens agree that, generally, essentials oils are slightly more active against Gram-positive than Gram-negative bacteria (Burt, 2004; Pellissari et al., 2009; Zivanovic et al., 2005). Burt (2004) suggests that this behaviour may be related to the presence of an additional external membrane surrounding the cell wall in Gram-negative bacteria, which restricts diffusion of hydrophobic compounds through its lipopolysaccharide covering. According to preliminary studies that evaluated the antibacterial activity of pure OEO (data no shown), the inhibitory effect of OEO incorporated into the alginate lms was lower than that found in pure OEO. The possible causes that would explain this result could be a partial loss of volatile compounds during lm preparation (Snchez-Gonzlez et al., 2010). It may also be due to a slower rate of diffusion of phenolic compounds into the agar as a result of the higher level of internal crosslinking achieved by the alginate matrix due to added CaCO3, or it may be due to possible interactions between the hydroxyl groups of phenolic compounds and alginate polymeric chains. These results prove that OEO can be immobilised in alginate lms and subsequently released, thereby inhibiting target microorganisms.

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