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Antimicrobial volatile essential oils in edible films for food safety

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Science against microbial pathogens: communicating current research and technological advances
______________________________________________________________________________
A. Méndez-Vilas (Ed.)

Antimicrobial volatile essential oils in edible films for food safety

Wen-Xian Du1, Roberto J. Avena-Bustillos2, Sui Sheng T. Hua3* and Tara H. McHugh1
1
Processed Foods Research and 3Plant Mycotoxin Research, USDA-ARS-WRRC, Albany, CA, USA
2
Department of Biological and Agricultural Engineering, University of California, Davis, Davis, CA USA
* Corresponding author: E-mail: Sylvia.Hua@ars.usda.gov Phone +1 5105595905

Plant-derived essential oils (EOs) and oil compounds usually have a relatively high vapor pressure and are capable of
reaching microbial pathogens through the liquid and the gas phase. Bioactivity of EOs in the vapor phase makes them
useful as possible fumigants for stored commodity protection. The use of edible films as carriers of natural antimicrobials
constitutes an approach for external protection of foods. These films can reduce surface microbial populations, enhance
oxygen-barrier, reduce the need for synthetic packaging materials, and improve their recyclability by simplifying their
structure. Incorporating antimicrobial compounds into edible films provides a novel way to improve the safety and shelf-
life of ready-to-eat foods. The evaluation of antimicrobial effectiveness of EOs in edible films can be done by different
methods depending on relevant applications. The overlay method represents the direct contact of the films on food
surfaces and the vapor phase method involves the inactivation of pathogens from a distance without direct contact of the
films with the contaminated food. The antimicrobial data obtained with vapors diffused from edible films can serve as a
guide for selection of appropriate levels of volatile EOs and their active constituents for incorporation into antimicrobial
edible films for non-direct contact applications on food. Edible films containing plant-derived volatile EOs provide new
ways to enhance microbial safety and shelf-life of foods.

Keywords edible film; antimicrobial; essential oils; volatile; food safety

1. Introduction
The demand for minimally processed, easily prepared and ready-to-eat ‘fresh’ food products, globalization of food
trade, and distribution from centralized processing pose major challenges for food safety and quality. Recent food-borne
microbial outbreaks are driving a search for innovative ways to inhibit microbial growth in foods while maintaining
quality, freshness, and safety. The increasing antibiotic resistance of some pathogens associated with foodborne illness
is another concern [1]. Therefore, there has been increasing interest in developing novel types of effective and non-
toxic antimicrobial compounds to protect the food against contamination and the consumer against infection.
Numerous studies have been published on the antimicrobial activities of plant essential oils (EOs) and their
constituents against foodborne pathogens [2, 3]. Recent research has been focused on incorporation of these naturally
occurring, food-compatible and safe compounds into foods to protect them against pathogenic bacteria. Development
of antimicrobial edible films to protect fresh and processed foods from human pathogens and extend the shelf life of
foods is becoming the new trend in food safety research. Antimicrobial edible films may provide an effective way to
control food-borne pathogens and spoilage microorganisms to thus enhance food safety and decrease product spoilage.
The use of edible films as antimicrobial carriers represents an interesting approach for the external incorporation of
plant EOs onto food system surfaces. The agents can then diffuse into the food to control target microorganisms. A
comprehensive update of experimental use of antimicrobial volatile EOs in edible film applications is provided in this
chapter. A brief description of how these antimicrobial edible films are produced and the methods used to determine
the antimicrobial activity and physical properties of these films are also given.

2. Edible films
Edible films are thin films prepared from edible material that act as a barrier to external elements (factors such as
moisture, oils, gases and vapors) and thus protect the product, extend its shelf life and improve its quality [4]. Edible
films can control moisture, oxygen, carbon dioxide, flavor and aroma transfer between food components or the
atmosphere surrounding the food. Generally, an edible film is defined as a preformed thin layer or solid sheets of edible
material placed on or between food components [5]. They can be used as film wraps or pouches for food. Different
food ingredients, derived from meats, cereals, nuts, fruits and vegetables, are being used to produce edible films for
strips and pouches. These films act as novel packaging systems and control the release of active compounds such as
antioxidants, flavors and antimicrobial agents [6-12]. The use of edible films in food protection and preservation has
recently increased since they offer several advantages over synthetic materials, such as being biodegradable and
environmentally friendly [13]. A greater emphasis on safety features associated with the addition of natural
antimicrobial agents is the next area for development in edible films technology [14].
For edible films to be used in foods, there are several requirements to be considered, such as appropriate gas and
water barrier properties; good mechanical strength and adhesion; reasonable microbial, biochemical and

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physicochemical stability; effective carrier for antioxidant, flavor, color, nutritional or antimicrobial additives; safe for
human consumption (free of pathogenic microorganisms and hazardous compounds); acceptable sensorial
characteristics; low cost of raw materials; and simple technology for production [15].

