1. Introduction
The tomato (
Solanum lycopersicum L.) is one of the many essential vegetables around the world, with a production of about 163 million tons per year and a high content of nutritious molecules including vitamin C, and E, β-carotene, lycopene, thiamin, riboflavin, and niacin, among others [
1,
2,
3].
However, the high production brings quality issues, especially in the postharvest stage, where tomato decay is a significant challenge in most developing countries since it is a very high perishable crop as a result of its high moisture content [
4,
5]. Developing countries also find severe problems in the postharvest tomato. Up to 30% of the tomato harvested crop may be lost during postharvest handling, mainly due to microbiological deterioration caused by fungus-like
Rhizopus stolonifer,
Alternaria alternata, and
Botrytis cinerea [
6,
7,
8,
9]. Some figures even account for 55% of losses of the total harvestable tomato per year, such as in the Australian market, for example [
10].
Colombia is not an exception, where the tomato production accounts for a total cultivated area of 4500 hectares. However, 50% of the harvesting tomato is lost because of fungal decay in postharvest stages [
11]. Fungicides are typically used to prevent fungal infection, like iprodione (Rovral), dichloran, fludioxonil, and fenhexamid, which eventually degrade into toxic compounds and generate pollution in the environment, complications in human health, and ultimately, resistant fungal strains [
12,
13]. Alternative strategies for fungal decay are proposed, like ozone (O
3), modified atmosphere packaging (MAP), ultraviolet-C (UV-C) light, gamma irradiation, and bioactive natural compounds [
14]. However, the uses of ultraviolet or gamma irradiation have grave concerns for human health, while ozone introduction is still costly.
A safer, cheaper, and environmentally friendly approach is found in the application of edible coatings to the surface of fruits. Usually, the layers are prepared from natural biopolymers such as polysaccharides and natural ingredients, taking advantage of their packaged structure based on a hydrogen-bonding network with an improved barrier to oxygen, moisture, and solute migration [
14,
15,
16] which makes them attractive in fruit applications.
Chitosan has been previously used as an edible coating in several fruits based on its excellent antimicrobial and biocompatibility properties [
16,
17]. However, its hydrophilic nature forces the introduction of hydrophobic compounds such as some essential oils, which also provide antioxidant, antibacterial, and antifungal properties to the food during the postharvest stage [
14,
18,
19,
20]. Although several chitosan-essential oil strategies have been reported to control fungal decay of tomatoes, many of them were applied during preharvest stages, with severe complications in the growth of the tree leaves. On the other hand, few studies have been addressed during the postharvest stage directly [
21,
22,
23,
24,
25,
26]. However, some of them were applied to cherry tomatoes, and others did not show complete fungal inhibition. For example, studies using coatings of chitosan–essential oils of (lemongrass) or
Thyme essential oil in combination with propolis were reported efficient in delaying the growth of
R. stolonifer and preserving the quality of fresh tomato (
Lycopersicon esculentum Mill.) fruit at room temperature (25 °C) storage [
25,
26]. In the same way, chitosan combination with starch demonstrated an excellent effect in weight loss and firmness conservation without microbial infection at room temperature [
27].
In recent years, chitosan-based nanoemulsions have emerged as an alternative to the conventional biofilms, presenting some advantages such as the allowance of a higher transfer area and higher reaction rates, a higher solubility, improved bioavailability, optical transparency [
28]. Moreover, they can limit the non-essential reactions with other components in the case of the food applications, as well as inhibit degradation during and after consumption [
29]. Different studies present the chitosan-essential oils based nanoemulsions as an alternative to avoid the decay of fruits, with the critical advantage not to generate changes in the organoleptic conditions of the foods where they are applied [
28].
Some studies have reported the effect of chitosan-based nanoemulsions incorporated with nutmeg seed essential oils and Zatariamuti flora essential oil in strawberries, with thyme essential oil in avocadoes, and with lemongrass essential oil in grape berries [
30,
31,
32,
33]. In general, the emulsions presented good antimicrobial activity and physicochemical property-preservation such as color, firmness, total soluble solids, and weight in the fruits where they were applied. Regarding tomatoes, Robledo et al. [
34] reported a decrease in the Botrytis cinerea growth in cherry tomatoes with the use of chitosan–thymol essential oil-based nanoemulsion as the coating. Despite all the information published, the study of the effects in the mold and microbial spoilage in postharvest stage on tomato var. “chonto,” as well as the impact on the postharvest quality, of chitosan-
Ruta graveolens essential oil (RGEO) coatings have not been reported yet.
