Antimicrobial Effects of Alginate-Based Film Containing Essential Oils for the Preservation of Whole Beef Muscle

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1 2364 Journal of Food Protection, Vol. 69, No. 10, 2006, Pages Copyright, International Association for Food Protection Antimicrobial Effects of Alginate-Based Film Containing Essential Oils for the Preservation of Whole Beef Muscle MOUNIA OUSSALAH, 1 STÉPHANE CAILLET, 1 STÉPHANE SALMIÉRI, 1 LINDA SAUCIER, 2 AND MONIQUE LACROIX 1 * 1 Research Laboratory in Sciences Applied to Food, Canadian Irradiation Center, Institut National de la Recherche Scientifique, Institut Armand-Frappier, Université du Québec, 531 Blvd des Prairies, Laval, Québec, Canada H7V 1B7; and 2 Département des Sciences Animales, Université Laval, Québec City, Québec, Canada G1K 7P4 MS : Received 8 March 2006/Accepted 5 May 2006 ABSTRACT Alginate-based edible films containing 1% (wt/vol) essential oils of Spanish oregano, Chinese cinnamon, or savory were immersed in 2% (wt/vol) or 20% (wt/vol) CaCl 2 solution and then applied to beef muscle slices to control the growth of Escherichia coli O157:H7 and Salmonella Typhimurium. Whole beef muscle surfaces were inoculated with one of these strains at 10 3 CFU/cm 2. During the 5 days of storage, samples of meat were obtained periodically for microbiological analysis. The availability of active compounds from essential oils present in films was evaluated by determination of total phenolic compounds for oregano- and savory-based films and of total aldehydes for cinnamon-based films during storage. After 5 days of storage, films containing oregano or cinnamon essential oils were the most effective against Salmonella Typhimurium regardless of the type of pretreatment used (2 or 20% CaCl 2 ). During the same period, meat inoculated with E. coli O157:H7 and coated with films treated with 2% CaCl 2 had significantly fewer bacteria (P 0.05) when oregano-based films were used than when cinnamon- and savory-based films were used. The E. coli O157:H7 concentration was higher at the end of the storage period when films were pretreated with 20% CaCl 2. Evaluation of the active compounds in films revealed that availability in oreganoand savory-based films was significantly more important (P 0.05) than that in cinnamon-based films regardless of the type of pretreatment used (2 or 20% CaCl 2 ). At the end of storage, release rates of 40, 60, and 77% were noted in oregano-, savory-, and cinnamon-based films in 2% CaCl 2 and rates of 65, 62, and 90% were noted in the same films in 20% CaCl 2. The use of natural compounds to control the growth of pathogenic bacteria has become preferred by customers and hence is of great interest to the meat industry (1). Essential oils extracted from plants or spices are rich sources of biologically active compounds such as flavonoids and phenolic acids, which exhibit a wide range of antimicrobial properties (5, 8). According to Conner and Beuchat (5), the active compounds present in essential oils could be responsible for the inactivation of a variety of enzyme systems in microbial cells, rendering them more sensitive to antimicrobial preservatives. In several studies, the antimicrobial activity of natural extracts has been demonstrated. Among these natural extracts, savory (S), oregano (O), and cinnamon (C) essential oils had bactericidal activity against a wide range of foodborne pathogens (5, 8). The incorporation of natural antimicrobial compounds into edible packaging films or coatings is a new method for control pathogenic growth and, thus, food contamination and quality loss. Antimicrobial packaging is part of the broader field of active packaging, which has become a major focus of food packaging research in the last decade (28) because biodegradability and environmental compatibility are assured (16). Antimicrobial packaging can play an important role in reducing the risk of pathogen development and extending the shelf life of foodstuffs (20). Biological * Author for correspondence. Tel: , Ext 4489; Fax: ; monique.lacroix@iaf.inrs.ca. coatings can be especially useful as carriers of antimicrobial compounds, allowing high concentrations of these compounds to be maintained on the food surface. The incorporation of organic acids and essential oils in edible film packaging or coatings contributed greatly to control of bacterial growth during the storage of meat (9, 21, 27). Active milk protein based film can allow progressive release of antimicrobial compounds (21). However, improved water resistance and mechanical properties have been obtained by developing films based on a mixture of alginic acid and polycaprolactone. Polycaprolactone has high water resistance, a