filmes antimicrobiano

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Research Signpost 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India Advances in Agricultural and Food Biotechnology, 2006: 193-216 ISBN: 81-7736-269-0 Editors: Ramón Gerardo Guevara-González and Irineo Torres-Pacheco 9 Incorporation of antimicrobial agents in food packaging films and coatings Pérez-Pérez C. 1 , Regalado-González C. 2 , Rodríguez-Rodríguez C. A. 1 Barbosa-Rodríguez J. R. 1 and Villaseñor-Ortega F. 1 1 Depto. Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y A. García Cubas S/N, Celaya, Gto. México, 38010 2 DIPA, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro Qro, México. 76010 Abstract Food quality and safety are major concerns in the food industry. Antimicrobial packaging can be considered an emerging technology that could have a significant impact on shelf life extension and food safety. Use of antimicrobial agents in food packaging can control the microbial population and target specific micro- organisms to provide higher safety and quality products. Many classes of antimicrobial compounds have been evaluated in film structures, synthetic polymers and edible films. The characteristics of some antimicrobial packaging systems are reviewed in this article. Correspondence/Reprint request: Dr. Pérez-Pérez C., Depto. Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y A. García Cubas S/N, Celaya, Gto. México, 38010. E-mail: [email protected]

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Page 1: filmes antimicrobiano

Research Signpost 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Advances in Agricultural and Food Biotechnology, 2006: 193-216 ISBN: 81-7736-269-0 Editors: Ramón Gerardo Guevara-González and Irineo Torres-Pacheco

9 Incorporation of antimicrobial agents in food packaging films and coatings

Pérez-Pérez C.1, Regalado-González C.2, Rodríguez-Rodríguez C. A.1 Barbosa-Rodríguez J. R.1and Villaseñor-Ortega F.1 1Depto. Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y A. García Cubas S/N, Celaya, Gto. México, 38010 2DIPA, PROPAC, Facultad de Química, Universidad Autónoma de Querétaro Qro, México. 76010

Abstract Food quality and safety are major concerns in the food industry. Antimicrobial packaging can be considered an emerging technology that could have a significant impact on shelf life extension and food safety. Use of antimicrobial agents in food packaging can control the microbial population and target specific micro-organisms to provide higher safety and quality products.Many classes of antimicrobial compounds have been evaluated in film structures, synthetic polymers and edible films. The characteristics of some antimicrobial packaging systems are reviewed in this article.

Correspondence/Reprint request: Dr. Pérez-Pérez C., Depto. Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico y A. García Cubas S/N, Celaya, Gto. México, 38010. E-mail: [email protected]

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Introduction There is a growing interest in edible coatings due to factors such as

environmental concerns, new storage techniques and markets development for under utilized agricultural commodities. Edible coatings and films prepared from polysaccarides, proteins and lipids have a variety of advantages such as biodegradability, edibility, biocompatibility, appearance and barrier properties. To control food contamination and quality loss, edible coating or biodegradable packaging has been recently introduced in food processing. Several applications have been reviewed with particular emphasis on the reduction of quality. The packaging can serve as a carrier for antimicrobial and antioxidant compounds in order to keep high concentration of preservatives on the food surfaces. Their presence could avoid moisture loss during storage, reduce the rate of rancidity causing lipid oxidation and brown coloration, reduce the load of spoilage and pathogen microorganism on the surface of foods and also, restricting the volatile flavor loss. The selection of the incorporated active agents is limited to edible compounds and safety is also essential.

Films with antimicrobial agents

There is a growing interest in edible coatings due to factors such as environmental concerns, need for new methods and opportunities for creating new markets for under utilized agricultural commodities with film forming properties. Edible antimicrobial coatings and films have a variety of advantages and constitute an innovation within the biodegradable active packaging concept. They have been developed in order to reduce and or inhibit the growth of microorganisms on the surface foods. The use of appropriate coatings can impart antimicrobial effectiveness. A polymer-based solution coating would be the most desirable method in terms of stability and adhesiveness of attaching a bacteriocin to a plastic film [3]. Low density polyethylene (LDPE) film was successfully coated with nisin using methylcellulose (MC)/ hydroxypropyl methylcellulose (HPMC) as a carrier. Nisin was found to be effective in suppressing S. aureus and L. Monocytogenes respectively [23]. The migration of bacteriocins attained the steady state within 3 days, but the level reached was too low in order to affect several bacterial strains spread on an agar plate media. Films placed in a phosphate buffer solution containing strains of M. flavus and L. monocytogenes showed a marked inhibition of microbial growth of both strains. Chi-Zhang et al. (2004) suggested that the combination of packaging material containing nisin used in conjunction with nisin – containing foods will provide an effective means of preventing L. monocytogenes growth; they concluded that the antimicrobial effectiveness of nisin strongly depends on the mode of delivery. The

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instantaneous and slow methods for adding nisin inhibited L. monocytogenes, but over time the bacteria developed tolerance to nisin. In contrast when antimicrobial was added slowly to the cells.

The efficacy of nisin- coated polymeric films such as PVC, linear low-density polyethylene (LLDPE), and nylon, in inhibiting Salmonella typhimurium on fresh broiler drumstick skin was studied by Natrajan and Sheldon (2000a). Low-density polyethylene (LDPE) films coated with a mixture of polyamide resin in i-propanol/ n-propanol and a bacteriocin solution showed an antimicrobial activity against Micrococcus flavus. Mauriello et al. (2005) showed the efficiency of Nisin coated onto a LDPE film to inhibit Micrococcus luteus ATCC 10240 and the microbiota of raw milk during storage and to examine the release of nisin from the activated film and the release of nisin was pH and temperature dependent.

According of Papadokostaki et al. (1997) a relationship between polymer structure and the transport of active molecules has been reported. Heat and γ- irradiation have been shown to produce cross-linking between protein molecules and improved physical and functional properties of edible films. The structure modification could increase the capacity of edible films to control the release of immobilized active compounds. Linear low-density polyethylene (LLDPE) film showed hydrophobic properties that rejected the hydrophilic nisin formulations to a greater extent than the other films and caused coalescence of the treatment solution droplets. The repulsion between the LLDPE film and the treatment solution may affect the overall inhibitory activity of the formulations by causing more localized inactivation of the target. An agar based film containing nisin was also studied. It was found that in this film, the degree of cross-linking depends on the agar concentration, which may affect the migration of nisin to the surface of a broiler drumstick skin [55]. Thus, 0.75% w/w compared with 1.25% w/w gels formed a more open and elastic network, allowing greater migration of the treatment components over time. The respective levels of bacterial inhibition exhibited by the films, especially after 96 h, appeared to support this postulation.

Incorporation of antimicrobial additives Antimicrobial additives have been used successfully for many years. The

direct incorporation of antimicrobial additives in packaging films is a convenient methodology by which antimicrobial activity can be achieved. The literature provides evidence that some of these additives may be effective as indirect fod additives incorporated into food packaging materials. Several agents have been proposed and tested for antimicrobial packaging using this method. However, the use of such packaging materials is not meant to be a substitute for good sanitation practices, but it should enhance the safety of food as an additional hurdle for the growth of pathogenic microorganisms.

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Oussala et al. (2004) studied the antimicrobial and antioxidant effects of milk protein based film containing 1.0 %(w/v) oregano, 1.0 % (w/v) pimento or 1.0 % oregano – pimento (1:1) essentials oils for the preservation on beef muscle to control the growth of pathogenic bacteria and increase the shelf life during storage at 4 °C, they showed that film containing oregano was the most effective against Escherichia coli O157: H7 and Pseudomonas spp, whereas film containing pimento oil seems to be the least effective against these two bacteria. Films containing oregano extracts, showed at the end of storage, a 0.95 log reduction of Pseudomonas spp level as compared to samples without film. A 1.12 log reduction of Escherichia coli O157: H7 level was observed in samples coated with oregano- based films.