2.1 Biopolymers used for edible films

Components of edible films can be divided into three categories: hydrocolloids, lipids, and composites. Hydrocolloids
include proteins and polysaccharides, such as starch, alginate, cellulose derivatives, chitosan, and agar. Lipids include
waxes, acylglycerols, and fatty acids [16]. Composites contain both hydrocolloid components and lipids. The choice of
formulation for edible film is largely dependent on its desired function such as biodegradability, edibility, aesthetic
appearance and good barrier properties against oxygen [14]. In addition, edible films can serve as a support for
antimicrobial, nutritional and antioxidant substances [17].
Depending on their composition, the functionality of edible film materials may vary, as each component confers
different properties on the composite matrix. Films made of polysaccharides or proteins usually have suitable
mechanical and gas barrier properties but show poor water vapor barrier properties. In contrast, films composed of
lipids exhibit good water vapor barrier properties but show poor mechanical strength and high oxygen permeability.
When such ingredients are combined, they could physically and/or chemically interact and may result in films with
improved properties [18]. For example, fruit based edible films can be made with excellent oxygen barrier properties,
but not very good moisture barrier properties. By combining fruit purees with various gelling agents (such as alginate)
the water barrier and tensile properties of fruit-based films may improve [19].
The development of films from water soluble polysaccharides has led to promising new types of materials for
preservation of fruits and vegetables because of the selective permeability of these biopolymers to O2 and CO2. The
ability of water soluble polysaccharides to reduce O2 and increase CO2 levels in internal atmospheres of coated fruits
and vegetables reduces respiration rates, thereby extending the shelf-life of fresh produce in a manner similar to
modified/controlled atmosphere storage [18].
Polysaccharides are most often used for edible films because their film-forming properties are derived from cellulose,
starch, alginate and their mixtures. A plasticizer is normally added to increase film flexibility. Plasticizers are additives
used to increase the flexibility or plasticity of polymers, and occasionally they are used only to facilitate the polymer
processing. The most commonly used plasticizers in starch-based films are polyols, such as sorbitol and glycerol. They
are frequently added into edible films to reduce the intermolecular forces and increase the mobility of the polymeric
chains, therefore improving flexibility [20]. Glycerol is often used to modify the mechanical properties of hydrophilic
films. It is a low molecular weight nonvolatile substance. Addition of glycerol into films reduces internal hydrogen
bonding between polymer chains while increasing molecular volume, resulting in an improvement in film flexibility
[21].

2.2 Edible film casting methods

Despite the growth in research on edible films, the extent of commercialization has not been as great as needed.
Processing, mechanical, and water barrier properties of edible films must be improved for practical use [22]. Edible
films are commonly produced via a solution casting process where the films are dried from 12 min with hot air to 12 h
at room temperature. Reductions in drying times enable the formation of films with no significant microbial
contamination. Knowledge of critical control points is necessary to reduce the risk of microbial growth. The quality of
the starting materials, as well as the use of heat and good sanitation during casting and drying, are necessary to insure
safety [23].
Most edible films made on a research scale have been cast using inefficient (time, space, energy) solution casting
technologies. Thus, for mass production of edible films more efficient methodologies have been developed. Recently
we reported significant differences in physical and antimicrobial properties of apple and tomato-based edible films
made by continuous casting under infrared heating in a pilot plant lab coater and by a batch drying process done
overnight under ambient air [9, 10]. The continuous method for film casting was more suitable for large scale
production of edible films than the batch method. Higher evaporation of volatile active antimicrobial compounds
during casting at high temperatures can be compensated during formulation to achieve a desired antimicrobial final
concentration in dried films [9, 10].