low melting point, and a low glass transition temperature and is highly processable (19). It also exhibited some desirable characteristics of a diffusion-controlled delivery system, including biodegradability, biocompatibility, commercial availability, and affordability (18). In this study, polycaprolactone was used as both a hydrophobic and emulsifying agent to stabilize the essential oils in the alginate matrix. In several studies, alginate has been suggested as an interesting film matrix because of its biocompatibility, low toxicity, and ability to form an insoluble film by ionotropic gelation (15). Alginate films are oxygen barriers, prevent lipid oxidation, and improve food flavor and texture (13). The aims of this study were (i) to evaluate the type of the pretreatment used (2% or 20% CaCl 2 ) and the antimicrobial potential of C, O, and S alginate-based films against

2 J. Food Prot., Vol. 69, No. 10 ANTIMICROBIAL EFFECTS OF FILM FOR PRESERVATION OF MEAT 2365 the growth of two important pathogenic bacteria (Escherichia coli O157:H7 and Salmonella Typhimurium) on beef muscle and (ii) to evaluate the effect of controlled release of main active compounds present in films on meat during storage. MATERIALS AND METHODS Film preparation. Alginate-based films were prepared according to a method developed in our laboratory. Sodium alginate and polycaprolactone diol (M n, 1,250) were purchased from Sigma-Aldrich Ltd. (Oakville, Ontario, Canada). The polycaprolactone was slowly solubilized in distilled water at 45 C for 15 min for a final concentration of 1% (wt/vol) in film-forming solution. Alginate was solubilized in distilled water at room temperature under agitation for 2htoobtain a final concentration of 3% (wt/ vol). The polycaprolactone solution was then mixed with the alginate solution. Glycerol was added to this mixture at a final concentration of 2% (wt/vol) (24). The alginate-polycaprolactone solution was then heat sterilized at 121 C for 30 min. Essential oils of oregano (Corydothymus capitatus), cinnamon (Cinnamomum cassia), and savory (Satureja montana) (Robert et Fils, Montréal, Québec, Canada) were added at a final concentration of 1% (wt/ vol). The film-forming solution was maintained under agitation for 1 min at room temperature. For each film-forming solution, 5 ml was deposited in petri dishes (50 by 11 mm; Sarstedt Inc., Montréal, Québec, Canada) and dried at ambient temperature at 40 to 50% relative humidity for 24 h to form a homogeneous and transparent film. After drying, the water-soluble film was rendered insoluble by immersion in solution (2% for 1 min or 20% for 20 min). CaCl 2 produced an insoluble gel by forming ionic bonds. The effects of 2 and 20% CaCl 2 treatment on the release of active compounds to beef muscle was compared. Preparation of bacterial strains. E. coli O157:H7 EDL 933 and Salmonella Typhimurium SL1344 (Institut National de la Recherche Scientifique, Institut Armand Frappier, Laval, Québec, Canada) stock cultures were maintained at 80 C in brain heart infusion (BHI; Difco, Becton Dickinson, Sparks, Md.) broth containing 10% (wt/vol) glycerol. Working cultures were prepared by subculturing 1 ml of each stock culture in 9 ml of BHI and incubated at 35 1 C through two successive 24-h incubation periods. Sample preparation. Beef from the semimembranosus (inside round, 15 kg) was purchased at a local grocery store (IGA, Laval, Québec, Canada) and transported to the Institut Armand Frappier and the Canadian Irradiation Center under refrigerated conditions in an ice-filled cooler. Beef muscle was cut into 390 pieces (300 pieces for microbiological analysis and 90 pieces for biochemical analysis) of equal thickness (7.5 mm) and diameter (47 mm) with a pastry cutter. The 390 beef pieces (10 pieces per package) were placed in copolymer sterile bags of 0.5-mm metallized polyester and 2-mm ethylene vinyl acetate (305 by 210 mm; Winpak, St-Léonard, Québec, Canada) and sealed under vacuum. The packages were stored overnight at 80 1 C until sterilized by irradiation with 25 kgy and at rate of kgy/h from a Co 60 source UC-15A (MDS-Nordion International Inc., Kanata, Ontario, Canada). After irradiation, 300 pieces of beef were inoculated with E. coli O157:H7 and Salmonella Typhimurium to obtain a final concentration of 10 3 CFU/cm 2 on the beef. Each sample of beef muscle was inoculated with 50 l ( CFU/ml), which was spread over the entire upper surface of each sample. Meat samples were then divided into two groups (films treated in 2 or 20% CaCl 2 solution), and each group was subdivided into five separate groups: (i) control group without films (M); (ii) alginate-based film coating without essential oil (W); (iii) alginate-based