The incorporation of 1.0% w/w potassium sorbate in low-density polyethylene films (0.4-mm thick) was studied by Han and Floros (1997), they found that potassium sorbate lowered the growth rate and maximum growth of yeast, and lengthened the lag period before mold growth became apparent. Potassium sorbate is active against yeast, mould and many bacteria. Contradictory results were obtained by Weng and Hotchkiss (1993) using low-density polyethylene films (0.05-µm thick) containing 1.0 % w/w sorbic acid, unable to suppress mold growth when brought into contact with inoculated media. A limited migration of potassium sorbate into water and cheese cubes occurs, probably because of the incompatibility of the polar salt with the nonpolar LDPE as was suggested by Weng and Hotchkiss (1993). Research developed by Devlieghere et al. (2000) confirm that ethylene vinyl alcohol/ linear low-density polyethylene (EVA/LLDPE) film (70 - mm thick) impregnated with 5.0% w/w potassium sorbate is unable to inhibit the growth of microorganisms on cheese and to extend its shelf life.

The choice of an antimicrobial agent is often restricted by the incompatibility of that agent with the packaging material or by its heat instability during extrusion [35, 83]. Polyethylene has been widely employed as the heat-sealing layer in packages, in some cases the copolymer polyethylene - comethacrylic acid was found to be preferable for this purpose. Weng et al. (1999) reported a simple method for fabricating polyethylene- co-methacrylic acid films (0.008- to 0.010-mm thick) with antimicrobial properties by the incorporation of benzoic or sorbic acids. The experimental results suggest that sodium hydroxide and preservative-treated films exhibit dominantly antimicrobial properties for fungal growth, presumably due to the higher amount of preservatives released from the films (75 mg benzoic acid or 55 mg sorbic acid per g of film) than hydrochloric acid and preservative-treated films.

Chen et al. (1996) found that chitosan films made from dilute acetic acid solutions inhibit the growth of Rhodotorula rubra and Penicillium notatum if the film is applied directly to the colony-forming organism. Since chitosan is

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soluble only in slightly acidic solutions, production of such films containing the salt of an organic acid (such as benzoic acid, sorbic acid) that is an antimicrobial agent is straightforward. However, the interaction between the antimicrobial agent and the film-forming material may affect the casting process, the release of the antimicrobial agent and the mechanical properties of the film. Chitosan films are easily prepared by evaporating from dilute acid solutions. A number of studies on the antimicrobial characteristics of films made from chitosan have been carried out earlier [13, 22, 60]. The antimicrobial efficiency of chitosan – hydroxy propyl methyl cellulose (HPMC) films, associated with stearic acid and chitosan – HPMC films chemically modified by citric acid as crosslinking agent were evaluated by Möller et al. (2004). Chitosan – HPMC based films, with and without stearic acid inhibited the growth of Listeria monocytogenes completely. On the other hand, a loss of antimicrobial activity after chemical cross linking modification was observed.

Chitosan edible films incorporating garlic oil was compared by Pranoto et al. (2005) with conventional food preservative potassium sorbate and bacteriocin nisin at various concentrations, showing an antimicrobial effect against Escherichia coli, Staphylococcus aureus, Salmonella typhimorium, Listeria monocytogenes and Bacillus cereus. Garlic oil incorporated into chitosan films led to an increase in its antimicrobial efficiency. However, the applications of garlic oil into chitosan films depend on the type of food were its flavor is not a problem. The films were physically acceptable in term of appearance, mechanical and physical properties. The incorporation of garlic oil into chitosan films has the desirable characteristics of acting as a physical and antimicrobial barrier to food contamination. Cooksey (2005) focussed on the use of chitosan to inhibit Listeria monocytogenes and chlorine dioxide sachets for the reduction of Salmonella on modified atmosphere packaged fresh chicken breast.

Antimicrobial agents as organic acids, bacteriocins and spice extracts have been tested for their ability to control meat spoilage [1, 40, 52]. Garlic oil is composed of sulfur compounds such as allicin, diallyl disulfide and dyallyl trisulfide that possess better antimicrobial activity than the corresponding ground form [58].

Begin and Calsteren (1999) showed that films containing antimicrobial agents with a molecular weight larger than that of acetic acid are soft and can be used in multi-layer systems or as a coating. Acetic acid diffusion was, however, not as complete as that of propionic acid when chitosan-containing films were used in contact with processed meats [60] in spite of the fact that in an aqueous medium, acetic acid diffused out of chitosan more rapidly than propionic acid [61]. These results suggest that the release of organic acids from chitosan is a complex phenomenon that involves many factors such as

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electrostatic interactions, ionic osmosis, and structural changes in the polymer induced by the presence of the acids.

According to Weng and Hotchkiss (1993), anhydrides are more compatible with Polyethylene than their corresponding free acids or salts, due to the lower polarity and higher molecular weight of the former compared to the latter. Hence, anhydrides may serve as appropriate additives to plastic materials for food packaging low density polyethylene (LDPE) films impregnated with benzoic anhydride completely suppressed the growth of Rhizopus stolonifer, Penicillium species and Aspergillus toxicarius on potato dextrose agar (PDA). Similarly, LDPE films that contained benzoic anhydride delayed mold growth on cheese. The rapid hydrolysis of benzoic anhydride to benzoic acid should not pose a safety concern, although at the time of their study benzoic anhydride did not have FDA approval.

Polyethylene (PE) films (0.010- to 0.015-mm thick) containing benzoic anhydride (20 mg benzoic anhydride per g of PE in the initial preparation) alone or in combination with minimal microwave heating, were effective in controlling microbial growth of tilapia fillets during a 14-day storage at 4 °C [42]. Shelf life studies of packaged cheese and toasted bread demonstrated the efficiency of LDPE film containing benzoic anhydride against mold growth on the food surface during storage at 6 °C [30]. No single antimicrobial agent can cover all the requirements for food preservation. Weng and Chen (1997) investigated a range of anhydrides for use in food packaging. It is known that for mold growth inhibition, the effectiveness of sorbic anhydride (10 mg sorbic anhydride per g of PE initial concentration) incorporated in PE films (0.10- to 0.12-mm thick) is much better with slow-growing (Penicillium species) than with fast-growing mold (Aspergillus niger). This is due to the time required for the PE to release sorbic acid to an inhibitory concentration.

Apart from organic acids and anhydrides, Imazalil has also been used with LDPE film. Weng and Hotchkiss (1992) showed that an Imazalil concentration of 2000 mg/ kg LDPE film (5.1 µm thick) delayed A. Toxicarius growth on potato dextrose agar, while LDPE film containing 1000 mg/kg Imazalil substantially inhibited Penicillium sp. growth and the growth of both of these molds on cheddar cheese. Little published data exist on the incorporation of bacteriocins into packaging films. Siragusa et al. (1999) highlighted the potential of incorporating Nisin directly into LDPE film for controlling food spoilage and enhancing product safety. Dobias et al. (2000) also studied the migration of benzoic anhydride, ethyl paraben (ETP) and propyl paraben (PRP) in LDPE films. It was found that the incorporation of these parabens in the polymer was more difficult than that of benzoic anhydride due to their higher volatilities. Devlieghere et al. (2000b) were probably the first to use hexamethylene-tetramine (HMT) as an antimicrobial packaging agent. The antimicrobial activity of the latter is believed to be due to the formation of

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formaldehyde when the film comes into contact with an acidic medium [50]. It was found that a LDPE film containing 0.5% w/w hexamethylene-tetramine exhibited antimicrobial activity in packaged cooked ham and therefore this agent is a promising material for food packaging applications.

