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Antimicrobial Edible Films and Coatings for Fresh andMinimally Processed Fruits and Vegetables: A ReviewSilvia A. Valencia-Chamorro a b , Lluís Palou a , Miguel A. del Río a & María B. Pérez-Gago a ca Centro de Tecnología Poscosecha, Instituto Valenciano de Investigaciones Agrarias (IVIA) ,46113 Moncada, Valencia , Spainb Departamento de Ciencia de Alimentos y Biotecnología, Escuela Politécnica Nacional , P. O.BOX 17 – 01 2759, Quito , Ecuadorc IVIA - Fundación AGROALIMED , 46113 Moncada, Valencia , SpainAccepted author version posted online: 23 May 2011.Published online: 20 May 2011.

To cite this article: Silvia A. Valencia-Chamorro , Lluís Palou , Miguel A. del Río & María B. Pérez-Gago (2011) AntimicrobialEdible Films and Coatings for Fresh and Minimally Processed Fruits and Vegetables: A Review, Critical Reviews in Food Scienceand Nutrition, 51:9, 872-900, DOI: 10.1080/10408398.2010.485705

To link to this article: http://dx.doi.org/10.1080/10408398.2010.485705

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Critical Reviews in Food Science and Nutrition, 51:872–900 (2011)Copyright C©© Taylor and Francis Group, LLCISSN: 1040-8398 / 1549-7852 onlineDOI: 10.1080/10408398.2010.485705

Antimicrobial Edible Filmsand Coatings for Fresh and MinimallyProcessed Fruits and Vegetables:A Review

SILVIA A. VALENCIA-CHAMORRO,1,2 LLUIS PALOU,1 MIGUEL A. DEL RIO,1

and MARIA B. PEREZ-GAGO1,3

1Centro de Tecnologıa Poscosecha, Instituto Valenciano de Investigaciones Agrarias (IVIA), 46113 Moncada, Valencia, Spain2Departamento de Ciencia de Alimentos y Biotecnologıa, Escuela Politecnica Nacional, P. O. BOX 17 – 01 2759, Quito,Ecuador3IVIA - Fundacion AGROALIMED, 46113 Moncada, Valencia, Spain

The use of edible films and coatings is an environmentally friendly technology that offers substantial advantages for shelf-lifeincrease of many food products including fruits and vegetables. The development of new natural edible films and coatings withthe addition of antimicrobial compounds to preserve fresh and minimally processed fruits and vegetables is a technologicalchallenge for the industry and a very active research field worldwide. Antimicrobial agents have been successfully addedto edible composite films and coatings based on polysaccharides or proteins such as starch, cellulose derivatives, chitosan,alginate, fruit puree, whey protein isolated, soy protein, egg albumen, wheat gluten, or sodium caseinate. This paper reviewsthe development of edible films and coatings with antimicrobial activity, typically through the incorporation of antimicrobialfood additives as ingredients, the effect of these edible films on the control of target microorganisms, the influence ofantimicrobial agents on mechanical and barrier properties of stand-alone edible films, and the effect of the application ofantimicrobial edible coatings on the quality of fresh and fresh-cut fruits and vegetables.

Keywords food preservatives, fresh-cut, barrier and mechanical properties, postharvest quality

INTRODUCTION

New edible films and coatings formulated with natural prod-ucts have been developed for fresh and processed food products.They constitute an environmentally-friendly technology thatmay enhance food quality, safety, stability, and the mechanical-handling properties by providing a semi-permeable barrier towater vapor, oxygen, and carbon dioxide between the food andthe surrounding atmosphere (Greener-Donhowe and Fennema,1994). Edible films and coatings can also be used as carriersof antioxidants, flavoring agents, coloring agents, growth reg-ulators, and antimicrobials that will improve food quality andsafety (Vojdani and Torres, 1990; Cuppet, 1994; Yaman and

Address correspondence to Marıa B. Perez-Gago, Centro de Tec-nologıa Poscosecha, Instituto Valenciano de Investigaciones Agrarias (IVIA),46113 Moncada, Valencia, Spain Tel.:(+34) 963424000; Fax number: (+34)96342400. E-mail: perez mbe@gva.es

Baymdirh, 2001; Coma et al., 2002). In fresh fruits and veg-etables, the creation of a moisture and gas barrier may lead toweight loss and respiration rate reductions with a consequentgeneral delay of produce senescence (Hagenmaier and Baker,1993; Debeaufort et al., 1998; Perez-Gago et al., 2002). Fur-thermore, the application of coatings may improve the visualquality by providing gloss to the coated commodities (Trezzaand Krochta, 2000). On the other hand, edible films and coatingsmay replace, to some extent, plastic packaging by natural andbiodegradable substances. Their use could lead to an importantreduction on the overall packaging requirements and, therefore,waste disposal problems.

In the last decade, a considerable amount of work has focusedon the development of films and coatings based on proteins andpolysaccharides with food additives from natural or syntheticsources to control microbial growth on fresh and processedfoods. Edible films and coatings containing antimicrobials, suchas some organic acids and their salts, parabens, chitosan, essen-tial oils, or natural plant extracts have been effective in delaying

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ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 873

the growth of contaminating microorganisms and maintainingthe quality during storage and distribution of fresh and fresh-cut horticultural products. This paper reviews existing literatureand the current state of the art on: (1) edible films preparedwith food additives as antimicrobial ingredients and their ef-fect on the control of target microorganisms; (2) the influenceof antimicrobial agents on mechanical and barrier properties ofstand-alone edible films; and (3) the effect of antimicrobial ed-ible coatings on the quality of fresh and minimally processedfruits and vegetables.

EDIBLE FILMS AND COATINGS: DEFINITION,COMPOSITION, AND FUNCTIONAL PROPERTIES

Films vs Coatings

Films are usually defined as a stand-alone thin layer of ma-terials that can be used as covers, wraps, or separation layers.However, the main use of stand-alone films is as testing struc-tures for determination of barrier, mechanical, solubility, andother properties provided by certain film materials. Coatingsinvolve the formation of films directly on the surface of theproduct they are intended to protect or enhance (Krochta, 2002).Therefore, edible coatings are considered part of the final foodproduct and should confer acceptable color, odor, taste, flavor,and texture to the coated product.

Edible Film and Coating Materials

According to their components, edible films and coatings canbe divided into three categories: hydrocolloids, lipids, and com-posites. Hydrocolloids include proteins and polysaccharides.Lipids include waxes, acylglycerols, and fatty acids. Compos-ites contain both hydrocolloid components and lipids (Greener-Donhowe and Fennema, 1994; Nisperos-Carriedo, 1994;Baldwin, 1999). Several other compounds such as plasticiz-ers and emulsifiers may be added to edible films and coatings toimprove their mechanical properties and form stable emulsionswhen lipids and hydrocolloids are combined. In addition, ediblecoatings and films can also act as carriers of food additives, in-cluding antioxidants, colorants, flavoring agents, and antimicro-bial compounds (Cuppet, 1994; Baldwin, 1999; Franssen andKrochta, 2000; Cha and Chinnan, 2004; Han and Gennadios,2005).

Highly polar polymers containing hydroxyl groups, such asproteins and polysaccharides, generally present a good barrier tooxygen at low relative humidity (RH) due to their tightly packed,ordered hydrogen-bonded network structure and low solubility(McHugh and Krochta, 1994). However, they form a poor mois-ture barrier due to their hydrophilic character. Film-formingpolysaccharide materials include starch and starch deriva-tives, cellulose derivatives, alginate, carrageenan, pectin, pul-

lulan, chitosan, and various gums (Han and Gennadios, 2005).Proteins that have received great attention for their capabil-ity of forming edible films and coatings include corn zein,wheat gluten (WG), soy protein, whey protein, casein, colla-gen/gelatin, pea protein, rice bran protein, cottonseed protein,peanut protein, and keratin (Baldwin and Baker, 2002; Han andGennadios, 2005). However, some considerations with respectto food intolerances such as wheat gluten intolerance (celiacdisease), or milk protein intolerance, allergies, or religious be-liefs/banning should be taken into account when protein-basedfilms and coatings are used. Lipids and resins, due to their hy-drophobic nature, are used in edible films and coatings to providea barrier to moisture. In addition, they are often used to pro-vide gloss to food surfaces (Greener-Donhowe and Fennema,1994). However, because lipids and resins are not polymers,they form films and coatings with poor mechanical proper-ties. Lipids and resins used for the preparation of lipid-basededible films and coatings include neutral lipids, fatty acids,waxes (beeswax (BW), candelilla wax, carnauba wax, rice branwax), and resins (shellac, wood rosin) (Rhim and Shellhammer,2005).

Composite films and coatings comprise hydrocolloid com-ponents and lipids, thus enhancing the advantages and lesseningthe disadvantages of each. A composite film can be producedas either a bi-layer or a stable emulsion. In bi-layer compositefilms, the lipid forms a second layer over the polysaccharideor protein layer. In emulsion composite films, the lipid is dis-persed and entrapped in the supporting matrix of protein orpolysaccharide (Shellhammer and Krochta, 1997; Perez-Gagoand Krochta, 2005). In composite edible films and coatings,the efficiency of lipid materials depends on the lipid structure,its chemical arrangement, hydrophobicity, physical state, andits interaction with other components of the film (Rhim andShellhammer, 2005).

Plasticizers are low molecular weight agents that are incor-porated into film-forming materials to decrease the intermolec-ular forces between polymer chains, which results in greaterfilm flexibility, elongation, and toughness. However, they canalso increase film permeability (Han and Gennadios, 2005).Plasticizers used for edible films and coatings include sucrose,glycerol, sorbitol, propylene glycol, polyethylene glycol, fattyacids, and monoglycerides. Water also acts as a plasticizer forpolysaccharide and protein edible films (Krochta, 1997). Thus,the film moisture content, as affected by RH, has a large effecton film properties.

Emulsifiers or surfactants are surface-active agents of am-phiphilic nature that interact at the water-lipid interface andreduce surface tension between the dispersed and continuousphases to improve the stability of the emulsion (Han and Gen-nadios, 2005). They are also used to ensure good surface wet-ting, spreading, and adhesion of the coating to the food surface.Common emulsifiers used on films and coatings are fatty acids,ethylene glycol monostearate, glycerol monostearate, esters offatty acids, lecithin, sucrose ester, and sorbitan monostearate orpolysorbates (tweens).

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874 S. A. VALENCIA-CHAMORRO ET AL.

Functions and Properties of Edible Films and Coatings

The main function of edible films and coatings is to offera protective barrier to moisture, oxygen, flavor, aroma, and/oroil between the food and the environment. In addition, ediblefilms and coatings may also maintain food integrity by pro-viding some mechanical protection. The protective function ofedible films and coatings may be enhanced with the addition ofantioxidants, antimicrobials, flavors, nutrients, etc. Given thatthe main interest in edible films and coatings is generally basedon their barrier and protective functions, most of the studiesare focused on determining these properties on stand-alone edi-ble films. Barrier properties commonly studied to determine theability to protect foods from the environment and from adjacentingredients are water vapor permeability (WVP) and oxygenpermeability (OP). Aroma and oil permeability are also veryimportant for many foods but have generally received less at-tention. The ability of edible films and coatings to protect foodagainst mechanical damage is usually assessed by determiningfilm tensile properties: (1) Young’s Modulus (YM), which deter-mines film stiffness as determined by ratio of pulling force/areato degree-of-film-stretch, (2) tensile strength (TS), which in-dicates the pulling force per film cross-sectional area requiredto break the film, and (3) elongation at break (E), which givesthe degree to which the film can stretch before breaking andit is expressed as percentage (Krochta, 2002). Other film prop-erties that have been typically investigated include film watersolubility, gloss, and color.

With respect to the evaluation of antimicrobial edible filmsand coatings, different methods have been used to examine theantimicrobial properties of stand-alone films against target mi-croorganisms. Antimicrobial assays include: (1) “agar difussiontest,” “zone of inhibition test,” or “disk diameter test,” in whichan antimicrobial film is placed over a lawn of the microorganismgrowing on an agar medium plate. Over time, the antimicrobialdiffuses from the film into the medium and kills the microor-ganism or inhibits its growth, creating a zone of clearing orinhibition. The results are expressed as the diameter or the areaof the zone of inhibition. This method is relatively simple andeasy to apply, but the quantitative zone measurements fromdifferent studies are difficult to compare because of the manyspecific conditions of the experiments including film size andproperties, antimicrobial, agar media, microorganism, tempera-ture, incubation time, etc.; (2) the “cell count method” or “logreduction assay” involves placing the film in a microorganismgrowing broth solution and removing samples from the solutionover time. The solution sample is then plated on agar media andcolonies are counted. This method gives a microorganism countthat can be used to measure log reduction due to the antimicro-bial film. As with the “agar difussion test,” the results of thisexperimental approach are not directly applicable to a coatedfood product because of the different experimental conditions.There are other experiments intended to determine the abilityof the film to release the antimicrobial ingredient. These testsmay provide information about (1) the antimicrobial release

rate or the amount of antimicrobial compound that is releasedover time; (2) the antimicrobial diffusion coefficient that gives aquantitative measurement of the rate at which the diffusion pro-cess occurs; and (3) the antimicrobial permeability coefficient(Franssen and Krochta, 2000; Nychas and Skandamis, 2000).

ANTIMICROBIAL FOOD ADDITIVES

Additives used to prevent biological deterioration are termedantimicrobials or preservatives. As they are allowed for foodcontact applications, this category comprises of natural or syn-thetic compounds with known and minimal toxicological effectson mammals and the environment. Antimicrobial compoundsinclude some inorganic (carbonates, bicarbonates, etc.) or or-ganic acids and their salts (propionates, sorbates, benzoates,etc.), parabens, chitosan, enzymes, bacteriocins, polypeptides,and essential oils or other natural extracts.