3. Physical properties of edible films containing plant EOs

The main desired characteristics of an ideal edible film would be low water vapor permeability and high mechanical
strength. The physicochemical properties of edible films (color, tensile strength, water vapor and oxygen permeability)
relate to coating enhancement of mechanical integrity of foods, inhibition of moisture loss and oxidative rancidity, and
final-product appearance [15]. A complete analysis of both antimicrobial and physicochemical properties is important
for predicting the behavior of antimicrobial edible films in food system [24, 25].

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3.1 Color
Color of the film may influence consumer acceptability of a product [26]. Addition of EOs in edible films may change
the native color of edible films. The degree of change is concentration-dependent. Du et al. [11] reported that darker
films were produced with the addition of cinnamon, allspice, and clove bud oils into apple film forming solutions, and
the L∗ , b∗ values as well as the whitish index of apple solutions increased as the concentration of the oils increased.
Sivarooban et al. [27] also reported that the incorporation of 1% grape seed extract into soy protein isolate films
significantly influenced the L∗ , a∗ , and b∗ values. Rhim et al. [28] reported that the addition of various compounds
that structurally bind with the film forming solutions changed the native color of the soy protein film.

3.2 Water vapor permeability

McHugh et al. [29] demonstrated that apple-based edible films were not very good moisture barriers and that addition
of lipids could potentially improve the water barrier properties of fruit-based films. Rojas-Graü et al. [6] found that
water vapour permeability decreased when the proportion of the hydrophobic compounds increased in apple-based
edible films, this effect being more prominent when oregano oil was used in the composition of the films.
Carvacrol addition to apple puree edible films resulted in significant decrease in film water vapor permeability.
Water vapor transfer generally occurs through the hydrophilic portion of the film; thus, water vapor permeability
depends on the hydrophilic-hydrophobic ratio of the film components [30]. Water vapor permeability increases with
polarity, unsaturation, and branching degree of the lipid, depending also on the water absorption properties of the polar
part of the film [31].
Essential oil’s chemical nature also plays an important role in the barrier properties of edible films. Differences
observed by addition of different plant EOs can be explained by their hydrophobicity. In this way, carvacrol, a phenolic
compound containing an alcohol group in its chemical structure seems to be a good barrier compared to aldehyde
compounds (e.g., cinnamaldehyde, citral) because the hydroxyl group has less affinity for water than for the carbonyl
groups. Carvacrol then offers the possibility not only to enhance antimicrobial efficiency but also to improve barrier
properties of edible films.

3.3 Oxygen permability

McHugh et al. [29] demonstrated that apple-based edible films are excellent oxygen barriers, particularly at low to
moderate relative humidities. An apple puree edible film was a good oxygen barrier exhibiting values of 22.6 ± 1.3
cm3-µm/m2-d-kPa. The oxygen permeability values of this film increased as higher amounts of plant EOs were
incorporated. McHugh and Krochta [32] indicated that films containing lipids exhibit relatively poor oxygen barrier
properties. Oil chemical nature plays a major role in the barrier properties of edible films. Lower oxygen permeability
was observed in films that contained oregano, lemon grass and cinnamon oils than in those that contained its
antibacterial compounds carvacrol, citral and cinnamaldehyde, respectively [6,7].

3.4 Tensile strength

Tensile strength is one of the most common indicators of the mechanical property of an edible film. It expresses the
maximum stress developed in a film specimen during tensile testing [33]. The incorporation of plant EOs in apple-
based edible films caused a significant increase in tensile strength, % elongation, and elastic modulus of the film. These
differences could be related to their different polarities. These results are in agreement with those obtained by Pranoto et
al. [34], who studied the physical and antibacterial properties of alginate edible film with garlic oil.
Elongation at break is a measure of the film stretchability prior to breakage [5]. Zivanovic et al. [35] studied the
antimicrobial and physicochemical properties of polysaccharide (chitosan) films enriched with EOs. They observed a
decrease in tensile strength and an increase in elongation percentage when the EOs were introduced into the films. This
behaviour also was observed by Begin and Van Calsteren [36].

