**2. Use of Edible or Antimicrobial-Containing Coatings (e.g., Chitosan-Based Coatings) on Fruits or Vegetables**

Edible coatings (ECs) and edible films belong to the modern food protection system; over the past few years, interest in the use of edible coatings for perishable foods has considerably increased due to their advantages and potential applications [4]. Edible coatings and edible films are terms that are often used interchangeably; however, a distinction is necessary.

An edible coating is defined as a thin layer of edible material applied to the surface of foods in addition to or as a substitution for natural protective coatings, able to form a barrier to moisture, oxygen and solute movement for the food [5–10].

On the other hand, edible film is defined as a thin layer of edible material formed on a product surface as a coating or placed (pre-formed) on or between food components [11].

Thus, an edible film is a thin skin, which has been pre-formed (for example, by casting a biopolymer solution separately from the food to form a film later applied to the food), whilst an edible coating is a suspension or an emulsion, which is applied directly to the food surface and later forms a film [12].

Edible coatings and films do not replace traditional packaging materials, but provide an additional factor to be applied for food preservation; ECs are consumed along with the food, thus the composition must conform to the regulations applied to the food product.

One of the advantages in using edible coatings and films is the reduction of water loss, considered one of the main factors in the deterioration of perishable foods. In fact, this thin layer protects fruits and/or vegetables against moisture loss, maintaining the texture and extending the shelf-life of the product, forming a protective barrier. On the other hand, when edible coatings are poor in water vapor barrier properties, a weight or moisture loss of the product could be recovered.

Numerous benefits result when edible coatings are applied, and these are summarized in Figure 1.

In addition, edible coatings may enhance sensory characteristics, can be consumed along with the food, provide additional nutrients and include quality-enhancing antimicrobials. Furthermore, they may reduce the cost and also the amount of traditional packaging used [13].

**Figure 1.** Main benefits of edible coatings.

#### *2.1. Edible Coating Materials*

Numerous varieties of fruits and vegetables are characterized by cuticle, a natural waxy layer on the surface, which, generally, has a low permeability to water vapor. To enhance the barrier properties of cuticle and/or substitute it when processing operations remove it, edible coatings could be applied.

Water and ethanol (or a blend of these compounds) are the solvents generally used for edible coating production.

Various coating materials could be added, but it is necessary that they possess some specific requirements (water solubility, hydrophobic of hydrophilic nature, easy formation of coatings, good sensorial properties) to obtain the resulting coating. In addition, an essential requirement is the plasticizing capacity to provide films with good flexibility properties; for this purpose, glycerol is generally used, followed by sorbitol, polyethylene glycol (PEG) and sugars [13].

Hydrocolloids have a good aptitude for forming continuous and cohesive matrices thanks to the hydrogen bonding of their polymeric chains.

Polysaccharides, proteins and lipids, alone or in combination, can also be used to produce edible films and coating. Lipids (together with waxes and fatty acids) do not have a suitable stand-alone filmmaking nature; they are usually opaque and relatively inflexible; the resulting films could also be quite fragile and unstable (rancidity) [9]. For this reason, lipids are generally incorporated into hydrocolloids-based films formulations [13].

The main polysaccharide, protein and lipid compounds used in edible coatings are reported in Table 1.

**Table 1.** Main polysaccharides, proteins and lipid compounds used for edible coatings.


**Table 1.** *Cont.*


Polysaccharides include starch, dextrins, pullulan, cellulose and derivatives, alginate, carrageenan, gums, pectins and chitosan. Polysaccharides render transparent and homogeneous edible films; these

films are oxygen, aroma and oil barriers (due to their tightly-packed, ordered hydrogen-bonded network structure and low solubility), but are not effective moisture barriers due to their hydrophilic nature. However, when applied in the form of high-moisture gelatinous coatings, they can retard the moisture loss of food [15].

Proteins can be obtained from animal sources, such as casein and whey protein (the main milk protein fractions: 80% and 20%, respectively), and from plant sources, such as zein, gluten and soy proteins [34].

Different proteins are able to form films and coatings; this ability depends on their molecular weight, conformations, electrical properties (charge *vs.* pH), flexibilities and thermal stabilities [18]. Nevertheless, proteins have been studied less extensively than polysaccharides.