film coating containing O essential oil; (iv) alginate-based film coating containing C essential oil; and (v) alginate-based film coating containing S essential oil. The other 90 pieces of beef reserved for the biochemical analysis were not inoculated but were divided into two groups (films treated in 2 or 20% CaCl 2 solution), and each group was subdivided into three separate groups (O, C, and S). Samples from each group were placed individually into sterile petri dishes (50 by 11 mm), and except for the control, all samples were covered on the upper surface with one of the corresponding films. Petri dishes were sealed hermetically and stored at 4 1 C. Samples were removed periodically (days 1, 2, 3, 4, and 5) for microbiological and biochemical analysis. Day 1 corresponded to the day of sample preparation. Enumeration of bacteria. Each beef muscle sample was homogenized for 2 min in 153 ml of sterile peptone water (0.1%, wt/vol; Difco, Becton Dickinson) using a Lab-blender 400 stomacher (Seward Medical, London, UK). Serial dilutions were prepared from this homogenate, and appropriate dilutions were spread plated on sterile petri plates containing tryptic soy agar (TSA; Difco, Becton Dickinson). The TSA plates were incubated for 24 h at 35 1 C, and results were expressed as log CFU per square centimeter of meat. Availability of active compound. To evaluate the availability of active compounds in the packaging films, 1 g of each film sample was removed with a scalpel. The thin strips were placed in 50 ml of ethanol solution and kept under agitation with a magnetic stir bar at room temperature for 30 min, and the extract was then filtered through m-pore-size filter paper (Whatman International Ltd., Maidstone, UK). The total phenolic compounds in each film extract was determined according to the Folin-Ciocalteu procedure (26). The availability of total aldehydes in C- based films was determined with the Kleber method using a phenylhydrazine reagent (2). Experimental design and statistical analysis. Experiments for microbiological analysis were done using a factorial design: 3 replicates, 5 treatments (control, film without essential oil, film with O, C, and S essential oils), 2 bacteria (E. coli and Salmonella Typhimurium), 2 kind of films (insolubilized in 2 or 20% CaCl 2 solution), and 5 storage periods (1, 2, 3, 4, and 5 days). Biochemical analyses were done using a factorial design: 3 replicates, 3 treatments (film with O, C, and S essential oils), 2 kind of films (insolubilized in 2 or 20% CaCl 2 solution), and 5 storage periods (1, 4, 5, 6, and 7 days). Analysis of variance and Duncan s multiple range tests were used to perform statistical analysis on all results. Differences between means were considered significant at P Stat- Packets Statistical Analysis software (Walonick Associates, Inc., Minneapolis, Minn.) was used for the analysis. For each measurement, three samples in each replication were tested. RESULTS AND DISCUSSION Antimicrobial effects. The effects of edible films on E. coli O157:H7 and Salmonella Typhimurium counts during storage are presented in Tables 1 and 2, respectively. E. coli O157:H7 counts were stable in M and W film samples for all storage periods (Table 1). The use of film containing essential oils significantly reduced the E. coli (P 0.05) on the meat surface compared with that on M samples

3 2366 OUSSALAH ET AL. J. Food Prot., Vol. 69, No. 10 TABLE 1. Effect of film composition on E. coli O157:H7 growth on meat during storage at 4 C E. coli counts (log CFU/cm 2 ) b Samples a Day 1 Day 2 Day 3 Day 4 Day 5 2% CaCl 2 M (control) a A c A b A b A c A W a A c A b A b A c A O a B c B b B b B a A C a C a B a AB a AB b A S a C b B a A a A b A 20% CaCl 2 M (control) a A a A a A b A b A W a A b B b B c B b A O a B a B a B a A a A C a C a B a B a A a A S a A a A a A b A b A a Alginate-based edible films were treated with a 2 or 20% (wt/vol) CaCl 2 solution. M, control (meat without film); W, meat plus film without essential oil; O, meat plus film containing Spanish oregano essential oil; C, meat plus film containing Chinese cinnamon essential oil; S, meat plus film containing winter savory essential oil. b Within each treatment and each column, means with the same lowercase letter are not significantly different (P 0.05). Within each row, means bearing the same capital letter are not significantly different (P 0.05). and on samples coated with W film during storage. C- and S-based films treated with 2% CaCl 2 were significantly more efficient (P 0.05) than O-based films until day 4 of storage, but the microorganism count in samples coated with O-based films was significantly lower (P 0.05) than the count in samples coated with C- and S-based