4. Antimicrobial volatile EOs

Plant-derived EOs, the odorous, volatile products of an aromatic plant’s secondary metabolism, normally formed in
special cells or groups of cells, are well-known antimicrobial agents that could be used to control food spoilage and
foodborne pathogenic bacteria [2]. They have long been served as flavouring agents in food and beverages, and due to
their versatile content of antimicrobial compounds, they possess potential as natural agents for food preservation [37].
The antimicrobial activity of plant EOs is assigned to a number of small terpenoid and phenolic compounds, which also
in pure form exhibit antibacterial or antifungal activity. Given the fact that consumers demand less use of chemicals on
minimally processed food products, more attention has been paid to the search for naturally occurring substances able to

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act as alternative antimicrobials and antioxidants. Plant EOs and their derivatives are becoming more popular as
naturally derived antimicrobial agents.
Recent studies have shown that EOs of oregano (Origanum vulgare), thyme (Thymus vulgaris), cinnamon
(Cinnamon casia), lemongrass (Cymbopogon citratus), and clove (Eugenia caryphyllata) are among the most active
against strains of E. coli [38-41]. Although the effectiveness of all these compounds has been widely reported, carvacrol
(a major component of the EOs of oregano and thyme) appears to have received the most attention from investigators.
Carvacrol is “Generally Regarded as Safe” (GRAS) and used as flavouring agent in baked goods, sweets, ice cream,
beverages and chewing gum [42]. Some plant EOs and their components are compatible with the sensory
characteristics of fruits and vegetables and have been shown to prevent bacterial growth. Among the complex
constituents of citrus EOs, the terpene citral is known to have strong antifungal properties [43]. In addition, cinnamon
oil and its active compound (cinnamaldehyde) also have been tested for their inhibitory activity against E. coli [44-46].
Friedman et al. [39] screened 120 naturally occurring plant-derived oils and oil compound for their antibacterial
activities against four species of foodborne pathogens. The most active oils in terms of BA50 values (% of oil in
phosphate buffer that killed 50% of the bacteria) are: Campylobacter jejuni (BA50, 0.003-0.009%): Marigold, ginger
root, jasmine, patchouli, Gardenia, cedarwood, carrot seed, celery seed, mugwort, spikenard, and orange bitter.
Escherichia coli O157:H7 (BA50, 0.046-0.14%): Oregano, thyme, cinnamon, palmarosa, bay leaf, clove bud, lemon
grass, and allspice. Listeria monocytogenes (BA50, 0.057-0.092%): Gardenia, cedarwood, bay leaf, clove bud, oregano,
cinnamon, allspice, thyme, and patchouli. Salmonella enterica (BA50, 0.045-0.14%): Thyme, oregano, cinnamon, clove
bud, allspice, bay leaf, palmarosa, and marjoram.
Friedman et al. [47, 44] showed that carvacrol, oregano, and cinnamaldehyde were effective antibacterials against
antibiotic-resistant Bacillus cereus, Campylobacter jejuni, E. coli, Salmonella enterica, and Staphylococcus aureus.
These compounds are candidates for incorporation into film formulations to reduce both non-resistant as well as
antibiotic-resistant pathogens in human foods.

5. Methods to measure the antimicrobial activity of EOs in edible films

The growth of microorganisms on the surface of a food is a key factor affecting the safety and/or spoilage of food
products [48]. The direct addition of an antimicrobial additive into foods might reduce its effectiveness, due to the
presence of substances that interact with it, so inactivating or reducing its antimicrobial effect [20]. The use of
antimicrobial films could be more efficient than directly using antimicrobials in the food. Since the antimicrobials
migrate selectively and gradually from film surface towards the surface of the food, maintaining the high concentrations
and a continued antimicrobial effect at the food surface during extended exposure [49]. Antimicrobial substances
incorporated into edible films can control microbial contamination of foods by reducing the growth rate of target
microorganisms, or by inactivating microorganisms by direct or indirect contact.
Overlay diffusion test is a commonly use method for determining the antimicrobial effect of edible film which
requires direct contact of films with pathogenic bacteria, while vapor phase diffusion test is a indirect contact assay that
is normally used for testing the antimicrobial activity of volatile compounds in edible films. Plant EOs are a potentially
useful source of antimicrobial compounds that can be incorporated into edible films. Evaporation of the EOs is effected
by external factors such as temperature, humidity, concentration and pressure [50]. Storage temperature can affect the
antimicrobial activity of EOs. Generally, increased storage temperature can accelerate the migration of the active agents
in the film, while refrigeration slows down the migration rate [51]. Factors such as the composition and solubility of
the oil, bacterial strain, the sources of antimicrobial samples used, and the method of growing and enumerating the
surviving bacteria can influence the antimicrobial activity test results of a plant oil [39, 52].