Generally, protein-based films have good oxygen, carbon dioxide and lipid barrier properties and mechanical properties; on the other hand, the poor water vapor resistance limits their application; this can be attributed to the inherent hydrophilicity of proteins.

As protein films are generally brittle and susceptible to cracking (due to the strong cohesive energy density of the polymers), an improvement of their properties could be attained by adding other components; for example, the addition of compatible plasticizers could improve their extensibility [35].

Edible lipid coatings include neutral lipids, fatty acids, waxes and resins. These compounds are effective in providing a moisture barrier and improving the surface appearance. Triglycerides or neutral lipids form a continuous stable layer on the food surface, thanks to their high polarity.

The growing interest addressed toward edible coatings leads to the formulation of new compositions consisting of blends of polysaccharides, proteins and lipids. The combination could be proteins and carbohydrates, proteins and lipids and carbohydrates and lipids [13], and it is based on the fact that each polymer has characteristic functions, that, when combined with each other, enhance the final functionality of the coating [15], improving the mechanical properties and, with emulsifiers, stabilizing composite coatings and improving coating adhesion.

Edible coatings can incorporate several compounds, such as: plasticizers (glycerol, acetylated monoglyceride, polyethylene glycol and sucrose), antimicrobials (bacteriocins (nisin)), enzymes (lysozyme, peroxidase and lactoperoxidase), essential oils (cinnamon, oregano, lemongrass, clove, rosemary, tea tree, thyme and bergamot), nitrites and sulfites [36], as well as synthetic antioxidants (butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, octyl gallate, dodecyl gallate, ethoxyquin, ascorbyl palmitate and tertiary butyl hydroquinone) and natural antioxidants (tocopherols, tocotrienols, ascorbic acid, citric acid, carotenoids). In these cases, edible films and coatings act as carriers of active compounds that, applied to the surface of fruits and vegetables, lead to the extension of shelf-life, the reduction of the risk of foodborne pathogenic microorganisms' growth on cut surfaces [37] and the improvement of the quality, stability and safety of coated foods [15].

#### *2.2. Edible Coating: A Focus on Chitosan*

Physical and chemical damage accrued during minimal processing of fruits and vegetables causes disruption of the plant tissues, and the exudates become ideal substrates for the growth of several microorganisms (pathogens, molds and bacteria). Natural biodegradable compounds with antimicrobial activity are recognized as safe (generally recognized as safe (GRAS)) and environmentally friendly, and chitosan is one such compound. Chitosan is able to create a semi-permeable film on the fruit surface, which results in limiting respiration and/or transpiration and in reducing weight loss [38,39]. Furthermore, its compatibility with other substances and its capability to induce host resistance to pathogens [40] have prompted its application as a coating on fruits and vegetables [41].

Chitosan has been widely used in controlling weight-loss in fresh strawberries (*Fragaria x ananassa*) and raspberries (*Rubus idaeus*), mango (*Mangifera indica*), litchi, blueberries and other fruit and vegetables [42]. Meng *et al.* [43] and Romanazzi *et al.* [44] reported that postharvest application of chitosan coating has a good control effect on decay of grapes. Chitosan owes its antimicrobial activity to its polycationic nature, which allows the interaction and formation of polyelectrolyte

complexes with acid polymers produced at the surface of microbial cells, increasing their permeability and causing cell death [45].

Several factors affect the antimicrobial activity: type of chitosan, degree of acetylation, molecular weight, concentration, target microorganism, pH of the medium and presence of other additives or food components [46].

Benhabiles *et al.* [47] demonstrated that by reducing the number of steps for the synthesis and chemical reagents, chitosan coating was effective at improving the quality of strawberries by delaying changes in weight loss and the appearance of molds. These authors used three different chitosan coatings (chitosan C1 obtained by the classical method, chitosan C2 without decoloration and chitosan C3 without the decoloration and deproteinization steps) on strawberries, and they observed the best quality for the strawberries coated with C3 (1%), which was obtained through a lesser number of steps.

In a recent work, chitosan (in acid and water solution) exhibited its antibacterial activity against *Burkholderia seminalis*, an apricot fruit rot pathogen [48]. Lou and his coworkers demonstrated that acid-solution chitosan at a concentration of 2 mg/mL inhibited *B. seminalis*, while water-solution chitosan showed limited inhibition activity.