films at day 5. A 1.32-, 1.47-, and 1.97-log reduction of E. coli O157:H7 was observed in meat covered with S-, C-, and O-based films, respectively, after 5 days of storage. Films treated with 2% CaCl 2 had higher antimicrobial activities than films treated with 20% CaCl 2. Meat covered with O- and C-based films treated with 20% CaCl 2 had significantly decreased E. coli O157:H7 counts only at day 4 of storage. At day 5, a 1-log reduction (P 0.05) was observed in O- and C-based films, but no significant difference (P 0.05) was observed on meat covered with S-based films between the days of storage, indicating microbial stability during the entire storage period. Results for Salmonella Typhimurium are presented in Table 2. At day 2 of storage, a significant increase (P 0.05) was observed for M samples and samples covered with W films regardless of the pretreatment used (2 or 20% CaCl 2 ). A significant 0.76-log decrease (P 0.05) was observed on meat covered with O-based films treated with 2% CaCl 2 at day 3 of storage, and a significant reduction (P 0.05) occurred in meat covered with C- and S-based TABLE 2. Effect of film composition on Salmonella Typhimurium growth on meat during storage at 4 C Salmonella counts (log CFU/cm 2 ) b Samples a Day 1 Day 2 Day 3 Day 4 Day 5 2% CaCl 2 M (control) a A c B d D c BC c A W a A b c B c BC c C d BC O a C b D a B a A a A C a C a B a AB a A a A S a C a B b A b A b A 20% CaCl 2 M (control) a A c B d B d B c A W a A b B c B c B d B O a B a A a A a A a A C a C a AB b B ab A a A S a C a A b B b B b B a Alginate-based edible films were treated with a 2 or 20% (wt/vol) of CaCl 2 solution. M, control (meat without film); W, meat plus film without essential oil; O, meat plus film containing Spanish oregano essential oil; C, meat plus film containing Chinese cinnamon essential oil; S, meat plus film containing winter savory essential oil. b Within each treatment and in each column, means with the same lowercase letters are not significantly different (P 0.05). Within each row, means with the same capital letter are not significantly different (P 0.05).

4 J. Food Prot., Vol. 69, No. 10 ANTIMICROBIAL EFFECTS OF FILM FOR PRESERVATION OF MEAT 2367 films at day 2. At the end of storage, O-, C-, and S-based films were able to reduce Salmonella Typhimurium by 0.98, 0.96, and 0.61 log CFU; thus, O- and C-based films were significantly more efficient (P 0.05) than S-based films. For films treated with 20% CaCl 2, O-, C-, and S-based films were more effective at controlling the growth of Salmonella Typhimurium on day 2. However, at the end of storage, O- and C-based films were more efficient against Salmonella Typhimurium, with a significant and 0.88-log reductions (P 0.05), respectively. In our study, the incorporation of essential oils at 1% (wt/vol) into the alginate film formulation allowed a significant decrease (P 0.05) in Salmonella Typhimurium and E. coli O157:H7 on beef muscle samples. Regardless of the pretreatment used (2 or 20% CaCl 2 ), the microorganism counts on meat coated with O-, C-, and S-based films were significantly lower (P 0.05) than that with W films and on M samples. Our data also suggest that O-, C-, and S-based films treated by immersion in 2% CaCl 2 were a little more efficient against E. coli O157:H7 than were films unsolubilized with 20% CaCl 2. The calcium seems to have a protective effect on E. coli O157:H7. Calcium plays a role in the development of bacterial competence, an inducible property of many bacterial species (32). In the presence of CaCl 2, the lipopolysaccharide (LPS) of certain E. coli strains is modified by induction of a phosphoethanolamine transferase, which might ensure resistance to environmental stresses such as exposure to certain detergents and antibiotics (12). Hauben et al. (10) found that the presence of divalent cations such as 0.5 mmol/liter CaCl 2 had a protective effect on E. coli by reducing high-pressure hydrostatic inactivation. However, LPS of gram-negative bacteria also can be stabilized by a regular array of calcium that serves to cross-link the negatively charged LPS molecules, and this phenomenon could perhaps produce a membrane less conducive to entry of the polar polyphenolic compounds and more resistant to external stresses. The reduction in Salmonella Typhimurium on the beef muscle coated with essential oil based films unsolubilized with 2 or 20% CaCl 2 was identical (P 0.05). In samples covered with films without essential oils, both bacteria seemed to use the contents of the film (e.g., alginate) as nutrients to maintain their growth. The essential oils evaluated in this study previously were found to be effective against pathogenic bacteria. Yuste and Fung (33) found