5.1 Overlay diffusion tests

Overlay diffusion test (zone of inhibition/agar diffusion assay) is a direct contact method using solid medium to
measure antimicrobial activity of EOs in edible films. For overlay diffusion tests, edible films with different
concentration of EOs were aseptically cut into 12-mm diameter discs and then deposited over the agar plate inoculated
with tested bacteria [9-12]. After 24 or 48 h of incubation, the inhibition radius around each film disc (colony-free
perimeter) was measured with a digital calliper (Fig 1a). The inhibition area (Fig 1b) was then calculated in mm2.
Antibacterial activity of soy protein edible films incorporated with 1- 5% oregano or thyme EOs was recently evaluated
against Escherichia coli, E. coli O157:H7, Staphylococcus aureus, Pseudomonas aeruginosa and Lactobacillus
plantarum by Emiroğlu et al. [53] using the overlay diffusion test. A visual screening method for anti-fungal activity of
EO edible films was recently developed by Hua et al. [54] using the overlay diffusion test. These scientists reported
that oregano oil in tomato film can inhibit both the growth and aflatoxin production of A. flavus (Fig 2).
A recent study on the contribution of vapors to the antimicrobial effect in the direct disc diffusion method indicated
that only the water-soluble components diffused across the agar while the re-deposition of the vaporised components on
the surface of the agar accounted for the remainder of the inhibition. It was found that for oils containing alcohol, ester,

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Inhibitory zone

(a) (b)
Fig. 1 (a) Inhibitory zone (antimicrobial effect) of S. enterica growth on a bacterial plate induced by an oregano oil-containing
tomato film measured with a digital caliper. (b) inhibitory zones of S. enterica growth around films with different concentrations of
oregano oil (0.5-1.5%) added to tomato puree solutions used to prepare the films.

ketone, oxide and hydrocarbons the major inhibition came from the vapors whereas for oils containing greater volumes
of aldehydes inhibition came from diffusion [55].
Minimum inhibitory concentrations (MICs) of antimicrobial edible films can be assessed by agar diffusion assay
with zone of inhibition observed, or the agar dilution method with visible growth observed, or broth dilution with
visible growth, optical density, absorbance or viable counts measured [2]. The MIC is determined as the lowest
concentration at which growth is inhibited. The major problem with determination of strength of antimicrobial activity
of edible films in this way is their hydrophobic nature which makes them insoluble in water based media [56]. The
other consideration when determining the MIC of edible films is that the absolute concentration of inhibition can be
anywhere between the lowest MIC and the next concentration where growth is observed.

Fig 2. Antifungal activity of edible films against Aspergillus flavus. The red
orange color (left) in the control plates (without film disks) indicate aflatoxin-
positive. When the nor mutant was grown in the presence of films impregnated with
oregano oil (right), they were practically colorless, which implied aflatoxin-
negative.

5.2 Vapor phase diffusion test

One advantage of EOs is their bioactivity in the vapor phase, a characteristic that makes them useful as possible
fumigants for stored commodity protection. Volatile compounds from plants usually have a relatively high vapor
pressure and are capable of approaching an organism through the liquid and the gas phase [57]. The antimicrobial
activity of EOs by vapor contact was first reported by Kellner and Kober [58]. They studied the effect of 175 EOs
against eight airborne bacteria and fungi. Inverted Petri plate technique was used to test of antimicrobial activity of EOs
in gaseous state [59]. A volatile compound contained in a cup or on a paper disc was exposed to the inverted agar
medium inoculated with a test organism. The size of the growth inhibitory zone after incubation is used as the measure
of vapor activity. This technique is convenient for qualitative analysis, but not for quantitative comparison of the vapor
activity of EOs [60].

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Most of existing methods for testing the antimicrobial activities of substances require direct contact between the
active agent and the microorganism (i.e. food), and thus are not relevant to many commercial products in which there is
little or no direct contact between the food and packaging material [61]. Vapor phase tests, which are not direct contact
assays, can be used to assess the protection provided by the antimicrobial volatile materials under no direct contact
conditions. For vapor-phase diffusion tests, edible films with different concentration of EOs were aseptically cut into
50 mm diameter discs and then placed on the lids of agar plates, which had been previously spread with bacterial
inoculum [11, 12]. The inoculated agar plate was inverted with dish on the top of each lid containing antimicrobial film
(Fig 3a). Parafilm was used to tightly seal the edge of each agar plate. All sealed and inverted plates were incubated at
35 °C. The growth of each pathogen on the agar plates was checked after incubation for 24 or 48 h. The inhibition
radius (absence of bacteria) on each agar plate (Fig 3b) was measured with a digital caliper. The values obtained were
used to calculate inhibition area in mm2.