In Japan, the ions of silver and copper, quaternary ammonium salts, and natural compounds are generally considered safe antimicrobial agents. Silver-substituted zeolite (Ag-zeolite) is the most common agent with which plastics are impregnated. The use of Ag-zeolite as an acceptable food additive in Europe has not been clarified [9]. However, recently, Ag-zeolites such as AgIONTM and Zeomic® received the approval of the FDA for use in food-contact materials. It retards a range of metabolic enzymes and has a uniquely broad microbial spectrum. As an excessive amount of the agent may affect the heat-seal strength and other physical properties such as transparency, the normal incorporation level used is 1 to 3% w/w. Application to the film surface (that is increasing the surface area in contact with the food) is another approach that could be investigated in the future [43]. Another interesting commercial development is Triclosan-based antimicrobial agents such as Microban®, Sanitized® and Ultra-Fresh®. Vermeiren et al. (2002) reported that LDPE films containing 0.5 and 1.0% w/w triclosan exhibited antimicrobial activity against S. aureus, L. monocytogenes, E. coli O157:H7, Salmonella enteritidis and Brocothrix thermosphacta in agar diffusion assay. The 1.0% w/w Triclosan film had a strong antimicrobal effect in in vitro simulated vacuum-packaged conditions against the psychrotrophic food pathogen L. monocytogenes. However, it did not effectively reduce spoilage bacteria and growth of L. monocytogenes on refrigerated vacuumpackaged chicken breasts stored at 7 °C. This is because of ineffectiveness towards microbial growth. Other compounds with antimicrobial effects are natural plant extracts. Recently, researchers developed certain antimicrobial films impregnated with naturally-derived antimicrobial agents [2, 18, 33, 39, 48, 77]. These compounds are perceived to be safer and were claimed to alleviate safety concerns [48].

It was reported that the incorporation of 1% w/w grapefruit seed extract in Low Density Polyethylene film (30 µm thick) used for packaging of curled lettuce reduced the growth rate of aerobic bacteria and yeast. In contrast, a level of 0.1% grapefruit seed extract yielded no significant effect on the rate of microbial growth in packaged vegetables, except for lactic acid bacteria on soybean sprouts [48]. Ha et al. (2001) studied grapefruit seed extract incorporated (by co-extrusion or a solution-coating process) in multilayered Polyethylene films and assessed the feasibility of their use for ground beef. They found that coating with the aid of a polyamide binder resulted in a higher level of antimicrobial activity than when incorporated by co-extrusion. A co-extruded film (15 µm thick) with 1.0% w/w grapefruit seed extract showed

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antimicrobial activity against M. flavus only, whereas a coated film (43 µm of LDPE with 3 µm of coating layer) with 1.0% w/w grapefruit seed extract showed activity also against E. coli, S. aureus, and Bacillus subtilis. Both types reduced the growth rates of bacteria on ground beef stored at 3 °C, as compared with plain PE film. The 2 investigated grapefruit seed extract levels (0.5 and 1.0% w/w) did not differ significantly in the efficacy of the film in terms of its ability to preserve the quality of beef. Chung et al. (1998) found that LDPE films (48 to 55 µm thick) impregnated with either 1.0% w/w Rheum palmatum and Coptis chinensis extracts or silver-substituted inorganic zirconium retarded the growth of total aerobic bacteria, lactic acid bacteria and yeast on fresh strawberries.

However, the study of An et al. (1998) showed that LDPE films (48 to 55 µm thick) containing 1.0% w/w R. palmatum and C. chinensis extracts or Ag-substituted inorganic zirconium did not exhibit any antimicrobial activity in a disk test against E. coli, S. aureus, Leuconostoc mesenteroides, S. cerevisiae, A. niger, Aspergillus oryzae, Penicillium chrysogenum [27]. A film containing sorbic acid showed activity against E. coli, S. aureus, and L. mesenteroides. The reasons for this unusual result are not clear. During diffusion assays, the antimicrobial agent is contained in a well or applied to a paper disc placed in the center of an agar plate seeded with the test microorganism. This arrangement may not be appropriate for essential oils, as their components are partitioned through the agar due to their affinity for water. Accordingly, broth and agar dilution methods are widely used to determine the antimicrobial effectiveness of essential oils [27].

According to Hong et al. (2000), the antimicrobial activity of 5.0% w/w Propolis extract, Chitosan polymer and oligomer, or Clove extract in LDPE films (30 to 40 µm thick) against Lactobacillus plantarum, E. coli, S. cerevisiae, and Fusarium oxysporum is best determined through viable cell counts. Overall, LDPE films with incorporated natural compounds show a positive antimicrobial effect against L. plantarum and F. oxysporum. Preliminarily studies by Suppakul et al. (2002) with LLDPE films (45 to 50 µm thick) containing 0.05% w/w linalool or methyl chavicol showed a positive activity against E. coli. Chiasson et al., 2004 showed that the bactericidal action of carvacrol against E. coli ATCC 25922 in ground beef was eliminated or reduced with the addition of another compound with high antiradical properties, such as ascorbic acid.

Edible films and various antimicrobial compounds incorporated in edible food packages have also been investigated recently [21, 71]. Rodrigues and Han (2000) investigated edible antimicrobial materials produced by incorporating Lysozyme, Nisin and Ethylenediamine tetracetic acid (EDTA) in whey protein isolate (WPI) films. Such Lysozyme or Nisin-containing films are effective in inhibiting Brochothrix thermosphacta but fail to suppress

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L. Monocytogenes. Combined efficacy of nisin and pediocin with sodium lactate, citric acid, phytic acid and potassium sorbate and EDTA were tested as possible sanitizer treatments for reducing the Listeria monocytogenes population of inoculated fresh – cut produce (Bari et al., 2005) Nisin – phitic acid and nisin – pediocin – phytic acid caused significant reductions of L. Monocytogenes on cabbage and broccoli but not on mung bean sprouts.

The possibility of modulating the release kinetics of active compounds such as lysozyme, nisin and sodium benzoate either by regulating the degree of cross-linking of films or by using multilayer structures, from crosslinked polyvinilacohol (PVOH) a highly swellable polymer, was evaluated by Buonocore et al. (2003, 2004). Micrococcus lysodeikticus, Alicyclobacillus acidoterrestris and Saccharomyces cerevisiae were used to test the antimicrobial efficiency of released active compounds; they showed that the release kinetics of lysozime and nisin depends of the cross-link of the polymer matrix whereas multilayer structures need to be used to control the release of sodium benzoate. Lysozime and nisin are both antimicrobial proteins effective against gram positive bacteria. However, the use of these antimicrobials in combination of chelating agents as EDTA displays increased effectivenesss against gram-negative bacteria [64].

The incorporation of EDTA in whey protein isolate (WPI) films improved the inhibitory effect on L. monocytogenes but had a marginal effect only on E. coli O157:H7. Coma et al. (2001) studied the moisture barrier and the antimicrobial properties of HPMC-fatty acid films (30-50 µm thick) containing Nisin (105 IU/mL) as the antimicrobial agent and its efficacy against Listeria innocua and S. aureus growth in food products. Stearic acid was chosen as the fatty acid because of its ability to reduce the rate of water vapor transmission. However, it impaired the effectiveness of the film against both strains. This may be explained by electrostatic interaction between the cationic Nisin and the anionic stearic acid. Nisin is a bacteriocin produced by Lactococcus lactis subsp. Lactis active against a broad spectrum of Gram-positive bacteria. Nisin has been widely used in the food industry as a safe and natural preservative and has been studied of its suitability to be incorporated into cellulose, whey protein isolate, soy protein isolate, egg albumen, wheat gluten hydroxyprophyl methyl cellulose and corn zein films [21, 45, 46].

Antimicrobial migration system Antimicrobial packaging is a promising form of active food packaging.

Microbial contamination of foods occurs primarily at the surface, due to post-processing handling; attempts have been made to improve safety and to delay spoilage by use of antibacterial sprays or dips. However, direct surface application of antibacterial substances onto foods have limited benefits because the active substances are neutralized on contact or diffuse rapidly from

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the surface into the food mass [69]. Antimicrobial food packaging materials have to extend the lag phase and reduce the growth rate of microorganisms in order to extend shelf life and to maintain product quality and safety [37]. This solution is becoming increasingly important, as it represents a perceived lower risk to the consumer [57].

Food packages can be made antimicrobial active by incorporation and immobilization of antimicrobial agents or by surface modification and surface coating. Incorporation of bactericidal or bacteriostatic agents into meat formulations may result in partial inactivation of the active substances by product constituents and is therefore expected to have only limited effect on the surface microflora. The use of packaging films containing antimicrobial agents could be more efficient, by slow migration of the agents from the packaging material to the surface of the product, keeping high concentration where they are needed. On the other hand, if an antimicrobial can be released from the packaging during an extended period, the activity can also be extended into the transport and storage phase of food distribution.