Chitosan has been proven to be a natural compound with antifungal activity for a wide varieties of postharvest fruits [38,49]. As reported by Bautista-Banos *et al.* [38], the level of inhibition of fungi is highly correlated to chitosan concentration. It is known that the polycationic nature of this compound is the key to its antifungal properties and that the length of the polymer chain enhances its antifungal activity. An additional explanation includes the possible effect that chitosan might have on the synthesis of certain fungal enzymes. El Ghaouth *et al.* [50] studied the antifungal effect of chitosan against *Botrytis cinerea* and *Rhizopus stolonifer*. These authors hypothesized that the mechanisms by which chitosan coating reduced the decay of strawberries appear to be related to its fungistatic properties, rather than to its ability to induce defense enzymes. A further confirmation of chitosan's ability to control fungal growth was reported by Park *et al.* [51]. They obtained a reduction of 2.5 and 2 log CFU/g in the counts of *Cladosporium* sp. and *Rhizopus* sp., respectively, on strawberries coated with a chitosan-based edible film, just after the coating application. A reduction in the counts of aerobic and coliform microorganisms during storage has been also reported. Chien *et al.* [52] investigated the effects of coating with low and high molecular weight chitosan on the decay of citrus and the maintenance of its quality. A concentration of 0.2% low molecular weight chitosan (LMWC) exhibited its antifungal activity in controlling the growth of *Penicillium digitatum* and *Penicillium italicum*. LMWC coating resulted also in being able to improve firmness, titratable acidity, ascorbic acidity and the water content for citrus stored at 15 ◦C for 56 days.

González-Aguilar *et al.* [53] have also reported a reduction in mesophilic aerobic microorganism count when fresh-cut papayas were coated with an edible coating based on chitosan of low and medium molecular weight. These researchers also observed a complete inhibition of yeast and molds throughout the storage (14 days at 5 ◦C). As reported by Ali *et al.* [54], chitosan preserved papaya fruit, delaying the ripening process by reducing the respiration rate. These results could be the reason for the delayed senescence and reduced tendency to decay [40]. Chitosan had also made improvements in the taste, peel and pulp color, texture and flavor of treated papaya fruit, but the sensory features of the papaya fruits coated with a 1.5% chitosan concentration demonstrated overall superiority after five weeks of storage.

Pilon *et al.* [55] made an alternative use of chitosan. They obtained chitosan nanoparticles and demonstrated that chitosan, used as a coating based on nanoparticles, reduced the microbial growth in fresh-cut apples. The samples treated with chitosan-tripolyphosphate (CS-TPP) nanoparticles (10 nm) showed higher antimicrobial activity against mesophilic and psychrotrophic bacteria, as well as molds and yeasts than conventional chitosan coating and control [55].

Numerous papers have demonstrated that chitosan-based coatings inhibit microbial growth on fresh produce, increasing shelf-life.

In a recent paper, Benhabiles *et al.* [56] reported that chitosan coatings and a chitosan derivative (N,O-carboxymethyl chitosan (NOCC)) coating improved the quality of tomato fruits (through delaying ripening, reducing weight loss and retaining fruit firmness) and extended the shelf-life. No microbial growth was observed during storage.

Assis and Pessoa [57] and Han *et al.* [58] proposed chitosan for extending the shelf-life of sliced apples and fresh strawberries, respectively. Chien *et al.* [52] reported the effectiveness of chitosan coating (at a concentration of 0.5%, 1% and 2% (w/v)) for prolonging the quality and extending the shelf-life of sliced mango fruit through a delay in the growth of mesophilic aerobic bacteria.

Durango *et al.* [59] and Devlieghere *et al.* [60] used a chitosan-based coating to cover carrots and lettuce, respectively, observing a reduction in the respiration rate and ethylene production, as well as a decrease in firmness loss. In particular, Durango *et al.* [59] controlled the growth of mesophilic aerobes, yeasts, molds and psychrotrophs of minimally-processed carrots during the first five days of storage at 15 ◦C using an edible yam starch coating containing chitosan. Campaniello *et al.* [28] observed that chitosan coating in combination with low temperature and suitable packaging was able to control browning and decay in strawberry fruits. Chitosan affected the microbial growth (psychrotrophic, lactic acid bacteria and yeasts) and did not affect the visual appearance. pH and thickness values remained unchanged by chitosan coating, whereas color was positively influenced.