The proposed study represents an excellent option to complement the antimicrobial activity of chitosan and extend the postharvest stability of tomato “chonto” under refrigeration conditions, improving the stability of tomatoes during 12 days of storage. Based on the vigorous antifungal activity of some RGEO components, the efficiency of CS + RGEO to increase the stability of fruits has been demonstrated by our group in guavas to control
Colletotrichum gloesporioides fungi growth and quality aspects [
16], cape gooseberries for microbial and quality assessment, and papayas [
35,
36,
37]. This is the first time that the application of CS + RGEO coatings is reported in Tomato var. “chonto” (
Solanum lycopersicum L.) to evaluate the effect in quality aspects and as a postharvest strategy. The study could be beneficial for farmers and producers in Colombia and developing countries, promoting their exportation capacity around the world.
4. Discussion
The present study used chitosan of medium molecular weight, taking advantage of excellent film-forming properties, superior mechanical characteristics, improved gas barrier, lesser flavor and aroma loss, and higher humidity resistance capacity than chitosan of low molecular weight [
35]. On the other hand, despite the controversy that chitosan of low molecular weight presents better antimicrobial activity due to electrostatic interactions with cell membranes of the microorganisms [
54], the activity of chitosan-medium molecular weight has also shown excellent antimicrobial activity due to adsorption on the cell surface for Gram-positive bacteria and fungi [
55]. Therefore, to maximize the antimicrobial and barrier properties of chitosan, as well as its biocompatibility, it was combined with the high antifungal and hydrophobic power derived from the terpenoid and ketone-type components of RGEO [
16,
37,
56,
57,
58,
59].
Preparation of stable and useful coatings usually is achieved using materials that are easily dissolved in water, while some additives are emulsified (like plasticizers and stabilizing agents) using surfactants, which in turn decrease the fruit ripening [
60]. In this study, the emulsions presented excellent stability without any separation phenomena when they were observed after six months. Regarding the viscosity of the emulsions, chitosan at acidic pH has a cationic structure with a high viscosity, usually obtained for medium molecular weight chitosan. However, with the introduction of the RGEO, unexpectedly, the viscosity decreases. Similar results have been collected for other studies [
49]. At the pH of chitosan solutions, several electrostatic interactions between chitosan chains and the main components of the essential oil occur, decreasing the net electric charge of the solution and leading to bigger droplet sizes, as reflected by the particle size measurements [
49,
61]. The chitosan interfacial adsorption on the oil droplets leads to stabilization of the emulsion [
61,
62].
Particle sizes increased with the RGEO content since chitosan chains are adsorbed on the oil droplet surfaces, including more and more oil droplets in a bridge mode until no more chitosan chains are available, leading to some flocculation of the oil droplets [
63]. Rheology studies observed bimodal distributions of the ζ-potential because of the chitosan adsorption on the surface of some oil droplets. Regardless, some oil droplets without any chitosan adsorbed [
62]. The particle charge affects the rheology of the emulsions by electroviscous effects, for instance, altering the viscosity and the droplet sizes [
64]. CS + RGEO 1.5% does not present a total solid percentage increase in comparison with the other emulsions. This behavior is probably related to the oil evaporation (lower chitosan adsorbed on the oil interfaces lead to oil evaporation in the analysis). Moreover, oil droplets are adsorbed in the hydrophobic region of the chitosan through van der Waals interactions and hydrogen bonds between hydroxides and amines of the CS and ketones present in the oil.
The effects of CS + RGEO coatings on the physicochemical properties of tomato fruits were evaluated. A lower consumption of organic acids related to a lower pH for tomatoes coated with F4 and F5 at the end of the storage at cold temperature was observed. Other authors reported similar trends with chitosan-based coatings with pH also ranging between 4.0 and 4.6 [
65,
66]. Changes in the internal atmosphere could be the cause of the differences in pH, generally showing some correspondence with the titratable acidity. Another factor of the discrepancy of F4 and F5 on day 12 could be intrinsic variations in the composition of the evaluated fruits, which depends on edaphic–climatic (environmental) and fertilization of the fruits (cultural) aspects [
66]. Similarly, the differences between F2 and F5 on the 3rd day are related to intrinsic variations instead of treatments themselves.
In the present study, despite that no clear trends were observed for SS during the experiments, no adverse effect in the SS was observed. Some authors have attributed the variations of the SS to changes on the electrical conductivity of soils derived from fertilization processes [
66,
67,
68,
69] or due to water flow restrictions derived from osmotic pressure effects of the high electrical conductivity [
70].
