Chitosan (CHI) of medium molecular weight (deacetylation degree 75–85%, batch 03318AJ) was obtained from Sigma-Aldrich Co. (St. Louis, USA). Origanum vulgare L. essential oil (OVEO) (Batch ORE001 density at 20°C: 0.90, refractive index at 20°C: 1.47) was supplied by Aromalândia Ind. Com. Ltda. (Minas Gerais, Brazil). The GC–MS analysis of the OVEO was performed following previously described procedures (Targino et al., 2016). A total of 22 different constituents were identified in the OVEO. The constituents present in the highest amounts in OVEO were carvacrol (64.42%), σ-cymol (12.50%), and γ-terpinene (6.78%). Other constituents, such as thymol (3.82%), linalol (2.85%), and α-pinene (1.69%) were found in minor amounts (Supplementary Table 1).

Mature, commercially available cherry tomato (Lycopersicon esculentum L.) fruit were purchased from EMPASA (Supplies and Services Company of Paraiba, João Pessoa, Brazil). Fruit of similar shape and color and without signs of fungal infection or mechanical damage were selected for the experiments. Prior to the tests, the cherry tomato fruit were disinfected via immersion in a sodium hypochlorite solution (1 mL/100 mL; pH 7.2) for 5 min, rinsed with sterile water and dried for 2.5 h in a Class II safety cabinet (de Sousa et al., 2013).

R. stolonifer URM 3728 and A. niger URM5162 were obtained from the Mycology Culture Collection of the Federal University of Pernambuco, Recife, Brazil. Stock cultures were grown in Sabouraud agar (Himedia, India) at 25°C for 7 days to allow for sufficient sporulation. Spores were collected in a sterile saline solution (0.85 g/100 mL NaCl), and the suspension was filtered through triple gauze to remove hyphal fragments. The number of spores was determined with a hemocytometer and the spore count was adjusted with a sterile saline to yield an inoculum of approximately 10⁶ spores/mL (Guerra et al., 2015).

Prior to the assays, each test fungal strain was tested for its capability to cause dark mold or soft rot infection in cherry tomato fruit by looking for a characteristic set of symptoms. After black mold or soft rot development, the strains were re-isolated and identified with taxonomical keys. Continuous re-inoculations and re-isolations on cherry tomato fruit were carried out to maintain the pathogenicity of the test fungal strains (Plascencia-Jatomea et al., 2014; Guerra et al., 2015).

CHI solutions were obtained by dissolving the powder (16 mg/mL) in acetic acid (1 mL/100 mL) with stirring (120 rpm) at room temperature (28°C) for 24 h (Sánchez-González et al., 2011). Successive serial dilutions (1:1) were performed in Sabouraud broth (Himedia, India) to obtain solutions of different concentrations (16, 8, 4, 2, and 1 mg/mL). The pH of the CHI solutions used in the in vitro antifungal activity assays was adjusted to 5.0 by adding 1 M NaOH. OVEO solutions were obtained by dissolving the substance (80 μL/mL) in Sabouraud broth containing Tween 80 [2%, v/v (Himedia, India)] as a emulsifying agent (Santos et al., 2012), and successive dilutions (1:1) in the same broth were prepared to obtain solutions of different concentrations (20, 10, 5, 2.5, 1.25, and 0.06 μL/mL). Tween 80 at the highest final concentration tested (2%, v/v) did not interfere with the fungal growth. For the combined application, CHI was diluted in acetic acid (1 mL/100 mL) (as described above) and OVEO was added at the desired concentration. The combined solution was obtained by stirring for 18 h at room temperature (Ojagh et al., 2010).

The MIC values of CHI and OVEO against A. niger and R. stolonifer were determined by macrodilution in broth. An aliquot of 1 mL of the spore suspension (approximately 10⁶ spores/mL) was inoculated in 4 mL of double-concentrated Sabouraud broth, and 5 mL of the CHI solutions at different concentrations (16–1 mg/mL) or OVEO (20–0.03 μL/mL) were added. The mixture was incubated at 25°C for 7 days. At the end of this period, the lowest CHI or OVEO concentration that caused inhibition of visible fungal growth was considered as the MIC (Santos et al., 2012).

