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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
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 microorganisms 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 lowdensity 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 γirradiation 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 lowdensity 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- comethacrylic 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 preservativetreated 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. Silversubstituted 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 foodcontact 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 coextruded 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 Agsubstituted 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 postprocessing 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 RhonePoulenc (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 heatpress 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
Food biotechnology
205
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 heatinactivated 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 vacuumpacked 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
206
Pérez-Pérez C. et al.
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 chitooligosaccharide side chain. The chito-oligosaccharide was introduced on
polyvinylacetate by cross-linking with the bifunctional compound, Nmethylolacrylamide. 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,
Food biotechnology
207
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
208
Pérez-Pérez C. et al.
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)
Food biotechnology
209
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
210
Pérez-Pérez C. et al.
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
Food biotechnology
211
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].
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