On the other hand, Barreto et al. [
24] indicated, for cherry tomatoes, the absence of a total soluble solid decreasing with tomatoes coated with chitosan-
Origanum vulgare essential oil as compared to the uncoated fruits. They argued a reduction in fruit metabolism effect from the glucose and fructose levels measured. Different trends were obtained by other authors, where a decrease then follows an initial increase in the SS values [
25,
71].
A decrease of TA in the different treatments was observed, which usually occurred in the fruit ripening after organic acid consumption for the synthesis of sugars during the metabolic pathways [
24,
50]. The trend was not clear, but the reduction of the TA was lower for CS+RGEO-coated samples, indicating that the coated tomatoes (
Table 2) suffered a slowdown of the metabolism by the barrier effect of the CS + RGEO coatings against oxygen, inhibiting the oxidation of the organic acids like ascorbic acid [
51]. Similar results have been reported for other chitosan-essential oil systems in tomatoes and cherry tomatoes [
65,
66]. Usually, the decomposition of the organic acids (citric, pyruvic, lactic, among others) is used as a substrate for metabolic biochemical reactions, for ATP synthesis, or even in enzymatic reactions [
24,
70,
72]. More moderate SS content and a higher TA are consistent with a reduction of the metabolism of the organic acids or intrinsic differences of the experimental units, as stated above. However, the coating of the fruits delays the ripening process similar to other studies, which is a beneficial result to control the postharvest decay of fruits [
25,
71]. The values obtained for MI could result in a higher acceptance of the consumer since a low level of titratable acidity and high content of soluble solids produces a better taste and aroma of tomatoes [
66].
The decay index and disease damage incidence are usually due to weight loss, but in some cases, fungal colonization is observed, which also deteriorates the quality of tomatoes. From the results of the
DI measurements, CS + RGEO 0.5% (F3) could be enough to delay the decay index and the incidence of the fungal infection. It is well known that chitosan-based coating reduces free radical presence, increases the disease resistance, and for its elicitor activity, induces the production of defense-related enzymes in fruits [
73,
74]. Moreover, essential oil addition, such as cinnamon and clove oils to the chitosan coatings, improves the antioxidant capacity of the fruits by inducing defense mechanisms to the fruits [
60]. In a previous study, the preservation of the antioxidant capacity of cape gooseberries using CS + RGEO coatings was demonstrated, which also had a positive influence on the deterioration index of the fruits [
35]. The preservative effect of CS + RGEO coating might be due to a free-radical scavenging ability of the essential oil [
35]. Additionally, some authors reported an increased stimulated activity of defense enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxide dismutase (POD) in plants by the application of essentials oils [
75]. This could account for the lower decay index of the fruits, which is regulated by the concentration of reactive oxygen species (ROS) [
76]. Finally, an increased antifungal activity due to the ketone components of RGEO affecting cell membranes contributes to the preservation [
37].
The weight is a parameter crucial for consumer acceptance and could be directly related to the decay of the fruit quality and fungal infections [
16]. The decrease in weight loss percentage in F3, F4, and F5 compared with the control is indicative of coatings efficiency for delay the gas exchanges due to a semi-permeable barrier effect that is reinforced against water by the hydrophobic character of the RGEO [
24,
46,
77]. Usually, changes in fruit weight are related primarily to water loss since the loss of volatile molecules responsible for aroma and flavor is practically undetectable in weight [
78]. On the other hand, firmness loss, which is correlated with the softening of the fruit, is considered one of the most important characteristics during fruit ripening [
79]. In this regard, it is a fact that fungi take advantage of colonization of the fruit by delivering cell wall degrading enzymes (such as polygalacturonase, pectin methylesterase, and β-galactosidase) during colonization and infection [
80,
81,
82]. Usually, chitosan-essential oil-based coatings reduce transpiration, providing turgor to the fruit cells, maintaining firmness [
16]. In this study, in treatments F4 and F5, when fungal growth was not detected, less firmness loss was observed. This behavior could be due to some components of the essential oil with the ability to oxidize the fungi enzymes related to the fungal decay of fruit [
16,
37].