A wide variety of antimicrobials have been added to ediblefilms and coatings to control microbiological growth and ex-tend produce shelf-life. Antimicrobials used for the formulationof edible films and coatings must be classified as food-gradeadditives or compounds generally recognized as safe (GRAS)by the relevant regulations. International regulatory agenciesare in charge of approving antimicrobials for the use on foods.In the European Union (EU), those compounds are regulatedby the EU Framework Directive 89/107 (EU, 1989) and in theUnited States (US) by the part 21CFR172 enacted by the USFood and Drug Administration (US FDA, 2009). Table 1 showscommon antimicrobial agents used on edible films and coatingsand their code numbers for food additives approved by the EU(E-Code) or the regulation numbers established by the US FDA(RegNum).

Antimicrobial Synthetic Chemical Agents

Organic acids are the most common synthetic antimicro-bial agents and include acetic, benzoic, citric, fumaric, lactic,malic, propionic, sorbic, succinic, and tartaric acid, among oth-ers. These acids typically inhibit the outgrowth of bacterial andfungal cells. Potassium sorbate (PS) and sodium benzoate (SB)are the two organic acid salts more widely used as antimicrobialfood additives. Benzoic acid is also called phenylformic acidor benzene-carboxylic acid. The antimicrobial activity of ben-zoic acid and SB is related to pH, and the most effective arethe undissociated forms. Therefore, the use of these preserva-tives has been limited to those products that are acid in nature(Chipley, 2005). Sorbic acid is a straight-chain unsaturated fattyacid. The carboxyl group of sorbic acid is highly reactive withcalcium, sodium or potassium, and results in the formation ofvarious salts and esters (Stopforth et al., 2005b). PS, the mostsoluble form of sorbate is well known for its potent antifungalactivity. Major mold species inhibited by PS belong to the generaAlternaria, Penicillium, and others. The antimicrobial action of

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ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 875

Table 1 Antimicrobial compounds used on edible films and coatings

Food preservatives

Chemical compounds E-CodeaNaturalcompounds

E-Code/RegNumb

Organic acids PolypeptidesAcetic E-260 Lysozyme E-1105Benzoic E-210 Peroxidase —Citric E-330 Lactoperoxidase —Lactic E-270 Lactoferrin —Malic E-296 Nisin E-234Propionic E-280 Natamycin E-235Sorbic E-200Tartaric E-334

Organic acid salts Plant extracts,essential oils,spices

Sodium acetate E-262(I) Cinnamon 182.10Sodium diacetate E-262(II) Capsicum 182.10Sodium benzoate E-211 Lemongrass 182.20Sodium citrate E-331(I) Oregano 182.10Sodium formate E-237 Rosemary 182.20Calcium formate E-238 Garlic 184.1317Sodium L-lactate E-325 Vanilla 182.10Sodium propionate E-281 Carvacrol 172.515Calcium propionate E-282 Citral 182.60Potassium sorbate E-202 Cinnamaldehyde 182.60Sodium L-tartrate E-335(I) Vanillin 182.60

Grape seedextracts

—

ParabensMethyl paraben E-218Ethyl paraben E-214Propyl paraben E-216Sodium salt of methyl

parabenE-219

Sodium salt of ethylparaben

E-215

Sodium salt of propylparaben

E-217

Mineral saltsSodium bicarbonate E-500(I)Ammonium bicarbonate E-237Sodium carbonate E-500(II)

OthersEDTA-CaNa2

c E-385

aE-Code = code number for food additives approved by the European Union.bRegNum = Regulation number in Title 21 of the U.S. Code of Federal Regu-lations where the chemical appears.cEDTA-CaNa2 = disodium calcium ethylenediaminetetraacetate.

sorbates is also pH dependent. In general, PS activity is greater atlow pH values, although sorbates may be effective at pH valuesas high as 7.0. In contrast, other common organic acid-basedantimicrobials, such as propionates or benzoates, only showconsiderable antimicrobial activity at low pH values such as5.0–5.5 and 4.0–4.5, respectively (Stopforth et al., 2005b). Sev-eral studies have also indicated increased antimicrobial effectswhen PS was combined with various phosphates. Combinationsof sorbate with benzoate or propionate may be used to expand

the range of inhibited microorganisms with reduced concentra-tions of each preservative (Stopforth et al., 2005b). Propionicacid is a naturally-occurring monocarboxylic acid. Salts of theacid have a slight cheeslike flavor. The antimicrobial activityof propionate salts is pH dependent, being also more effectiveat low pH because of the higher activity of the undissociatedform. Propionic acid is primarily inhibitory to molds; however,some yeasts and bacteria have also been satisfactorily controlled(Doores, 2005).

Parabens are the alkyl esters of para-hydroxybenzoic acid.The alkyl chain length of parabens determines their water sol-ubility. The shorter the alkyl chain length, the higher the watersolubility of parabens. Parabens are inhibitory to either severalgram-positive and gram-negative bacteria or molds, althoughfungi are generally more susceptible to parabens than bacteria(Davidson, 2005). For both bacteria and fungi, the inhibitory ac-tivity generally increases as the alkyl chain length of parabensalso increases. The optimum pH for effective antimicrobial ac-tivity of parabens is in the range 3.0–8.0.

Natural Antimicrobial Agents

Natural antimicrobial agents include chitosan, polypetides,and plant oils, extracts, and spices. Chitosan is a polysaccharideprepared by deacetylation of chitin. It is composed of β-1,4linked glucosamine units and N-acetyl glucosamine residues. Itis obtained by the alkaline deacetylation of chitin, the most abun-dant component of the shells of crustaceans (Coma et al., 2002;No et al., 2007). It has been suggested that chitosan antimicrobialactivity may come from its positive charges that would interferewith the negatively charged residues of macromolecules on thecell surface, rendering membrane leakage (Sebti et al., 2007).Likewise, chitosan may also act as an elicitor of plant defenseresponses that include synthesis of antimicrobial phytoalexinsin fresh produce. Functional properties and antimicrobial effectsof chitosan are related to its deacetylation degree and molecularweight. Chitosan inhibits the growth of a wide variety of fungi,yeasts, and bacteria. Due to its film forming property, chitosanis used to prepare films and coatings (No et al., 2007).

Nisin, a hydrophobic protein, is a low-molecular-weightpolypeptide produced by the bacterial dairy starter Lactococ-cus lactis subspecies lactis. Nisin has a broad spectrum of ac-tivity against gram-positive bacteria, but do not significantlyinhibit gram-negative bacteria, yeasts, or molds (Thomas andDelves-Broughton, 2005). Nisin was proved to be non-toxicand classified as GRAS by the US FDA in 1969. Since then, ithas been widely used in the food industry as a safe and naturalpreservative (Sebti et al., 2007).

Natamycin is a tetraene polyene macrolide. It is a natural an-tifungal agent produced by Streptomyces natalensis. Natamycinhas no effect on bacteria, but it is active against nearly all moldsand yeasts. Natamycin is usually applied as a surface treatmentfor hard cheese and dry or ripened sausages (Ture et al., 2009b).

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876 S. A. VALENCIA-CHAMORRO ET AL.

Lactoperoxidase is a hemoprotein present in milk, tears, andsaliva. The lactoperoxidase system consists of three compo-nents: lactoperoxidase, thiocyanate, and hydrogen peroxide. Thelast compound serves as a substrate for lactoperoxidase in oxi-dazing thiocyanate and iodide ions, resulting in the generation ofhighly reactive oxidazing agents (Naidu, 2003). The lactoperox-idase system has shown the ability to inhibit different bacteria,fungi, parasites, and viruses, and for that reason it is considereda broad-spectrum natural antimicrobial (Stopforth et al., 2005a).Killing of cells is usually greater at low pH and, low temperatureand with iodide ions as the electron donor (Naidu, 2003).

Lactoferrin is an iron-binding, bioactive glycoprotein of thetransferrin family that contributes to the control of iron in biolog-ical fluids. Lactoferrin inhibits microorganisms by binding ironand making it unavailable for microbial development (Stopforthet al., 2005a).

Lysozyme is an enzyme comprising 129 amino acids cross-linked by disulfide bonds (Cagri et al., 2004). Lysozyme ex-hibits antimicrobial activity against vegetative cells of a widevariety of organisms, including numerous foodborne pathogensand spoilage microorganisms. Gram-negative bacteria are gen-erally less sensitive than Gram-positive bacteria to lysozyme,mainly as a result of protection of the cell wall by the outermembrane (Johnson and Larson, 2005). The rate of cell cataly-sis by lysozyme depends upon the pH of the medium, showinga bell-shape with a maximum at pH 5.0 and inflections at pH3.8 and 6.7 (Naidu, 2003).

Plants, herbs, and spices, as well as their derived essential oilsand substances isolated from different extracts, contain a largenumber of compounds that are known to inhibit the metabolicactivity of bacteria, yeasts, and molds (Lopez-Malo et al., 2005).For instance, it has been proved that essential oils of angelica,anise, carrot, cardamom, cinnamon, cloves, coriander, dill weed,fennel, garlic, nutmeg, oregano, parsley, rosemary, sage, or thy-mol are inhibitory to various spoilage or pathogenic bacteria,molds, and yeasts (Cagri et al., 2004).

ANTIMICROBIAL EDIBLE FILMS: ANTIMICROBIAL,BARRIER, AND MECHANICAL PROPERTIES

Studies on antimicrobial, barrier, and mechanical propertiesshould be combined to correctly predict the behavior of an an-timicrobial edible film (McHugh and Krochta, 1994; Cagri et al.,2001). The properties of these films are strongly influenced bythe type and concentration of the antimicrobial compound andthe nature of the film matrix.

Antimicrobial Activity of Edible Films

The antimicrobial activity of different polysaccharide-based(Table 2) and protein-based (Table 3) edible films containingantimicrobials against important target pathogens is presented.Results reported in different studies are difficult to compare

mainly due to differences in experimental conditions such as filmcomposition, antimicrobial agent and concentration, strain, andconcentration of the target microorganism and analytical methodused to determine the film antimicrobial activity. For this reason,the antimicrobial activity of the films is reported in the tablesas inhibition (+) or no inhibition (−) of the target pathogenicmicroorganism with no dependence on the magnitude of theinhibition, as concluded by the authors of the different studies,according to their experimental conditions.

Much research work reports the addition of antimicrobialagents to different film matrixes and their effect on the antimi-crobial activity against target pathogens. Polysaccharide-basedfilms containing antimicrobial agents that have been evaluatedfor this purpose include those prepared with starch, cellulosederivates, chitosan, alginate, and fruit-puree. Regarding protein-based films, there are available studies on those prepared withwhey protein isolated (WPI), soy protein, and soy protein iso-lated (SPI), egg albumen (EA), WG, and sodium caseinate. Fromthe broad variety of target microbes, human pathogens of thegenus Listeria, Escherichia, and Salmonella have been the mostwidely studied.

Cellulose-Based Edible Films

Some studies (Table 2) report the antimicrobial effect of nisinin hydroxypropyl methylcellulose (HPMC) films against Lis-teria monocytogenes ATCC 15313, Staphylococcus aureus IP58156, Kocuria rhizophila ATCC 9341, and Aspergillus niger.The addition of 15% stearic acid to HPMC films decreasedfilm inhibitory activity by 70 and 40% for L. monocytogenesand S. aureus, respectively. This phenomenon was explainedby electrostatic interactions between the cationic nisin and theanionic fatty acid, which decreased nisin desorption from thefilm (Sebti et al., 2002). Similarly, a 3-fold reduction of filmantimicrobial activity against K. rhizophila was observed when18% milkfat was added to the HPMC film (Sebti et al., 2007).Incorporation of chitosan to HPMC films at concentrations aslow as 0.1% (w/v) showed a complete inhibition of the fungusA. niger (Sebti et al., 2007). On the other hand, when nisin wasadded to cross-linked HPMC film (98% cross-linking level withcitric acid), no antimicrobial activity against the bacterial strainMicrococcus luteus 270 was observed (Sebti et al., 2003). Theauthors concluded that HPMC could potentially graft nisin viaester bonds from the nisin C-terminal carboxylic acid group andcellulosic hydroxyl group. In addition, the primary amine groupfrom the N-terminal position and from the lysine residues couldreact on the carboxylic function available on citric acid to formamine bonds. In both cases, nisin desorption could be stronglyreduced, limiting film antimicrobial activity.

Valencia-Chamorro et al. (2008) (Table 2) evaluated thein vitro activity against the citrus fruit postharvest pathogensPenicillium digitatum and Penicillium italicum of stand-aloneHPMC-lipid edible composite films containing food preserva-tives, such as mineral salts, organic acid salts and their mix-tures, parabens and their mixtures, and other GRAS compounds.

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3 ,104 ,1

05sp

ores

/mL

+(V

alen

cia-

Cha

mor

roet

al.,

2008

)

Am

mon

ium

bica

rbon

ate

2.0%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04sp

ores

/mL

+A

mm

oniu

mbi

carb

onat

e2.

0%P.

ital

icum

103 ,1

04 ,105

spor

es/m

L−

Pota

ssiu

mso

rbat

e,so

dium

benz

oate

2.0,

2.5%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04 ,105

spor

es/m

L+

Sodi

umac

etat

e,so

dium

diac

etat

e,ca

lciu

mpr

opio

nate

,cal

cium

form

ate,

sodi

umL

-lac

tate

,so

dium

L-t

artr

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04 ,105

spor

es/m

L−

Sodi

umpr

opio

nate

,sod

ium

form

ate,

calc

ium

form

ate

2.0,

1.0,

1.0%

P.di

gita

tum

103

spor

es/m

L+

Sodi

umpr

opio

nate

,sod

ium

form

ate,

calc

ium

form

ate

2.0,

1.0,

1.0%

P.it

alic

um10

3 ,104 ,1

05sp

ores

/mL

−

Pota

ssiu

mso

rbat

e+

sodi

umpr

opio

nate

1.5

+0.

5%P.

digi

tatu

m10

3 ,104 ,1

05sp

ores

/mL

+

Pota

ssiu

mso

rbat

e+

sodi

umpr

opio

nate

1.5

+0.