In vitro antifungal effects of CHI and OVEO were measured by observing the inhibition of radial mycelial growth, spore germination and sporulation of the test fungal strains. The inhibition of the radial mycelial growth of A. niger and R. stolonifer was determined using the poisoned solid substrate technique. For this method, 2-mm disks taken from a 7-days fungal culture (grown on Sabouraud agar at 28°C) were placed on the center of Petri dishes containing Sabouraud supplemented with only CHI (4 mg/mL) or OVEO (1.25 μL/mL) or CHI (4 mg/mL) combined with OVEO (5, 2.5, and 1.25 μL/mL). The material was incubated at 28°C for 7 days and the radial mycelial growth (mm) was measured using calipers every 24 h over a 72 h period. Cultures cultivated in growth media without addition of CHI or OVEO were similarly tested (controls). The results were expressed as inhibition rates (percentage) of mycelial radial growth (mm) relative to the control (de Oliveira et al., 2014).

To assess the effects on spore germination, 0.1 mL aliquots of the A. niger or R. stolonifer spore suspensions (10⁶ spores/mL) obtained from a 7-day-old culture cultivated on Sabouraud agar at 28°C were placed in tubes with 0.1 mL of the solution containing CHI (4 mg/mL) or OVEO (1.25 μL/mL) or CHI (4 mg/mL) combined with OVEO (5, 2.5, and 1.25 μL/mL). The mixture (0.1 mL) was placed on a glass slide and statically incubated in a moist chamber for 24 h at 28°C. The slides were treated with lactophenol cotton blue stain and observed under the light microscope. On each slide, 200 spores were counted. As controls, spore suspensions obtained from fungal cultures grown in media without the addition of CHI and/or OVEO were similarly assayed. The results were expressed as the percent inhibition of spore germination by comparing the number of germinated spores in the media supplemented with CHI and/or OVEO with those obtained in the control assay (Guerra et al., 2015).

Sporulation by A. niger and R. stolonifer was evaluated according to a previously described procedure (de Sousa et al., 2013). The fungi were grown for 7 days at 25°C on Sabouraud agar or under the same conditions in Sabouraud agar supplemented with CHI (4 mg/mL) or OVEO (1.25 μL/mL) or CHI (4 mg/mL) combined with OVEO (5, 2.5, and 1.25 μL/mL). From each Petri dish containing the mycelium, three 8 mm-mycelium plugs were taken from the central and peripheral regions using a copper awl. The plugs were transferred to individual test tubes containing 10 mL of a mixture (1:1) of saline (NaCl 0.89% w/v) and Tween 80 solution (0.1% v/v). After vigorous shaking of the mixtures, the spores were counted in a Neubauer chamber. As a control, mycelium plugs not exposed to CHI and/or OVEO were similarly assayed. The results were expressed as the number of total spores relative to the control treatment.

The fruit (n = 80) was first immersed in an inoculum solution (10⁶ spores/mL) of A. niger or R. stolonifer for 1 min and then placed in a Class II biosafety cabinet for 1 h (25°C). Then, the fruit were immersed in 500 mL of a solutions composed of only CHI (4 mg/mL) or OVEO (1.25 μg/mL) or CHI (4 mg/mL) combined with OVEO (1.25 μg/mL), with mild shaking for 1 min. The fruit were air-dried and stored in a polyethylene container. One fruit group was stored at 25°C (room temperature), and the other at 12°C—the temperature commonly applied during transportation and market storage of cherry tomato fruit (Li et al., 2016). In the control experiment, CHI and OVEO solutions were replaced with sterile distilled water. Each treatment included 40 fruit. Fruits stored at room temperature were sampled at 1, 4, 8, and 12 days; those stored at cold temperature were sampled at 1, 6, 12, 18, and 24 days. The fruit were examined for characteristic signs of Rhizopus soft rot and Aspergillus black mold (Feng and Zeng, 2007; Liu et al., 2007). The results are expressed as the storage time needed for visible signs of fungal infection to appear and the disease incidence (percent of infected fruit) at the assessed time intervals (Guerra et al., 2015).