Additionally, fruits coated with CS + RGEO presented less color change, with an increase in the
b *, and
a * coordinates. The increase could be associated with the fact that lycopene (related to the red color) and β-carotene (compared with the orange color) achieve their concentrations peaks in the full ripening [
83]. With red color increasing in tomatoes, a decrease in the L * value was also observed, indicating the darkening of the red color. The intensification generally occurs during the ripening of the tomatoes, as is shown with the results of Δ
E. It is evident from the matrix of color differences between F1 with F3, F4, F5 that the color change above 5.39 to the control can be perceived by consumers and is associated with a higher ripening stage than coated tomatoes [
76]. The chromophore degradation molecules like lycopene could be the main reason for the loss in color attributes, which could be delayed by coatings [
27,
33]. Metabolic reactions allow the color of the fruit to increase its intensity after chlorophyll degradation and lycopene synthesis [
30]. From the results of Δ
E, it is evident that coating has a beneficial effect on the reduction of color changes in tomatoes. A color change above 5.39, which was found in the control batch, can be perceived by consumers and could be associated with a higher ripening stage of the tomatoes [
84]. The chromophore degradation molecules like lycopene could be the main reason for the loss in color attributes, which could be delayed by coatings [
25,
37]. Metabolic reactions allow the color of the fruit to increase its intensity after chlorophyll degradation and lycopene synthesis [
35].
The quality and shelf life of tomatoes and climacteric fruits usually are reduced due to their high vulnerability to spoilage microorganisms such as bacteria, molds, and yeast [
74]. Microbial spoilage in tomatoes is due primarily to fungal attacks of
Rhizopus stolonifera,
Aspergillus niger,
Penicillium expansum, and
Botrytis cinerea causing soft-rot, black, blue/green, and grey rot mold, respectively [
85]. When thin chitosan-essential oil coatings, including antimicrobial agents, are applied on the surface of the fruits, they are very active in inhibiting spoilage microbial growth, especially against fungal colonization [
14,
37,
55,
86,
87]. From the gas chromatography-mass spectrometry (CG-MS) analysis of RGEO [
16], the relative amount demonstrated that the predominant amounts in the essential oil were: 2-undecanone (42.6%), 2-nonanone (23.5%), 2-decanone (4%), 2-nonanol (3%), 2-dodecanone (2.9%), and 2-tridecanone (2.5%) as the main components, accounting for the 78.5% of the essential oil. All those components are oxygenated terpenes, with five ketones and one alcohol. Strong antibacterial and antifungal activity has been previously reported for the two main parts (2-undecanone and 2-nonanone) of the
R. graveolens essential oil [
88]. Despite this, some controversy remains on the chitosan antimicrobial effects depending on the source, molecular weight, and deacetylation degree [
14,
74]. In the present work, chitosan did not show antibacterial activity. The strong effect in the current work was directly dependent on the RGEO content. Usually, the primary mechanism considered for antimicrobial activity of chitosan depends on the electrostatic interaction between the positively-charged amino groups of chitosan and the negatively-charged carboxylate groups of bacterial cell membranes, disrupting the cell [
74]. However, the previous effect strongly depends upon the cell membrane composition. In our case, only the diffusion of the RGEO components inside the bacteria cell caused cell growth inhibition. This could be related to the capacity of the essential oil inhibiting enzymatic reactions of membrane synthesis. Moreover, essential oils also have the ability to affect permeability capacity of the membrane by bonding ergosterol, disrupting the microbial mitochondria by affecting enzyme mitochondrial bacteria and affecting the level of reactive oxygen species (ROS), which oxidizes protein, DNA, and lipids inside the cell [
14].
In this study, we found that the antifungal activity of CS+RGEO is strongly dependent on RGEO content. The antifungal mechanism of essential oils might be related to the diffusion inside the cells, affecting cell membrane synthesis, mitochondrial function, DNA destruction, cell lysis, or inhibiting the sporulation and germination of spoilage fungi [
14,
89]. Previous studies on tomatoes and cherry tomatoes account for suitable antifungal activities of different chitosan–essential oil treatments [
22,
23,
24]. Still, our present work demonstrates that low-temperature treatment, in combination with CS + RGEO treatments with a minimum of 1.0% of RGEO, were able to inhibit fungi growth on tomato fruits in situ completely.
The results of the hedonic evaluations showed low scores in flavor for F3, F4, and F5. This could be influenced by bitter herbal flavors probably caused by the essential oil presence. Higher organic acid content was created by the better barrier performance of the CS + RGEO-coated tomatoes, which simply could be removed by a washing procedure or peel removal. However, in the texture, regarding aroma and gloss attributes, there were no significant differences during the treatments, which indicates that the characteristics were preserved between the first and sixth days of storage and profiling the CS+RGEO coatings postharvest procedures for tomato var. “chonto.”