5%P.

ital

icum

103 ,1

04 ,105

spor

es/m

L−

Sodi

umbe

nzoa

te+

pota

ssiu

mso

rbat

e2.

0+

0.5%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04 ,105

spor

es/m

L+

Sodi

umbe

nzoa

te+

sodi

umpr

opio

nate

2.5

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

3 ,104

spor

es/m

L+

Sodi

umbe

nzoa

te+

sodi

umpr

opio

nate

2.5

+0.

5%P.

ital

icum

105

spor

es/m

L−

Sodi

umsa

ltof

met

hylp

arab

en1.

0,1.

5%P.

digi

tatu

m,P

.ita

licu

m10

3 ,104 ,1

05sp

ores

/mL

+So

dium

salt

ofet

hylp

arab

en,

sodi

umsa

ltof

prop

ylpa

rabe

n1.

0,1.

0%P.

digi

tatu

m,P

.ita

licu

m10

3 ,104 ,1

05sp

ores

/mL

+

Sodi

umsa

ltof

met

hylp

arab

en+

sodi

umsa

ltof

prop

ylpa

rabe

n1.

0+

0.5%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04 ,105

spor

es/m

L+

ED

TA1.

5%P.

digi

tatu

m10

3 ,104

spor

es/m

L+

ED

TA1.

5%P.

ital

icum

104 ,1

05sp

ores

/mL

−2-

deox

y-D

-glu

cosa

0.5%

P.di

gita

tum

,P.i

tali

cum

103 ,1

04 ,105

spor

es/m

L−

MC

d—

—R

hodo

toru

laru

bra,

Peni

cill

ium

nota

tum

0.1

mL

ofsu

spen

sion

−(C

hen

etal

.,19

96)

Pota

ssiu

mso

rbat

e,so

dium

benz

oate

2.0%

R.r

ubra

,P.n

otat

um0.

1m

Lof

susp

ensi

on+

MC

Nat

amyc

in1.

5m

g/10

gfil

mso

lutio

nA

.nig

er10

4sp

ores

/mL

−(T

ure

etal

.,20

09b)

1.0

mg/

10g

film

solu

tion

P.ro

quef

orti

i10

6sp

ores

/mL

+N

atam

ycin

+ro

sem

ary

extr

act

0.5

+1.

5m

g/10

gfil

mso

lutio

nA

.nig

er,P

.roq

uefo

rtii

104 ,1

06sp

ores

/mL

−

(Con

tinu

edon

next

page

)

877

Dow

nloa

ded

by [

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vers

ity L

ibra

ry U

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ht]

at 0

7:35

09

Oct

ober

201

3

Tabl

e2

Ant

imic

robi

alac

tivity

ofed

ible

poly

sacc

hari

de-b

ased

com

posi

tefil

ms

cont

aini

ngan

timic

robi

alag

ents

(Con

tinu

ed)

Film

mat

rix

Ant

imic

robi

alag

ent

Con

cent

ratio

nTa

rget

path

ogen

Path

ogen

inoc

ulat

ion

Ant

imic

robi

alac

tivity

aR

efer

ence

Nat

amyc

in+

rose

mar

yex

trac

t1.

5+

1.5

mg/

10g

film

solu

tion

A.n

iger

104

spor

es/m

L+

Chi

tosa

n-M

CPo

tass

ium

sorb

ate,

sodi

umbe

nzoa

te4.

0%R

.rub

ra,P

.not

atum

0.1

mL

ofsu

spen

sion

+(C

hen

etal

.,19

96)

Chi

tosa

n-H

PMC

——

L.m

onoc

ytog

enes

300

cells

+(M

olle

ret

al.,

2004

)C

hito

san

——

R.r

ubra

,P.n

otat

um0.

1m

Lof

susp

ensi

on−

(Che

net

al.,

1996

)Po

tass

ium

sorb

ate,

sodi

umbe

nzoa

te2.

0%R

.rub

ra,P

.not

atum

0.1

mL

ofsu

spen

sion

−

Chi

tosa

nN

isin

250

µg/

mL

A.n

iger

,K.r

hizo

phil

aA

TC

C93

4110

3 ,104

spor

es/m

L+

(Seb

tiet

al.,

2007

)C

hito

san

——

L.m

onoc

ytog

enes

37ce

llpe

rPe

trid

ish

−(C

oma

etal

.,20

02)

Chi

tosa

n—

—L

.mon

ocyt

ogen

es30

0ce

lls+

(Mol

ler

etal

.,20

04)

Chi

tosa

nG

arlic

oil

100

µL

/gS.

aure

us,L

.mon

ocyt

ogen

es,B

acil

lus

cere

us,

105 ,1

06C

FUe /m

L+

(Pra

noto

etal

.,20

05a)

400

µL

/gE

sche

rich

iaco

li,S

alm

onel

laty

phim

uriu

m10

5 ,106

CFU

/mL

−

Pota

ssiu

mso

rbat

e10

0m

g/g

S.au

reus

,L.m

onoc

ytog

enes

,B.c

ereu

s10

5 ,106

CFU

/mL

+20

0m

g/g

E.c

oli,

S.ty

phim

uriu

m10

5 ,106

CFU

/mL

−N

isin

51,0

00IU

/gS.

aure

us,L

.mon

ocyt

ogen

es,B

.cer

eus

105 ,1

06C

FU/m

L+

(Pra

noto

etal

.,20

05a)

204,

000

IU/g

E.c

oli,

S.ty

phim

uriu

m10

5C

FU/m

L−

Chi

tosa

n—

—F

usar

ium

mon

ilif

orm

e,F.

prol

ifer

atum

,Asp

ergi

llus

ochr

aceu

s10

3sp

ores

+(S

ebas

tien

etal

.,20

06)

Chi

tosa

n—

—A

.nig

er10

2sp

ores

/Pet

ridi

sh+

(Seb

tiet

al.,

2007

)Ta

pioc

a-st

arch

Pota

ssiu

mso

rbat

e0.

3%Z

ygos

acch

arom

yces

bail

ii5

×10

6C

FU/m

L+

(Flo

res

etal

.,20

07b)

Sago

star

chL

emon

gras

soi

l0.

4%E

.col

iO15

7:H

710

5 ,106

CFU

/mL

+(M

aizu

raet

al.,

2007

)Pe

ast

arch

Gra

pese

edex

trac

ts1.

0%L

.mon

ocyt

ogen

es,S

.aur

eus,

Ent

eroc

occu

sfa

eciu

m,E

.fa

ecal

is,B

roch

othr

ixth

erm

osph

acta

106

CFU

/mL

+(C

orra

les

etal

.,20

09)

20.0

%S.

typh

imur

ium

,E.c

oli

−So

dium

algi

nate

Gar

licoi

l0.

2%S.

aure

us,B

.cer

eus

105 ,1

06C

FU/m

L+

(Pra

noto

etal

.,20

05b)

Sodi

umal

gina

teL

acto

pero

xida

se2.

0%E

.col

i,L

iste

ria

inno

cua,

Pse

udom

onas

fluor

esce

nces

3—

4lo

g 10

CFU

/mL

+(Y

ener

etal

.,20

09)

0.4%

E.c

oli,

S.ty

phim

uriu

m10

5 ,106

CFU

/mL

−A

lgin

ate-

appl

epu

ree

Ore

gano

oil/

carv

acro

l0.

1%E

.col

iO15

7:H

710

5C

FU/m

L+

(Roj

as-G

rau

etal

.,20

07a)

Lem

ongr

ass

oil/

citr

al0.

5%C

inna

mon

oil/

cinn

amal

dehy

de0.

5%A

pple

pure

eO

rega

nooi

l0.

1%E

.col

iO15

7:H

710

5C

FU/m

L+

(Roj

as-G

rau

etal

.,20

06)

Lem

ongr

ass

oil

0.5%

Cin

nam

onoi

l0.

5%To

mat

opu

ree

Car

vacr

ol0.

75%

E.c

oliO

157:

H7

105

CFU

/mL

+(D

uet

al.,

2008

)

a Ant

imic

robi

alac

tivity

:‘+

’=

inhi

bitio

n;‘−

’=

noin

hibi

tion.

b HPM

C=

hydr

oxyp

ropy

lmet

hylc

ellu

lose

.c IU

=in

tern

atio

nalu

nits

;1µ

gco

rres

pond

sto

40IU

.d M

C=

met

hylc

ellu

lose

.e C

FU=

colo

nyfo

rmin

gun

its.

878

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at 0

7:35

09

Oct

ober

201

3

Tabl

e3

Ant

imic

robi

alac

tivity

ofed

ible

prot

ein-

base

dco

mpo

site

film

sco

ntai

ning

antim

icro

bial

agen

ts

Film

mat

rix

Ant

imic

robi

alag

ent

Con

cent

ratio

nTa

rget

path

ogen

Path

ogen

inoc

ulat

ion

Ant

imic

robi

alac

tivity

Ref

eren

ce

WPI

aSo

rbic

acid

0.75

%L

iste

ria

mon

ocyt

ogen

es,E

sche

rich

iaco

liO

157:

H7,

Salm

onel

laty

phim

uriu

mD

T10

4

0.1

mL

+(C

agri

etal

.,20

01)

p-am

imob

enzo

icac

id0.

75%

L.m

onoc

ytog

enes

,E.c

oliO

157:

H7,

S.ty

phim

uriu

mD

T10

40.

1m

L+

WPI

Lac

tofe

rrin

0.1

g/g

Peni

cill

ium

com

une

105

spor

es−

(Min

and

Kro

chta

,200

5)L

acto

ferr

inhy

drol

ysat

e0.

1g/

g−

Lac

tope

roxi

dase

59m

g/g

film

+W

PIL

acto

pero

xida

se0.

5%L

.mon

ocyt

ogen

es10

3C

FUb /m

L+

(Min

etal

.,20

05b)

WPI

Lac

tope

roxi

dase

3.0%

Salm

onel

laen

teri

ca,E

.col

iO15

7:H

710

8C

FU+

(Min

etal

.,20

05a)

WPI

Ore

gano

oil

2.0%

E.c

oliO

157:

H7,

Stap

hylo

cocc

usau

reus

,Sa

lmon

ella

ente

ridi

tis,

L.

mon

ocyt

ogen

es,L

acto

baci

llus

plan

taru

m

108

CFU

/mL

+(S

eydi

man

dSa

riku

s,20

06)

Gar

licoi

l3.

0%E

.col

iO15

7:H

7,S.

aure

us,S

.en

teri

diti

s,L

.mon

ocyt

ogen

es,L

.pl

anta

rum

+

Ros

emar

yoi

l4.

0%E

.col

iO15

7:H

7,S.

aure

us,S

.en

teri

diti

s,L

.mon

ocyt

ogen

es,L

.pl

anta

rum

−

WPI

,SPI

c ,EA

d ,W

Ge

Nis

in4.

0IU

f /film

disk

L.m

onoc

ytog

enes

103

CFU

/g+

(Ko

etal

.,20

01)

SPI

Nis

in+

citr

icac

id20

5IU

/gpr

otei

n+

2.6%

L.m

onoc

ytog

enes

,Sal

mon

ella

gam

inar

a,E

.col

iO15

7:H

710

6C

FU+

(Esw

aran

anda

met

al.,

2004

)

Nis

in+

lact

icac

idN

isin

+m

alic

acid

Nis

in+

tart

aric

acid

SPI

Gra

pese

edex

trac

ts1.

0%L

.mon

ocyt

ogen

es10

6C

FU/m

L+

(Siv

aroo

ban

etal

.,20

08)

Nis

in10

,000

IU/g

Sodi

um-E

DTA

g0.

16%

Gra

pese

edex

trac

ts1.

0%S.

typh

imur

ium

,E.c

oliO

157:

H7

106

CFU

/mL

−G

rape

seed

extr

acts

+ni

sin

+so

dium

-ED

TA

1.0%

+10

,000

IU/g

+0.

16%

L.m

onoc

ytog

enes

,S.t

yphi

mur

ium

,E.c

oli

O15

7:H

7+

Sodi

umca

sein

ate

Sodi

umla

ctat

e40

.0%

L.m

onoc

ytog

enes

102

CFU

/cm

2+

(Kri

sto

etal

.,20

08)

Pota

ssiu

mso

rbat

e25

%N

isin

0.07

5%

a WPI

=w

hey

prot

ein

isol

ated

.b C

FU=

colo

nyfo

rmin

gun

its.

c SPI=

soy

prot

ein

isol

ated

.d E

A=

egg

albu

men

.e W

G=

whe

atgl

uten

.f IU

=in

tern

atio

nalu

nits

;1µ

gco

rres

pond

sto

40IU

.g so

dium

-ED

TA=

sodi

umet

hyle

nedi

amin

etet

raac

etat

e.

879

Dow

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by [

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7:35

09

Oct

ober

201

3

880 S. A. VALENCIA-CHAMORRO ET AL.

These food additives had shown antifungal activity against thesepathogens when applied as aqueous solutions (Palou et al., 2002;Montesinos-Herrero et al., 2009). The determination of the an-tifungal activity of the films was based on the agar diffusiontest, in which the diameter of the inhibition area surrounding afilm disk placed on contaminated agar media was measured andcompared to that observed with control films prepared withoutthe corresponding food preservative. Films containing sodiumsalt of parabens at a concentration of 1% or their mixtures(1.5%) were the most effective in inhibiting the growth of bothP. digitatum and P. italicum. Among all organic acid salts tested,only films containing PS (2%) and SB (2.5%) clearly inhibitedthe growth of both pathogens. Surprisingly, no additive or syn-ergistic effects for mold inhibition were observed with filmscontaining mixtures of food preservatives if compared to theuse of single antifungal compounds. It was reported that theantimicrobial activity of the films containing food preservativeswere strongly influenced by the type of antimicrobial compound(size, shape, and polarity), its concentration, and the nature ofthe film matrix.