The uncoated or coated cherry tomato fruit were evaluated for weight loss, firmness, total soluble solids (TSS), titratable acidity (TA), lycopene content and color at the same time intervals evaluated for fungal infection. To determine the weight loss of the cherry tomato fruit during storage, the fruit weight loss during each assessed period was calculated as a percentage of the initial weight (de Oliveira et al., 2014). The firmness was measured in a TA-XT2 Texture Analyzer (Stable Micro Systems, United Kingdom) using a 3 mm diameter probe (1/8). The results were expressed as N/mm (Guerra et al., 2015). Prior to the TSS, TA and lycopene analysis, 10 fruit from each group were randomly chosen, macerated and homogenized to remove the required amount of sample for each experiment. The TSS was determined using a digital refractometer (HI96801, Hanna Instruments, São Paulo, Brazil), and the results were expressed as °Brix (Ali et al., 2010). The TA was determined via titration with 0.1 N NaOH to the phenolphthalein-end point, and the results are expressed as a percent of anhydrous citric acid (Aquino et al., 2015). The peel color was measured at three different equatorial points of the fruit using the CIELab system (L^* a^* b^*). The chroma (C^*ab) and hue angle (h^*ab) were measured in a Minolta colorimeter (CR-300 Co., Osaka, Japan) using a 10-mm quartz cuvette according to the International Commission on Illumination (CIE, 1986). A D65 illuminant (standard daylight) at a 10° angle was used for the CIELab color scale (L^*a^*b^*). Lycopene content in the cherry tomato fruit was determined according to procedures described by Li et al. (2016). Lycopene was extracted using a mixture of hexane:acetone:ethanol (2:1:1). The lycopene content of cherry tomato fruit was measured spectrophotometrically at 472 nm using an extinction coefficient (E%) of 3450. The results were expressed in mg/100 g of fruit.

Sugars and organic acids in the cherry tomato fruit were determined at the 1st and 12th day of storage at room temperature or at the 1st, 12th, and 24th days of storage at cold temperature. From the fruit-macerate obtained for the physicochemical analyses, an aliquot of 2 g was homogenized in ultra-pure water (10 mL) for 10 min using a mini-Turrax apparatus. The mixture was centrifuged (4000 × g, 15 min, 4°C), and the supernatant was collected and filtered through a 0.45 mm membrane. Sugars (xylose, glucose, fructose and sucrose) and organic acids (ascorbic, acetic, citric, formic, lactic, malic, propionic and succinic acids) were measured in the extract by HPLC using a Varian Waters chromatograph (model 2690, Varian, California, USA) equipped with a binary solvent system “valve Rheodyne” with a 20-μL loop at a temperature of 65°C, coupled with a diode array detector (Varian 330) at wavelengths of 220–275 nm, a pumping system with high pressure gradient setting (VARIAN 230) and GALAXIE Chromatography Data System processing software. The other analytic conditions were as follows: Agilent Hi-Plex H column (7.7 × 300 mm, 8 μm); mobile phase 0.009 H₂SO₄; and flow rate, 0.7 mL/min. The HPLC sample peaks were identified by comparing their retention times with those of organic acid and sugar standards (Sigma Aldrich®). The results were expressed in mg per 100 g of fresh weight (Zeppa et al., 2001).

