Antimicrobial volatile essential oils in edible films for ... · PDF fileAntimicrobial...

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

1Processed Foods Research and 3Plant Mycotoxin Research, USDA-ARS-WRRC, Albany, CA, USA 2Department of Biological and Agricultural Engineering, University of California, Davis, Davis, CA USA * Corresponding author: E-mail: [email protected] 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



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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(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].

Inhibitory zone



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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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., EnterobacteriaceaeStrawberry 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

Table 1 Effect of essential oil at 3% level against E. coli O157:H7 in tomato edible film.

Test Overlay Vapor phaseDisc diameter 12 mm 50 mm Inhibition zone Perimetral Circular

Essential oil (mm2) (+/-)*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 inhibition, + : complete inhibition, +/-: Not growth of E. co li on the center o f plate,

but some bacter ia growth on the edge of agar plate.



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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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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