There are few studies in the literature on methylcellulose(MC) films containing antimicrobial agents. MC films contain-ing 2% organic acid salts, PS, or SB, were very active againstthe microorganisms Rodotorula rubra and Penicillium notatum,providing clear inhibitory zones around film disks plated in agarmedia (Chen et al., 1996). The addition of natamycin and rose-mary extract, alone or in combination, to MC films showeddifferent antimicrobial effects against A. niger and Penicilliumroquefortii. The minimum inhibitory concentration (MIC) val-ues of natamycin were 2 and 1 mg per 10 g of film solu-tion against A. niger and P. roquefortii, respectively. Rosemaryextract did not show any inhibitory antifungal activity alone;however, it acted synergistically with natamycin to prevent thegrowth of A. niger. Thus, although concentrations of natamycinof 1.5 mg per 10 g of film solution were not effective against A.niger, the combination of this compound at this concentrationwith rosemary extract satisfactorily inhibited the growth of thismold (Ture et al., 2009b).

Chitosan-Based Edible Films

Chitosan ability to form edible films and its antimicrobialactivity against a broad spectrum of microbes makes it one ofthe most studied biopolymers. Results show that the antimicro-bial activity of chitosan films depends primarily on the strainof the target microorganism and the assay conditions (Table 2).Coma et al. (2002) reported no inhibition from chitosan films de-posited on agar medium inoculated with L. monocytogenes after24 hours of incubation. However, chitosan films showed 100%inhibition of L. monocytogenes for at least 8 days when the bac-tericidal activity was measured by epifluorescence techniques.The authors stated that under the conditions tested, chitosanwas incapable of diffusing through the adjacent agar medium,indicating the importance of the test methodology. These re-searchers also observed a decrease in the antibacterial effect of

chitosan with time, which was attributed to a decrease in theavailability of amino-groups of chitosan. Moller et al. (2004)reported that a minimum of 1% chitosan content was requiredin chitosan films to maintain a significant anti-listerial activityusing an agar plate method, whereas the kind of solvent (water,aqueous acetic acid, and ethanol) used to prepare the film didnot influence the anti-listerial activity of the chitosan films.

Chitosan films containing 2% of PS or SB did not clearlyinhibit the growth of R. rubra and P. notatum in agar diffu-sion tests (Chen et al., 1996). These authors concluded thatthe interaction between chitosan and the preservatives inhibitedtheir release. An increase in the concentration of preservativesto 5% resulted in clear inhibitory zones in the agar with chi-tosan films, indicating that the binding sites for additives werepresumably saturated at this high concentration. Pranoto et al.(2005a) improved the antimicrobial activity of chitosan filmsagainst pathogenic bacteria through the addition of antimicro-bial agents, such as garlic oil, PS and nisin. The addition ofgarlic oil up to levels of at least 100 µL/g of chitosan, PS at100 mg/100 g, or nisin at 51,000 IU/g revealed an importantantimicrobial effect against S. aureus, L. monocytogenes, andBacillus cereus. However, these films did not show inhibitoryactivity against Escherichia coli and Salmonella typhimurium.They suggested that this behavior was due to the higher sen-sitivity of Gram-positive bacteria to the antimicrobial agents.Chitosan films have also been effective for the control of moldssuch as Fusarium moniliforme, Fusarium proliferatum, and As-pergillus ochraceus. A combination of chitosan and polylacticacid also presented considerable antifungal activity (Sebastienet al., 2006).

Several workers have satisfactorily modified chitosan activityby means of blending with other polymers to improve the filmphysical properties. The similarity of cellulose and chitosan inprimary structures has facilitated the formation of homogeneouscomposite films. Moller et al. (2004) reported that 1% of chi-tosan in chitosan-HPMC composite films was effective againstL. monocytogenes. The incorporation of stearic acid into thefilm-forming solution did not influence the anti-listerial activityof the film. However, when films were cross-linked with citricacid the anti-listerial activity of the film was lost, probably dueto a chemical reaction of the amino group, which is eventu-ally responsible for the anti-listerial activity. In another study,chitosan-MC films containing 4% PS or SB induced clear in-hibitory zones in the agar medium around the film disk whentested against R. rubra and P. notatum (Chen et al., 1996).

Starch-Based Edible Films

Different types of starch-based films containing antimicro-bial agents such as PS or natural compounds like lemongrassor grape seed extracts (GSE) have shown antimicrobial activ-ity against different pathogens (Table 2). Sago starch-alginatefilms containing lemongrass oil at concentrations of 0.1 to0.4% exhibited a clear inhibition zone around the film diskagainst E. coli. The zone of inhibition increased significantly as

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ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 881

lemongrass oil content increased in the presence of glycerol.This result was attributed to the increased solubility of lemon-grass oil in the matrix and the consequent more uniform disper-sion of the oil in the film (Maizura et al., 2007).

Pea starch films containing GSE greatly inhibited the growthof all Gram-positive bacteria tested, whereas the films were noteffective against the Gram-negative bacteria S. typhimurium andE. coli (Corrales et al., 2009). Bacterial growth inhibition wasprobably due to the presence of secondary metabolites such aspolyphenols in the seed extracts. These researchers assumed thatthese polyphenols could penetrate the semipermeable Gram-positive bacterial membrane reacting in the cytoplasm with cel-lular proteins. In contrast, the lipidic wall of Gram-negative bac-teria represented an impassable barrier for extracted polyphenolsto get into the cytoplasm.

Alginate-Based Edible Films

Sodium alginate films containing lactoperoxidase or garlicoil presented significant antimicrobial activity against differentpathogenic bacteria (Table 2; Pranoto et al., 2005b; Yener etal., 2009). However, the incorporation of garlic oil to sodiumalginate films reduced the antimicrobial effect of garlic oil ap-plied alone. At 0.1% (v/v), garlic oil in the nutrient broth de-creased viable cell counts of B. cereus, S. aureus, E. coli, andS. typhimurium by 5.61, 4.30, 2.28, and 1.24 log cycles, respec-tively. When garlic oil was incorporated to sodium alginatedfilms, a concentration of 0.2% was needed in agar diffusiontest to observe a clear inhibitory zone against S. aureus andB. cereus; while even at a concentration of 0.4% garlic oil thegram-negative bacteria S. typhimurium and E. coli were not ef-fectively inhibited. These results were consistent with an in vitrotest in nutrient broth, in which E. coli and S. typhimurium weremore resistant to garlic oil than S. aureus and B. cereus (Pranotoet al., 2005b).

Fruit-Based Edible Films

Different fruit-based films prepared with plant essential oilsor their major constituents have been effective to control micro-bial growth (Table 2). In recent work, edible tomato films con-taining carvacrol were effective to inhibit the microbial growthof E. coli. Antimicrobial assays with tomato films indicated thatcarvacrol levels were approximately of 0.75% when added totomato purees before film preparation. HPLC analysis of thefilms indicated that the carvacrol concentration and bactericidalactivity of the films remained unchanged over a storage periodof up to 98 days at 5 or 25◦C (Du et al., 2008). In other research,the antimicrobial activities of oregano essential oil in applepuree edible films, against E. coli O157:H7 were significantlyhigher than that of cinnamon or lemongrass oils (Rojas-Grau etal., 2006). Similar results were reported by this research groupwith alginate-apple puree edible films containing plant essen-tial oils such as oregano oil, cinnamon oil, or lemongrass oil,or oil compounds such as carvacrol, cinnamaldehyde, or citral.

Among all of them, carvacrol and oregano oils exhibited thestrongest antimicrobial activity against E. coli at a concentra-tion of 0.1%, whereas a concentration as high as 0.5% of theother compounds was required to inhibit the microbial growthon agar plates (Rojas-Grau et al., 2007a).

Protein-Based Edible Films

Among films prepared with proteins, WPI films have beentested with a great number of antimicrobial agents includingorganic acids and their salts, polypeptides, essential oils, natu-ral extracts, and other antimicrobial compounds (Table 3). WPIfilms containing sorbic acid or p-aminobenzoic acid were ef-fective against L. monocytogenes, E. coli, and S. typhimurium(Cagri et al., 2001). In general, as the concentration of the an-timicrobial agent in the film increased (range 0.5–1.5%), theactivity of the film in agar diffusion tests also increased. Sincethe undissociated form of weak acids at low pH increased theability to penetrate the cytoplasmatic membrane of the bacteria,a pH adjustment to 5.2 using lactic or acetic acids significantlyincreased the antimicrobial effect of these films. According todifferent research (Min et al., 2005a,b; Min and Krochta, 2005;Seydim and Sarikus, 2006), WPI films presented high antimicro-bial activity against P. comune, L. monocytogenes, Salmonellaenterica, and E. coli irrespective of the concentration of polypec-tic antimicrobials like lactoperoxidase or lactoferrin, and the mi-crobial test used. Furthermore, WPI films containing oregano orgarlic oils were effective against E. coli, S. aureus, Salmonellaenteriditis, L. monocytogenes, and Lactobacillus plantarum,while films containing rosemary oil had no activity against thesame pathogenic bacteria (Seydim and Sarikus, 2006).

Ko et al. (2001) studied the effect of nisin incorporated tofilm protein matrixes such as WPI, SPI, EA or WG. Amongthe tested film matrixes, WPI films containing nisin were themost effective in reducing L. monocytogenes counts, whereasWG films showed the lowest antimicrobial activity. These re-sults correlated with film surface hydrophobicity, indicating thatnisin was more active for inhibition of L. monocytogenes in hy-drophobic films, such as those made with WPI, than in lesshydrophobic films, such as those prepared with WG. As nisinconcentration increased from 4.0 to 160 IU per film disk, theinhibitory activity of all tested films progressively increased. Inaddition, edible films containing nisin in an acidic environmentexerted a greater inhibitory effect against the pathogens becausenisin is more active at acidic conditions (Klaenhammer, 1993).

The incorporation of nisin and organic acids (citric, lac-tic, malic, or tartaric acid) to SPI films was tested in orderto improve the film antimicrobial activity against L. monocyto-genes, Salmonella gaminara, and E. coli O157:H7 (Eswaranan-dam et al., 2004). The antimicrobial activity of these filmswas expressed in terms of the inhibition zone in agar platesand the log number of survivors. With SPI films, only L.monocytogenes was inhibited by the combined effect of nisinand organic acids at all concentrations tested (range 0.9–2.6%(w/w)) and S. gaminara and E. coli were only inhibited by the

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882 S. A. VALENCIA-CHAMORRO ET AL.

combination of nisin with citric, malic or tartaric acids at con-centrations of 1.8 or 2.6%. Lactic acid, however, only slightlyinhibited S. gaminara and E. coli. On the other hand, films with2.6% organic acid without nisin similarly inhibited L. monocy-togenes and their anti-salmonella activity was lower than thatof the nisin-organic acid combination. In a recent work, nisin,GSE, ethylenediaminetetraacetic acid (EDTA) and their com-binations were added to SPI films and tested against L. mono-cytogenes, S. typhimurium, and E. coli O157:H7 (Sivaroobanet al., 2008). L. monocytogenes was more sensitive to variouscombinations of antimicrobials than the other two pathogens.The authors pointed out that both phenolics and nisin act uponthe cytoplasmic membrane of the bacteria, thus the additive orsynergistic effect of combinations of these compounds couldenhance the inhibitory activity against L. monocytogenes. Thelower inhibitory activity of combined GSE and nisin againstS. typhimurium and E. coli O157:H7 might be related with thestructure of the cell membrane.

Barrier and Mechanical Properties of Antimicrobial EdibleFilms

The barrier and mechanical properties of films depend ba-sically on intrinsic factors such as film composition, thickness,and preparation techniques, but also on other secondary fac-tors like the test conditions. Therefore, the incorporation ofadditional ingredients including antimicrobial food additives orother agents into edible films may cause significant changeson the mechanical and barrier properties that need to be ex-amined when new films are developed (Greener-Donhowe andFennema, 1994). Tables 4 and 5 show the mechanical and bar-rier properties of edible films containing antimicrobial agents.Properties of controls refer to those of the same films preparedwithout the addition of antimicrobial agents. Considering all thedifferent factors that affect film properties, it is very difficult tocompare the performance of different films in different researchstudies. However, because such comparison is important, it willbe made when possible in this review.

In the literature, polysaccharide-based edible films contain-ing antimicrobial agents presented a wide range of WVP values(1–177 g mm/m2 day kPa) depending on film composition andRH gradient. Some HPMC, chitosan, alginate, or sago starchfilms presented WVP values lower than 35 g mm/m2 day kPa ata �RH around 50–0% or 0–100%. WVP values were higher insome HPMC-lipid, MC, pea starch or tomato puree films (50–90g mm/m2 day kPa), and the highest WVP values were those ofHPMC-lipid films containg PS, alginate-apple puree, and applepuree films (>120 g mm/m2 day kPa) (Table 4). The high WVPvalues for MC films could be related to high RH during the WVPmeasurements (50–100%), since the WVP of hydrophilic filmsis highly influenced by RH. However, alginate-apple puree filmspresented a high WVP, even at �RH similar to other polysac-charide films. Differences among HPMC-lipid films may be dueto either interactions between the antimicrobial agent and the

film matrix or small differences in film composition. Similarlyto WVP, the mechanical properties YM, TS, and E of any filmclearly depend on the type of film matrix and film composition.It is usual to find in the literature that edible films containingantimicrobial agents had YM, TS, and E values lower than 30MPa, 60 MPa, and 74%, respectively. Exceptions are tomatopuree-, MC-, or some HPMC-lipid based films that had YMvalues higher than 130 MPa, or pea starch films with TS valueshigher than 500 MPa.