Antimicrobial agents may be incorporated into the packaging materials and migrate into the food through diffusion and partitioning [37]. Besides diffusion and equilibrated sorption, some antimicrobial packaging uses covalently immobilized antibiotics or fungicides, or active moieties such as amino groups [69]. A great number of substances, which can be bound to polymers to impart antimicrobial properties such as Acetic acid [60, 61]; Allyl isothiocyanate [9, 49]; Benzoic acid [14, 85, 86]; Benzoic anhydride [83]; Chitosan [8, 39]; Carvacrol [15]; EDTA [71]; Eugenol, Geraniol, Linalool, Terpineol and Thymol [73]; Imazalil [82]; Lactic acid [75]; Lauric acid [28, 38, 59, 61, 65]; Nisin [3, 72]; Sodium benzoate [13]; Sorbic acid [86]; Palmitoleic acid [59]; Phenolic compounds [6, 41]; Potassium sorbate [13, 35, 36 70, 81]; Propionic acid [60, 61]; Sorbic acid [12]; Sorbic acid anhydride [84]. Allyl isothiocyanate is currently not approved by the FDA for use in the U.S.A. [9] due to a safety concern that synthetic compound may be contaminated with traces of the toxic allyl chloride used in the manufacturing process [19]. In Japan, the use of Allyl isothiocyanate is allowed only when this compound is extracted from a natural source [44].

Antimicrobial materials have been known for many years. Antimicrobial films can be classified in 2 types: (1) those that contain an antimicrobial agent that migrates to the surface of the food and this would require a molecular structure large enough to retain activity on the microbial cell wall even though bound to the plastic. Such agents are likely to be limited to enzymes or other antimicrobial proteins, and (2) those that are effective against surface growth of microorganisms without migration. Non edible packaging films may contain any type of food additives. Some chemical agents naturally exist in plants or fermented products. However, they mainly are chemically synthetized.

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Commercial antimicrobial systems have had relatively used, specially in Japan, as was suggested by Brody et al., 2001. Some of the trade names and manufacturers used for packaging films are MicroGardTM produced by Rhone-Poulenc (U.S.A.) and Piatech Daikoku Kasei Co. (Japan). In other hand, the concentrates and extracts commercializes MicroFreeTM by DuPont (U.S.A.) [9, 78]; as well as Microban® by Microban Products (U.S.A.) with a wide range of products such as cutting boards and dishcloths which contain triclosan (2,4,4’-trichloro–2’- hydroxydiphenyl- ether) also used in soaps, shampoos and toothbrushes [9, 78]; Ultra-Fresh® by Thonson Research Associates (Canada), Novaron® by Milliken Co. (U.S.A.), Sanitized® Sanitized AG / Clariant (Switzerland) [78]. Antimicrobial extracted from natural seeds as Grapefruit seed [48] produces by Extract CitrexTM Quimica Natural Brasileira Ltd. (Brazil) and mustard seeds [9] WasaOuro® by Green Cross Co. (Japan); Nisaplin® (Nisin) by Integrated Ingredients (U.S.A.) [9, 72].

The production of a nisin containg cellophane based coating was used in the packaging of chopped meat. The developed bioactive cellophane reduced significantly the growth of the total aerobic bacteria through 12 days of storage at 4° C, would result in an extension of the shelf life of chopped meat under refrigeration temperatures [32]. Cutter (1999) investigated the effectiveness of triclosan incorporated plastic against populations of food borne pathogenic bacteria as well as bacteria associated with meat surface. Plate overlay assays indicated that plastic containing 1500 ppm of triclosan inhibited the growth of Brochotrix thermosphacta ATTC 11509, Salmonella typhimorium ATTC 14028, Staphylococcus aureus ATTC 12598, Bacillus subtilis ATTC 6051, Shigella flexneri ATCC 12022, Escherichia coli ATCC 25922 and several strains of Escherichia coli O157:H7. However the same did not effectively reduce bacterial populations on refrigerated, vacuum-packaged meat surfaces. The presence of fatty acids might diminish the antibacterial activity of triclosan incorporated plastic on meat surfaces.

Triclosan is also not accepted by US regulatory authorities for food contact materials [9]. In Europe, the legislative status of Triclosan is unclear. Their uses for food contact application are allowed in EU countries, with a quantitative restriction on migration of 5 mg/ Kg of food. Triclosan does not appear on the EU directive list of approved food additives that may be used in the manufacturing of plastics intended for food contact materials [78]. No European regulations exist currently on the use of active and intelligent packaging. Packages intended for food contact applications are required to belong to a positive list of approved compounds, and an overall migration limit from the material into the food or food simulant was set at 60 mg/kg. This is incompatible with the aim of active packaging, especially when the system is designed to release active ingredients into the foods.

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Bacteriocins into packaging films to control pathogenic organisms

Incorporation of bacteriocins into packaging films to control food spoilage and pathogenic organisms has been researched for the last decades. Antimicrobial packaging film prevents microbial growth on food surface by direct contact of the package with the surface of foods. For this reason, the antimicrobial packaging film must contact the surface of the food so that bacteriocins can diffuse to the surface. The gradual release of bacteriocins from a packaging film to the food surface may have an advantage over dipping and spraying foods with bacteriocins. In the latter processes, antimicrobial activity may be lost or reduced due to inactivation of the bacteriocins by food components or dilution below active concentration due to migration into the foods [5].

Two methods have been commonly used to prepare packaging films with bacteriocins [5]. One is to incorporate bacteriocins directly into polymers. Such as incorporation of nisin into biodegradable protein films [64]. Two packaging film-forming methods, heat-press and casting, were used to incorporate nisin into films made from soy protein and corn zein in this study. Both film-forming methods produced excellent films and inhibited the growth of L. plantarum. Cast films exhibited larger inhibitory zones than the heat-press films when the same levels of nisin were incorporated. Incorporation of EDTA into the films increased the inhibitory effect of nisin against E. coli. Siragusa et al. (1999) incorporated nisin into a polyethylene-based plastic film that was used to vacuum-pack beef carcasses. Nisin retained activity against Lactobacillus helveticus and B. thermosphacta inoculated in carcass surface tissue sections. An initial reduction of 2- log10 cycles of B. thermosphacta was observed with nisin-impregnated packaged beef within the first 2 day of storage at 4 °C. After 20 day of refrigerated storage at 4 or 12 °C, B. thermosphacta populations from nisin-impregnated plastic-wrapped samples were significantly less than control (without nisin). Coma et al. (2001) incorporated nisin into edible cellulosic films made with hydroxypropylmethylcellulose by adding nisin to the film-forming solution. Inhibitory effect could be demonstrated against L. innocua and S. aureus, but film additives such as stearic acid, used to improve the water vapor barrier properties of the film, significantly reduced inhibitory activity. It was noted that desorption from the film and diffusion into the food required further optimization for nisin to function more effectively as a preservative agent in the packaged food. Another method to incorporate bacteriocins into packaging films is to coat or adsorb bacteriocins to polymer surfaces.

Examples include nisin/methylcellulose coatings for polyethylene films and nisin coatings for poultry, adsorption of nisin on polyethylene, ethylene

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vinyl acetate, polypropylene, polyamide, polyester, acrylics, and polyvinyl chloride [5] demonstrated that nisin adsorbed onto silanized silica surfaces inhibited the growth of L. monocytogenes. Nisin films were exposed to medium containing L. Monocytogenes and the contacting surfaces were evaluated at 4 hour intervals for 12 hours. Cells on surfaces that had been in contact with high concentration of nisin (40000 IU/mL) exhibited no signs of growth and many displayed evidence of cellular deterioration. Surfaces contacted with a lower concentration of nisin (4000 IU/ mL) had a smaller degree of inhibition. In contrast, surfaces contacted with films of heat-inactivated nisin allowed L. Monocytogenes to grow L. innocua and S. aureus (along with L. lactis subsp. lactis) were also used in a study by Scannell et al. (2000) of cellulose-based bioactive inserts and antimicrobial polyethylene/ polyamide pouches. Lacticin 3147 and nisin were the tested bacteriocins. Although Lacticin 3147 adhered poorly to plastic film, nisin bound well and the bioactive film made with nisin was stable for 3 months with or without refrigeration. Bacterial reductions of up to 2-log10 CFU/g cycles in vacuum-packed cheese were seen in combination with modified atmosphere packaging (MAP) with storage at refrigeration temperatures. Cellulose-based bioactive inserts were placed between sliced products of cheese and ham under Modified Atmospere Packaging (MAP). Inserts with immobilized nisin reduced L. Innocua (starting inocula of 2 to 4 x 105 CFU/g) by >3 log10 CFU/g in cheese after 5 day at 4 °C, and by approximately 1.5 log10 CFU/g in sliced ham after 12 day, while S. aureus (starting inocula of 2 to 4 x 105 CFU/g) was reduced by 1.5 and 2.8 log10 CFU/g in cheese and ham, respectively.