Phenolic compounds in the cherry tomato fruit were determined at the same storage time intervals as the sugar and organic acid analyses. An aliquot of 5 g was homogenized in methanol (50 mL) for 4 h, stirred at room temperature on a shaker table, filtered through Whatman no. 4 paper, and dried by evaporation. The phenolic extracts obtained in this way were then dissolved in water. Phenolic acids and flavonoids were measured using a Shimadzu Prominence LG2OAT HPLC equipped with a photodiode array detector (SPDM2O) and a reversed-phase column (Shimpack CLCODS, 4.6 mm × 250 mm × 5 mm). For the benzoic and cinnamic acid derivatives (2,4-dihydroxybenzoic, 3,4-dihydroxybenzoic, ferulic, ellagic, trans-cinnamic, p-coumaric, syringic), the mobile phase consisted of a mixture of 2% aqueous acetic acid in water (A) and acetonitrile: methanol (2:1) (B) at a flow rate of 1 mL/min. A gradient elution was used, starting with 20% B up to 15 min, 30% B at 20 min, 40% B at 30 min and isocratic at 40% B up to 45 min. The flavonoids (myricetin, quercetin, catechin, rutin, kaempferol, hesperetin, naringenin, chrysin) were separated using a mobile phase consisting of 1% aqueous acetic acid (A) and methanol (B) at a flow rate of 1 mL/min. The mobile phase was delivered using the following solvent gradient: 0–3 min 40% B; 5–15 min 45% B; 17–25 min 50% B; 27–35 min 55% B; and 35–40 min 70% B. The injection volume was 10 μL. The identification and quantification of phenolic compounds was based on the retention times, the UV spectra and a chromatographic comparison (co-injection) with authentic markers from Sigma Aldrich®. The results were expressed as changes in mg per g of fresh weight during the storage time (Borges et al., 2011).

Uncoated or coated cherry tomato fruit were subjected to sensory tests at the 1st, 6th, and 12th days of the cold temperature storage (to ensure the microbiological safety of the samples offered to the panelists). The acceptance and preference tests were performed with 100 untrained panelists after approval from an Ethics Research Committee (protocol 712.884/2014). The tests were performed in individual booths under controlled temperature and lighting conditions. Each panelist received the uncoated or coated cherry tomato fruit served on white plates codified with a three-digit number in a blinded and random sequence. The panelists were advised to eat a salty biscuit and drink water between samples to avoid aftertaste interference. For the acceptance of attributes (appearance, flavor, texture, color, aftertaste and overall evaluation), a 9-point structured hedonic scale was used, ranging from one “dislike very much” to nine “like very much.” For the preference test, the panelists were oriented to select the most and least appreciated samples considering the overall evaluation. The purchase intent was assessed using a 5-point structured hedonic scale ranging from one “certainly would not buy” to five “certainly would buy” (de Sousa et al., 2013).

All analyses were performed in triplicate on three different occasions. The data were analyzed using Sigma Stat software 2.03 (Jandel Scientific Software, San Jose, California). The results were evaluated by ANOVA followed by Tukey's post-hoc test or Student's t-test. The results were presented as the means ± standard deviation, and a probability p ≤ 0.05 was considered significant.

CHI and OVEO exhibited MICs of 8 mg/mL and 10 μL/mL, respectively, against both R. stolonifer and A. niger. CHI was tested at 4 mg/mL (1/2 MIC) in inhibition assays of mycelial growth, spore germination and sporulation because this is the lowest concentration needed to form a viscous solution with coating features for application in cherry tomato fruit (Guerra et al., 2015).

The incorporation of CHI at 4 mg/mL combined with OVEO at 5, 2.5, or 1.25 μL/mL, or OVEO at 10 μL/mL in growth media resulted in a fungicidal effect toward R. stolonifer and A. niger with the complete inhibition of fungal mycelial growth throughout the 48 h-incubation time. During the same period of incubation, the incorporation of only CHI at 4 mg/mL or OVEO at 1.25 μL/mL in growth media inhibited less than 50% of the mycelial growth of both R. stolonifer and A. niger.

The application of CHI at 4 mg/mL combined with OVEO at 5, 2.5, or 1.25 μL/mL strongly inhibited the spore germination of R. stolonifer (75–84%) and A. niger (90–100%). Lower inhibition (p ≤ 0.05) of the spore germination of R. stolonifer or A. niger was observed when assays were performed with only CHI at 4 mg/mL (≤50%) or OVEO at 1.25 μL/mL (≤30%) (Supplementary Table 2).