that cinnamon added to apple juice with nisin contributed greatly to the inactivation of Salmonella Typhimurium and E. coli O157:H7 after 3 and 7 days of storage, respectively. Skandamis et al. (25) found that 0.8% O essential oil eliminated most of the Salmonella Typhimurium population on meat. Chorianopoulos et al. (4) found that savory species from Greece have bactericidal activities against Salmonella and E. coli O157:H7. The antimicrobial properties of the essential oils tested in this study were not reduced by their incorporation in alginate-based films; their effects were prolonged in the films. Pranoto et al. (23) found that the edible alginatebased films containing garlic oil exhibited an antimicrobial activity on Staphylococcus aureus and Bacillus cereus. Application of milk protein based edible films containing 1% (wt/vol) O essential oil allowed a significant (P 0.05) 1.12-log reduction of E. coli O157:H7 after 7 days of storage (21). Chemical analysis of essential oils provided by the manufacturer revealed that O essential oil was composed of carvacrol (76%) and thymol (4.47%), two phenolic monoterpenes. Tested C essential oil was rich in aldehydes: cinnamaldehyde (65%) and methoxy-cinnamaldehyde (21%). S essential oil was composed of the phenolic monoterpenes carvacrol (5.70%) and thymol (42.71%) and of nonoterpenic hydrocarbons: -terpinene (8.63%) and p-cymene (11.68%). The antimicrobial activities of essential oils are predominately related to their main components (7). Cinnamaldehyde is one of the most active compounds present in C essential oil (3). The antimicrobial potential of O and S essential oils is related to their high concentrations of phenolic compounds, i.e., carvacrol and thymol (14). However, the hydroxyl group present in the structure of phenolic compounds confers antimicrobial activity, and the relative position of the hydroxyl group is very important for the effectiveness of these components (6), which can explain the differences in antimicrobial activity between carvacrol and thymol. Our results support this hypothesis; O essential oils had higher antimicrobial activity than did S essential oils. Burt (3) stated that carvacrol, thymol, and cinnamaldehyde have been identified as antibacterial agents on Salmonella Typhimurium and E. coli O157:H7, with MICs of to 0.05% (vol/vol) in vitro, whereas a higher concentration of around 0.05 to 2% (vol/wt) is needed to achieve the same effect in meat. Carvacrol and thymol create cell membrane permeability, provoking a release of the cell constituents, a decrease in the ATP activity in the cells, and a decrease of the intracellular ph (30). Cinnamaldehyde is a electronegative compound that interferes in biological processes involving electron transfer and reacts with vital nitrogen components (e.g., proteins and nucleic acids) and therefore inhibits the growth of microorganisms (31). Active compound availability in films applied to meat. The availability of the main active compounds in O-, C-, and S-based films was determined during the whole storage period at 4 C (Table 3). The main active compounds are phenolic compounds (carvacrol and thymol) in O- and S-based films and aldehydes (trans-cinnamaldehyde and methoxycinnamaldehyde) in C-based films. The initial percentage of active compound was considered 100% in O-, C-, and S-based films regardless of the type of the pretreatment used (2 or 20% CaCl 2 ). During the storage period, a progressive release of these active compounds was noted for the two types of films. Films treated with 20% CaCl 2 had a significant decrease (P 0.05) in these active compounds during storage regardless of the type of film used. During the entire storage period, the availability of aldehydes in C-based films was significantly lower (P 0.05) than that of the phenolic compounds in O- and S-based films. The availability of phenolic compounds in S-based films was significantly higher (P 0.05) than that in O- based films. However, no significant difference (P 0.05)