(a) (b)
Fig. 3 (a) Vapor phase test setup. (b) vapor phase inhibitory zone (bacterial colony free spot area) of apple puree edible films
containing 0.5% to 3.0% allspice oil against S. enterica. Top left in each picture illustrates apple film with 0.5% allspice oil, showing
no inhibition of bacteria, similar to control films without added EOs.

Du et al. [11, 12] studied the effect of allspice, cinnamon and clove bud oil in apple film, as well as allspice, garlic
and oregano oil in tomato film on antimicrobial activities against E. coli O157:H7, Salmonella enterica, and Listeria
monocytogenes using overlay and vapor phase methods. The results of the study show that these plant-derived EOs can
be used to prepare fruit and vegetable-based antimicrobial edible films with good physical properties for food
applications by both direct contact and indirectly by vapors emanating from the films. Rojas-Graü et al. [7] evaluated
the antimicrobial activity of EOs and active compounds in apple films against target pathogen microorganisms using
overlay test. They found that the order of antibacterial activities were as follows: carvacrol > oregano > citral >
cinnamaldehyde > lemon grass > cinnamon oil in apple-based edible films. Evaluation of physicochemical properties
of films made from apple slurries revealed no adverse effect of the additives on water vapor permeability properties.
The incorporation of these bactericidal compounds caused a significant increase in tensile strength, percentage
elongation, and elastic modulus of the films. Du et al. [62] evaluated the antimicrobial effect of 17 EOs incorporated in
tomato edible films against E. coli O157:H7 using overlay and vapour phase diffusion tests (Table 1). Among the EOs
tested, six EOs showed antimicrobial effect against E. coli O157:H7 at 3% level using overlay test. The order of
antibacterial activities was: oregano > clove bud > allspice > vanilla > thyme > bay leaf. Vapor phase test showed these
oils also had antimicrobial effect against E. coli O157:H7 in the vapor phase except vanilla which did not show any
antimicrobial effect in the vapor phase.

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Table 1 Effect of essential oil at 3 % level against E. coli O15 7:H7 in to mato edible film.
Test Overlay Vapor phase
Disc diameter 12 mm 50 mm
Inhibition zone Perimetral Circular
2
Essential oil (mm ) (+/-)*
Control 0 -
All spice 122.2 ± 17.7 +/-
Bay laurel oil 0 -
Bay leaf oil (West indian) 66.2 ± 28.0 +/-
Cedarwood oil (himalayan) 0 -
Citral (cis and trans) 0 -
Clove bud oil 128.8 ± 10.9 +/-
Hexanal 0 -
Trans-2-hexenal 0 -
Laurinaldehyde 0 -
Lemomgrass oil 0 -
Orange oil (bitter) 0 -
Orange oil (Mandarin) 0 -
Oregano oil (origanum) 231.2 ± 26.5 +/-
Palmarosa oil 0 -
Thyme oil (red) 68.4 ± 9.2 +/-
Vanilla oil (oleoresin) 81.5 ± 51.0 -
Vanillin 0 -
* – : no t inhibitio n, + : co mplete inhibition, +/-: No t gr owth of E. co li on the center o f plate,
bu t some b acteria growth on the ed ge o f agar plate.

6. Applications of antimicrobial edible films in foods

A potential application of edible films is as a controlled release matrix for volatile active antimicrobial compounds from
natural plant EOs. Edible films can then be incorporated into conventional food packaging systems [63] with a dual
purpose as edible and antimicrobial component. Applications (Table 2) of edible films to meat, poultry, fish, fresh
fruits and vegetables, and tree nuts have received increasing interest because films can serve as carriers for various
antimicrobials that can maintain fresh quality, extend product shelf life and reduce the risk of pathogen growth. Edible
films with antimicrobial properties could prolong the shelf life and safety of foods by preventing growth of pathogenic
and spoilage microorganisms as a result of their lag-phase extension and/or their growth rate reduction [51]. Moreover,
antimicrobials imbedded in films can be gradually released on the food surface, therefore, requiring smaller amounts to
achieve the target shelf life [16].