The efficacy of bacteriocins coatings on the inhibition of pathogens has also been demonstrated in other studies. Research in development of antimicrobial packaging applications on meat has become promissing. Dawson et al. (2002) evaluated the effect of lauric acid and nisin impregnated soy based films on the growth of Listeria monocytogenes on turkey Bologna. Films containing lauric acid and nisin completely eliminated detectable cells from a 106 culture after 8 h of exposure. Nisin films reduced cell number from 106 to 105 after 21 days. Meanwhile, the films containing only lauric acid reduced L. monocytogenes culture from 106 to 102 after 48 h. Hoffman et al. (2001) studied the antimicrobial effects of corn zein films impregnated with nisin, lauric acid and EDTA. Their results showed that L. monocytogenes cell numbers decreased by greater than 4 logs after 48 h of exposure to films containing Lauric acid or nisin alone. No cells were detected for L. monocytogenes to any film combination that included lauric acid. Films with EDTA and lauric acid, and EDTA-lauric acid and nisin were bacteriostatic. However, there was a 5 log increase in cells exposed to control within 24 h.

Nisin incorporated polymers may control the growth of undesirable bacteria, thereby extendig the shelf life and enhancing the microbial safety of

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meats as was suggested by Cutter et al. (2001) when tested several combinations using nisin - EDTA blended with polyethylene (PE) and polyethylene oxide (PEO). It appears that PE + PEO + nisin or PE + nisin + EDTA were more effective for reducing Brochothrix termosphacta to 0.30 log 10 CFU/cm2, as compared to polymers composed of PE + nisin. Coating of pediocin onto cellulose casings and plastic bags has been found to completely inhibit growth of inoculated L. monocytogenes in meats and poultry through 12 wk storage at 4° C. Coating of solutions containing nisin, citric acid, EDTA, and Tween 80 onto polyvinyl chloride, linear low density polyethylene, and nylon films reduced the counts of Salmonella typhimurium in fresh broiler drumstick skin by 0.4- to 2.1-log10 cycles after incubation at 4° C for 24 h. The inclusion of nisin based treatments into either calcium alginate or agar gels that were subsequently applied to contaminated broiler drumstick skin yielded significant Salmonella typhimurium population reduction between 1,8 to 4,6 log cycles [54, 55].

The application of polymers on solid or semisolid foods could increase the antimicrobial and antioxidant efficiency by maintaining high concentrations of active molecules on the food surface, where microbial growth mainly occurs. Immobilization of organic acids in edible coatings based on calcium alginate gel or whey protein has also been used to control Listeria monocytogenes on beef tissue [12, 75]. Besides diffusion and sorption, some antimicrobial packaging systems utilize covalently immobilized antimicrobial substances that suppress microbial growth. Appendini and Hotchkiss (1997) investigated the efficiency of Lysozyme immobilized on different polymers. It is known that cellulose triacetate (CTA) containing Lysozyme yields the highest antimicrobial activity. The viability of Micrococcus lysodeikticus was reduced in the presence of immobilized Lysozyme on CTA film. Scannell et al. (2000) showed that PE/polyamide (70:30) film formed a stable bond with Nisin in contrast to Lacticin 3147. Nisin-adsorbed bioactive inserts reduced the level of L. Innocua and S. aureus in sliced cheese and in ham.

Ozdemir et al. (1999) introduced (by chemical methods) functional groups possessing antimicrobial activity into polymer films with the purpose of preventing the transfer of the antimicrobial agents from the polymer to the food. Cho et al. (2000) synthesized a new biopolymer containing a chito-oligosaccharide side chain. The chito-oligosaccharide was introduced on polyvinylacetate by cross-linking with the bifunctional compound, N-methylolacrylamide. It was found that the growth of S. aureus was almost completely suppressed by this means. Surface amine groups formed in polymers by electron irradiation were also shown to impart antimicrobial effectiveness [20, 63]. By contrast, irradiation at 248 nm did not change the surface chemistry or initiate conversion of the amide [63]. Paik et al. (1998) and Shearer et al. (2000) observed a decrease in all bacterial cells, including S. aureus,

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Pseudomonas fluorescens, and E. faecalis in bulk fluid when using an antimicrobial nylon film. The results indicate that this decrease is more probably to be due to the bactericidal action than to surface adsorption [68]. Although the mechanism of the reduction in the bacteria population remained uncertain, electrostatic attractive forces between the positively charged film surface and the negatively charged E. coli and S. aureus were presumed to be the reason for this effect [74].

Further research is needed to characterize the antimicrobial active groups on the irradiated film surface and the mechanism of antimicrobial action. Ionomers, with their unique properties such as a high degree of transparency, strength, flexibility, stiffness and toughness, as well as inertness to organic solvents and oils, have also drawn much attention as food packaging materials. Halek and Garg (1989) successfully incorporated the Benomyl fungicide into ionomer films via its carboxyl groups. Unfortunately, Benomyl is not an approved food preservative. Weng et al. (1997) investigated application of antimicrobial ionomers combined with approved food preservatives. Anhydride linkages in the modified films were formed by reaction of acid/or base-treated films with benzoyl chloride. The antimicrobial activity was characterized in terms of the release of benzoic acid, which was higher in the base treated version indicating the superiority of the latter. The antimicrobial effect of modified ionomer films was further demonstrated by their ability to inhibit the growth of Penicillium species and A. niger.

Factors involved in the manufacturing of antimicrobial films

According to Han (2000), several factors must be taken into account in the design or modelling of the antimicrobial film or package. It is clear that the selection of both the substrate and the antimicrobial substance is important in developing an antimicrobial packaging system and the physico-mechanical properties of the package could be modified.

1. Chemical nature of films, process conditions and residual antimicrobial activity

The effectiveness of an antimicrobial agent applied by impregnation may deteriorate during film fabrication, distribution and storage [37]. The choice of the antimicrobial is often limited by the heat lability of the component during the extrusion and also the shearing forces and pressures involved in the process conditions [36]. In order to minimize this problem, Han (2000) recommended using master batches of the antimicrobial agent in the resin for preparation of antimicrobial packages; for instance 1% potasium sorbate in a low density

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polyethylene (LDPE) film inhibited the growth of yeast on agar plates. A master batch could be produced, by mixing the LDPE resin and potassium sorbate powder, extruded and pelletized. These pellets can be added later to LDPE resin to prevent heat decomposition [35]. Also, all operations such as lamination, printing and drying as well as the chemicals used (adhesives and solvents) should also be characterized quantitatively. In addition, some of the volatile antimicrobial compounds may be lost during storage. All these parameters should be evaluated.