Pushkala *et al.* [61] investigated chitosan-based powder coating on radish shreds, demonstrating the favorable effects of two different forms of chitosan (purified chitosan (CH) and chitosan lactate (CL)) on shelf-life extension of radish shreds by a minimum of 3 d over the control. Both CH- and CL-coated samples enhanced the microbial quality and sensory acceptability of the radish shreds, exhibiting a lower degree of weight loss, respiration rate, titrable acidity, % of soluble solids, a higher content of phytochemicals, moisture and pH, compared to control samples. The treated samples also exhibited lower exudate volume, lesser browning and lower microbial load compared to the control.

Sometimes, a chitosan-only coating demonstrated certain defects (including limited inhibition to some microorganisms that led fruit to decay and a poor coating structure); thus, chitosan was combined with other substances to improve its performance [62]. Chitosan coatings combined with organic acids are easy to handle, biodegradable and cause no harm to the coated fruit and/or vegetable [62]. For example, Yu *et al.* [63] combined 1% phytic acid (known as inositol hexakisphosphate (IP6), inositol polyphosphate) with 1% chitosan to preserve fresh cut lotus root. This composite coating decreased the weight loss rate and malondialdehyde (MDA) content of fresh-cut lotus root, postponed browning, restrained the activities of peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) and maintained the content of vitamin C and polyphenol at a relatively high level.

Chitosan combined with natamycin significantly decreased fresh melon decay and weight loss caused by *Alternaria alternata* and *Fusarium semitectum*, two strains of spoilage fungi [64].

Zhang *et al.* [65] reported that chitosan was able to inhibit the growth of *Botrytis cinerea* and *Rhizopus* sp. by increasing the activities of various defense enzymes, such as β-1,3-glucanase (in orange, strawberries and raspberries) and phenylalanine ammonia-lyase (PAL) (in strawberries andtable grapes).

As expected, the antimicrobial activity of chitosan is also dependent on the food matrix; generally, the antimicrobial activity of chitosan is higher at a low pH because more of its amino groups are protonated; thus, it is able to interact with the negatively-charged surfaces inhibiting bacterial growth. Since the antimicrobial activity of chitosan is dependent on the charges on chitosan and the electrostatic forces, each food component could influence these interactions, affecting its activity.

Regarding this issue, Devlieghere *et al.* [60] published an interesting work on the interaction of chitosan and food components. The authors evaluated the effect of different food components (starch, proteins, NaCl and fat) on the antimicrobial activity of chitosan, following the growth of *Candida lambica*, a spoiling yeast strain, in a laboratory medium.

The authors reported that the higher concentrations of starch (30% (w/v)) inhibited the antimicrobial activity of chitosan, hypothesizing that it was due to a protective effect of starch or to electrostatic interactions when the starch was charged by modification. Proteins influenced the antimicrobial activity of chitosan depending on the pH of the medium, as their charges depend on the combination of the iso-electric point (IEP) of the proteins and the pH of the medium. If the pH is lower than the IEP, proteins and chitosan are positively charged; thus chitosan can exert its antimicrobial activity, as the interactions between both will be restricted. If the pH is higher than the IEP of the protein, the antimicrobial activity of chitosan is inhibited: proteins are negatively charged and neutralize most of the positive charges on the chitosan; thus, it cannot interact with the negatively-charged microbial surfaces [60].

NaCl reduces the antimicrobial activity of chitosan, because it interferes with the electrostatic forces between chitosan: Cl− ions neutralize the positive charges on the chitosan, and on the other hand, the Na<sup>+</sup> ions compete with chitosan for the negative charges on the cell surface. Devlieghere *et al.* [60] observed an improved solubility of chitosan by adding NaCl at different concentrations to the medium. The authors explained that this behavior was probably due to a shielding effect of NaCl against the positive charges, which led to a coiled structure of chitosan and less interactions with components in the media. Finally, the influence of fat on the antimicrobial activity of chitosan was found negligible.

The addition of the essential oils to enhance chitosan antimicrobial action is common.