Cellulose-Based Edible Films

Sebti et al. (2002; 2003) studied the effect of stearic acidand the degree of cross-linking level of HPMC-nisin films onthe film mechanical properties and WVP. Citric acid was usedas the cross-linking agent to produce films with 0–98% cross-linking level. The addition of 15% (w/w) of stearic acid im-proved the film moisture barrier, but reduced the mechanicalresistance with a decrease in film elasticity and extensibility.The negative effect of lipid addition to different polymer ma-trixes has been repeatedly observed in many research works andit is usually attributed to the partial replacement of the polymerby the lipids in the film matrix, which flavors the disruption ofthe film. Contrary to what was expected, an increase in cross-linking decreased YM, which might be explained by a higherheterogeneity of the space between cross-links related to the es-ter bond rate that induced the formation of cracks and the worstmechanical properties of cross-linked films.

Valencia-Chamorro et al. (2008) showed that the barrier andmechanical properties of antimicrobial HPMC-lipid compositefilms depended on lipid composition (BW and shellac propor-tions) and the properties of the food preservative. HPMC-lipidfilms containing PS alone or combined with sodium propionate(SP) exhibited higher WVP than similar films containing othersalts of organic acids, concluding than PS modified the HPMC-lipid film structure in a greater extend than the other saIts tested.On the other hand, films containing parabens had the lowestWVP, which was attributed to interactions of parabens with thepolymer matrix. The differences in film mechanical propertieswere mainly related to the different chemical structure of thefood preservatives used as antimicrobial ingredients. Films con-taining PS and the mixture PS + SP showed lower YM andTS and higher E values than the rest of films, which indicateda higher degree of film flexibility. In contrast, the addition ofparabens resulted in an important increase of YM and TS valuesand a reduction of E, reducing flexibility and conferring stiff-ness to the films. The higher flexibility of films containing PSwas attributed to the straight chain molecular structure of thiscompound, which allows it to penetrate more easily into the filmmatrix than parabens (benzene rings), conferring more mobilitybetween the HPMC chains.

The essential oil of Melaleuca alternifolia, also known astea tree essential oil (TTO), has been investigated as a possibleantimicrobial agent, showing a wide spectrum of action againstfungi, yeasts, viruses, and bacteria. Sanchez-Gonzalez et al.

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Tabl

e4

Mec

hani

cala

ndba

rrie

rpr

oper

ties

ofpo

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com

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ms

cont

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Mec

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ties

Wat

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Film

mat

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Ant

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alag

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Con

cent

ratio

nY

Ma

(MPa

)T

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Pa)

Ec

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Pe(g

mm

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day

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�R

Hf

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(cm

3

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Ref

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ce

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7

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CC

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)C

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30.0

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mor

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)So

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—(S

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.,20

06)

(Con

tinu

edon

next

page

)

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ober

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3

Tabl

e4

Mec

hani

cala

ndba

rrie

rpr

oper

ties

ofpo

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ride

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com

posi

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cont

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Mec

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ober

201

3

Alg

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a YM

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e WV

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perm

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f �R

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rela

tive

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grad

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i Con

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.

885

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ober

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3

Tabl

e5

Mec

hani

cala

ndba

rrie

rpr

oper

ties

ofpr

otei

n-ba

sed

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mpo

site

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icro

bial

agen

ts

Mec

hani

calp

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Film

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imic

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Con

cent

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ce

WPI

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trol

——

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.855

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oet

al.,

2001

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isin

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mL

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,200

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ate

10%

44.4

3.6

57.2

239.

3W

PIC

ontr

ol—

—24

4.1

—34

.855

–100

—(K

oet

al.,

2001

)N

isin

0.2

mg/

mL

—24

4.4

—38

.2SP

IC

ontr

ol—

—8.

6—

41.3

Nis

in0.

2m

g/m

L—

10.4

—42

.5E

AC

ontr

ol—

—1.

8—

57.8

Nis

in0.

2m

g/m

L—

1.4

—52

.8W

GC

ontr

ol—

—1.

8—

63.4

Nis

in0.

2m

g/m

L—

2.0

—51

.6W

PIC

ontr

ol—

21.9

2.3

147.

2—

—22

8.8

(Min

and

Kro

chta

,20

05)

Lac

tope

roxi

dase

59(m

g/g

film

)23

.22.

314

0.3

231.

7W

PIC

ontr

ol—

25.8

1.1

119.

5—

—27

0.1

(Min

etal

.,20

05a)

Lac

tope

roxi

dase

150

(mg/

gfil

m)

17.2

2.1

129.

514

1.0

WG

Con

trol

—28

.82.

122

4.8

164.

450

–100

—(T

ure

etal

.,20

09a)

Sodi

umca

sein

ate

Con

trol

—24

00.0

63.0

3.0

1.4

53–7

9—

(Kri

sto

etal

.,20

08))

Sodi

umla

ctat

e10

%14

00.0

38.0

3.0

2.8

53–7

2Po

tass

ium

sorb

ate

40%

250.

08.

020

.09.

453

–60

10%

2350

.070

.05.

02.

353

–75

Sodi

umca

sein

ate

Nis

in25

%90

0.0

28.0

28.0

3.3

53–7

1—

(Kri

sto

etal

.,20

08)

0.07

5%22

00.0

63.0

4.0

1.4

53–7

80.

0075

%—

——

1.6

53–7

6

Con

trol

sar

efil

ms

with

outa

ntim

icro

bial

agen

t.A

bbre

viat

ions

are

thos

ede

scri

bed

inTa

bles

3an

d4.

886

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3

ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 887

(2009) studied the effect of this essential oil on HPMC-basededible films. Results showed that the higher the TTO content, thelower the WVP and the moisture sorption capacity. In general,the addition of TTO into the HPMC matrix led to a significantdecrease in gloss and transparency and a decrease in the tensilestrength and elastic modulus of the composite films. The prop-erties of the films were related to their microstructure, whichshowed that the presence of TTO caused discontinuities associ-ated with the formation of two phases in the matrix, whereas acontinuous structure was observed for the HPMC film.

The addition of low concentrations of natamycin to MC filmsslightly increased YM and decreased E values, while it did notmodify TS value. Similarly, TS was not affected by the addi-tion of low concentrations of natamycin plus rosemary extract.However, the incorporation of high concentrations of natamycin(10 or 20 mg per 10 g of film solution) resulted in a significantdecrease of film TS (Ture et al., 2009a). These changes wereattributed to the weakening of some of the chemical bonds inthe polymer structure. In another study, the mechanical prop-erties of MC-chitosan films did not significantly change by theaddition of PS or SB (Chen et al., 1996).

Chitosan-Based Edible Films

In chitosan films (Table 4), WVP values increased as the con-centration of the antifungal ingredients PS or nisin increased,while an increase of garlic oil (up to 400 µL/g of chitosan)did not affect the film WVP (Pranoto et al., 2005a). The addi-tion of PS and nisin contributed to extend the intermolecularinteraction and lose the compactness of the structure, which en-hanced moisture diffusion through the film. Nevertheless, thisbehavior was not observed when garlic oil was added to thechitosan film, probably due to its hydrophobic character. In thesame experiments, the addition of PS or nisin produced higherreduction of TS than the incorporation of garlic oil. The au-thors confirmed that the incorporation of additives other thancross-linking agents generally reduced TS values. In contrast, Evalues increased with addition of PS or nisin to chitosan films,such increase being higher with nisin than with PS. Similarly toTS, garlic oil did not significantly affect E value.

Starch-Based Edible Films

Among the research conducted with antimicrobial starch-based edible films (Table 4), a recent work by Corrales et al.(2009) showed that GSE added to pea starch films significantlyincreased film E and decreased film TS by 50%. This was at-tributed to the chemical disposition of flavonoids and phenolicacids from GSE with amylose chains that lose intermolecularinteractions of starch because of repulsive charges of the GSEacids. Film WVP did not significantly change if compared tothe control film. However, the reduced polarity of these films,due to the minor polarity of the GSE compounds, acceleratedthe absorption of oxygen to the film surface, resulting in anincrease in OP if compared to control films.

Flores et al. (2007a) observed no effect on WVP as PS wasincorporated to tapioca-starch-glycerol edible films, whereasfilm YM and E decreased. These workers reported that the gela-tinization/drying method used to prepare the films significantlyaffected the barrier and the mechanical and antimicrobial sta-bility of the films. It was concluded that short gelatinization anddrying times were optimal for producing films of better antimi-crobial stability. However, these films showed poor mechanicaland moisture barrier properties.

Alginate-Based Edible Films

The addition of increasing amounts of garlic oil to alginate-based edible films greatly modified the film mechanical andbarrier properties. TS and E values were reduced by incorpo-ration of garlic oil at 0.3 and 0.4% (v/v), respectively (Table4). Considering that unpeeled films were dipped in a calciumchloride solution to help to form a network between polymerchains, the presence of garlic oil in the alginate film probablyinterfered with ionic interactions facilitated by calcium ions,causing an important reduction of TS at the higher garlic oilconcentration. WVP values of the films were not affected bygarlic oil incorporation at a concentration range of 0.1–0.3%.However, film WVP was significantly higher with a garlic oilconcentration of 0.4%. In spite of the hydrophobic character ofgarlic oil, the increase in film WVP was attributed to an exten-sion of the intermolecular interactions in the structural matrix,which enhanced moisture diffusion through the film (Pranoto etal., 2005b).

In alginate-apple puree films, the presence of plant essentialoils or oil compounds did not significantly affect WVP andOP of the films, but modified tensile properties. Only a slightdecrease in WVP was reported with the addition of 0.5% (v/v)cinnamaldehyde (Rojas-Grau et al., 2007a). Since water vaportransfer generally occurs through the hydrophilic portion ofthe film and depends on the hydrophilic-hydrophobic ratio ofthe film components, these authors suggested that the amountof essential oils or oil compounds were not enough to reducefilm WVP. In general, the addition of antimicrobial agents tofilms significantly reduced TS and increase E values. In thisresearch, films containing oregano oil and carvacrol presentedlower TS values, and films with carvacrol had the highest Evalue. YM was reduced in all films containing essential oilsor oil compounds, but no significant differences were reportedamong films. In other work by this group with apple pureeedible films (no alginate), they reported that the addition ofessential oils decreased WVP and increased OP, but did notsignificantly alter the tensile properties of the films even thoughthe concentrations were similar to those reported in previoustrials (Rojas-Grau et al., 2006). While the effect of essential oilson WVP was important for oregano oil, which at a concentrationof 0.1% (w/w) induced a significant decrease in film WVP,it was not for lemongrass and cinnamon oils, with which aconcentration of 0.5% (w/w) was required to reduce WVP. Theeffect of the incorporation of essential oils on film OP can be

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888 S. A. VALENCIA-CHAMORRO ET AL.

explained by their nonpolar character, which makes them lesseffective oxygen barriers.

Fruit-Based Edible Films

Du et al. (2008) reported two different film casting methods(batch- and continuous-cast) to develop tomato puree ediblefilms with the addition of carvacrol as antimicrobial agent (Ta-ble 4). YM and TS decreased with the addition of carvacrol.Batch-cast films presented lower YM and TS and higher Evalues than continuous-cast tomato films. To explain this be-havior in the batch-cast films, the authors pointed out that thecontinuous-cast films had a higher density. WVP was signifi-cantly higher for batch-cast films than for continuous-cast films.The addition of carvacrol increased WVP of batch-cast films.The differences were attributed to the higher casting temper-atures for continuous-cast films that increased the evaporationof both carvacrol and water, reducing the amount of interstitialspaces for molecular diffusion.

Protein-Based Edible Films

Mechanical and barrier properties of edible films based onthe addition of antimicrobial agents to proteins WPI, SPI, EA,or WG have been reported (Table 5). Among them, WPI hasbeen more extensively studied as a structural matrix for antimi-crobial edible films. Cagri et al. (2001) investigated the effectof incorporating p-aminobenzoic acid or sorbic acid on me-chanical and barrier properties of WPI films. The addition ofp-aminobenzoic acid or sorbic acid increased E and decreasedTS. Films containing sorbic acid presented lower TS and higherE than those containing p-aminobenzoic acid. It was suggestedthat the straight chain of sorbic acid could more easily pen-etrate into WPI films than p-aminobenzoic acid, which has abenzene ring. Consequently, sorbic acid may allow higher mo-bility between WPI chains resulting in lower YM and TS andgreater flexibility of the films. Film WVP increased with theaddition of p-aminobenzoic acid or sorbic acid, probably due tothe hydrophilic character of both antimicrobial agents. More-over, these compounds weakened chain packing in the film toproduce a looser structure, which increased water mobility.

The addition of 59 mg of dry basis lactoperoxidase systemper g of WPI film, which exhibited the most efficient inhibitioneffects in a microbial test, did not significantly modify the filmmechanical properties and OP, suggesting that the lactoperox-idase system did not change the structure of WPI films (Minand Krochta, 2005). Nevertheless, an increase of lactoperoxi-dase system higher than 0.15 g/g film (dry basis) in WPI filmsworsened the tensile properties and improved the oxygen bar-rier properties, suggesting the formation of protein aggregatesin lactoperoxidase system-WPI films due to the presence of glu-conolactone (Min et al., 2005a). Ozdemir and Floros (2008a;2008b) studied the effect of plasticizer (sorbitol), lipid (BW),and antimicrobial agent (PS) concentrations on the mechanical,barrier, optical, and sensory properties of WPI films. Film WVP

decreased as protein and BW concentration increased, but itincreased as sorbitol and PS concentration also increased. Onthe other hand, YM, TS, and E were significantly influencedby protein, sorbitol, and PS concentrations. As in other ediblefilms, the addition of PS decreased YM and TS and increasedE. The curvilinear increasing of E with increasing PS was con-sidered as an indicator of the fact that PS interacted with somecomponents in the mixture.