The sporulation of the tested fungal strains was completely inhibited by CHI at 4 mg/mL combined with OVEO at 5, 2.5, or 1.25 μL/mL. Similar to the results observed in the assays of mycelial growth inhibition, there were no differences (p > 0.05) in the inhibition of the spore germination or the sporulation of both fungi induced by the combination of CHI with OVEO at the different tested concentrations (Supplementary Table 2).

Considering that CHI combined with the different concentrations of OVEO showed similar in vitro antifungal effects against both A. niger and R. stolonifer, the assays with cherry tomato fruit were performed with a CHI-coating containing OVEO at 1.25 μL/mL, the lowest OVEO concentration assayed.

Cherry tomato fruit artificially contaminated and coated with CHI-OVEO presented visible signs of soft rot after 8 days of storage at room temperature, and after 21 days of storage at cold temperature (Table 1). The first visible signs of black mold on fruit coated with CHI-OVEO were observed after 8 and 15 days of storage at room and cold storage temperature, respectively. Uncoated cherry tomato fruit or coated fruit with only CHI or OVEO presented the first visible signs of black mold and soft rot in a shorter period of storage at room or cold temperatures (Table 1).

The incidence of black mold on cherry tomato fruit coated with CHI-OVEO at the end of the storage at room and cold temperature was 19 and 10%, respectively; whereas for the uncoated fruit the incidence was greater than 85% (Table 1). At the end of the storage time at room and cold temperatures, the incidence of soft rot on cherry tomato fruit coated with CHI-OVEO was 12 and 5%, respectively. In uncoated cherry tomato fruit, the incidence of soft rot was greater than 87% in fruit stored at room or cold temperature (Table 1). The incidence of black mold or soft rot on fruit coated with only CHI or OVEO was ≥50% for storage at either room or cold temperature (Table 1).

The physicochemical parameters of uncoated and coated cherry tomato fruit were evaluated during storage at room (25°C) and cold temperatures (12°C). The weight loss of the cherry tomato fruit coated with CHI-OVEO was lower (p ≤ 0.05) than that observed for uncoated fruit or fruit coated with only CHI or OVEO (Table 2).

The firmness decreased (p ≤ 0.05) with increasing storage time in coated and uncoated cherry tomato fruit. The firmness of fruit coated with CHI-OVEO was higher (p ≤ 0.05) in all assessed storage time points when compared with that of uncoated fruit or fruit coated with only CHI or OVEO (Table 2).

The TA and TSS decreased in uncoated fruit (p ≤ 0.05) stored at both room and cold temperatures, whereas no changes (p > 0.05) were observed in TA or TSS in fruit coated with CHI-OVEO (Table 2).

The content of lycopene decreased (p ≤ 0.05) in uncoated and coated fruit during storage at room or cold temperature. However, cherry tomato fruit coated with CHI-OVEO showed a minor (p ≤ 0.05) decrease of lycopene when compared with the other fruit groups (Table 2).

The uncoated and coated cherry tomato fruit were predominantly red throughout storage at room and cold temperatures (Supplementary Table 3). The a^* values decreased (p ≤ 0.05) over time in uncoated fruit stored at both temperatures, whereas they did not change in fruit coated with CHI-OVEO. The b^* values decreased (p ≤ 0.05) in uncoated and coated fruit during storage at room and cold temperature. The lightness values (L^* value) were higher (p ≤ 0.05) in fruit coated with CHI-OVEO or those coated only with CHI at all-time intervals assessed in both room and cold temperature storage (Supplementary Table 3).

The sugar present in the highest (p ≤ 0.05) amounts in uncoated or coated cherry tomato fruit was xylose (Figure 1). This sugar decreased in all fruit groups during storage at room or cold temperature; however, the greater (p ≤ 0.05) decrease was observed in uncoated fruit compared with fruit coated with CHI-OVEO.