The active agents embedded into composite films may be released into or absorb substances from the packaged food or its surrounding environment. The interactions that can occur between the food product, the film or coating used as packaging and the surrounding environment are governed by different mass transfer processes: migration, adsorption, absorption and permeation [66]. For example, through these processes, the essential oils included in the coating could be transferred to the product and modify its organoleptic and nutritional characteristics by interacting with peptides, vitamins, *etc.* It is recognized that some important food components, such as vitamins, minerals and other nutrients, can also be sequestered with the consequent modification of food properties and functionality. In addition, when transferred from the coating to the food to pursue their protective action (antioxidants, antimicrobials, *etc.*), these compounds could also lose their effectiveness. It is, in fact, well recognized that generally, the efficacy of various antimicrobial compounds may be reduced by food components; for example, essential oils (EOs) and/or their components have a significant antimicrobial activity *in vitro*, but higher amounts are required (1%–3%) to achieve the same effect in foods. The presence of fat, protein, carbohydrate, water, salt, antioxidants, preservatives, other additives and pH reaction strongly influence the effectiveness of the natural compounds in foods.

High levels of fat and/or protein in foodstuffs protect bacteria from the action of EOs in some way. Some authors suggested that the fat provides a protective layer around the bacteria, or the lipid fraction could absorb the antimicrobial agent, thus decreasing its concentration and effectiveness in the aqueous phase [67]. Patrignani *et al.* [68] reported that the low fat content of vegetables may contribute to the successful use of EOs. Due to their lipophilic nature, EOs could share fat, missing the microbial target.

The pH is an important factor affecting the activity of oils. At a low pH, the hydrophobicity of some EOs increases due to their ability to dissolve more easily in the lipid phase of the bacterial membrane, thus enhancing the antimicrobial action.

Carbohydrates in foods do not protect bacteria from this action, whilst high water and/or salt level seems to facilitate the action of EOs.

The antibacterial activity of chitosan-based films combined with lemon (LO), tea tree (TTO) or bergamot essential oils (BO) was tested against two Gram-positive bacteria (*Staphylococcus aureus* and *L. monocytogenes*) and one Gram-negative bacteria (*Escherichia coli*) [69]. CH-EO composite films exhibited a significant antimicrobial activity against the three pathogens tested. The nature and concentration of

the EOs, the film matrices and the interactions between CH and EOs affected the antimicrobial activity of the films. When TTO was added, CH exhibited the highest antimicrobial activity.

Chitosan-cinnamon oil coating extended the shelf-life of sweet pepper: after storage at 8 ◦C for 35 days, samples treated with chitosan-oil coatings showed a lower percentage of infected peppers and good sensory acceptability [70]. Sessa *et al.* [71] studied a novel approach to preserve vegetable products through modified chitosan edible coatings containing nanoemulsified natural antimicrobial compounds (lemon, mandarin, oregano or clove essential oils). A modified chitosan edible coating combined with lemon essential oil resulted in a remarkable increase in antimicrobial activity, with respect to other essential oils. This combination prolonged the shelf-life of rucola leaves from 3 to7 days, in comparison to the untreated samples.

Moreover, the modified chitosan containing the nanoemulsified antimicrobial caused a significantly longer shelf-life also in comparison to a coating made of modified chitosan or essential oil alone. Thanks to this novel treatment, it was possible to prolong the shelf life of rucola leaf vegetables to about 10–14 days, without alteration of the organoleptic properties of the product, preventing the loss of firmness and color changes and preserving palatability during storage.

More recently, Randazzo *et al.* [72] used chitosan-based and methylcellulose-based films added to several EOs derived from citrus fruits (orange, mandarin and lemon) to perform the antilisterial assay and concluded that chitosan films containing essential oil from lemon were the most effective at reducing *L. monocytogenes* counts.

A chitosan-methyl cellulose-based film was used as a coating on cantaloupe fruit. The application of a chitosan (1.5% w/v)/methyl cellulose (0.5% w/v) film on fresh-cut cantaloupe reduced populations of *E. coli* inoculated on fresh-cut cantaloupe by more than 5 log CFU/piece in 8 days at 10 ◦C. Furthermore, a reduction of 3 log CFU/piece of *Saccharomyces cerevisiae* in 4 days of storage at 10 ◦C was reported [73].

Krasaekoopt and Mabumrung [74] observed that the incorporation of 1.5% and 2% chitosan in the methylcellulose coating, applied on fresh-cut cantaloupe, produced a better microbiological quality in the final product. This coating reduced the growth of mesophilic aerobes, psychrotrophs, lactic acid bacteria, yeast and molds and prevented the multiplication of *E. coli* and *Salmonella* strains.