Ko et al. (2001) studied the effect of nisin on mechanical andbarrier properties of different protein film matrixes (WPI, SPI,EA, or WG). Theoretically, a decrease in the WVP of the proteinfilms was expected due to the hydrophobic character of nisin.However, film WVP was not affected by nisin addition, whichmight be due to the low concentration of nisin incorporated intothe film forming solution. The addition of nisin only affectedthe TS of WPI films, whereas no effect on SPI, EA, or WGwas observed. The increase in TS of WPI films was attributedto possible rearrangements of disulfide and hydrophobic bonds,more protein-protein interactions, or the electrostatic interactionbetween molecules of nisin and protein. The lower hydropho-bicity of the other protein films compared to that of WPI filmsmay have resulted in a lower number of potential hydrophobicbonds between nisin and protein molecules, which may explainthe differences in mechanical properties between WPI and theother protein films. The incorporation of natamycin into WGfilms was studied by Ture et al. (2009a). They concluded thatthe antimicrobial did not cause major changes on the mechanicalproperties of the films. However, the incorporation of a mixtureof natamycin and rosemary extract into the films decreased TSand E, whereas WVP was not affected by the addition of theseantimicrobial agents alone or in combination.

Kristo et al. (2008) investigated the effect of the additionof an increasing concentration of sodium lactate, PS, and nisinon the mechanical and barrier properties of sorbitol-plasticizedsodium caseinate films. The addition of sodium lactate (0–40%dry basis) and PS (0–25% dry basis) to the films increased filmWVP. Films containing PS presented lower WVP than filmswith sodium lactate. The addition of an increasing concentrationof both antimicrobials to sodium caseinate films resulted in areduction of YM and TS, and an increase of E, suggesting thatboth antimicrobials acted as a plasticizer for those films. Incontrast, the addition of nisin did not cause significant changesin WVP or the tensile properties of sodium caseinate films,probably due to the low concentration of nisin in the films.

ANTIMICROBIAL EDIBLE COMPOSITE COATINGSAPPLIED TO FRUITS AND VEGETABLES

Nowadays, many commercial edible coatings for use on freshand fresh-cut fruits and vegetables are available on the marketto reduce weight loss, physiological disorders, and maintainproduce quality. Most of them are assigned to maintain thequality of citrus and apples and, to a lesser extent, mangoes,papayas, pomegranates, avocados, or tomatoes (Olivas et al.,

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3

Tabl

e6

Ant

imic

robi

aled

ible

com

posi

teco

atin

gsap

plie

dto

fres

hor

min

imal

lypr

oces

sed

frui

tsan

dve

geta

bles

Hor

ticul

tura

lpro

duct

Coa

ting

Ant

imic

robi

alag

ent

Con

cent

ratio

nTa

rget

path

ogen

Path

ogen

inoc

ulat

ion

Ant

imic

robi

alac

tivity

Ref

eren

ce

CIT

RU

SFR

UIT

S“V

alen

cia”

oran

ges

Shel

lac

(pH

=7.

3)E

than

ol12

.0%

Esc

heri

chia

coli

,E

nter

obac

ter

aero

gene

s10

6C

FU/c

m2

+(M

cGui

rean

dH

agen

mai

er,

2001

)Sh

ella

c(p

H=

9.0)

Eth

anol

5.2%

E.c

oli,

E.a

erog

enes

106

CFU

/cm

2+

Shel

lac

(pH

=9.

0)Pa

rabe

n0.

1%E

.col

i,E

.aer

ogen

es10

6C

FU/c

m2

+

“Val

enci

a”or

ange

sH

PMC

-lip

idPo

tass

ium

sorb

ate

2.0%

Peni

cill

ium

digi

tatu

m,P

enic

illi

umit

alic

um

105

spor

es/m

L+

(Val

enci

a-C

ham

orro

etal

.200

9a,b

)So

dium

benz

oate

2.5%

+C

alci

umpr

opio

nate

,cal

cium

form

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

Pota

ssiu

mso

rbat

e+

sodi

umpr

opio

nate

1.5

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+

Sodi

umbe

nzoa

te+

pota

ssiu

mso

rbat

e2.

0+

0.5%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L+

Sodi

umbe

nzoa

te+

sodi

umpr

opio

nate

2.5

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+

Sodi

umm

ethy

lpar

aben

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L+

2-de

oxy-

D-g

luco

se0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

–“O

rtan

ique

”H

PMC

-lip

idSo

dium

bica

rbon

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

hybr

idm

anda

rins

Pota

ssiu

mso

rbat

e2.

0%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+So

dium

benz

oate

2.5%

+So

dium

acet

ate,

sodi

umdi

acet

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

Sodi

umpr

opio

nate

,Sod

ium

form

ate

2.0,

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

Pota

ssiu

mso

rbat

e+

sodi

umpr

opio

nate

1.5

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+

Sodi

umbe

nzoa

te+

pota

ssiu

mso

rbat

e2.

0+

0.5%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L+

Sodi

umbe

nzoa

te+

sodi

umpr

opio

nate

2.5

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+

Sodi

umsa

ltof

met

hylp

arab

en1.

0%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+So

dium

salt

ofm

ethy

lpar

aben

+so

dium

salt

ofpr

opyl

para

ben

1.0%

P.di

gita

tum

105

spor

es/m

L+

ED

TA1.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

–2-

deox

y-D

-glu

cose

0.3%

P.di

gita

tum

105

spor

es/m

L+ (C

onti

nued

onne

xtpa

ge)

889

Dow

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ded

by [

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at 0

7:35

09

Oct

ober

201

3

Tabl

e6

Ant

imic

robi

aled

ible

com

posi

teco

atin

gsap

plie

dto

fres

hor

min

imal

lypr

oces

sed

frui

tsan

dve

geta

bles

(con

tinu

ed)

Hor

ticul

tura

lpro

duct

Coa

ting

Ant

imic

robi

alag

ent

Con

cent

ratio

nTa

rget

path

ogen

Path

ogen

inoc

ulat

ion

Ant

imic

robi

alac

tivity

Ref

eren

ce

“Cle

men

ules

”H

PMC

-lip

idSo

dium

bica

rbon

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

clem

entin

em

anda

rins

Am

mom

ium

bica

rbon

ate

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

Pota

ssiu

mso

rbat

e2.

0%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

+So

dium

benz

oate

2.5%

+So

dium

salt

ofm

ethy

lpar

aben

1.0%

P.di

gita

tum

105

spor

es/m

L–

Sodi

umsa

ltof

ethy

lpar

aben

1.0%

P.di

gita

tum

,P.i

tali

cum

105

spor

es/m

L–

Sodi

umsa

ltof

prop

ylpa

rabe

n1.

0%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

–So

dium

salt

ofm

ethy

lpar

aben

+so

dium

salt

ofpr

opyl

para

ben

1.0

+0.

5%P.

digi

tatu

m,P

.ita

licu

m10

5sp

ores

/mL

–

“Mur

cott”

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orC

hito

san

—0.

2%P.

ital

icum

,Bot

rydi

plod

iale

cani

dion

,Bot

ryti

sci

nere

a10

5sp

ores

/mL

+(C

hien

etal

.,20

07)

Lem

ons

Chi

tosa

n—

1m

g/m

LP.

digi

tatu

m3-

mm

myc

eliu

mpl

ug+

Ben

ham

ou,

2004

POM

EFR

UIT

S“R

edD

elic

ious

”ap

ple

Chi

tosa

n—

0.25

,0.5

,1.0

,2.

0%Pe

nici

lliu

mex

pans

um10

4sp

ores

/mL

+(D

eC

apde

ville

etal

.,20

02)

“Gal

a”ap

ple

Chi

tosa

n—

2.0%

B.c

iner

ea,P

.exp

ansu

m10

4sp

ores

/mL

+(W

uet

al.,

2005

)“F

uji”

appl

epi

eces

App

lepu

ree-

algi

nate

Ore

gano

oil,

lem

ongr

ass,

vani

llin

0.5%

L.i

nnoc

ua10

5C

FU/m

L+

(Roj

as-

Gra

uet

al.,

2007

b)“F

uji”

appl

epi

eces

Alg

inat

eC

inna

mon

,clo

ve,l

emon

gras

ses

sent

ialo

ils,

cinn

amal

dehy

de,e

ugen

ol,

and

citr

al

0.3,

0.7%

E.c

oliO

157:

H7

108

CFU

/mL

+(R

ayba

udi-

Mas

silia

etal

.,20

08a)

TR

OPI

CA

LA

ND

SUB

TR

OPI

CA

LFR

UIT

SM

ango

,ban

ana

Chi

tosa

n-gl

ycer

ol—

——

Nat

ural

infe

ctio

n+

(Kitt

uret

al.,

2001

)B

anan

aC

hito

san

Chi

tosa

nC

inna

mon

extr

act

1% 1%-

5g/

LC

olle

totr

ichu

mm

usae

,F

usar

ium

sp.,

Las

iodi

plod

iath

eobr

omae

5×

103

spor

es/m

L(m

ixtu

re3

path

ogen

s)+

(Win

etal

.,20

07)

Pine

appl

eC

hito

san-

MC

Van

illin

0.9

gE

.col

i,Sa

ccha

rom

yces

cere

visi

ae10

5C

FU/m

L+

(San

gsuw

anet

al.,

2008

)Pa

paya

Chi

tosa

n—

1.5%

Col

leto

tric

hum

gloe

ospo

rioi

des

106

spor

es/m

L+

(Bau

tista

-Ban

oset

al.,

2003

)

890

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09

Oct

ober

201

3

Fres

h-cu

tpap

aya

Chi

tosa

n—

0.02

mg/

mL

Mes

ophi

licto

talc

ount

,yea

stan

dm

old

coun

tN

atur

alin

fect

ion

+(G

onza

lez-

Agu

ilar

etal

.,20

08)

“Ham

i”m

elon

Chi

tosa

nN

atam

ycin

20m

g/L

Alt

erna

ria

alte

rnat

a,F

usar

ium

sem

itec

tum

Nat

ural

infe

ctio

n+

(Con

get

al.,

2007

)Fr

esh-

cutm

elon

Alg

inat

eM

alic

acid

,cin

nam

on,

palm

aros

aan

dle

mon

gras

ses

sent

ialo

ils,a

ndth

eir

mai

nac

tive

com

poun

ds

0.3,

0.7%

S.en

teri

tidi

s10

8C

FU/m

L+

(Ray

baud

i-M

assi

liaet

al.,

2008

b)

TAB

LE

GR

APE

S“I

talia

”C

hito

san

(dis

solv

edin

acet

icac

id)

—0.

1,0.

5,1.

0%B

.cin

erea

105

spor

es/m

L+

(Rom

anaz

ziet

al.,

2002

,20

07,2

009)

“Tho

mps

omSe

edle

ss”

“Aut

umn

Seed

less

”C

hito

san

(in

acet

icac

id)

Eth

anol

0.1,

0.5%

10.0

,20.

0%B

.cin

erea

105

spor

es/m

L+

“Tho

mps

omSe

edle

ss”

“Aut

umn

Seed

less

”“C

rim

son

Seed

less

”

Chi

tosa

n(i

ndi

ffer

enta

cids

)—

1.0%

B.c

iner

ea10

5sp

ores

/mL

+

“Red

glob

e”C

hito

san

Gra

pefr

uits

eed

extr

act

1.0%

0.1%

B.c

iner

ea10

5sp

ores

/mL

+(X

uet

al.,

2007

)

—C

hito

san

—1.

0,2.

5%C

olle

totr

ichu

msp

.4-

mm

myc

eliu

mpl

ug+

(Mun

ozet

al.,

2009

)“C

rim

son

Seed

less

”A

loe

vera

gel

—10

0%(d

ilute

d1:

3in

dist

illed

wat

erM

esop

hilic

tota

lcou

nt,y

east

and

mol

dco

unt

Nat

ural

infe

ctio

n+

(Val

verd

eet

al.,

2005

)B

ER

RIE

SSt

raw

berr

ySt

arch

(mix

ture

sof

corn

and

pota

to)

Pota

ssiu

mso

rbat

e0.

2g/

L—

Nat

ural

infe

ctio

n+

(Gar

cıa

etal

.,19

98)

Stra

wbe

rry

HPM

CPo

tass

ium

sorb

ate

0.3%

Cla

dosp

oriu

msp

.,R

hizo

pus

sp.

1.1

×10

4sp

ores

/mL

+(P

ark

etal

.,20

05)

Stra

wbe

rry

Chi

tosa

n—

—C

lado

spor

ium

sp.,

Rhi

zopu

ssp

.1.

1×

104

spor

es/m

L+

(Par

ket

al.,

2005

)Po

tass

ium

sorb

ate

0.3%

Cla

dosp

oriu

msp

.,R

hizo

pus

sp.

Stra

wbe

rry

Chi

tosa

n—

10,1

5m

g/m

LB

.cin

erea

,Rhi

zopu

sst

olon

ifer

2.0

×10

5sp

ores

/mL

+(E

l-G

haou

thet

al.,

1992

a)(C

onti

nued

onne

xtpa

ge)

891

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3

Tabl

e6

Ant

imic

robi

aled

ible

com

posi

teco

atin

gsap

plie

dto

fres

hor

min

imal

lypr

oces

sed

frui

tsan

dve

geta

bles

(con

tinu

ed)

Hor

ticul

tura

lpro

duct

Coa

ting

Ant

imic

robi

alag

ent

Con

cent

ratio

nTa

rget

path

ogen

Path

ogen

inoc

ulat

ion

Ant

imic

robi

alac

tivity

Ref

eren

ce

Stra

wbe

rry

Chi

tosa

n—

——

Nat

ural

infe

ctio

n+

(Var

gas

etal

.,20

06)

Stra

wbe

rry,

rasp

berr

ies

Chi

tosa

n-vi

tam

inE

orC

hito

san-

calc

ium

lact

ate

and

calc

ium

gluc

onat

e

——

—N

atur

alin

fect

ion

+(H

anet

al.,

2004

)

Fres

h-cu

tstr

awbe

rry

Chi

tosa

n(i

nci

tric

acid

)—

1%—

Nat

ural

infe

ctio

n+

(Cam

pani

ello

etal

.,20

08)

OT

HE

RFR

UIT

SA

ND

VE

GE

TAB

LE

SPe

ach

Chi

tosa

n—

5,10

mg/

mL

Mon

ilin

iafr

ucti

cola

105

spor

es/m

L+

(Lia

ndY

u,20

00)

Swee

tche

rry

Alo

eve

rage

l—

100%

(dilu

ted

1:3

indi

still

edw

ater

Mes

ophi

licto

talc

ount

,yea

stan

dm

old

coun

tN

atur

alin

fect

ion

+(M

artın

ez-

Rom

ero

etal

.,20

06)

Tom

ato

Chi

tosa

n—

—B

.cin

erea

Nat

ural

infe

ctio

n+

(El-

Gha

outh

etal

.,19

92b)

Chi

tosa

n—

10g/

LA

.alt

erna

ta2.