Table 2 Applications of antimicrobial EOs in food system to control pathogens and spoilage bacteria.
Food Type Base materials for edible films EOs Target microorganisms Ref.
Ground beef Apple puree Carvacrol C. perfringens [65]
Ground beef patties Soy protein Oregano, thyme Coliform, Pseudomonas spp. [53]
Whole beef muscle Milk protein-based Oregano, pimento E. coli O157:H7, Pseudomonas spp. [67]
Fresh beef Sorbitol-plasticized whey protein Oregano Pseudomonads, lactic acid bacteria [68]
Bologna (meat) Chitosan-based Cinnamaldehyde Enterobacteriaceae, S. liquefaciens [49]
Bologna slices Chitosan Oregano L. monocytogenes, E. coli O157:H7 [35]
Chicken breast Apple Carvacrol E. coli O157:H7 [66]
Cold smoked sardine Gelatin based Oregano, rosemary Total spoilage bacteria [71]
Cod fish Bovine-hide gelatin and chitosan Clove H2S-producer microorganisms, [70]
Pseudomonas spp., Enterobacteriaceae
Strawberry Whey protein isolate Oregano Mold, spoilage bacteria [62]
Spinach Apple puree, tomato puree Carvacrol, cinnamaldehyde E. coli O157:H7 [62]
Almond Apple puree, tomato puree Cinnamon, allspice, thyme, Aspergillus flavus [54]
oregano

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6.1 Fresh and minimally processed fruit and vegetable

Applications of edible films to fresh fruits and vegetables have also received increasing interest because films can serve
as carriers for various antimicrobials that can maintain fresh quality, extend product shelf life and reduce the risk of
pathogen growth. The most important quality attributes contributing to the marketability of fresh produce include
appearance, color, texture, flavor, nutritional value, and microbial safety. These quality attributes are determined by
plant variety, stage of maturity or ripening, and the pre- and postharvest conditions [64]. Fresh fruits undergo vigorous
biological reactions after harvest and their respiration accelerates the natural loss of fruit tissue. Therefore fruits tend to
lose water, change appearance, texture and quality after harvest and thus reduce in commercial value.
For use on fresh fruits and vegetables, the main desirable characteristics of edible films would be good barrier
properties, odorless, tasteless, and transparent. Edible polymer films may be formed as either food coatings or stand-
alone film wraps and pouches. They have potential for use with food as moisture, gas and/or aroma barriers. The
potential benefits of using edible films in the fresh produce industry include: to provide a moisture barrier on the surface
of produce to decrease moisture loss; to provide a sufficient gas barrier to control gas exchange between the fresh
produce and its surrounding atmosphere (create a modified atmosphere), in an effort to slow respiration, delay
deterioration and protect the fresh produce from brown discoloration and texture softening during storage; to restrict the
exchange of volatile compounds between the fresh produce and its surrounding environment by providing gas barriers,
which prevents the loss of natural volatile flavor compounds and color components from fresh produce and the
acquisition of foreign odors; to protect from physical damage of produce caused by mechanical impact, pressure,
vibrations, and other mechanical factors; and to act as carriers for other functional ingredients, such as antimicrobial and
antioxidant agents, nutraceuticals, and color and flavor ingredients for reducing microbial loads, delaying oxidation and
discoloration, and improving quality and shelf-life of fresh produce [64]. Appropriately formulated edible films can be
utilized for fresh produce to meet challenges associated with stable quality, market safety, nutritional value, and
economic production cost.
Du et al. [62] evaluated the antimicrobial effect of edible films and pouches containing EOs on the mold growth of
strawberry during cold storage. They found that whey protein isolate film with 1% oregano oil can increase the shelf
life of strawberry during cold storage. The antimicrobial effect of carvacrol and cinnamaldehyde in apple and tomato
edible film against E. coli O157:H7 in spinach was also evaluated by these scientists. They found these antimicrobial
EO edible film can inhibit the initial growth of E. coli O157:H7 in spinach at day 1, but failed to reduce the counts of
this pathogenic bacterium after 5 days of cold storage. These results indicated that edible films and pouches containing
essential oils and their constituents can be used as release system for volatile antimicrobial agents to improve the safety
and shelf life of fresh-cut fruits and vegetables.