Cherry tomato fruit coated with CHI-OVEO exhibited an increase in the amount of glucose (p ≤ 0.05) during storage at room and cold temperatures, whereas the amount of this sugar in other fruit group decreased (p ≤ 0.05) (Figure 1). No changes in the amount of fructose (p > 0.05) were observed in fruit coated with CHI-OVEO stored at room or cold temperature. Uncoated fruit or fruit coated with only CHI or OVEO stored at both temperatures showed decreases (p ≤ 0.05) in fructose contents (Figure 1). Sucrose amounts decreased (p ≤ 0.05) in all fruit groups during storage at both room and cold temperatures (Figure 1).

Ascorbic and citric acids were the organic acids detected in highest (p ≤ 0.05) amounts in all fruit groups stored at both room and cold temperatures (Figures 2, 3). The decrease (p ≤ 0.05) in ascorbic acid was greater in uncoated fruit or fruit coated with only OVEO or CHI than that in fruit coated with CHI-OVEO during storage at both room and cold temperature (Figures 2, 3). The amounts of citric acid decreased (p ≤ 0.05) in all fruit groups during cold storage, whereas it increased (p ≤ 0.05) in fruit coated with CHI-OVEO during storage at room temperature (Figures 2, 3).

During storage at both room temperature or cold temperatures, pyruvic, succinic, propionic and lactic acids amounts decreased (p ≤ 0.05) in uncoated cherry tomato fruit and in fruit coated with only CHI or OVEO. No changes in the amounts of these organic acids were observed in fruit coated with CHI-OVEO stored at room or cold temperatures (Figures 2, 3). The increases observed in the amounts of formic and acetic acids were higher (p ≤ 0.05) in uncoated fruit than those detected in fruit coated with CHI-OVEO during storage at room or cold temperature (Figures 2, 3).

During storage at room and cold temperatures, uncoated and coated fruit exhibited decreases in the amount of 2.5-dihydroxybenzoic acid. A minor decrease (p ≤ 0.05) in the amount of this phenolic acid was observed in fruit coated with CHI-OVEO (Table 3). The amount of 4-dihydroxybenzoic, salicylic, syringic and caffeic acids increased (p ≤ 0.05) in all fruit groups during storage at both temperatures (Table 3). However, cherry tomato fruit coated with CHI-OVEO always exhibited the most significant increase (p ≤ 0.05) in the levels of these compounds (Table 3). A decrease (p ≤ 0.05) of sinapinic acid and the increase (p ≤ 0.05) of p-coumaric and trans-cinnamic acids amounts were similar (p > 0.05) among the fruit groups (Table 3). Vanillic acid amounts increased (p ≤ 0.05) only in fruit coated with CHI-OVEO during storage at room or cold temperature (Table 3). Ellagic acid amounts decreased (p ≤ 0.05) in uncoated fruit and fruit coated with only CHI or OVEO stored at both temperatures. No changes (p > 0.05) in the amount of this acid were observed in fruit coated with CHI-OVEO (Table 3).

Regarding flavonoids, rutin amounts decreased (p ≤ 0.05) in uncoated fruit or fruit coated with only CHI or OVEO stored at room or cold temperatures; however, they did not change (p > 0.05) in fruit coated with CHI-OVEO (Table 3). Catechin and myricetin amounts increased in all fruit groups during storage at both temperatures; however, the greatest increases (p ≤ 0.05) occurred in fruit coated with CHI-OVEO (Table 3).

The uncoated and coated cherry tomato fruit received similar scores (p > 0.05) for flavor, texture, aftertaste and overall evaluation at the different storage times assessed (Supplementary Table 4). The scores fell between “liked slightly” or “liked moderately” for all sensory parameters tested. Fruit coated with CHI-OVEO received higher scores (p ≤ 0.05) for appearance, taste and color (corresponding to “liked very much”) in tests performed after 6 and 12 days of storage. When the panelists were asked to indicate the intent to purchase the cherry tomato fruit, the responses were generally “possibly purchase” for all fruit groups and assessed storage periods.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.