5×

105

spor

es/m

L+

(Red

dyet

al.,

2000

)C

hito

san

—0.

5,1.

0%B

.cin

erea

,P.e

xpan

sum

5×

103

spor

es/m

L+

(Liu

etal

.,20

07)

Chi

tosa

n—

—B

.cin

erea

105

spor

es/m

L+

(Bad

awy

and

Rab

ea,2

009)

—C

hito

san

—1.

0,2.

5%C

olle

totr

ichu

msp

.4-

mm

myc

eliu

mpl

ug+

(Mun

ozet

al.,

2009

)H

PMC

Sorb

icac

id0.

4%Sa

lmon

ella

mon

tevi

deo

—+

(Zhu

ang

etal

.,19

96)

Squa

shsl

ices

Chi

tosa

nO

leor

esin

sol

ive

1.0%

L.m

onoc

ytog

enes

Nat

ural

infe

ctio

n+

(Pon

ceet

al.,

2008

)R

osem

ary

Cap

sicu

m

Abb

revi

atio

nsar

eth

ose

desc

ribe

din

Tabl

es2,

3,an

d4.

892

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ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 893

2008). However, no commercial edible coatings are found toinhibit microbial growth on fruits and vegetables. Table 6 out-lines research on antimicrobial edible coatings applied to dateto fresh or minimally processed fruits and vegetables, includingthe target pathogen and an assessment of their antimicrobial ac-tivity. A summary of what has been reported in the literature isgiven below.

Citrus Fruits

Postharvest green and blue molds, caused by the pathogens P.digitatum (Pers.:Fr.) Sacc. and P. italicum Wehmer, respectively,are the most economically important postharvest diseases ofcitrus fruits worldwide, but especially in those production areascharacterized by low summer rainfall, such as Spain, California,or Israel (Eckert and Eaks, 1989). Commercial decay control oncitrus fruit has been obtained for many years by the use of con-ventional synthetic chemical fungicides such as imazalil, thi-abendazole, or sodium ortho-phenyl phenate (Eckert and Eaks,1989). More recently, new reduced-risk chemical fungicidessuch as pyrimethanil, fludioxonil, or azoxystrobin have beendeveloped (Smilanick et al., 2006; Kanetis et al., 2007). How-ever, consumer concerns about human health and environmentalcontamination are leading researchers worldwide to increase theefforts to find non-polluting alternatives for postharvest decaycontrol (Palou et al., 2008). Among them, increasing attention isbeing devoted to the development of antifungal edible coatingsto control diseases and preserve fruit safety.

In the literature, many works report the effect of edible coat-ings on storability and postharvest quality of citrus fruits (Ha-genmaier et al., 2002; Hagenmaier and Shaw, 2002; Perez-Gagoet al., 2002; Hagenmaier, 2004; Porat et al., 2005; Navarro-Tarazaga and Perez-Gago, 2006; Navarro-Tarazaga et al., 2007;2008; Rojas-Argudo et al., 2009). However, there is not muchpublished information on edible coatings containing antimicro-bial agents to control citrus postharvest diseases or prevent fruitcontamination by human pathogens (Table 6). Besides theirantimicrobial activity, the effects of these coatings on fruit qual-ity also need to be assessed. Valencia-Chamorro et al. (2009a)studied the efficacy of HPMC-lipid edible composite coatingscontaining antifungal food additives (mineral salts, organic acidsalts and their mixtures, parabens and their mixtures, and otherGRAS compounds) to control green and blue molds on “Va-lencia” oranges and “Ortanique,” and “Clemenules” mandarinsartificially inoculated with P. digitatum and P. italicum and in-cubated at 20◦C for 7 days. In every cultivar, no reduction ofdisease incidence (number of decayed fruit) or severity (lesiondiameter) was observed on fruit coated before fungal inocula-tion (preventive activity). Among all the tested coatings, thosecontaining PS, SB, SP, and their mixtures were the most effec-tive to reduce the incidence and severity of both green and bluemolds when oranges or mandarins were coated 24 hours af-ter fungal inoculation (curative activity). For example, HPMC-lipid edible composite coatings containing SB reduced green

mold incidence and severity by 86 and 90%, respectively, on“Clemenules” mandarins treated and incubated at 20◦C for 7days. On “Ortanique” mandarins, the mixture of PS and SPcaused a synergistic effect for incidence reduction of both green(78%) and blue (67%) molds. On “Valencia” oranges, PS- andSB-based coatings reduced by more than 90% the incidence andseverity of both molds after 7 days of incubation at 20◦C. Thesereductions on “Valencia” oranges, however, were lower afterlonger incubation periods at 20◦C, which indicated that the an-tifungal action of the coatings was fungistatic rather than fungi-cidal. In general, irrespective of their formulation and antifungalingredient, the inhibition activity of the coatings was higher on“Valencia” oranges than on “Ortanique” or “Clemenules” man-darins. It was suggested that these differences were probablyrelated to different susceptibility of the fruit host to infectionsby Penicillium spp. In subsequent works, the effect of the mosteffective HPMC-lipid composite coatings on fruit incubated at20◦C (i.e., coatings containing PS, SB, SP, and their mixtures) onthe antifungal activity and physico-chemical and sensory qualityof coated fruit cold-stored at 5◦C for up to 2 months was studiedon “Valencia” oranges (Valencia-Chamorro et al., 2009b) and“Ortanique” and “Clemenules” mandarins (Valencia-Chamorroet al., 2010; Valencia-Chamorro et al., 2011). During long-termcold storage of all coated cultivars, the incidence of blue moldwas higher than that of green mold. The inhibitory activityof the coatings containing organic acid salts and their mix-tures was strongly dependent on the susceptibility of each citruscultivar to penicillium decay. Hence, it was higher on “Valen-cia” oranges than on “Ortanique” hybrid mandarins, and higheron this cultivar than on “Clemenules” clementine mandarins.Specifically, the most effective coatings to inhibit green andblue molds on “Valencia” oranges, “Ortanique” mandarins, and“Clemenules” mandarins were those containing PS+SP, SB,and SB+PS, respectively, as antifungal ingredients. All coat-ings significantly reduced weight loss and maintained firmnessof coated “Ortanique” and “Clemenules” mandarins, but did notreduce weight loss of “Valencia” oranges. All coatings modi-fied the gas composition in the internal atmosphere of coatedoranges and mandarins, but did not induce off-flavors. In gen-eral, although the coatings did not improve rind gloss, the overallsensory quality of all coated citrus fruit was reported as accept-able.

Chien et al. (2007) studied the effects of low and high molec-ular weight chitosan coatings (0.05–0.2%) on the antifungal ac-tivity against P. digitatum and P. italicum and the quality ofcoated “Murcott” tangor fruit. Low molecular weight chitosan(0.2%) exhibited effective antifungal activity against both moldsand significantly retarded the loss of fruit water content, firm-ness, and titratable acidity during storage. Moreover, the per-formance of low molecular weight chitosan was equiparable tothat of the synthetic chemical fungicide thiabendazole. Workingwith lemons, Benhamou (2004) observed that the application ofchitosan to wounded fruits prior to inoculation with P. digitatumresulted in a near absence of fungal development in the woundsthat were bordered by a reddish scar. This worker suggested

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894 S. A. VALENCIA-CHAMORRO ET AL.

that the treatment had the ability to induce the transcriptionalactivation of defense genes leading to the accumulation of struc-tural and biochemical compounds at strategic sites. Likewise,significant reduction of postharvest penicillium decay and delayof fruit senescence during long-term cold storage of differentcitrus species and cultivars have been observed after the appli-cation of certain chitosan formulations (El-Ghaouth et al., 2000;Chien and Chou, 2006).

Shellac formulations at various pH and ethanol concentra-tions with and without parabens were applied to “Ruby Red”grapefruit and “Valencia” oranges over a carboxymethyl cellu-lose layer that facilitated shellac adherence (McGuire and Ha-genmaier, 2001). The results showed that a shellac formulationat pH 9.0 with 5.2% ethanol was more toxic to the coliformbacteria Enterobacter aerogenes and E. coli than a formulationat pH 7.25 with 12% ethanol. Paraben addition to the shellacformulation at pH 9.0 further inhibited coliform growth.

Pome Fruits

Aqueous solutions of chitosan applied to fresh apples at con-centrations from 0.25 to 2% significantly induced resistance topostharvest blue mold, caused by the fungus Penicillium expan-sum (de Capdeville et al., 2002). Similar results were obtainedin later work by Wu et al. (2005). The severity of blue moldand also that of gray mold, caused by Botrytis cinerea, wasconsiderably reduced in artificially inoculated “Gala” apples bypreventive 2.0% chitosan treatments.

Rojas-Grau et al. (2007b) incorporated lemongrass andoregano oils and vanillin into apple puree-alginate edible coat-ings to extend the shelf-life of fresh-cut “Fuji” apples. It wasreported that all antimicrobials significantly inhibited the growthof psychrophilic aerobes, yeasts and molds. Coatings containinglemongrass or oregano oils exhibited the strongest antimicrobialactivity against Listeria innocua. In addition, the coatings re-duced the respiration rate and ethylene production of coatedfresh-cut apples. The addition of calcium chloride to the coat-ings effectively maintained fruit firmness and color, while coat-ings containing lemongrass caused severe softening. Coatingscontaining vanillin were the best in terms of sensory quality.

In a later work, cinnamon, clove, and lemongrass essentialoils and their active compounds cinnamaldehyde, eugenol, andcitral, respectively, were investigated as antimicrobial agentsin an alginate-based edible coating applied to fresh-cut “Fuji”apples (Raybaudi-Massilia et al., 2008a). The coatings also con-tained malic acid, N-acetyl-L-cysteine, glutathione, and calciumlactate as quality stabilizing compounds. The addition of es-sential oils at 0.7% (v/v) or their active compounds at 0.5%into the coating reduced E. coli O157:H7 population by morethan 4 log CFU/g (colony forming units) and extended the mi-crobiological shelf-life by more than 30 days. However, thoseconcentrations affected the physicochemical characteristics offresh-cut apples and limited their shelf-life. Lemongrass and cin-namon (0.7%), citral (0.5%), and cinnamaldehyde (0.5%) were

the most effective compounds for extending the microbiologi-cal shelf-life, whereas lemongrass, cinnamon, and clove at 0.3%(v/v) best maintained the physicochemical characteristics of theproduct.

In another study, whole apples were coated with soy proteincoatings containing malic or lactic acid (Eswaranandam et al.,2006). The main objective of this work was to evaluate the ef-fect of the coatings on the sensory quality of the fruit, withoutstudying the antimicrobial effect of the coatings. In general, or-ganic acids incorporated to films did not adversely affect thesensory properties of coated apples after cold storage. In a pre-vious work, these authors reported the in vitro antimicrobialactivity of soy films containing malic or lactic acid (agar diffu-sion test) (Eswaranandam et al., 2004). However, even thoughthe results from in vitro assays are a good approach to evaluatethe potential of antimicrobial films, the actual antimicrobial ac-tivity on coated produce could considerably differ from that ofstand-alone films, due to important factors such as the type ofsurface of the produce, the diffusion rate of the antimicrobial tothe coated produce, or the fruit storage conditions.

Tropical and Subtropical Fruits

Chitosan-based composite coatings have shown the ability todelay ripening and extend the shelf-life of banana and mango.These coatings significantly retarded color development, re-duced weight loss and respiration rate, maintained firmness,and reduced titratable acidity of coated fruits compared to un-coated controls. The application of an additional 1% chitosanto the fascicle region reduced the incidence of molds (Kitturet al., 2001). The antifungal activities of chitosan alone or incombination with cinnamon extract were evaluated against ba-nana crown rot, caused by mixed infections of the pathogensColletotrichum musae, Fusarium sp., and Lasiodiplodia theo-bromae. Crown rot development during storage at 13◦C for 7weeks was significantly reduced by chitosan and, to a higherextent, by the combined treatment. Chitosan delayed ripeningas in terms of peel color, firmness, and soluble solids content.The addition of cinnamon extract showed no negative effectson fruit quality (Win et al., 2007). Chitosan coatings containingnatamycin were also effective in controlling decay of “Hami”melons caused by natural infections of Alternaria alternata andFusarium semitectum. This coating also improved the qual-ity properties of coated fruit (Cong et al., 2007). On papaya,chitosan-based coatings reduced by about 40% the postharvestdisease antracnose caused by Colletotrichum gloeosporioides(Bautista-Banos et al., 2003).

Sangsuwan et al. (2008) evaluated the inhibitory effect of chi-tosan/MC stand-alone films, with and without vanillin, againstE. coli and Saccharomyces cerevisiae on wrapped fresh-cut can-taloupe and pineapple. Both films inhibited the growth of E. coliand S. cerevisiae on fresh-cut cantaloupe, the film with vanillinbeing more effective. However, it took a longer time for thisfilm to show the inhibitory effect than for chitosan/MC filmswithout vanillin. The use of films containing vanillin showed a

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ANTIMICROBIAL EDIBLE COATINGS FOR HORTICULTURAL PRODUCE 895

different response in cantaloupe and pineapple. In low pH fruitlike pineapple, the vanillin film was more effective to inhibitmicroorganisms than in cantaloupe, which was attributed to ahigher release rate of vanillin out of the film. In general, thequality attributes of coated fresh-cut cantaloupe and pineapplewere reported as acceptable in this study. However, the ap-plication of the antimicrobial film reduced the ascorbic acidcontent in pineapple, remaining after storage only 10% of itsoriginal content. Chitosan coatings were also effective in sup-pressing mesophilic microorganisms and the growth of moldsand yeasts on fresh-cut papaya, as assessed by plate counts(Gonzalez-Aguilar et al., 2009). However, the effect of the coat-ings depended on chitosan molecular weight and its concentra-tion. Medium molecular weight chitosan coatings at 0.02 g/mLresulted in the highest antimicrobial activity, maintained thehighest color values, and decreased the activity of the enzymespolygalacturonase and pectin methylesterase, thus mantainingfruit firmness.