5 2368 OUSSALAH ET AL. J. Food Prot., Vol. 69, No. 10 TABLE 3. Availability of total phenolic compounds or aldehydes in films containing essential oils during meat storage at 4 C Availablity of active compounds (%) b Film a Day 1 Day 2 Day 3 Day 4 Day 5 2% CaCl 2 O a C b B c B c A c A S a C a A b B b A b A C a D a C a B a A a A 20% CaCl 2 O a C b B b B b A b A S a C c B c B b A c A C a E a D a C a B a A a Alginate-based edible films were treated with a 2 or 20% (wt/vol) CaCl 2 solution. O, film containing Spanish oregano essential oil; S, film containing winter savory essential oil; C, film containing Chinese cinnamon essential oil. b Within each treatment and each column, means with the same lowercase letter are not significantly different (P 0.05). Within each row, means with the same small cap letter are not significantly different (P 0.05). was noted between the concentration of phenolic compounds present in O- and S-based films at day 4. These data suggest that the migration of active compounds in C- based films to beef muscle is more important than migration in the O- and S-based films. Releases of these compounds of 65, 62, and 90% were recorded from O-, S-, and C-based films, respectively, after 5 days of storage. The availability of main active compounds present in films treated with 2% CaCl 2 also was determined (Table 3). Between days 1 and 5, a significant decrease (P 0.05) in the availability of these compounds was observed in all films. The concentrations of these compounds remained stable between days 2 and 3 in O- and S-based films, but a progressive loss of aldehydes was observed in C-based films at the third day of storage. A significant (P 0.05) decrease in availability of active compounds occurred on the fourth day of storage for three films. However, the concentration of active compounds was then stable until the last day of storage. According to these results, it seems that even for the films treated with 2% CaCl 2, active compounds present in C essential oil more easily migrated to the beef muscle than did those of O and S essential oils at all storage times. Components of S-based films migrated more than did those of O-based films during the same period. A release of 60, 40, and 77% of the active compounds was noted for the S-, O-, and C-based films, respectively, at the end of the storage period. The comparison of films treated with 2 and 20% CaCl 2 revealed similarity in the releases of the main active compounds during the storage period. Between days 2 and 4, a mean release of 39% of the aldehydes was observed in the C-based films treated in 2 and 20% of CaCl 2. The phenolic compounds released from S-based films treated with 20% CaCl 2 were similar to those from films treated with 2% CaCl 2. Between day 2 and 4, releases of 62 and 74% were observed, respectively. In O-based films between day 2 and 4, a more important release was noted in films treated with 2% CaCl 2 than in films treated with 20% CaCl 2. Many interactions may occur during oil diffusion from polymer to beef muscle. In particular, water uptake of meat may cause the polymer to swell (22) and migration rates to change continuously because of a time-dependent relaxation process resulting from swelling stress (17). The oil diffusion would also be expected be related to oil composition and polarity due to the structural configuration of the constituent compounds and their functional groups. The hydrophobicity of these compounds, which is related to their structure, could have an influence on the migration to beef muscle (29). The high release rate of C oil can be explained by the polar character of trans-cinnamaldehyde and 2-methoxycinnamaldehyde, which are the main components of C oil, implying a better affinity with water present in beef muscle than observed for carvacrol and thymol, the main components of O and S oils. Helander et al. (11) found that the solubility of cinnamaldehyde in water at 20 C was higher than that of carvacrol and thymol. Better insolubilization treatment of films (20 versus 2% CaCl 2 ) provides the same level of availability of active compounds in O- and S-based films the end of storage. The release of active compounds in C-based films was faster than that in the O- and S-based films during the first 3 days of storage. There was no significant difference (P 0.05) in mechanical properties between films treated with 2% CaCl 2. For films treated with 20% CaCl 2, the puncture resistance of S-based films (68.11 N/mm) was significantly higher (P 0.05) than that of O- and C-based films (57.65 and N/mm, respectively) (data not shown). These results indicate that the release of active compounds is a complex phenomenon. The incorporation of essential oils into alginate-based edible films helped to reduce E. coli O157:H7 and Salmonella Typhimurium growth on beef muscle during 5 days of storage. The active compounds were maintained better in the films treated with 2% CaCl 2. Thus, foods coated with films treated with 20% CaCl 2 would have a shorter shelf life than foods coated with films treated with 2% CaCl 2. The treatment of the films in 2 and 20% CaCl 2 changes the structure to form a perfect obstruction, which allows controlled release of the active compounds to the beef muscle during storage. The use of edible films containing O, C, and S essential oils as a preservation for beef is promising.

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