2. Characteristics of antimicrobial substances and foods

Food components significantly affect the effectiveness of the antimicrobial substances and their release. The mechanism and kinetics of growth inhibition are generally studied in order to permit mathematical modeling of microbial growth [37]. Physico chemical characteristics of food could alter the activity of antimicrobial substances. The pH of a product affects the growth rate of target microorganisms and changes the degree of ionization (dissociation/ association) of the most active chemicals, and could change the antimicrobial activity of organic acids and their salts [37]. Weng and Hotchkiss (1993) reported that Low density polyethylene film containing benzoic anhydride was more effective in inhibiting molds at low pH values. Rico- Pena and Torres (1991) found that the diffusion of sorbic acid decreased with an increase in pH. Foods with different chemical characteristics are stored under different environmental conditions, which may cause different patterns of microflora growth. Aerobic microorganisms can exploit headspace O2 for their growth. The antimicrobial activity and chemical stability of incorporated active compounds could be influenced also by the food aw. Moreover, each food has its own micloflora. Vojdani and Torres (1989a) showed that the diffusion of potassium sorbate through polysaccharide films increases with aw; this has a negative impact on the amount available for protection. Rico-Pena and Torres (1991) found that potassium sorbate diffusion rates in MC/HPMC film containing palmitic acid were much higher at higher values of aw. The release kinetics of antimicrobial agents has to be designed to maintain the concentration above the critical inhibitory concentration with respect to the contaminating microorganism.

3. Chemical interaction of additives with film matrix

During incorporation of additives into a polymer, the polarity and molecular weight of the additive have to be taken into consideration. Since LDPE itself is non polar, additives with a high molecular weight and low polarity are more compatible with this polymer (Weng and Hotchkiss 1993). Furthermore, the molecular weight, ionic charge and solubility of different additives affect their rates of diffusion in the polymer [23]. Wong et al. (1996)

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compared the diffusion of ascorbic acid, potassium sorbate, and sodium ascorbate in calcium-alginate films at 8, 15, and 23 °C. They found that ascorbic acid had the highest and sodium ascorbate the lowest diffusion rate at all studied temperatures. These findings were attributed to the different ionic states of the additives.

4. Storage temperature

The storage temperature may also affect the antimicrobial activity of chemical preservatives. Generally, increased storage temperature can accelerate the migration of the active agents in the film and deteriorate the protective action of antimicrobial films, due to high diffusion rates in the polymer [79, 80, 87]. The temperature conditions during production and distribution have to be predicted to determine their effect on the residual antimicrobial activity of the active compounds. The diffusion rate of the antimicrobial agent and its concentration in the film must be sufficient to remain effective throughout the shelf life of the product [23]. Weng and Hotchkiss (1993) stated that low amounts of benzoic anhydrides in LDPE might be as effective at refrigeration temperatures as high levels at room temperature. 5. Mass transfer coefficients and modeling

The simplest system is the diffusional release of active compounds from the package into the food. Use of a multilayer package has the advantage that the antimicrobial can be added in one thin layer and its migration and release controlled by the thickness of the film. Control of the release rates and migration amounts of antimicrobial substances from food packaging is very important. A mass transfer model of the migration phenomena can be used to describe the migration of active substances through food packaging systems consisting of one or several layers. By ussing, mathematical modeling of the diffusion process could permit prediction of the antimicrobial agent release profile and the time during which the agent remains above the critical effectiveness concentration. With a higher diffusivity and much larger volume of the food component compared to the packaging material, a semi-infinite model in which the packaging component has a finite thickness and the food component has infinite volume could be practical [37]. The initial and boundary conditions that could be used in mass transfer modeling have been identified.

6. Physical properties of packaging materials

Antimicrobial agents may affect the physical properties, processability or machinability of the packaging material. The performance of the packaging

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materials must be maintained after the addition of the active agents, even thougth the heterogeneous formulations. Han and Floros (1997) reported no significant differences in the tensile mechanical properties before and after the incorporation of potassium sorbate in LDPE films, but the transparency of the films decreased with the addition of the potassium sorbate. Weng and Hotchkiss (1993) reported no differences in opacity and strength of LDPE film when increased the concentration from 0.5 to 1.0% benzoic anhydride. Similar results were reported for naturally-derived plant extracts such as propolis at 5.0% and clove at 5.0% [39], R. palmatum at 1.0% and Capsicum chinensis at 1.0% [2, 18]. Presence of nisin on LDPE film coated with MC/HPMC difficulted the heat-seal efficiency [23]. Dobias et al. (2000) reported statistically significant differences between the physical properties of films without antimicrobial agents and with different agents at concentrations of 5 g/kg and 10 g/kg.

Perspectives

Antimicrobial packaging is a promising form of active food packaging and an emerging technology. A new approach in food packaging regulations is needed. The current applications of antimicrobial food packaging are rather limited, although promising. This is because of the legal status of the tested additives [78]. The innovative food packaging concepts that have introduced as a response to the continuos changes in current consumer demands and market trends. The need to package foods in a versatile manner for transportation and storage, along with the increasing consumer demand for fresh, convenient, and safe food products presages a bright future for antimicrobial packaging. However, more information is required on the chemical, microbiological and physiological effects of these systems on the packaged food especially on the issues of nutritional quality and human safety [31]. So far, research on antimicrobial packaging has focused primarily on the development of various methods and model systems, whereas little attention has been paid to its preservation efficacy in actual foods [37]. The major potential food applications of antimicrobial films include meat, fish, poultry, bakery goods, cheese, fruits and vegetables [47]. Research is essential to identify the types of food that can benefit most from antimicrobial packaging materials. It is likely that future research into a combination of naturally-derived antimicrobial agents, biopreservatives and biodegradable packaging materials will highlight a range of antimicrobial packaging in terms of food safety, shelf-life and environmental friendliness [21, 57, 71]. The reported effectiveness of natural plant extracts suggests that further research is needed in order to evaluate their antimicrobial activity and potential side effects in packaged foods. An additional challenge is in the area of odor/flavor transfer by natural plant extracts to packaged food products. Thus, research is needed to determine

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whether natural plant extracts could act as both an antimicrobial agent and as an odor/flavor enhancer. Moreover, in order to secure safe food, amendments to regulations might require toxicological and other testing of compounds prior to their approval for use [78].

References 1. Abougroun HA, Cousin NA and Judge MD. 1993. Extended shelf life of

unrefrigerated prerigor cooked meat. Meat Sci. 33: 207 – 229. 2. An DS, Hwang YI, Cho SH, Lee DS. 1998. Packaging of fresh curled lettuce and

cucumber by using low density polyethylene films impregnated with antimicrobial agents. J Korean Soc Food Sci Nutr 27(4): 675 -681.

3. An DS, Kim YM, Lee SB, Paik HD, Lee DS. 2000. Antimicrobial low density polyethylene film coated with bacteriocins in binder medium. Food Sci. Biotechnol. 9(1):14-20.

4. Appendini P, Hotchkiss JH. 1997. Immobilization of lysozyme on food contact polymers as potential antimicrobial films. Packag. Technol. Sci. 10(5): 271 - 279.

5. Appendini P, Hotchkiss JH. 2002. Review of antimicrobial food packaging. Innovative Food Sci. and Emerging Technol. 3: 113 – 126.

6. Barbosa – Rodríguez JR., Ayala – Vidal A., Zurita – Olvera L., Guevara - González R., Villaseñor – Ortega F. and Pérez – Pérez C. 2005. Propiedades antimicrobianas de películas de Caseinato de sodio con extractos fenolicos provenientes de plantas de cascalote (Caesalpinia cacalaco) y quebracho (Schinopsis balansae). 7mo. Congreso Internacional de Inocuidad de Alimentos. Guadalajara, Jal. Nov 11- 12.

7. Bari, M. L., Ukuku, D. O., Kawasaki T., Inatsu Y., Isshiki K., Kawamoto S. 2005. Combined efficiency of nisin and pediocin with sodium lactate, citric acid, phytic acid, and potassium sorbate and EDTA in reducing the Listeria monocytogenes population of inoculated fresh-cut produce. J. Food Prot. 68(7): 1381- 1387.

8. Begin A, Calsteren M-RV 1999. Antimicrobial films produced from chitosan. Int J Biol Macromol 26(1): 63 - 67.

9. Brody AL, Strupinsky ER, Kline, LR. 2001. Active packaging for food applications. Lancaster: Technomic Publishing Co., Inc. 218 p.

10. Buonocore G. C., Del Nobile, M. A., Panizza A., Corbo M. R. and Nicolais L. 2003. A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications. J. Control Release. 90(1): 97 – 107.

11. Buonocore G. C., Sinigaglia M., Corbo M. R., Bevilacqua A., La Notte E. and Del Nobile M. A. 2004. Controlled release of antimicrobial compounds from highly swellable polymers. J. Food Prot 67(6): 1190- 1194.