6.2 Meat and meat products

Recent studies showed that carvacrol in apple films inhibited the growth of Clostridium perfringens during chilling of
cooked ground beef [65], inactivated E. coli O157: H7, and inhibited the formation of carcinogenic heterocyclic amines
during grilling of hamburger beef patties [66]. Emiroğlu et al. [53] reported that soy protein edible films incorporated
with oregano, thyme, or their mixture did not have significant effects on total viable counts, lactic acid bacteria and
Staphylococcus spp. when applied on ground beef patties whereas reductions (p < 0.05) in coliform and Pseudomonas
spp. counts were observed. Ouattara et al. [49] reported that the growth of Enterobacteriaceae and S. liquefaciens on
drier surfaces of meat (bologna) was delayed or completely inhibited when chitosan-based antimicrobial films
containing cinnamaldehyde were used for the application. Oussalah et al. [67] tested the antibacterial effects of milk
protein-based edible films containing EO from oregano or pimento against Escherichia coli O157:H7 and Pseudomonas
spp. in preserving whole beef muscle. They found the film that contained the added oregano extract to be the most
effective, achieving reductions of around 1 log unit for each of these bacterial species at the end of storage compared
with uncoated samples. Other workers [35] combined the properties of a chitosan film with the EO of oregano and
observed it to be efficacious against both L. monocytogenes and E. coli O157:H7. In that study, of several film
formulations used to coat the surface of inoculated bologna slices, pure chitosan by itself displayed bactericidal activity,
but higher activity was achieved by chitosan film enriched with oregano extract. Zinoviadou et al. [68] also reported
that the maximum specific growth rate of total flora (total viable count) and pseudomonads in film wrap beef cuts were
significantly reduced (p < 0.05) by a factor of two with the use of oregano oil containing whey protein films (1.5% w/w
oil in the film forming solution), while the growth of lactic acid bacteria was completely inhibited. Those results
pointed to the effectiveness of oregano oil containing whey protein films to increase the shelf life of fresh beef.

6.3 Poultry
Friedman et al. [66] discovered that carvacrol in apple films inhibited the growth of E. coli O157:H7 on the surfaces of
raw chicken breast. Avena-Bustillos et al. [69] further evaluated the effect of adding carvacrol (the active ingredient of
oregano EO) and cinnamaldehyde (the active ingredient of cinnamon oil) to apple- and tomato-based film-forming
solutions on sensory properties of wrapped cooked chicken. Preference tests indicated that baked chicken wrapped with

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A. Méndez-Vilas (Ed.)

tomato and apple films containing 0.5% carvacrol or cinnamaldehyde were equally preferred over chicken wrapped
with tomato or apple films without the plant antimicrobials. The consumers preferred carvacrol-containing tomato film
chicken wraps over the corresponding apple film wraps. These findings suggest that edible films containing
antibacterial EOs can be used to protect raw chicken pieces against bacterial contamination without adversely affecting
sensory preferences of cooked wrapped chicken pieces.

6.4 Fish
Gómez-Estaca et al. [70] reported that when the complex gelatin-chitosan film incorporating clove EO was applied to
cod fish during chilled storage, the growth of microorganisms was drastically reduced in gram-negative bacteria,
especially enterobacteria, while lactic acid bacteria remained practically constant for much of the storage period.
Gómez-Estaca et al. [71] reported that the stability of cold-smoked sardine muscle was improved by coating the muscle
with functional gelatin- based edible films. Films enriched with oregano or rosemary extracts were able to slow lipid
oxidation, but they failed to slow microbial growth. Microorganism counts and oxidation indices were kept well below
those in the other sample batches tested for at least two weeks of chilled storage under no vacuum-packaging conditions
when the combinations of high pressure and coating with film enriched with an oregano extract were used.

6.5 Tree nuts

Hua et al. [54] reported that tomato and apple edible films containing 3% cinnamon, allspice, thyme, and oregano oil
can inhibit the growth of A. flavus (aflatoxin producing fungi) in almond during storage. These results suggested that
the use of edible films as carriers of natural antimicrobials constitutes an approach for external protection of almonds.
The films can reduce surface microbial populations and enhance oxygen-barrier in stored almonds.

7. Concluding remarks
Edible films containing plant-derived volatile EOs provide new ways to enhance microbial safety and shelf-life of foods
by direct and/or indirect contacts of the antimicrobials in the films with the food. The antimicrobial data obtained with
vapors diffused from the edible films can serve as a guide for selection of appropriate levels of volatile EOs and their
active constituents for incorporation into antimicrobial edible films.

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