Raybaudi-Massilia et al. (2008b) studied the effect of malicacid and essential oils of cinnamon, palmarosa, and lemongrassand their main active compounds as natural antimicrobial sub-stances incorporated into an alginate-based edible coating onthe shelf-life and safety of fresh-cut melon. The coating con-taining malic acid was effective to improve the shelf-life offresh-cut melon from both the microbiological (up to 9.6 days)and physicochemical (more than 14 days) points of view in com-parison with non-coated fresh-cut melon samples. The incorpo-ration of the essential oils or their active compounds into thecoating prolonged the microbiological shelf-life by more than21 days in some cases, probably due to an enhanced antimicro-bial effect of malic acid and the essential oils. However, somephysicochemical characteristics, such as firmness and color, andalso some sensory quality attributes were adversely affected,causing a significant reduction of fresh-cut melon shelf-life. Incontrast, when malic or lactic organic acids incorporated to soyprotein coatings were applied to fresh-cut cantaloupe, they didnot adversely affect the sensory properties of the fruit after coldstorage (Eswaranandam et al., 2006).

Table Grapes

The effectiveness of pre- and postharvest treatments withchitosan (0.1, 0.5, and 1.0%) to control the gray mold fungusB. cinerea on table grapes was investigated by Romanazzi etal. (2002). In postharvest treatments, small bunches dipped inchitosan solutions and inoculated with the pathogen showeda reduction of incidence, severity, and nesting of grey mold,in comparison with control fruit. The activity of the enzymephenylalanine ammonia-lyase (PAL) in the skin of table grapeberries sprayed with 1.0% chitosan was 2-fold higher than inthe untreated control. In further studies, Romanazzi et al. (2007)found that the combination of reduced doses of chitosan (0.5%)and ethanol (10 or 20%) improved the control of gray moldon artificially inoculated table grapes compared to their appli-

cation alone, and the effect was at least additive and at timessynergistic.

Similarly, the integration of chitosan treatments with a grape-fruit seed extract showing antimicrobial properties lead to a syn-ergistic effect in reducing postharvest gray mold of “Redglobe”grapes challenged with B. cinerea. Moreover, the treatments,alone or combined, significantly improved important grape qual-ity attributes such as weight loss, flesh firmness, rachis andberry appearance, shatter, cracking, or sensory flavor (Xu et al.,2007). More recently, Romanazzi et al. (2009) studied the in-fluence of the utilization of different acids to dissolve crab-shellchitosan on its ability to control gray mold. Among 15 acidstested, chitosan acetate was the most effective treatment whicheffectively reduced gray mold at both cold and ambient stor-age temperatures and did not injure the grape berries. In recentwork by Munoz et al. (2009), single grape berries treated withaqueous solutions of 1.0 and 2.5% chitosan were artificially in-oculated with Colletotrichum sp. and incubated at 24◦C. Lesiondiameters after 10 days were significantly reduced by chitosanapplications.

Another natural edible coating that showed significant an-timicrobial activity on table grapes was a gel from the plantAloe vera. When applied at 100% purity (diluted 1:3 in distilledwater) to naturally infected “Crimson Seedless” table grapes, anet reduction of the population of yeasts and molds present in thefruit was obtained after 28 days of cold storage at 1◦C (Valverdeet al., 2005). Furthermore, the coating was effective to maintainthe rachis condition and reduce grape softening, color changes,and weight loss. Additional benefits from the application ofthe Aloe vera gel were the retention of functional properties ofcoated table grapes during cold storage. While the contents oftotal phenolics and ascorbic acid in coated “Crimson Seedless”grapes were effectively maintained after 35 days of storage at1◦C followed by 4 days of shelf-life at 20◦C, with a higher re-tention of total antioxidant activity, the loss of these compoundswas clear in control uncoated berries, which showed an accel-erated ripening process during storage revealed by a significantincrease in anthocyanin content (Serrano et al., 2006).

Berries

Starch-based coatings containing PS reduced the microbialcounts of “Selva” strawberries, extending the storage life ofcoated fruit to up to 28 days from an average period of only 14days that lasted uncoated fruits. The addition of citric acid to thecoating enhanced the antimicrobial action of PS. The coatingalso reduced fruit weight loss and satisfactorily maintained fruitquality (Garcıa et al., 1998).

Several studies reported the effect of chitosan coatings onthe antimicrobial activity and quality of strawberries (Table 6).Chitosan-based coatings containing calcium or vitamin E wereused to extend the shelf-life of fresh and frozen strawberriesand raspberries (Han et al., 2004). The coatings decreased de-cay incidence and weight loss, improving the storability and

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enhancing the nutritional value of fresh and frozen fruits. Parket al. (2005) found that chitosan-based coatings reduced weightloss of strawberries during storage and observed an importantantifungal activity against Rhizopus sp. and Cladosporium sp. onartificially inoculated fresh strawberries. Although a significantsynergic inhibition of microbial growth was noticed in in vitrotests when PS was incorporated into chitosan, no significant syn-ergistic inhibitory effects were reported for prevention of fun-gal development on fresh strawberries. Likewise, El-Ghaouthet al. (1992a) found considerable reductions on postharvest de-cay of strawberry caused by the fungi B. cinerea and Rhizopusstolonifer. The addition of oleic acid to chitosan coatings en-hanced the antimicrobial activity, improved water resistance,and reduced the respiration rate of cold-stored “Camarosa”strawberries. However, chitosan-oleic acid coatings decreasedaroma and flavor of coated samples. In order to avoid unpleasantchanges in sensory attributes, it was recommended to incorpo-rate oleic acid in a chitosan:oleic ratio lower than 4:1 (Vargaset al., 2006). In another study, starch, carrageenan, and chitosanwere used to optimize coating composition and properties. Theoptimized coatings were applied to fresh strawberries to deter-mine the fruit microbiological and quality properties (Ribeiroet al., 2007). Calcium chloride added to the coatings decreasedthe microbial growth rate on treated fruit. The lowest microbialgrowth rate was found on strawberries coated with chitosan andcalcium chloride. This chitosan-calcium chloride coating wasalso the most effective in reducing weight loss and firmness lossof coated strawberries. Campaniello et al. (2008) found that theapplication of low molecular weight chitosan at 1% inhibitedthe growth of yeasts and mesophilic and psychrotrophic bacteriaon fresh-cut strawberries, particularly when the samples werepackaged in a modified atmosphere with low oxygen level. Asthe coating was invisible and did not affect the visual appear-ance and the overall sensorial quality of coated strawberries, theauthors proposed it as a convenient technology to extend theshelf-life of minimally processed strawberries.

Other Fruits and Vegetables

Some antimicrobial coatings have also been applied to otherhorticultural produce such as peach, cherry, tomato, or squash toevaluate both their antimicrobial activity and effects on productquality (Table 6).

Work by Li and Yu (2000) showed that the application of chi-tosan to peaches artificially inoculated with Monilinia fructicolasignificantly reduced the incidence of brown rot and delayed dis-ease development if compared with water-treated control fruit.Chitosan-treated peaches were firmer and had higher titratableacidity and vitamin C content than control peaches. Martınez-Romero et al. (2006) found that after 16 days of cold storageat 1◦C plus 1 day at 20◦C of shelf-life, the application of Aloevera gel (100% purity; diluted 1:3 in distilled water) to naturallyinfected sweet cherries cv. “Cerezas de la Montana de Alicante”reduced the populations of mesophilic aerobics and yeast and

molds from 4.7 and 3.1 log CFU/g, respectively, on uncoatedcontrol fruit to 2.0 and 1.2 log CFU/g, respectively, on Aloe-treated sweet cherries. This coating also reduced cherry soften-ing, weight loss and respiration rate, and largely contribute to theretention of skin color, stem freshness, and sensory attributes.

Chitosan-based coatings reduced the respiration rate andethylene production, consequently delaying ripening of coatedtomatoes. Coated fruit also showed less postharvest decay thancontrol fruit (El-Ghaouth et al., 1992b). In another study withtomatoes, chitosan coatings greatly reduced postharvest blackrot caused by the pathogen A. alternata (Reddy et al., 2000).According to Liu at el. (2007), the application of chitosan sig-nificantly controlled tomato gray and blue molds, caused byB. cinerea and P. expansum, respectively. An increment of chi-tosan concentration from 0.5 to 1.0% substantially reduced theincidence of these diseases. The authors observed that chitosantreatment induced a significant increase in the fruit enzymaticactivity and enhanced the content of phenolic compounds incoated fruit, which lead to an important increment of fruit dis-ease resistance. Furthermore, Badawy and Rabea (2009) re-ported that chitosan of different molecular weight inhibited thegrowth of B. cinerea both in in vitro and in vivo assays. In ad-dition to its direct antifungal activity, chitosan showed the po-tential to elicitate defence mechanisms on tomato fruit. Anotherimportant postharvest disease of tomato, antracnose, caused byColletotrichum sp., was also significantly inhibited by chitosanapplications (Munoz et al., 2009).

HPMC-based coatings containing sorbic acid at a concentra-tion of 0.4% enhanced the inactivation of Salmonella montev-ideo on the surface of tomatoes. However, these coatings causeda chalky appearance of the fruit surface, limiting its potential forcommercial application (Zhuang et al., 1996). The addition ofcitric or acetic acid to HPMC film formulations did not enhancethe inactivation of S. montevideo on the surface or core tissueof tomatoes. Treating with HPMC coatings delayed changes inthe color and the firmness of tomatoes.

Film forming solutions made of sodium caseinate, chitosanor carboxymethyl cellulose containing 1% of oleoresins (olive,rosemary, onion, capsicum, garlic, or oreganum) showed limitedantimicrobial activity against L. monocytogenes in in vitro stud-ies. Similarly, in in vivo studies, chitosan coatings enriched withrosemary and olive oleoresin applied to butternut squash did notshow a significant antimicrobial effect. The coatings, however,did not induce deletorious effects on the sensory acceptabilityof coated squash (Ponce et al., 2008).

CONCLUDING REMARKS

The use of edible films and coatings is an environmentally-friendly technology that offers substantial advantages for shelf-life increase of many food products including fruits andvegetables. The development of new natural edible films andcoatings with either inherent microbicidal activity or the addi-tion of antifungal ingredients (food preservatives, essential oils,

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antagonistic microorganisms, etc.) in order to provide, on theone hand, significant fruit senescence retardation and, on theother hand, effective control of postharvest diseases and overallmicrobiological safety of fresh and minimally processed fruitsand vegetables is a technological challenge for the industry anda very active research field worldwide. To date, most of theresearch on this subject has been focused on the developmentof antimicrobial edible films and the effect of composition andpreparation techniques on film antimicrobial, barrier, and me-chanical properties. In comparison, relatively few studies reportthe application of antimicrobial edible coatings to fresh or fresh-cut horticultural products and their effect on physico-chemical,physiological, or microbiological properties. Coatings devel-oped for one fruit species or cultivar may not be appropriatefor another because of important differences in either crucialinherent fruit attributes or issues related to commodity valueand commercialization. Skin and/or flesh physical, physiolog-ical, and biochemical properties or the rates of moisture andgas exchange (especially respiration and ethylene release) arefactors that will clearly influence the performance of particularedible coatings. The antimicrobial activity will also depend oncontamination sources and pressure, and in the case of freshfruit, on the potential incidence of postharvest diseases, all is-sues related to climatic and local conditions and preharvest andpostharvest commodity handling. Furthermore, in contrast topolluting conventional pesticides and sanitizers, typically char-acterized for a biocidal mode of action, with high persistenceand curative activity, the mode of action of natural antimicro-bials is rather fungistatic and not so persistent, which may re-strain their use as stand-alone treatments and make their per-formance more variable and dependent on species, cultivar, andthe physical and physiological condition of the treated produce.Finally, the economical impact of the commercial use of antimi-crobial edible coatings will be determined by their productionand application costs in opposition to the benefits from occu-pying certain increasing market shares (high value markets forhealthier or environmentally friendly produce). Despite the sub-stantial research progress, a strong impulse in the developmentof this novel technology is required in the future for commer-cial application, since natural antimicrobial edible coatings ortheir integration with other non-polluting physical, chemical,or biological postharvest treatments are emerging concepts inhorticultural technology that may fulfil consumer demand forsafe products avoiding the use of contaminating chemicals as ameans of preservation.

For all these reasons, new oriented research effortsshould focus on the development of tailor-made coatingsbased on the selection of the most appropriate film form-ing constituents and active ingredients to suit their appli-cation to commercially important fresh and minimally pro-cessed fruits and vegetables according to specific industryneeds. Moreover, research is also needed to evaluate thereal impact of such postharvest treatments from the pointof view of both commercial feasibility and consumer accep-tance.

ACKNOWLEDGEMENTS

The authors thank the Spanish “Ministerio de Ciencia a In-novacion” (MICINN) and “Instituto Nacional de Investigaciony Tecnologıa Agraria y Aliementaria” (INIA) and the Eu-ropean Union FEDER program for funding research in thistopic (projects RTA-2006-00114-00-00, RTA-2008-00074-00-00 and RTA-2009-00135-00-00). The doctorate program ofSilvia Valencia-Chamorro was supported by the ProgrammeAlβan, the European Union Programme of High Level Schol-arships for Latin America, scholarship No. E05D060018EC.

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