12. Cagri A., Ustunol Z., Ryser E. T. 2001. Antimicrobial, mechanical and moisture barrier properties of low pH whey protein based edible films containing p-aminobenzoic or sorbic acids. J. Food Sci. 66: 865 – 870.

13. Chen MC, Yeh GHC, Chiang BH. 1996. Antimicrobial and physicochemical properties of methylcellulose and chitosan films containing a preservative. J Food Proc Preserv 20(5): 379 - 390.

Page 20: filmes antimicrobiano

Pérez-Pérez C. et al. 212

14. Chen MJ, Weng YM, Chen W. 1999. Edible coating as preservative carriers to inhibit yeast on Taiwanese- style fruit preserves. J Food Safety 19(2): 89 - 96.

15. Chiasson F., Borsa J., Ouattara B., and Lacroix M. 2004. Radiosensitization of Escherichia coli and Salmonella typhi in ground beef. J. Food Prot. 67: 1157 – 1162.

16. Chi-Zhang Y., Yam K. L., Chikindas M. L. 2004. Effective control of Listeria monocytogenes by combination of nisin formulated and slowly released into a broth system. Int. J. Food Microbiol. 90(1): 15 – 22.

17. Cho YW, Han SS, Ko SW. 2000. PVA containing chitooligosaccharide side chain. Polymers 41(6): 2033 - 2039.

18. Chung SK, Cho SH, Lee DS. 1998. Modified atmosphere packaging of fresh strawberries by antimicrobial plastic films. Korean J Food Sci Technol 30(5): 1140 -1145.

19. Clark GS. 1992. Allyl isothiocyanate. Perf Flav 17: 107-109. 20. Cohen JD, Erkenbrecher CW, Haynie SL, Kelley MJ, Kobsa H, Roe AN, Scholla

MH. 1995. Process for preparing antimicrobial polymeric materials using irradiation. U.S. Patent 5428078.

21. Coma V., Sebti I., Pardon P., Deschamps A. and Pichavant F. H. 2001. Antimicrobial edible packaging based on cellulosic ethers, fatty acidsmand nisin incorporation to inhibit Listeria innocua and Staphylococcus aureus. J. Food Prot. 64(4): 470 - 475.

22. Coma V., Martial-Gros A., Garreau S., Copinet A. and Deschamps A. 2002. Edible antimicrobial film based on chitosan matrix. J. Food Sci. 67(3): 1162-1169.

23. Cooksey K. 2000. Utilization of antimicrobial packaging films for inhibition of selected microorganism. In: Risch SJ, editor. Food packaging: testing methods and applications. Washington, DC: American Chemical Society. p 17 - 25.

24. Cooksey K. 2005. Effectiveness of antimicrobial food packaging materials. Food Addit. Contam. 22(10): 980 – 987.

25. Cutter C. N. 1999. The effectiveness of triclosan incorporated plastic against bacteria on beef surfaces. J. Food Prot. 62: 474 – 479.

26. Cutter C. N., Willet J. L., and Siragusa G. R. 2001. Improved antimicrobial activity of nisin incorporated polymer films by formulation change and addition of food grade chelator. Lett. Appl. Microbiol. 33(4): 325 – 328.

27. Davidson PM, Parish ME. 1989. Methods for testing the efficacy of food antimicrobials. Food Technol 43(1):148 - 155.

28. Dawson P. L., Carl G. D., Acton J. C. and Han I. Y. 2002. Effect of lauric acid and nisin impregnated soy-based films on the growth of Listeria monocytogenes on turkey bologna. Poult Sci. 81(5): 721 – 726.

29. Devlieghere F, Vermeiren L, Jacobs M, Debevere J. 2000. The effectiveness of hexamethylene tetramine – incorporated plastic for the active packaging of foods. Packag technol Sci. 13(3): 117-121.

30. Dobias J, Chudackova K, Voldrich M, Marek M. 2000. Properties of polyethylene films with incorporated benzoic anhydride and/or ethyl and propyl esters of 4-hydroxybenzoic acid and their suitability for food packaging. Food Add Contamin 17(12): 1047 - 1053.

31. Floros JD, Dock LL, Han JH. 1997. Active packaging technologies and applications. Food Cosmet Drug Packag 20(1):10-7.

Page 21: filmes antimicrobiano

Food biotechnology 213

32. Guerra N. P., Macias C. L., Agrasar A. T. and Castro L. P. 2005. Development of a bioactive packaging cellopane using Nisapln as biopreservative agent. Lett Appl Microbiol. 40(2): 106 – 110.

33. Ha JU, Kim YM, Lee DS. 2001. Multilayered antimicrobial polyethylene films applied to the packaging of ground beef. Packag Technol Sci 14(2):55-62.

34. Halek GW and Garg A. 1989. Fungal inhibition by a fungicide coupled to an ionomeric film. J Food Safety 9(3):215 - 222.

35. Han JH, Floros JD. 1997. Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. J Plastic Film Sheeting 13(4):287 - 298.

36. Han JH, Floros JD. 1999. Modeling anntimicrobial activity loss of potassium sorbate against Baker’s yeast after heat process to develop antimicrobial food packaging materials. Food Sci Biotechnol 8(1):11-4.

37. Han JH. 2000. Antimicrobial food packaging. Food Technol 54(3): 56 - 65. 38. Hoffman K. L., Han I. Y., and Dawson P. L. 2001. Anti microbial effects of corn

zein films impregnated with sisin,lauric acid and EDTA. J Food Prot 64(6): 885 – 889.

39. Hong SI, Park JD, Kim DM. 2000. Antimicrobial and physical properties of food packaging films incorporated with some natural compounds. Food Sci Biotechnol 9(1): 38 - 42.

40. Hotchkiss JS. 1995. Safety considerations in active packaging. In Rooney, ML (Ed.) Active food packaging. Pp 238 – 253. Glasgow, Blackie Academic and Professional.

41. Hotchkiss JH. 1997. Food-packaging interactions influencing quality and safety. Food Add Contamin 14(6): 601 - 607.

42. Huang LJ, Huang CH, Weng YM. 1997. Using antimicrobial polyethylene films and minimal microwave heating to control the microbial growth of tilapia fillets during cold storage. Food Sci Taiwan 24(2): 263 - 268.

43. Ishitani T. 1995. Active packaging for food quality preservation in Japan. In: Ackerman P, Jagerstad M, Ohlsson T, editors. Food and food packaging materials-chemical interactions. Cambridge: Royal Society of Chemistry. p 177 - 188.

44. Isshiki K, Tokuoka K, Mori R, Chiba S. 1992. Preliminary examination of allyl isothiocyanate vapor for food preservation. Biosci Biotech Biochem 56(9):1476 - 1477.

45. Janes, M. E., Kooshesh, S. and Johnson, M. G. 2002. Control of Listeria monocytogenes on the surface of refrigerated, ready to eat chiken coated with edible zein film coatings containing nisin and / or calcium propionate. J. Food Sci. 67(7): 2754 – 2757.

46. Ko, S., Janes, M. F. Hettiarachchy, N. S. and Jonhson, M. G. 2001. Physical and chemical properties of edible films containing nisin and their action against Listeria monocytogenes. J. Food Sci. 66 (7): 1006 – 1011.

47. Labuza TP, Breene WM. 1989. Applications of «active packaging» for improvement of shelf-life and nutritional quality of fresh and extended shelf-life foods. J Food Proc Preserv 13(1):1-69.

48. Lee DS, Hwang YI, Cho SH. 1998. Developing antimicrobial packaging film for curled lettuce and soybean sprouts. Food Sci Biotechnol 7(2):117 - 121.

49. Lim LT, Tung MA. 1997. Vapor pressure of allyl isothiocyanate and its transport in PVDC/PVC copolymer packaging film. J Food Sci 62(5):1061 - 1066.

Page 22: filmes antimicrobiano

Pérez-Pérez C. et al. 214

50. Luck E, Jager M. 1997. Antimicrobial food additives: characteristic, uses, effects. 2nd ed. Berlin: Springer. 260 p.

51. Mauriello G., De Luca E., La Storia A., Villani F. and Ercolini D. 2005. Antimicrobial activity of a nisin activated plastic film for food packaging. Lett. Appl. Microbiol. 41: 464 – 469.

52. Miller AJ., Call JE and Whiting RC. 1993. Comparison of organic acid salts for Clostridium botulinum control in an uncured turkey product. J. Food Prot. 56: 958 – 962.

53. Möller H., Grelier S., Pardon P., and Coma V. 2004. Antimicrobial and physycochemical properties of chitosan – HPMC – based films. J. Agric. Food Chem. 52(21): 6581 – 6591.

54. Natrajan N, Sheldon BW. 2000a. Efficacy of nisin coated polymer films to inactivate Salmonella typhimurium on fresh broiler skin. J Food Prot 63(9):1189 - 1196.

55. Natrajan N, Sheldon BW. 2000b. Inhibition of Salmonella on poultry skin using protein- and polysaccharide- based films containing a nisin formulation. J Food Prot 63(9):1268 -1272.

56. Nattres F. M., Yost C. K. and Baker L. P. 2001. Evaluation of the ability of lysozyme and nisin to control meat spoilage bacteria. Int. J. Food Microbiol. 70(1): 111 – 119.

57. Nicholson MD. 1998. The role of natural antimicrobials in food packaging biopreservation. J Plastic Film Sheeting 14(3): 234 - 241.

58. Nychas, G. J. E. 1995. Natural antimicrobials from plants. In Gould, G. W. (Ed.) New methods of food preservation. Pag: 59 – 89. Glasgow. Black Academic and Professional.

59. Ouattara B, Simard RE, Holley RA, Piette GJP, Begin A. 1997. Antibacterial activity of selected fatty acids and essential oils against 6 meat spoilage organisms. Int J Food Microbiol 37(2-3):155 - 162.

60. Ouattara B, Simard RE, Piette G, Begin A, Holley RA. 2000a. Inhibition of surface spoilage bacteria in processed meats by application of antimicrobial films prepared with chitosan. Int J Food Microbiol 62(1-2): 139 - 148.

61. Ouattara B, Simard RE, Piette G, Begin A, Holley RA. 2000b. Diffusion of acetic and propionic acids from chitosan-based antimicrobial packaging films. J Food Sci 65(5):768 - 773.

62. Oussalah, M., Caillet S. Salmieri S., Saucier L. and Lacroix M. 2004. Antimicrobial and antioxidant effects of milk protein based film containig essential oils for the preservation of whole beef muscle. J. Agric. Food Chem. 52: 5598 – 5605.

63. Ozdemir M, Yurteri CU, Sadikoglu H. 1999. Physical polymer surface modification methods and applications in food packaging polymers. CRC Crit Rev Food Sci Nutr 39(5): 457 - 477.

64. Padgettt T., Han I. Y. and Dawson P. L. 1998. INcorporation of food grade antimicrobial compounds into biodegradable packaging films. J. Food Prot 61(10): 1330 – 1335.

65. Padgett T., Han IY., and Dawson PL. 2000. Effect of lauric acid addition on the antimicrobial efficacy and water permeability of corn zein films containing nisin. J. Food Proc. Preserv. 24: 423 – 432.

Page 23: filmes antimicrobiano

Food biotechnology 215

66. Papadokostaki K. G., Amanratos S. G., and Petropoulos J. H. 1997. Kinetics of release of particules solutes incorporated in cellulosic polymer matrices as a function of solute solubility and polymer swellability. I. Sparingly soluble solutes. J. Appl. Polym. Sci. 67: 277 – 287.

67. Pranoto Y., Rakshit S. K. and Salokhe V. M. 2005. Enhancing antimicrobial activity of chitosan films by incorporating garlic oil,potassium sorbate and nisin. Lebensmittel-Wissenschaft und Technologie. 38: 859-865.

68. Paik JS, Dhanasekharan M, Kelley MJ. 1998. Antimicrobial activity of UV-irradiated nylon film for packaging applications. Packag Technol Sci 11(4):179 -187.

69. Quintavalla S. and Vicini L. 2002. Antimicrobial food packaging in meat industry. Meat Science. 62: 373 – 380.

70. Rico-Pena DC, Torres JA. 1991. Sorbic acid and potassium sorbate permeability of an edible methylcellulose- palmitic acid film: water activity and pH effects. J Food Sci 56(2): 497 - 499.

71. Rodrigues ET, Han JH. 2000. Antimicrobial wheyprotein films against spoilage and pathogenic bacteria. Proceedings of the IFT Annual Meeting; Dallas, Tex.; June 10-14. Chicago, Ill.: Institute of Food Technologists. p 191.

72. Scannell AGM, Hill C, Ross RP, Marx S, Hartmeier W, Arendt EK. 2000. Development of bioactive food packaging materials using immobilised bacteriocins Lacticin 3147 and Nisaplin. Int . Food Microbiol 60(2-3): 241 - 249.

73. Scora KM, Scora RW. 1998. Effect of volatiles on mycelium growth of Penicillium digitatum, P. Italicum and P. ulaiense. J Basic Microbiol 38(5-6): 405 -413.

74. Shearer AEH, Paik JS, Hoover DG, Haynie SL, Kelley MJ. 2000. Potential of an antibacterial ultravioletirradiated nylon film. Biotechnol Bioeng 67(2):141 -146.

75. Siragusa G. A. and Dickinson J. S. 1992. Inhibition of Listeria monocytogenes on beef tissue by application of organic acids immobilized in a calcium alginate gel. J. Food Sci. 57 : 293 – 296.

76. Siragusa GR, Cutter CN, Willett JL. 1999. Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat. Food Microbiol 16(3): 229 - 235.

77. Suppakul P, Miltz J, Sonneveld K, Bigger SW. 2002. Preliminary study of antimicrobial films containing the principal constituents of basil. World Conference on Packaging: Proceedings of the 13th Intl.Assoc. of Packaging Res. Inst., Michigan State Univ., East Lansing, Mich., June 23-28. Fla.: CRC Press LLC. p 834 -839.

78. Vermeiren L, Devlieghere F, Debvere J. 2002. Effectiveness of some recent antimicrobial packaging concepts. Food Add Contamin 19(Suppl.):163 - 171.

79. Vojdani F, Torres JA. 1989a. Potassium sorbate permeability of polysaccharide films: chitosan, methylcellulose and hydroxypropyl methylcellulose. J Food Proc Eng 12(1): 33 - 48.

80. Vojdani F, Torres JA. 1989b. Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose multi-layer films. J Food Proc Preserv 13(6): 417 - 430.

81. Vodjani F and Torres J.A. 1990. Potassium sorbate permeability of methylcellulose and hydroxypropyl methylcellulose coatings. Effect of fatty acid. J. Food Sci. 55: 841 – 846.

Page 24: filmes antimicrobiano

Pérez-Pérez C. et al. 216

82. Weng YM, Hotchkiss JH. 1992. Inhibition of surface molds on cheese by polyethylene film containing the antimycotic imazalil. J. Food Prot. 55(5): 367 - 369.

83. Weng YM, Hotchkiss JH. 1993. Anhydrides as antimycotic agents added to polyethylene films for food packaging. Packag. Technol. Sci. 6(3): 123 - 128.

84. Weng YM, Chen MJ, Chen W. 1997. Benzoyl chloride modified ionomer films as antimicrobial food packaging materials. Int. J. Food Sci. Technol. 32(3): 229 - 234.

85. Weng YM, Chen MJ. 1997. Sorbic anhydride as antimycotic additive in polyethylene food packaging films. Lebensm. Wiss. Technol. 30(5): 485 -487.

86. Weng YM, Chen MJ, Chen W. 1999. Antimicrobial food packaging materials from poly(ethylene-comethacrylic acid). Lebensm. Wiss. Technol. 32(4): 191 - 195.

87. Wong DWS, Gregorski KS, Hudson JS, Pavlath AE. 1996. Calcium alginate films: Thermal properties and permeability to sorbate and ascorbate. J. Food Sci. 61(2): 337 - 341.